Immunofluorescence Tests: Direct and Indirect

Immunofluorescence tests
 There are two type of fluorescent antibody tests

(FAT):
Direct and
A.
B. Indirect
Immunological tech….cont
Immunofluorescence tests…cont
A. Direct fluorescent antibody tests

(Direct FAT)
+ is used to detect and identify unknown antigen
in specimens
- E.g. Viral, bacterial, and parasitic antigens
+ It is called direct test because the fluorescent
dye is attached, or labeled, directly to the
antibody.
Immunofluorescence tests…cont
+ The fluorochrome used is usually

fluorescein isothyocynate (FITC), which
gives a yellow-green fluorescence.
+ A fluorescent substance is one that, when
absorbing light of one wavelength, emits
light of another (longer) wavelength.
Procedure: Direct FAT
 A tissue or smear containing the organism

(antigen) is fixed to a glass slide and incubated
with the fluorescent antibody (antibody chemically
linked to FITC).
 It is then washed to remove unbound antibody.
 Examined by dark-field illumination under a
microscope with UV light source.
 The antigenic particles that have bound the
labeled antibodies are seen to fluoresce brightly.
Procedure: Direct FAT……cont
 Direct FAT can be used;
 To identify bacteria when the numbers are very

low,
 It may also be used to detect viruses growing in

tissue culture or tissues from infected animals
such as rabies virus in the brains of infected
animals or antigens of HIV on the surface of
infected cells.
B. Indirect Fluorescent Antibody Test
 In the IFAT, unlabelled antibody combines with

antigen and the antigen antibody complex is
detected by attaching a fluorescent-labeled
antispecies globulin to the antibody. The antibody,
therefore, is labeled indirectly. Fluorescent-labeled
antihuman globulin is used if the antibody is of
human origin.
B. IFAT………cont
 The indirect FAT is used in two main ways:
 To detect and identify unknown
antigen in specimens
 To detect antibodies in a patient's

serum using a known antigen
(microorganism).
I. Indirect FAT to detect antigen
 A slide preparation of the specimen is made and unlabelled
specific antibody is added.
 After allowing time for the antigen and antibody to

combine, the preparation is washed leaving only antibody
that has combined with the antigen on the slide.
 A fluorescent labeled anti-species globulin is added and

allowed to combine with the antibody. The excess is
washed from the slide.
 The preparation is examined by fluorescence

microscopy using the correct filters. The antigen
antibody complex will be seen fluorescing
brightly.
II. Indirect FAT to detect Antibody
 In this test, a known antigen is placed on the slide

and the patient's serum is added.
 The preparation is then washed and fluorescent-

labeled antihuman globulin is added to
demonstrate the antigen-antibody reaction. The
preparation is examined by fluorescence
microscopy using the correct filters.
Radio Immunoassay (RIA)
 One of the most sensitive techniques for detecting
antigen or antibody is RIA
 1st developed by two endocrinologist called
S.A. Berson and
Rosalyn Yalow, in 1960 to determine levels of

insulin-anti-insulin complexes in diabetics
A. Conventional RIA
• is a competitive immunologic procedure
• It measures very low concentrations of
antigens (or antibodies) by using radioactively
labeled antigens as competitors.
• Radioactive isotopes such as 3H , 14C, 35S, 30P
or 125I can be used for labeling
• Radioactive isotopes are molecules with

unstable nuclei and therefore emit radiation
spontaneously.
 It is highly sensitive method to detect low
concentration of unknown (unlabeled) antigen
 RIA is used to assay:
 Hormones,
 Drugs,
 Enzymes,
 Microbial antigens
e.g.
• hepatitis B antigen,
• carcinoembryonic and α- feto protein

antigen.
 It also used to the detection of antibody
 RIA technique utilizes three components
+ Patient antigen
the specific compound we wish to determine.
+ Labeled antigen
the same compound as above to which is attached a
radioactive label.
+ Antibody
specific for the sample and labeled antigen.
 There are two assay approaches in conventional
RIA
• Liquid phase Assay
• Solid phase Assay

 Liquid phase assay:
 The sample, labeled antigen and the specific

antibody are added to the mixture in a
solution form.
 After completion of incubation with the

ligand of interest (analyte), a bound-free
separation step is performed using different
techniques.
 Solid phase assay
In this assay,
 the specific antibody is added either in a suspension or
 the antibody is covalently bound to the inside wall of the
reaction tube.
 Separation of the bound-free fraction is realized by
centrifugation or magnetic separation followed by decanting
the supernatant or by simply pouring off the reaction mixture if
coated tube is used.
 The bound fraction is then washed adequately with appropriate
buffered wash solution and made ready for counting.
B. Immunoradiometric Assay (IRMA) (Sandwich
Immunoassay)
 Developed with the objective of solving the

problems associated with conventional RIA
 Reading assignment (Read

more about IRMA)
Western blot test (WB)
• Used as a confirmatory test
• Very specific for HIV
• Samples that give a negative result are reported
as negative
• Antibodies to only a selection of viral proteins may
yield an indeterminate Western blot
• Bands corresponding to p24 and p55 are detected
early in sero conversion followed by glycoprotein
bands
Southern blotting
 Named in E.M. Southern, who 1st described it in 1975
 Used to detect specific DNA sequences
 Used in clinical lab to detect gene mutations known to

cause disease
 Used to detect carriers of the fragile X syndrome and

sickle cell anemia for genetic counseling
Northern blotting
• Very similar with southern blotting

•
• Used to detect RNA
• Not used in clinical laboratory mostly

Agglutination tests
In district laboratories, agglutination tests are
frequently used
 because compared with other serological tests,

they are
 simpler to perform
 require no special equipment
 usually less expensive.

Agglutination tests…cont
 The fundamental and most commonly used reaction in

medical serology laboratory
 Mostly used in clinical lab to demonstrate the interaction
of antigen and antibody
 By broad definition, agglutination is simply the
clumping of cells into aggregates, often as a result
of the combination of an antibody's binding sites
with antigen binding sites of the cells
 Agglutination tests can be performed:
i. On slides or tiles
ii. In tubes
iii. In micro titration plates
i. Slide or tiles agglutination tests
Agglutination can be either
Rapid and easily performable techniques
Gives a reaction in minutes or even seconds.
Not usually as sensitive as tube or micro titration
techniques.
Specificity depends on the reagent used.
active or
passive.
Active agglutination slide tests
 Direct agglutination of bacterial antigen with its

corresponding antibody.
E.g. the slide agglutination of salmonella,

shigella or vibrio cholera using specific antibody,
or the agglutination of leptospiral antigen by
leptospiral antibodies present in a patients
serum in acute leptosprosis.
Slide agglutination tests are used:
To identify bacteria from cultures are difficult to

standardize and control.
To check auto-agglutination (false agglutination)

Passive agglutination slides and tile tests
 Specific antibody or known antigen is attached to inert

particles or cells.
 When the known antigen or antibody combines with its

corresponding-antibody or antigen in the specimens the
particles or cells are used only to show that an antigen
antibody reaction has occurred. Their role in these
reactions is therefore passive.
The substances and cells used as carriers in passive slide
agglutination test include:
 Latex particles
 Carbon particles
 Stabilized staphylococcal cells
ii. Tube agglutination tests
 In tube tests, agglutination occurs in a larger volume of
fluid and therefore in an environment that can be more
fully controlled.
 Tube tests are usually more sensitive than slide tests.
iii. Micro titration agglutination tests

 Are performed in micro iteration plates
 Now replaced several tube agglutination tests because
they are more sensitive, more economical, and easier to
perform, and usually give quicker results.
Haemagglutination inhibition antibody test
(HAI)
This technique is used:
• To detect antibodies against
• arboviruses,
• influenza viruses,
• measles, viruses, and
• rubella virus.
These viruses are able to agglutinate red cells
because they posses heaemagglutinins on their
outer surfaces.
Reverse passive haemagglutination test (RPHA)
• This technique is used
• to identify viruses that do not
haemagglutinate.
• It performed by reacting viral specimens with red
cells coated with specific viral antibody.
• If the corresponding antigen is present, the red
cells will be agglutinated.
Precipitation tests
 A precipitation reaction may be defined as

 the visible result of an antigen antibody reaction
between a soluble antigen and its antiserum.
 In addition to these two substances,

electrolytes are necessary to bring the process
to its desired conclusion and pH and
temperature of the mixture also have an effect.
 Antigen and antibody molecules are bound
together in lattice of alternate molecules if the
reaction is successful.
• Unlike agglutination rxns ppt rxn involve a small,
soluble antigen.
• When the antibody antigen rxn occurs, a few small,
soluble lattice complexes form followed by a long
period and are less visible than agglutination.
Precipitation techniques
 are used to detect and identify antigens in
 specimens
 extracts and
 cultures
 are used to detect and quantify antibodies in

serum.
 Compared with aggt. tests, ppt techniques
require more experience in their performance
and interpretation.
 Some tests have a low sensitivity.

Types of precipitation test
There are three main types of precipitin techniques:
 Tube precipitation test
 Gel diffusion tests
 Counter immunoelectrophoresis tests

Gel diffusion tests
 When an antibody and its antigen are placed in
different regions of an agar gel, they diffuse
toward each other and form an opaque band of
precipitate at the junction of their diffusion
fronts.
 When both antigen and antibody diffuse through
the agar this is referred to as double diffusion.
 When only the antigen or antibody diffuse, with

the corresponding antigen or antibody is being
contained in the agar, this is called single
diffusion.
Double gel diffusion (Ouchterlony)
 Antigen and antibody diffuse towards each other

and where they meet in optimal proportion a
visible line of precipitation forms.
 The thickness of the line of precipitation is a semi
quantitative measure of the amounts of antigen
and antibody that combine.
Example:
 Elek gel technique

 used to detect toxogenic strains of
C. diphtheria
 Biken test
 used to detect toxin- producing fecal
E. coli (ETEC)
 If two antigens are present in the solution that can
be recognized by the antibody, two precipitin bands
form independently. This results three basic
reaction patterns:
 Identity
 Non identity
 Partial identity
Single gel diffusion
 In this technique, specific antibody is incorporated
into the agar gel and wells are cut to contain the
antigen, which diffuses radially.
 A ring of precipitation forms around a well that
contains the corresponding antigen.
 The higher the concentration of antigen, the larger
the ring of precipitation will be formed.
Counter immunoelectrophoresis (CIE)
Also called
 countercurrent-electrophoresis (CEP)
 Immuno electroosmophoresis (IEOP)
 Electro immunodiffusion.
Complement fixation Tests
 In general, complement fixation tests (CFT) are
best performed in reference laboratories where
facilities exist for the careful standardization and
control of reagents, which these tests require.

Complement Fixation
 Methodology
 Ag mixed with test serum to be assayed for Ab
 Standard amount of complement is added
 Erythrocytes coated with Abs is added
 Amount of erythrocyte lysis is determined
Ag No Ag
Ag
Patient’s
serum
Ag
Tertiary binding tests
 Tertiary binding tests measure the

consequences of immune responses in vivo.
 These tests are much more complex than
primary and secondary tests but their results
reflect the practical significance of the immune
response.
 E.g. measurement of the protective effects of antibody.
Factors affecting antigen antibody reaction
 specificity
 cross reactivity
 temperature
 pH
 ionic strength
 concentration
 intermolecular specificity
Review questions
 Write the difference between precipitation and
agglutination tests
 How does the zonal reaction affects test
results?
 Write the advantage and disadvantage of
serological test compared with other laboratory
techniques for infectious disease.
Reference
1. Tizard. Immunology an introduction,4th

edition ,Saunders publishing,1994
2. Naville J. Bryant Laboratory Immunology and

Serology 3rd edition. Serological services
Ltd.Toronto,Ontario,Canada,1992
3. Mary Louise .Immunology and Serology in

Laboratory medicine 3rd edition
chapter Three
Serologic techniques
Outline
 Introduction
 Materials necessary for basic serologic tests
 Collection, preparation and preservation of
serologic al tests
 Shipment of serological specimens
 Complement inactivation
 Dilution
 Serial dilution
Learning Objective
At the end of this chapter, the students should
be able to:
1. List material and equipment for serological tests
2. Collect, preserve and prepare serological
specimens
3. Run complement inactivation procedure and
state its importance
4. Run serial dilution, determine end point and
titer.
Definition
 Complement is a group of non-immunoglobulin plasma
proteins that are sequentially activated by Ag–Ab
complexes
 Dilution is the act of making a weaker solution from a
strong solution.
 Serial dilution The systematic re-dilution of a fluid
number of times is called a Serial dilution
 Titer is the reciprocal of the highest dilution showing a
positive reaction
Materials necessary for basic
serologic tests
Glassware
 Dirty glassware easily affects serological tests.
 After using all the glassware (test tube, beaker,
pipette, etc) they should be soaked in detergent
for several hours and rinsed several times in tap
water.
 Finally, allow drying by placing in a dry oven or
dust free place. Test tubes and pipettes should
not be scratched or broken, which will interfere
with the reading of a test.
Glassware's and plastic wares
Materials necessary… cont
Types of glassware include:

 Test tubes
 Glass slides
 Serological pipette with a size of 10ml, 5ml, 2ml
and 1ml.
Constant Temperature Device

 Incubators and water baths are used in
serological tests. These materials are
electrically operated and have thermostat that
hold the temperature within the required
limits. These devices should be checked prior
to use by a thermometer.
Rotating Machine
 Rotating machines are required to facilitate
antigen antibody reactions. Such machines have
a flat plate, which rotate at a prescribed rate of
speed. A knob located on the front of the
machine controls the number of revolutions per
minute.
Collection, Preparation And Preservation
Of Specimens For Serologic tests
 Specimens that are used for serologic test

include: serum, plasma and cerebrospinal fluid.
 Serum or plasma samples could be obtained from
venous blood, which can be collected by the
laboratory personnel.
 CSF should be collected by a physician or trained
nurse.
Collection, Preparation… cont
Serum or plasma sample collection
 Collect 2-3ml of venous blood from a patient using a sterile
syringe and needle.
 If serum is required, allow the whole blood to clot at room
temperature for at least one hour,
 Centrifuge the clotted blood for 10 minutes at 2000 rpm.
 Transfer the serum to a labeled tube with a paster pipette
and rubber bulb.
 Plasma samples are obtained by treating fresh blood with
anticoagulant,
 Centrifuge and separate the supernatant.
Collection, Preparation… cont
 The specimen should be free from hemolyzed

blood.
 Finally, seal the specimen containing tube; the
tube should be labeled with full patient's
identification (age, sex, code number, etc).
 The test should be performed within hours after
sample collection, if this could not be done
preserve it at -20oc.
Shipment of serological specimens
 Most health center and clinic laboratories are

limited in the diagnostic procedures that can be
carried out and have to ship serologic specimens
to other laboratories.
 Before shipment, the following things should be
considered.
 Don't ship whole blood unless the tests to be
performed require whole blood.
 Don't inactivate serum or plasma inactivate.
Shipment of serological specimens….cont
Serum, plasma, and CSF should be handled as follows:

 Collect and process specimens under sterile
conditions.
 Ship specimens by the fastest route as soon after
collection as possible.
 Don't ship whole blood unless the test to be
performed required whole blood. Remove cells from
plasma and clot from serum before shipment.
Shipment of serological specimens….cont
 Don't inactivate serum or plasma before mailing.

 Keep the specimen and packing container in the
refrigerator until time of shipment.
 Shipment is requires several days preserve by
refrigeration in transit. First, freeze the
specimen; then pack and ship in a well-insulated
container with dry ice.
Complement inactivation
 Complement is a group of non-immunoglobulin

plasma proteins that are sequentially activated by
Ag–Ab complexes (or directly by microbial
constituents) and cause irreversible damage to
membrane of cellular target
Complement inactivation… cont
 Complement molecules circulate in the blood in

an inactive form but activation of the first
complement component sets in motion a ripple
effect. As each component is activated, it acts in
turn on the next component in a precise
sequence called complement cascade.
 Some tests need inactivated serum. Others do

not.
 Inactivation may be important since complement
promotes lysis of erythrocytes and can contribute
to false test results in tests using RBCs.
 Some complement components may also cause
false agglutination in some tests.
 Complement components can be inactivated by of

three mechanism
 Spontaneous decay
 Enzymatic degradation of C4, C3 and C5
rapidly decay
 Stoichiometric inhibition
 The complement in serum must be inactivated

usually by stoichiometric inhibition for most
serological testing.
 To inactivate complement, place tubes of serum
in hot water bath (56c) for 30min
 If the protein complement is not inactivated it will
promote lysis of the red cells and other types of
cells and can therefore produce invalid results
 Complement is also known to interfere with

certain tests for syphilis.
 Serum samples to be tested more than 4 hours
after inactivation should be reheated at 560c for
10 minutes and allowed to cool to room
temperature
Dilutions
 Dilution is the act of making a weaker solution

from a strong solution.
 Adding a diluent such as water or saline, which
contains none of the material being diluted, is
used to do this.
 Dilutions are usually expressed as 1 unit of the
final solution.
Dilutions …..cont
DILUTION TECHNIQUES
 Dilutions can be used in the laboratory to change
the concentration of the body fluids, such as
serum so that it is consistent with the range of an
assay.
 Making dilutions can also be necessary to prepare
reagents and standards.
 Dilution has two parts: diluents and solute.
 A dilution involves adding of a substance the
diluent to other substances, the solute.
 Dilutions show the relative amount of the solute in
the dilute solution.
 It is an indicator of concentration, not volume.
Dilutions …..cont
 EXPRESSION OF DILUTION
Dilution is usually expressed as:
a to b
a:b
a/b
Whereas;
a, is the volume of the original materials that was
diluted e. g. serum (solute)
b , is the total volume to which it was diluted. It

contains a and diluent b.
Dilutions …..cont
 The dilution factor is the inverse of the dilution

statement. For a 1:10 dilution, the dilution factor is 10.
For a : b dilution the dilution factor is b.
Dilutions …..cont
TECHNIQUE
 two liquids of very different compositions (density,
or surface tension) is required
 An exact volume of concentrated solute is added
to a calibrated flask or container, and then diluent
is added to the required volume.
 Adequate mixing must take place to ensure
homogeneity
Dilutions …..cont
E.g.,
if you want to prepare 1:10 dilution
 Take 1 ml solute
1st
 Take 9 ml solvent
2nd
 Then mix
Dilutions …..cont
METHOD
 Add 1-ml solute into10 ml graduated volumetric
flask and then add water up to the 10-ml mark or
graduation of the flask.
Dilutions …..cont
 When a solution is diluted with water, its concentration is
decreased and its volume is increased. But the total amount of
solute remains constant.
 Mathematical expressions of the dilutions are;
C i V i = C fV f Where,
Ci is initial concentration
Vi is initial volume.
Cf final concentration
Vf is final volume.
 Dilution increases volume but does not change the solute
Dilutions …..cont
SERIAL DILUTIONS
 The systematic re-dilution of a fluid number of
times is called a "Serial dilution".
 Serial dilutions are most commonly employed in
serological procedure to obtain quantitative
estimations of antigen or antibody content.
Dilutions …..cont
 Serial dilutions are a unique type of dilution

techniques.
 In serial dilution, all dilutions, except the 1st are
prepared from the previous dilution and all
dilutions made after the initial dilution are the
same.
Dilutions …..cont
 Serial dilutions are used to prepare sets of
standard solutions and are also used to prepare
patient's samples to analyze components that can
exist over a wide concentration range, such as
antibody titers.
 Serial dilutions must be prepared with care as
errors can be compounded during the serial
technique.
Dilutions …..cont
0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.5 0.5
0.5 0.5 0.5 0.5

0.9 0.5 0.5 0.5 0.5 0.5
initial
Tube 1 2 3 4 5 6 7 8 9 10
Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120
Dilution
factor 10 20 40 80 160 320 640 1280 2560 5120
Dilutions …..cont
An example of the serial dilution is as follows: -

 Into each of ten test tubes is measured 0.5 ml of saline 1/2 ml
of serum is placed in the 1st tube and mixed.
 Since there is 0.5 ml of serum in a total volume of 1.0 ml; a
0.5:1 or a 1:2 dilution exists in the first tube.
 Now, 0.5 ml of this solution is removed and mixed with the 0.5
ml of saline in the 2nd tube; this gives another 1:2 dilution, but
since the 0.5 ml of solution put into the 2 nd tube is already a
1:2 dilution of the serum, the dilution of serum in the 2 nd tube
is one half that of the 1st tube or 1/2 of ½ =1/4 or 1:4.
 This and, by applying the above reasoning, the dilutions of
serum are found to be (1/2)10 = 1/1024 or 1: 1024 in the 10 th
tube.
Dilutions …..cont
Class work
Q. Calculate the volume of serum
in 2nd tube and next respective
tubes?
Dilutions …..cont
Procedure of serial dilution determining the dilutions:
1. Add 0.9 ml of saline to the first tube using a 1ml pipette.
2. Add 0.5 ml of saline to the remaining 6 tubes.
3. Add 0.1 ml serum to the first tube; wipe of the pipette tip.
4. Draw up 0.5ml and allow to drain into tube 2
5. Add 0.5 ml from the first tube to the second tube and Mix it
6. Transfer 0.5 ml from second tube to third tube
7. Continue transferring 0.5 ml until the last tube is reached.
8. Remove 0.5 ml from the last tube and discard it.

Dilutions …..cont
THE TITER
 The titer (Fr. Titer = standard) may be defined as the
quantity of a substance required to produce a
reaction with a given volume of another substances
or the amount of one substances required to
correspond with a given amount of another
substances.
 It is also defined as the reciprocal of the highest
dilution showing a positive reaction (agglutination,
hemolysis, etc,).
Dilutions …..cont
 In clinical serology titer is usually referred to as a

measure of the number of antibody molecules per
unit volume of the original serum and gives and
indication of the antibody concentration in the
patient’s serum.
Dilutions …..cont
 An antibody titer of serum is the highest dilution
of serum that will give a reaction with antigen.
 For example, if the last tube showing a ratio contains
1ml. Volume, and the serum in this tube is 1 part in a
total of 640 parts, the titer is 640 units/ml of serum, or
1:640.
 Generally a maximum dilution of a specific
antibody that gives a measurable reaction with a
specific antigen; usually expressed as the respect
of that dilution is called titer
Review question
Try the following problems

 For ASO titer, tube 1 contains 0.8ml of saline,
tubes 2 to 5 contain 0.5ml of saline; 0.2ml of
serum is added to tube 1, and serial dilutions
using 0.5ml are carried out in the remaining
tubes. What is the dilution in each tube?
 Explain the shipment of specimen and complement
inactivation.
Chapter Four
Common Serologic tests for
bacterial and parasitic
infections
Outline
Introduction
The stage of syphilis
Primary syphilis
Secondary syphilis
Latent syphilis
Late (tertiary) syphilis
Immunologic manifestation
Diagnostic evaluation
Dark field microscopy
Serologic tests
Antigen of syphilis
Antibodies in syphilis
The relation between serologic tests and
stages of syphilis
Syphilis serology
 At the end of this chapter, the students
should be able to:
 Describe the etiology and sign and symptoms of
primary, secondary, latent and late (tertiary)
syphilis
 Discuss the principle and clinical applications of
the qualitative and quantitative VDRL procedure
and RPR card test
 Describe specific and non specific Treponemal
antibodies
Definition
 Primary chancre is a stage syphilis appears
at the site of inoculation, initially painless papule
that quickly erodes and becomes indurated
Introduction
 Reported in the medical literature as early as

1495.
 In 1905 it was discovered that syphiluis was
caused by a spirochete type of bacteria,
 T. pallidum (originally called spirochaeta pallida).
 The first diagnostic blood test, the Wassermann
test, was developed in 1906.
Introduction
 Syphilis is a chronic systemic disease, which
leads to lesions on the body.
 It is derived from a Greek word "syphilos"
meaning crippled, maimed (heart victim).

ORIGIN AND HISTORY
Schools of thought exist regarding the origin of
syphilis.
1. Pre-Columbian (Unitarian) theory
 Syphilis was present in Europe before the
voyage of Columbus
 probably first appeared in tropics (central
Africa) in mild form and carried to Europe by
merchants.
2. Columbian theory
 Syphilis was endemic in Haiti and was
subsequently carried to Europe by Columbus
crew.
ORIGIN AND HISTORY…
Both theories are not satisfactory
 The disease become subject of medical literature
in the closing years of the 15th century i.e. 1495
onwards
Etiology and Transmission
 Is a systematic infection caused by the spirochate
Treponema pallidium
 Transmitted by:
 Mainly Sexual contact (Venereal syphilis)
 Less commonly via the placenta (congenital
syphilis) OR
 By accidental inoculation from infectious
material
e.g. fresh blood transfusion
Etiology and Transmission…
 T. pallidum
order :- Spirochaetalis
family :- Treponemataceae
genera:- Borrelia, Treponema, Leptospira
 The genera Treponema additional 3 human

pathogens:
- T.p.pertenue =Yaws
- T.p.endemicum =endemic syphilis
- T.p.carateum =Pinta
 All are morphological identical and this diseases
are collectively known as other treponematoses.
Etiology and Transmission…
 hours to days after penetrates intact mucosa or

abraded skin travel via lymphatic's to general
circulation and disseminates throughout body (all
organs including CNS)
 Incubation period directly proportional to size of
inoculums
 Host has immune response resulting inflammation
responsible for clinical manifestation
Stages of syphilis
Infection with T. pallidium
Primary Syphilis Primary Chancre
Secondary Syphilis Secondary lesions

60%
40% Relapse Trans placental
Transmission
Latent Syphilis
Congenital Syphilis
Tertiary Syphilis Persistent Asymptomatic
Cardiovascular
Gumma Tabes Dorsalis
Syphilis
(20 Yrs)
(5 Yrs) (10-15Yrs)
Stages of syphilis…….cont
Primary chancre
 Appears at the site of inoculation, initially painless
papule that quickly erodes and becomes indurated
 appears as a hard erythematus nodule about 1cm
in diameter with regional lymph node enlarged.
 Persists for some weeks and heals
 In women it commonly develops in the vulva or
cervix
 In HIV-infected patients, may see multiple or
atypical chancres, or no primary lesion
Chancres of primary syphilis

Secondary syphilis
 Always wide spread erythematus skin about 1cm in
diameter with regional lymph node enlarged.
 Ulcerates with a clear rim
 The ulcer is painless, persists for some weeks and
heals
 In women it commonly develops in the vulva or
cervix
Secondary syphilis…. cont
 Secondary syphilis (2-8 weeks after primary
inoculation)
 Protean symptoms, may include:
 Rash (macular, maculopapular, or pustular, or
condyloma lata)
 Generalized lymphadenopathy
 Constitutional symptoms (fever, malaise,
anorexia, arthralgias, headache)
 CNS symptoms
Secondary syphilis…. cont

 Symptoms last days-weeks
 In advanced HIV infection, may be more
severe or progress more rapidly
 Distinguish from primary HIV infection
Secondary syphilis
Rash of secondary syphilis

Secondary syphilis
Rash of secondary syphilis

Secondary syphilis
Rash and ulcerations

of secondary syphilis
Tertiary syphilis
 Appears irregularly over succeeding years and may
cause series and permanent damage by means of
chronic inflammation.
 If untreated about 25% died directly by late syphilis
Tertiary syphilis….cont
3 basic forms of late syphilis

 Gumma- necrotic masses appear in skin, liver,
testes and bones
 Cardiovascular lesions- lesions on the veins,
valves and muscles of the heart.
 Neurosyphilis - meningo vascular
- general paralysis
- tabes dorsalis
–degeneration of posterior column of the spinal
cord
Tertiary syphilis….cont
 Latent syphilis: no overt signs/symptoms, though
relapse of manifestations of secondary syphilis may
occur
 Late syphilis: neurosyphilis, cardiovascular
syphilis, gummatous syphilis; or slowly progressive
disease in any organ system
Neurosyphilis
 Neurologic complications or neurosyphilis may
occur earlier or progress more rapidly in HIV-
positive patients
 Meningitis, meningovascular, or parenchymatous
disease similar in HIV-uninfected patients
 Concomitant uveitis and meningitis more
common in HIV-positive patients
 Asymptomatic neurosyphilis (CSF with elevated
protein, lymphocytosis, or positive serologic test, in
absence of symptoms): not a late complication or
manifestation
Congenital syphilis
 Pregnancy does not alter the course of syphilis in
adults
 Transmission and adverse outcomes highest with
early syphilis
 Syphilis may increase risk of perinatal HIV
transmission to infants = congenital syphilis
 Screening:
 At first prenatal visit in all women; in high-
prevalence areas or high-risk women, repeat at
28 weeks and at delivery
Morphology
 T. pallidum is seen microscopically as a closed
coiled, thin, delicate regular spiral organism
varying in length from 6 to 15m and consisting
of 8 to 24 coils.
 The width of the spiral is seldom more than
0.25.m.
 The cytoplasm is surrounded by a trilaminar
cytoplasmic member, which in turn is surrounded
by a delicate peptidoglycan layer providing some
structural rigidity.
Morphology
 A lipid rich outer membrane that contains relatively
few integral membrane proteins surrounds this layer,
although a putative surface exposed porin molecule
was recently described.
 Six Endo-flagella wind around the cell body in a space
between the inner cell wall and the outer membrane
and may be the elements responsible for motility.
 The only known natural host for T. pallidum is the
human.
Morphology…..cont
 T. pallidum can infect many animals but only

humans, higher apes, and a few laboratory
animals regularly develop syphilitic lesions.
Morphology…..cont
Morphological x-ics may be studied microscopically.
Usual methods:
 Dark field (Dark ground)
 Indian ink (Negative stain) Wet preparation

 Phase contrast microscopy
 Electron microscopy
 Silver stain Dry preparation
 Fluorescent stain (DF)
Morphology…..cont
When examined by DFM or II wet prep. T.p
appears:
 close coiled
 thin (0.25- 0.3 μm and 6-14 μm long
 8-14 regular tightly wound deep spirals
 exhibit quick and abrupt movement
 rotates relatively slowly about longitudinal

axis (like a corkscrew) sometimes bending
without loosing its regular shape
Morphology…..cont
 lengthening & shortening like spring

 their coils may be distorted when obstructed or
attached to heavier materials
 Elctronmicroscopy revealed that T.pallidum

multiplies by transverse fission = which usually
happens every 30 hours
Morphology…..cont
Morphology…..cont
METABOLISM
Nutritional requirements of cultivable treponems:
 Glucose or other fermentable CHOs- 10 source of
energy
 Multiple amino acids
 Nucleotides (purines and pyrimidines)
 Bicarbonate
 Coenzyme
 At least one exogenous fatty acid
 not recovered in blood stored at 4 0C for 48 hrs.
 May remain up to 5 days in tissue specimen
 Suspension frozen at -70 0C with glycerol/

cryoprotective agent can be kept viable for
years.
IMMUNE RESPONSE
 Infection with T. pallidum involves both CMI and
humoral immune response.
 Antigens
Wasserman Ag- phospholipid diphosphatidyl
glycerol = cardiolipin
Is a normal constituent of host tissue
 Antibodies
Wasserman Antibody=Anticardiolipin=Reagin
 Is an Ab to Ags of treponemal proteins as carriers
and cardiolipin as immunogenic determinant.
IMMUNE RESPONSE
Treponemal Antigens
From one or more pathogenic species
Shared by many/different strains, spps, sub
spps or specific to spps or sub spps
Produce anti-treponemal Abs = Abs to
components of treponems
Treponemal Antibodies could be:

- Non specific directed against proteins common
to pathogenic/non-pathgenic treponems
- Specific directed to pathogenic treponems only
Test for syphilis
1.Tests that detect the etiologic agent
2. Serologic tests for syphilis
1.Tests that detect the etiologic agent

 Dark field (Dark ground)
 Indian ink (Negative stain)
 Phase contrast microscopy
 Electron microscopy
 Silver stain
 Fluorescent stain (DF)
Test for syphilis… cont
2. Serologic tests for syphilis

 More than 200 tests developed and only few are
used currently.
 Generally grouped into TWO, based upon the
type of Ag used and Ab detected
A. Reagin tests for syphilis (Non-

treponemal/Non-specific tests)
B. Treponemal tests for syphilis (Specific tests)

Lesion
Yes No
Negative
DAF Non treponemal
Reactive Non-reactive
Positive Titer Repeat
Treat
Treponemal test
Non reactive
Reactive
False
Treat Positive
Test for Syphilis
Standard Non Standard
Non specific Specific

Treponemal and
Non treponemal
Non treponemal Treponemal
TP- complement test

Standard
Flocculation CF TPI
ELISA
TPH ABS
Kolimer EIA
Wassermann
RPCF
Western Blot
TPA agglutination
Tube Slide Card test SRTD (syphilis rapid
TP. Methylene blue tests test device)
VDRL VDRL RPR
FTA-ABs
USR
Kliane
PCT-plasma Crit
Khan
Mazzini
Hinton
DIAGNOSTIC EVALUATION
 The diagnosis of syphilis depends on clinical skills,
demonstration of microorganism in a lesion, and
serologic testing.
 A wide variety of diagnostic procedures for syphilis
is available.
Dark field microscopy
 For symptomatic patients with primary syphilis,
dark field microscopy is the test choice.
 A dark field examination is also suggested for
immediate results in cases of secondary syphilis
with a VDRL titer follow-up test.
 The purpose of all tests for syphilis is to detect in

the blood of patients with syphilis, either reagin
or specific antibodies.
 Therefore tests for syphilis are of two main types.
+ Standard non-treponemal tests: - which use either
cardioipin or lipoid extracts as antigens and detect
reagin. (E.g.,VDRL, RPR)
+ Standard treponemal tests: - which use treponemal
antigens and peak up specific antibodies. ( E.g., FTA-
ABS, and TPI)
Standard non- Treponemal Tests for Syphilis

 A non- treponemal test employs an antigen (E.g.,
cardiolipin-lecithin),
 are used to detect an antibody like substances or
“reagin” antibody,
 are not 100% specific for syphilis antibodies,
 but are highly sensitive for syphilis
 advantages of being practical, inexpensive and
widely available.
 basically of two types:
I. Flocculation (tube or slide) and
II. Complement fixation tests.
Flocculation Tests
a. Slide flocculation tests,
 needs small amount of clinical specimen and
antigen suspension
 are rapidly performed
 results are usually read microscopically
 It utilizes cardiolipin, lecithin, cholesterol
antigen and heat inactivated serum.
 Performed on slide or tube
E.g., (VDRL)
b. Tube flocculation tests

 are performed in test tubes
 requires large quantities of specimen and antigen
suspension
 are more complicated
 are read with or without magnification.
Eg.,
 Kliane flocculation Test
 Khan flocculation Test
 VDRL
 Mazzini test
 Hinton (serum) test

c. Card flocculation tests:
Rapid reagin tests

 RPR (rapid plasma reagin)
 USR- unheated serum reagin
 PCT- plasma CriT
 RPR (Teardrop) card test
 RPR (18-mm circle) card tests
II. Complement fixation Test

 Complement components are thermo labile
proteins found in normal serum.
 It is also found in other animals.
 It is destroyed at 56C for 30 minutes.
 But up on standing at 7-370C, it may regain part
of its activity.
 Therefore, previously heated serum must be
reheated for 10 min. at 560C before a test can be
performed.
Complement Fixation
Serum with Serum without Abs

Antibodies
Antigen binds Unbound Antigen

Day 1
to antibodies
Complement
Unbound Complement
binds to Ag/Ab
complex
Hemolysin sensitized Hemolysin sensitized

red blood cells RBCs serve as an
indicator
serve as an indicator
Day 2
No lysis Positive Lysis Negative

SEROLOGICAL TECHNIQUE
RPR (Rapid reagain card test for syphilis)

Principle
 Destructive syphilitic lesions cause tissue damage.
Circulating antibodies called reagain are produced
against some of the tissue components. The rapid
regain card test uses a modified form of the VDRL
(Vernal Disease Research Laboratory) antigen
called cardiolipin in suspension with carbon
particles. When cardiolipin antigen reacts with
reagain antibody in patient’s serum the carbon
particles in the suspension clump together.
Materials
 The following are provided in the test kits:
 Reagin antigen suspension
 Reagin positive control serum
 Reagin negative control serum
 Reagin test card
 Dispensing bottle and needle
 Dropper tubes
 Mixing sticks and etc
Qualitative test method
Procedure (RPR)
 Let the reagents and specimens warm up to room
temperature.
 Dispense one drop of negative control serum on to
one circle on the test card using a disposable
dropper tube.
 Repeat step two with the positive control serum
using a clean dropper tube.
 Dispense on drop of each sample serum or plasma
on to one circle on the card using a clean dropper
tube for each specimen.
Procedure (RPR)
 Spread all the drops to cover the whole area of the
circles using the mixing sticks.
 Mix with reagain antigen suspension in the
dispensing bottle. Hold the bottle vertically and
dispense one drop on to each test sample. Do not
mix again.
 Place the card on the rotor for 8 minutes at
100rpm.
Reading the results
 Negative result:
The carbon particles remain in an even

suspension = Non reactive
 Positive result:
The carbon particles clump together =

Reactive
For the test to be valid the negative control must be non-
reactive and the positive control must be reactive
Reporting results of a qualitative test
 Qualitative results should be reported as reactive
or Non-reactive
Quantitative test method
1. Dispense 1 drop of 0.85% saline on to circles 1 to
5 on the test card
2. Dispense 50µl of patient serum or plasma on to
the first circle and mix by filling and discharging
the pipette at least 6 times (do not make
bubbles). You now have a 1 in 2 dilution in the
first circle.
3. Transfer 50µl from the first circle to the second
circle and repeat the mixing procedure.
4. Continue the dilution procedure to circles 3, 4
and 5 and discard the last 50µl.
You know have the following dilutions:
Circle 1 2 3 4 5
Dilution ½ ¼ 1/8 1/16

1/32
5. Starting at circler number 5 uses a mixing stick to
spread all the drops to cover the whole area of the
circles.
6. Mix the regain antigen suspension in the dispensing

bottle. Hold the bottle vertically and dispense one
drop on to each test sample. Do not mix again.
7. Place the card on the rotor for 8 minutes at

100rpm.
Reporting the results of a quantitative test
 The titer reported in a quantitative test is the
highest sample dilution to show a reactive
(positive) result.
 In the example below the result would be
reported as:
 Reactive to 1/8
If the highest dilution (1 in 32) is reactive proceed
as follows:
8. Prepare a 1in 16 dilution of the sample by adding
0.1ml of serum to 1.5ml of 0.85% saline and mix
well.
9. Dispense 1 drop of saline on to circles 6 to 10 on
the test card using a dropper tube.
10.Dispense 1 drop of the 1 in 16 dilution on to circle
number 6 and proceed as before (step 3 and 4)
making doubling dilutions up to well number 10
You know have the following dilutions:
Circle 6 7 8 9 10
Dilution 1/32 1/64 1/128 1/256 1/512
11. starting at well number 10 spread all the drops as

before
12. Mix the reagin antigen suspension in the dispensing
bottle. Hold the bottle vertically and dispense one drop
on to each circle. Do not mix again
13. place the card on the rotor for 8 minute at
100rpm
14. report the titer as the highest dilution to show a

positive result
Limitation of the test
 The rapid regain card test is a non-specific test
for syphilis. A positive reaction indicates tissue
damage such as that caused by destructive
syphilitic lesions.
 False negative reactions can occur in the early
and later stages of syphilis when there is not a lot
of tissue damage.
 False positive reactions can occur due to other
diseases which result in tissue damage such as
malaria, leprosy, viral infections, autoimmune
diseases and many other conditions including
pregnancy.
 It is very important that reactive specimens are
checked by another test procedure which is
specific for syphilis.
 In the CPHL this is done by the Wellcosph HA test
which is specific for antibodies to T. pallidum, the
bacterium which causes syphilis.
VDRL TEST
Slide Qualitative Test
Sample: serum
Principle
 During the period of infection with syphilis,
reagin, a substance with the properties of an
antibody, appears in the serum affected patients.
Reagin has the ability to combine with a colloidal
suspension extracted from animal tissue and
clump together to form visible masses, a process
known as flocculation.
Procedure:
1. Pipette 0.05ml or 1drop of inactivated serum into
one ring of the ringed glass slide.
2. Add one-drop (1/60ml) antigen suspension onto
each serum.
3. Rotate slide for 4 minutes. (If rotated by hand on
a flat surface, this movement should roughly
circumscribed a 2 inch/5mm diameter circle).
4. Tests are read immediately after rotation
microscopically with a 10x ocular and a 10x
objective.
Reading and reporting of results
 Tests are read microscopically with low power
objective at 10x magnification, which appears
short rod forms. Aggregation of these particles
into large or small clumps is interpreted in
degrees of reactivity.
Reporting system
No clumping or very slight roughness : Non-reactive (NR)

Small clumps : Weakly reactive (WR)
Medium and large clumps : Reactive (R)
Standard Treponemal tests for syphilis
1. T. pallidum immobilization test (TPI)
2. RPCF test ( Reiter protein complement fixation test)
3. Fluorescent Treponnema antibody Absorption

test (FTA-ABS-test)
4. Treponoma pallidum Methylene Blue tests.
5. Treponomal pallidum agglutination test.
6. Treponemal pallidum complement fixation test.

1. T. pallidum immobilization test (TPI)
Principle:
Treponema pallidum immobilization test is

the most specific and extremely valuable test
for syphilis. It becomes positive after the
second week of infection. The test is however
quite complicated and relatively costy to
perform. The test, where available, is used for
reference and to rule out false-positive sero
reactors of other tests.
Method:
 Patient’s serum is placed in a test tube with living
spirochetes and complement.
 After incubation in an atmosphere free of 02, slide
preparations are made and examined by dark field
illumination.
 The spirochetes will be immobilized by syphilitic
serum but will be actively motile in normal serum.
The TPI test has its greatest value in confirming
syphilis or ruling out biological false positive
reaction.
 It requires live treponemas from infected animals
and is difficult to perform.
 It does not distinguish the various treponematoses
(i.e. yaws, pinta, bejel)
 It fails to detect early syphilis
 It cannot be used as an index of therapeutic
response.
 It is ineffective when the patient is on antibiotics.
Advantage:
 On the positive side, the test is the one of choice
for spinal fluids, especially for detecting
neurosyphilis when reagin tests give non-reactive
results.
2. RPCF test ( Reiter protein complement fixation
test)
 RPCF is a test with treponemal antigen
 is performed for the diagnosis of syphilis
 less specific and sensitive than TPI
 the test is simple to perform and quite cheap
3. Fluorescent Treponnema antibody
Absorption test (FTA-ABS-test)
 A modified form of fluorescent treponemal
antibody test (FTA-Test) with treponemal antigen
employing indirect immunofluorescence
 FTA-used for diagnosis of syphilis
 It is a specific and sensitive test
Principle
 The FTA-ABS test is a direct method of
observation. Although not recommended for
screening, it is the most sensitive serologic
procedure in the detection of primary syphilis.
 This test is recommended as a confirmatory test
for syphilis.
 It is recommended for screening test.
 A reagin test such as the VDRL or RPR is not
recommended for screening.
Stage
Primary Secondary Late
Non Treponemal (Reagin tests)
VDRL 70% 99% 1%
RPR 80% 99% 0%
Specific Treponemal test
FTA-ABS 85% 100% 95%
TPHA-TP 65% 100% 95%
TPI 50% 97% -

Non standard non treponemal and treponemal
test
 ELISA
 CAPTIA-syphilis G test
 CAPTIA-syphilis M test
 Syphilis Rapid test device
ELISA
 The ELISA immuno assays is available for both non-
treponemal and treponemal tests.
 The ELISA non-treponemal assaya uses the VDRL
antigen affixed to a micro titer plate.
 At least two different treponemal ELISA assay are
commercially available in kit form.
 These are the CAPTIA-syphilis G and the CAPTIA-
syphilis M tests (trinity Bio tech).
 CAPTIA-syphilis G test
 used to detect anti-treponemal antibody
 used to measure IgG of Treponemal infection
 CAPTIA-syphilis M test
 used to detect anti-treponemal antibodies
 This test is particularly useful for diagnosis of

congenital syphilis.
 Babies whose mothers are infected with syphilis
cannot be diagnosed using the tests that
measure IgG antibodies.
 IgM antibodies do not cross the placenta, the
identification of anti-T. pallidum antibodies in the
newborn sera indicates congenital syphilis
 Syphilis Rapid test device
 it is a rapid qualitative chromatographic immunoassay
that uses the affinity of protein A for IgG antibodies to test
for treponemal antibodies
 Protein A binds to the Fc region of most subclasses of IgG.
 One of the advantages of this test is that dilutions are not
required and the prozone phenomenon is not an issue as
it is for tests whose end points are flocculation or
agglutination.
Q. Discuss the difference between RPR and VDRL?
Next topic is Agglutination tests for Febrile diseases

4.2. Agglutination tests
for Febrile diseases
4.2.1 Salmonella
 Serologic diagnosis
4.2.2 Rickettisia
 Serologic diagnosis
Salmonella
 Are often pathogenic for humans or animals

 transmitted from animal and animal product to
humans
 Three main diseases are
 Enterocolitis (enteritis)
 Septicemia (systemic infection)
 Enteric fever (typhoid fever)
Morphology
 Microscopic
+ Vary in length
+ Gram –Ve
+ Rod shape bacilli
+ Most isolates are motile
+ has peritrichous flagella

 Culture
+ Grow readily in simple media
+ Never ferments lactose or sucrose
+ Form acid and some times gas from glucose and

mannose
+ Produce H S gas
2
 Refrigerator
+ Survive freezing in water for long periods
+ Resistant to certain chemicals
- Brilliant green
- Sodium tetrathionte
- Sodium deoxycholate
+ Such compounds are useful for inclusion in media

to isolate salmonella from feces
+ It inhibits other enteric bacteria
 Classification
 More than 2500 serotypes
 Four serotypes can cause enteric fever
+ S. paratyphi A (Serogroup A)
+ S. paratyphi B (serogroup B)
+ S. choleraesusis (serogroup C1)
+ S. typhi (serogroup D)
 Antigenic variation
+ Salmonella has
 O-antigen ……… somatic

 H-antigen……… flagellar antigen
 Vi- antigen …… capsular antigen
+ Organism may lose H antigens and become
non-motile
+ Loss of O antigen is associated with a
change from smooth to rough colony form

Pathogenesis
 Chiefly pathogenic to Source of infection
animals and reserved for •Water
human infections:
 Pigs
•Milks and dairy
 Rodents products (ice, cream,
 Cattle cheese)
 Pets
•Shellfish
 Poultry
•Dried or frozen eggs
•Meats and meat
products
 Rout of entry
+ Oral rout with contaminated food and drink
+ the mean dose to produce clinical or sub
clinical infection in humans is 105-108

(perhaps as few as 103 S. typhi organisms)
 Host factors
+ Host factors may contribute to resist
salmonella infections.
- Gastric acid
- Normal intestinal microbial flora
- Local intestinal immunity

A. The enteric fever (Typhoid fever)
 Produced by only S. typhi
 The ingested salmonella reach the small intestine
From which they enter the lymphatics
Then go to blood stream From this to many
organs including intestine The organism
multiply in intestinal lymphoid tissue And
excreta stool
 Incubation period is 10-14 days after that it
causes
+ Fever
+ Malaise
+ Headache
+ Constipation
 Fever raises to high and the spleen and liver
become enlarged
 Rose spots-usually on skin of the abdomen or
chest
 WBC is normal or low
 Some time intestinal hemorrhage and perforation
at pre-antibiotic era
 Mortality rate is 10-15%
Clinical disease induced by salmonellae
Enteric fever Septicemia Entercolitis
Incubation 7-20 days Variable 8-48 hours
period
Fever Gradual; then high Rapid raise then Usually low

plateau with spiking ‘septic’
typhoidal state temp
Duration of Several weeks Variable 2-5 days
disease
GI symptoms Often early Often none Nausea vomiting
constipation; later, diarrhoea at on
bloody diarrhea set
Blood Positive for 1st to 2nd Positive during Negative
cultures weeks of disease high fever
Stool culture Positive from 2nd Infrequently Positive soon

week on; negative positive after onset
earlier on disease
Laboratory Diagnosis
 Specimen
 Blood for Culture must be taken repeatedly
 Bone marrow cultures may be useful
 Urine cultures may be positive after the 2nd
week
 Stool specimens also must be taken repeatedly
 Bacteriologic method
1. Differential medium cultures
+ EMB
+ MacConkey agar
+ Deoxycholate medium
+ Bismuth sulfite medium
2. Selective media cultures
+ SS agar media
+ Hektoen enteric agar
+ XLD
+ Deoxycholate citrate agar
3. Enrichment cultures
+ Selenite F or
+ Tetrathionate broth
 Serological method
- Widal Test
 New serological methods
- Typhidot (better)
- Dipstick test
Widal test is a serological test widely used for diagnosis

of enteric fever.
 It is suspension of killed S. typhi as Ag. to detect
ant-S-typhi antibody
 it has many limitations as a diagnostic tool.
However, it used to carry out sero-surveys in a
community to know the endemicity of any infection
 Widal test measures titres of serum agglutinins against
somatic (O) and flagellar (H) antigens which usually
begin to appear during the 2nd week.
 In the absence of recent immunization, a high titer of
antibody to O antigen > 1:640 is suggestive but not
specific.
 While using Widal test for the diagnosis of enteric
fever, several factors need to be considered for
interpretation. For example:
+ Endemicity of enteric fever in an area of
investigation
+ Administration of antibiotics
+ Immunization with any typhoid vaccine or a

previous infection or exposure
 The stage of the disease at the time of
collection of sample.
Early in the disease low antibody titers

are found. The antibodies start rising
after 1st of illness and do so until ¾
week.
Infection with any other gram-negative

bacteria may give a false positive reaction.
To make any logical conclusion in the diagnosis
of enteric fever on the basis of widal test, one
must submit a paired serum sample.
 The 1st sample taken early in the disease
 The 2nd sample at least 2 weeks later.
Significance of Widal test
 In enteric fever endemic areas Widal test are
very important to diagnose S. typhi.
 When facilities for culturing are not available,
the Widal test if performed and interpreted with
care can be of value in the diagnosis of typhoid
fever.
 Widal test for typhoid and paratyphoid fever is
an agglutination test.
 The typhoid bacillus causes two types of
agglutinins to be produced. The agglutinins are
called:
- Flagella (H) agglutinins
- Somatic (O) agglutinins.
 The patient serum is tested for those O and H
antibodies against the antigen suspensions.
 Salmonella antigen suspensions are
commercially available from different
manufacturer.
 The antigens are stable for rapid slide (screen)
testing and tube testing.
NB: Before use, the suspensions must be allowed to

warm to room temperature and be well mixed. The
test must be adequately controlled.
 Methods of widal tests
+ Tube method
+ Slide method
Procedures
Slide method
+ Modification of tube test method by Welch and
Mickle at 1936
Specimen: serum/plasma
 Take clean slide

 Add a drop of serum, which is obtained, from non-
hemolyzed blood.
 Add a drop of antigen suspension, which is non-
expired,
 Mix well antigen suspension and serum.
 Look for agglutination.
Positive Result
Negative result
Tube method
 Used to confirm slide test method
Sample: serum/plasma
Procedure
1. For each antigen arrange 10 small test tubes in
a rack.
2. Place 0.9ml of saline in the 1st tube and 0.5ml
in the remaining 9 tubes.
3. Add 0.1ml of fresh cell-free serum to the 1st
tube.
4. Mix and transfer 0.5ml to tube
2,3,4,5,6,7,8,and tube 9. From tube 9 discard
0.5ml.
+ Tube 10 will contain only saline and will
serve as a negative control (antigen
control)
5. Mix antigens well and add 0.5ml to each tube.
Mix by gently shaking the tubes.
6. The final dilutions are 1:20, 1:40, 1:80,
1:160, etc.
7. Incubate the tubes at 37oC for 2-4 hrs
8. Read the negative control at the end of the
incubation period.
+ It should have no agglutination.
9. Read the test one row of antigen at a time. For
reading a white light shining vertically above
the tube is best and using a black background.
10. Shake the tubes gently.
The H type of agglutination is easily broken
up and may be missed. The agglutination is
more granular and not so fragile.
11. Report the highest dilution with definite clumps.
Tube dilution
0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.5
0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
initial
Tube 1 2 3 4 5 6 7 8 9 10
Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120
Dilution
factor 10 20 40 80 160 320 640 1280 2560 5120
Reporting of Widal Reactions
 The Widal test is reported by giving the titer

for both O and H antibodies.
 The antibody titer is taken as the highest

dilution of serum in which agglutination
occurs.
 The type of agglutination seen with O

reactions is granular while that seen with H
reactions. Both slide and tube tests are more
easily read against a dark background.
If no agglutination occurs the test should be reported
as:
 S. typhi O titer less than in 20 (O 1:20)
 S. typhi H titer less than in 20 (H 1:20)
Interpretation of the Widal Reaction

 A Negative test does not necessarily mean the
patient is not infected. Reaction occurs in infected
patients about
 50% during the 1st week
 80% in the 2nd week,
 90-95% in the 3rd or 4th week
 Positive reactions with O antigen occur earlier in
the disease than the reaction with the H
antigen. H antigen reactions may remain
sometimes for years.
 A positive reaction occurs after typhoid
vaccination and lasts for 1-5 years.
 In endemic regions, natural agglutinins may be
present in the serum.
 Some persons will not produce antibodies
because of other diseases.
E.g.
+ agammaglobinemia (absence of
globulin in the serum),
+ leukemia, and
+ carcinoma (malignant tumor)
 Taking antibiotics may cause a decrease in the
titer.
 No one titer can be considered diagnostic for
typhoid fever. Arise in titer over a period of time
is significant and of diagnostic value.
Causes of positive widal result
 Chronic salmonellosis, associated with
Schistosomial infection
 Vaccination with typhoid vaccine
 the patient being tested has typhoid fever
 cross-reaction with non-typhoidal Salmonella.
 variability and poorly standardized commercial
antigen preparation
 infection with malaria or other
enterobacteriaceae
 other diseases such as dengue
 Chronic liver disease
 Immunological disorder like
+ rheumatic fever,
+ multiple mayeloma,
+ nephritic syndrome, and
+ ulcerative colitis.
 Causes of negative Widal agglutination tests
 absence of infection by S typhi
 the carrier state
 an inadequate inoculums of bacterial antigen in

the host to induce antibody production
 technical difficulty or errors in the performance
of the test
 previous antibiotic treatment
 variability in the preparation of commercial

antigens
Source of error
 Hemolyzed blood
 Expired antigen suspension
 Dirty slides or tubes
 Failure in performing technique
 Failure in reading of technique or
interpretation
References
1. Neville J. Bryant (Ed.) Laboratory Immunology and Serology 3rd
Ed. 1992
2. Monica Cheesbroguh (Ed), Medical Laboratory Manual for

Tropical Countries (Vol. II)
3. Francess Talaska Fischbach (Ed), A Manual of Laboratory and

Diagnostic Tests, 2nd edition.
4. Geo. F. Brooks, Janet S. Butes, Stephen A. Morse, Medical

Microbiology 23rd edition, 2004
5. Jacqueline Stanley Essential of immunology and serology, 2002

4.2.2 Agglutination test
for rikettsianceae
Rickettsiaceae
 The genus rickettsia contains several species and sub
species.
 Although classified as bacteria, rickettsia resemble viruses
in that they are mostly obligate parasites and are unable to
survive as free living organisms.
 They are about the size of the largest viruses and can just
be seen with the light microscope.
 Rickettsia resembles bacteria also by virtue of their
morphology and microscopic visibility.
 Unlike viruses, however, rickettsia contains both
RNA and DNA, multiply by binary fission, have
cell wells that contain muramic acid, possess
enzymes, and show sensitivity to antiseptic and
antibodies.
 Species in the genus rickettsia have been sub
divided into three groups of antigenically related
microorganisms:
 typhus group
 scrub group
 spotted group
Organism Disease Host Method of transmission
R. prowazeki Louse born typhus. Man Through the feaces of louse
(Endemic typhus)
R. typhi Mourine typhus (Ende Rats Rates---Fleas/lice ----rat
mic typhus) --- Fleas/lice----human
Rats, field mice, v
R. tsutsugamushi Scrubing typhus (mite Bite of infected larvae mite
oles & quail, mite
typhus)
Opossums
R. rickettsia Spotted typhus Bite of infected tick
domestic & wild
dogs wild rabbits,
rodents.
R. conori Fever boutonneus Dog Bite of infected tick
R. siberica Asian tick typhus Dog Bite of infected tick
R. conori pjiperi African tick typhus Dog Bite of infected tick
Serologic diagnosis
 The most reliable and useful serological technique
for diagnosing rickettsial infections is the indirect
fluorescent antibody (IFA) test. It is of value not
only in diagnosing acute infections but also in
serological epidemiological studies. Another
important test is CFT test, which is not sensitive
as IFA test.
Weil-Felix reaction test
 Weil-Felix is an agglutination test for various
rickettsial infections (as typhus and tsutsugamushi
disease) using particular strains of bacteria of the
genus Proteus that have antigens in common with
the rickettsiae to be identified Weil, Edmund
(1880-1922), and Felix, Arthur (1887-1956),
Austrian bacteriologists.
 During World War I Felix served as a bacteriologist
charged with the diagnosing of typhus in the
Austrian army.
 As a result of his work he and Weil developed an
agglutination test for typhus in 1916.
 The Weil-Felix reaction test, developed by Weil
and Felix (1916), is based on the fact that certain
strains of Proteus vulgaris and P. mirabilis share
antigens with several of the rickettsia species
that produce febrile disease, such as typhus.
 Three strains of Proteus species have been found
to be useful in diagnosing rickettsial diseases;
these have been labeled OX-2, OX-19 and OX-K.
A test for diagnosis of typhus and certain other
rickettsial diseases.
The blood serum of a patient with suspected

rickettsial disease is tested against certain strains
of (OX-2, OX-19, OX-K).
The agglutination reactions, based on antigens

common to both organisms, determine the
presence and type of rickettsial infection.
 In 1915, Weil and Felix showed that serum of
patients infected with any member of the typhus
group of diseases contains agglutinins for one or
more strains of OX Proteus.
 In cases of typhus fever the reaction usually
appears before the sixth day and reaches its
height in the second week.
 Weil-Felix reaction is an agglutination test based
on the cross-reactions, which occur between
antibodies, produced in acute rickettsial infections
and the OX-19 and X-2 strains of Proteus vulgaris
and the OX-K strains of Proteus mirabilis.
 In a Weil-Felix reaction a strong agglutination
test with strain OX-19 may indicate epidemic or
endemic typhus, and a weak agglutination may
indicate Rocky Mountain spotted fever,
Mediterranean fever and South African tick fever,
while agglutination with Proteus strain OX-K
indicates scrub typhus.
Disease
Proteus group used and
Organism
degree of reaction
OX-19 OX-2 OX-K
Typhus group
Endemic typhus,Brill’s disease ++++ +/- 0
R. prowazeki
Murine typhus ++++ +/- 0
R. typhi (R. mooseri)
Scrub typhus group

0 0 ++++/-
R. tsutsugamushi Scrub typhus
(R.orientalis )
Spotted fever group:

---- +/++++ 0
R. conori +/++++
R. conoripeiperi ---- +/++++ +/++++ 0
R. siberica Asian tick fever +/++++ +/++++ 0
R. rickettsia Rocky mountain spotted fever +/++++ +/++++ 0
 The Weil-Felix reactions are non- specific and
cannot be fully relied on to diagnose acute
rickettsial infections. False negative Weil-Felix
reactions are common especially with R.
tsutsugamushi infections.
 False positive Weil-Felix reactions are may also
occurs in Proteus infections, relapsing fever,
brucellosis, rat bite fever, infectious,
mononucleosis, and other acute febrile illness.
Class activity
Discuss factors that influencing

Widal and Weil Felix reaction test.
4.2.3 Serology of streptolysisn O
and ASO
Streptolysin O (SLO)
 Streptolysin O (SLO) is a bacterial toxin produced
by virtually all strains of S. pyogenes.
 It is one of two extra cellular hemolysis (or cyto
lysins), the other being Streptolysin S (SLS).
 SLO is released during infection as indicated by
antibody production to it.
 The toxin is a protein with a molecular weight of
approximately 70,000, which in its reduced state
brings about the lysis of red and white blood cells.
Properties of Streptolysin O
 SLO is so called because of its oxygen liability,
and it is quit distinct from SLS.
 It is hemolytically in active in the oxidized form
and is characteristic of a group of cytolytic toxins
known as the oxygen labile toxins.
 Toxins are produced by several different gram
positive bacteria and possess a number of
common properties they are activated by
sulfhydry (SH) compounds.
 The addition of SLO into culture causes
hemomlysis of erythrocytes and toxic effect on
mammalian cells.
 It also cardiotoxic.
 SLO may cause interstitial carditis in
experimental animals
Importance of the Antistreptolysin O Reaction
 Streptolysin O is antigenic, eliciting the formation
of antibodies that effectively neutralize its
hemolytic action.
 Streptococcal infection particularly in cases of
rheumatic fever and glomerulonephirits
SLO SLS
 SLO is so called because of its oxygen liability.  SLS is so called because of its
 Its molecular weight is 70,000 oxygen stability.
 SLO is synthesized only by growing streptococci  It’s molecular weight is 2,800
 In oxidized form, it is hemolytically inactive.  Both growing and resting cells
 Several different gram-positive bacteria produce it. synthesize it.
 Like other oxygen labile toxins, SLO is activated by SH  SLS can be transferred
compounds and is antigenically related, and its biologic among the various carriers
activity is completely inhibited by low concentrations of and finally to the surface of
cholesterol and certain related sterols. mammalian cells.
 SLO hemolyzes erythrocytes.  SLS is inhibited by lecithin and
 It is carditoxic beta lipoproteins, but not by
 Only cells that contain membrane cholesterol are cholesterol.
susceptible to the toxin; therefore, membrane
cholesterol is the binding site of SLO.
 It is inactivated by the membrane lipid fraction that
contains cholesterol.
 The mechanism that results in cell lysis is unknown.
Tests for antistreptolysin O
 The most widely used test for SLO is the
neutralization test used to detect ASO in serum.
 ASO is important in the investigation of post-
streptococcal diseases.
 Most complications develop at a stage when it is
not possible to isolate Group-A streptococcus in
culture.
 ASO test is based on the fact that ASO can be
specifically fixed to SLO in vitro, where it will
neutralize its hemolytic activity
 The test therefore, by doubling dilution, estimates
the amount of antibody that, in the presence of a
constant dose of SLO, can completely inhibit
hemolysis of a given number of red cells.
 In the interpretation of ASO titers, many
variables, including age, the severity of the
infection, and the individuals, ability to respond
immunologically to the toxin, must be taken into
account, because no set “normal” titer has been
established.
 Todd, whose name is still used to express the
levels of antibody titer, developed the original
ASO test procedure.
 One Todd unit is that amounts of antibody that
completely neutralizes two and one-half minimal
hemolytic doses of SLO.
 Most healthy adults have ASO titers of 125 Todd
units (or less).
 Children, however, show fluctuating ASO titers
from 5 to 125 Todd units; the usual titer normally
decreases after 50 years of age.
 One IU is equivalent to 1.04 Todd units.
 Commercially available tests for the investigation
of raised ASO antibody levels are of two types:
 ASO latex slide agglutination test
 ASO micro titration or tube hemolysis tests
to titrate the antibody.
ASO latex slide agglutination test
Principle
 If polystyrene latex particles are coated with
streptolysin O antigen visible agglutination will be
exhibited in the presence of the corresponding
antistreptolysin O antibody.
 If the patient’s serum contains more than 200
lU/ml ASO antibody, the excess antibody will
agglutinate the antigen in the latex reagent.
 If no agglutination occurs the antibody level is
below 200lU/ml.
 When the antibody level is greater than 200lu/ml,
further testing is required to estimate the
approximate titer of the antibody.
Procedure
Qualitative slide method
1. Allow each component to reach room temperature.

2. Gently shake the latex reagent to disperse the particles.
3. Place a drop of undiluted serum on to the circle of the
test slide using the disposable pipettes provided.
4. Add one drop of the latex reagent next to the drop of
serum.
5. Spread the latex reagent and serum sample over the
entire area of the test circle.
6. See for agglutination by tilting the test slid for 2 min.
Interpretation
 Agglutination indicates a positive result which
200IU/ml and no agglutination indicates a

negative result, which are 200IU/ml provided
that the controls have given the expected results.
ASO titration
Principle
 A constant amount of streptolysin O antigen
reagent (reduced form) is added to a series of
dilution of the patient’s serum. Following a period
of incubation, group O washed human or rabbit red
cells is added. The tubes are then examined for
lysis of the red cells. Hemolysisi occurs in those
tubes in, which there is insufficient antibody to
neutralize the antigen.
 The highest dilution of serum showing no
hemolysis is the ASO titer (the titer of ASO
antibody in the serum is directly proportional to the
reciprocal of the serum dilution).
Procedure
 Always follow the manufacturer instructions, which
is written over the manual
Results
 The titer is expressed as the reciprocal of the
highest dilution showing microscopic agglutination.
4.3.4 Serological diagnosis of
toxoplasmosis
 Toxoplasmosis is caused by a protozoan toxoplasma
gonidi , a member of sporozoa .
 The tachyzoite directly destroys cells and has a predilection

for paranchymal cells and those of reticuloendothelial cells .
 Humans are relatively resistant, but a low grade lymph

node infection resembling infectious mononucleosis may
occur.
 When a tissue cyst ruptures releasing many bradyzoites , a

local hypersensitivity reaction may cause inflammation,
blockage of blood vessels and cell death near the damage
cyst.
 The organism in humans produces either
congenital or postnatal toxoplasmosis.
 Congenital infection develops only when non

immune mothers are infected during pregnancy.
Postnatal toxoplasmosis is much less Sever.
 Congenital infection leads to still births,

intracerebral calcification and psychomotor
disturbance when the mother is infected
 Most human infections are asymptomatic cases.

But fatal infections may present in patients with
AIDS.
Laboratory diagnosis
 Direct visualization
+ biopsy specimen from, brain, liver,

myocaridium ,lymph node, or from body fluids
CSF, amniotic fluid peritoneal fluid by using
direct fluorescent antibody stains
 Tissue culture ,mouse inoculation
 Serological tests
Serological tests
 Elevation of toxplasma antibodies indicates

infection
 Antibodies can be demonstrated within the first two

weeks of infection
 The antibodies fall slightly but persist for months
 Both IgG and IgM antibodies can be detected

 The presence of IgM indicates active infection
 The test procedures that detect antibodies
include
- Indirect fluorescent antibody
- Indirect heam agglutunation
- ELISA
- Complement fixation
- Sabin Feldman dye test
Sabin Feldman dye test
 Laboratory cultured live T.gonidi + patient

serum then add methylen blue
 The organism is unstained In the presence of
positive serum.
 Not routinely performed for diagnostic

purpose
Summary
 Serologic tests of bacterial infections like
(syphilis, typhoid fever, paratyphoid fever,
typhus), Anti-streptolysin O, parasitic infection
(Toxoplasmosis) is very crucial activity in medicine
world to list factors affecting each patient and
medication.
 On top of that it is important to know how to read,
interpret and report the serological lab result.
Chapter Five
Common serologic
tests for viral infection
Learning Objective of the course
 At the end of this chapter the students
should be able to:
1. Describe and /or perform serologic tests of viral

infections (HIV/AIDS, Hepatitis, Infectious
mononucleosis),
2. Identify factors affecting each serologic test and
minimize this effect
3. Explain how to read, interpret and report the
results of each serologic test.
Out line
5.1. HIV serology
5.2. Serology of dengue virus
5.3. Serology of H. pylori
5.4. Serology of Hepatitis Viruses
5.5. Serology of Infectious mononucleosis (IM)

Definition of terms
 HIV is a human immunodeficiency virus which
causes chronic diseases with other infectious
agent.
5.1. HIV Serology
 Several laboratory methods are available to screen
blood, diagnose infection, and monitor disease
progression in individuals infected by HIV.
 HIV tests can be classified into:
+ detect antibody
+ identify antigen
+ detect or monitor viral nucleic acids, and
+ estimate of T-lymphocyte numbers (cell
phenotyping).
 The isolation of HIV, its nucleic acid and methods
used to detect HIV antigen are mainly used to
detect early HIV infection before antibodies
develop
HIV Antibody Tests

+ Based on a multi test algorithm for detecting
antibodies to HIV by using screening and
confirmatory tests.
Common HIV Antibody
Tests
A. Enzyme Linked Immunosorbent Assays
(ELISA)
B. Rapid Tests
C. Western Blot
A. Enzyme Linked Immunosorbent Assays
(ELISA)
 ELISA rely on a primary antigen-antibody interaction
 Generation of ELISA
- First generation assays
- Second generation assays
- Third generation assays
- Fourth generation assays

 First generation assays
+ based on purified HIV whole viral lysates
+ have poor sensitivity and specificity
 Second generation assay

+ used HIV recombinant proteins/or synthetic
peptides which enabled the production of assays
+ capable of detecting HIV-1 and HIV-2.
+ It has had improved specificity but has similar
overall sensitivity to that of first generation assay.
 Third-generation assays
+ Used recombinant antigens and/or peptides
and similar recombinant antigens and
peptides conjugated to a detection on
enzyme or hapten that could detect HIV-
specific antibodies.
+ could detect IgM early antibodies to HIV
infection
+ in addition to IgG
 Fourth generation assays

+ are very similar to third-generations test
+ but have the ability to detect simultaneously

HIV antibodies and antigens.
Characteristics of ELISA
+ are best performed at a regional or national
laboratory
+ Why?
- Because they require well-trained and
skilled laboratory technicians,
- technologically advanced equipment
(incubators, Washers and
spectrophotometers) that requires
maintenance and a constant source of
electricity.
• ELISA is most efficient for laboratories
 process a large number of specimens (100
or more) daily or
 for batch testing which is common in HIV
surveillances.
• Because of test design, they are not suitable or
cost-effective to run on a small number of
specimens.
• laboratory processes at least 50 specimens
each day on a regular basis ELISA may still be
more appropriate than rapid tests
Performing an ELISA
 ELISA can be performed with serum, plasma,

urine, oral fluids, or dried blood spots
 Can take from 2 to 4 hours to perform
(including specimen preparation and dilution)
and an additional 3 to 4 hours if a screening
result has to be confirmed
 Manufacturer’s instructions provided with the
specific ELISA used should be followed.
General steps for performing an ELISA
1. Dilute the specimen in the specimen buffer and
put it in a micro well plate containing HIV
antigen already bound to the plate.
2. Incubate the plate as per protocol and then
wash as indicated.
3. Add antihuman immunoglobulin-enzyme
conjugate, which will react with the HIV specific
antibody if present
4. Incubate
5. Wash the plate, add the substrate and incubate
as prescribed
6. Add a stopping solution to terminate the
enzyme reaction and read the absorbance of
the solution using spectrophotometer.
 A positive reaction has occurred if the specimen in
the specimen well changes color or becomes
colored
 Positive reaction indicates the presence of HIV-

specific antibody in the specimen.
 The reaction is best read quantitatively with an

ELISA plate using spectrophotometer (ELISA
reader).
Surface structure of HIV
 Critical to the success of conducting an ELISA:
 Use of test kits that are not expired
 Calibrated and well-maintained equipment
 Adherence to dilution and incubation times

described in the manufacturer’s instructions.
 Use of deionized water
 Use of a spectrophotometer to read results

accurately and objectively
 Training with the technology being used
 Consistent source of power without outages

that would affect the storage of reagents or
the functioning of equipment
Quality control
Run controls with the patient sample as per the manufacturers’ instructions.
Principle
As its name suggests, the ELISA uses an enzyme
system to show the specific combination of an antigen
with its antibody. The enzyme system consists of:
+ An enzyme, which is labeled, or linked, to
specific antibody or antigen

+ A substrate, which is added after the antigen
antibody reaction. This substrate is acted on

(usually hydrolyzed) by the enzyme attached to
the antigen antibody complex, to give a color
change.
+ The intensity of the color gives an indication of
the amount of bound antigen or antibody.

Ways of performing ELISA:
1. Double antibody technique, to detect antigen.
2. Indirect technique, to detect antibody.
1. Double antibody ELISA

• Specific antibody is coated on the surface of the
well of a micro titration plate (or a test tube), and
the specimen is added. After a period of
incubation during which the antibody takes up
(cutpurse) the antigen in the specimen, the well is
washed leaving the antigen attached to the
antibody.
• Enzyme labeled specific antibody (often the same
antiserum as that coating the well except it is
enzyme linked) is added to detect the presence of
the antigen.
1. Double antibody ELISA (antigen test)
Add Specimen
Containing Ag
2. Indirect ELISA
 In this technique, known antigen is attached to
the inside surface of the well and patient’s
serum is added. After incubation and washing,
enzyme labeled antihuman globulin is reacted
with the antibody that has attached to the
antigen.
 The presence and concentration of antibody
that has reacted with the antigen is shown by a
change in color when the substrate is added.
 The intensity of the color is directly proportional
to the concentration of antibody in the serum.
Antibody test
(Specimen)
B. Rapid Tests
General Description
 Most HIV rapid tests contain antigens to HIV-1
and HIV-2 and detect antibodies to both.
 A positive test result is indicated by clumping, a
spot dot or line depending on the test format.
 The sensitivity and specificity of the latest
generation of rapid tests are similar to those of
ELISA.
 Many rapid tests are under evaluation or are
currently in use in developing countries for
screening, diagnostic and surveillance purposes.
Characteristics of Rapid Tests
 Rapid tests are useful for small laboratories that

routinely perform fewer than 100 HIV tests per day,
for laboratories without electricity or equipment, and
for geographic areas with limited laboratory
infrastructure.
 In some instances, even if a laboratory performs
more than 100 tests per day but only during a limited
time in a year, rapid tests may be more appropriate
than ELISA.
 A result can usually be obtained in less than 45
minutes, and it is easy to interpret.
 Training is required to correctly perform the test
and interpret the results.
 The test kits generally contain all reagents
needed to run the assay, no additional reagents
or equipment is required.
 Many rapid tests do not require electricity, special
equipment, refrigeration, or highly skilled staff
although a few require refrigeration for heat-
sensitive reagents.
 Sensitivity approaches 100%; specificity is >99%
 Negative tests can be reported as negatives
 Positive results should be confirmed
 Useful in situations where immediate results are

important to manage decisions
 Current HIV1/2 Test Algorithms
(Reading Assignment)
Previous test algorithm
Blood
Blood Sample
Sample
Test 1 (Determine)
Non-reactive
Non-reactive Reactive
Reactive
Report
Report Negative
Negative Test 2 (Capillus)
Non Reactive
Reactive
Non Reactive
Reactive
Report
Report Positive
Positive
Test
Test 33 (Unigold)
(Unigold)
Reactive
Reactive Result
Result Non-reactive
Non-reactive Result
Result
Report
Report Positive
Positive Report
Report Negative
Negative
previous test algorithm
Blood Sample
Test 1 (KHB)
Non-reactive Reactive
Report Negative Test 2 (Stat Pak)
Non Reactive Reactive

Report Positive
Test 3 (Unigold)
Reactive Result Non-reactive Result
Report Positive Report Negative

Add 1 drop
specimen to well
Add 4 drops of
wash solution
Positive Negative
Read results in 10 -12 minutes

+ Has sensitivity of 100%
+ Specificity 99.7-100%
Determine HIV1/2
+ Is an immuochromatographic test
+ Detects antibody of HIV1/2 antigens on
serum, whole blood, plasma
+ Uses recombinant antigen
 Test method
+ Sample added to sample application pad
+ Samples are serum, plasma, whole blood
+ Result read after 15 minute
+ Sensitivity 99.4-100%
+ Specificity 99.6-100%
Determine
Another rapid test
 SD bioline HIV1/2 3.0 imunochromatographic test

+ 3rd generation immunochramatic test
+ Detects antibodies for HIV1and 2
+ Uses serum, whole blood, plasma
+ Uses recombinant antigen
 Test method
+ Sensitivity 100%
+ Specificity 99.3%
Ora quick
CLIA-waived for finger stick, whole blood, oral fluid;

moderate complexity with plasma
Store at room temperature
•Screens for HIV-1 and 2
•Results in 20 minutes
Obtain finger stick specimen…
Insert loop into vial and stir
Collect oral fluid specimens by swabbing gums
with test device. Gloves optional; waste not
biohazardous
Insert device; test develops in 20 minutes
Read results in 20 –40 minutes
Reactive Control
Positive Negative
Multi-spot kit
Stat pak
Reactive Control
Positive Negative
Sure check
C. Western blot
 Separates proteins electrophoretically
 False positive may detected
Next topic is serology of Dengue virus

Serology of Dengue
fever
5.2. Serology of dengue
fever
 Viral disease transmitted by Aedes
mosquitoes, usually Aedes aegypti ,that bites
humans only during day-time
 Dengue fever is characterized by sudden onset
after an incubation period of 3-14 days
(most commonly 4-7 days) of high fevers,
severe frontal headache, and joint and muscle
pain.
 The disease is usually self-limited, although
convalescence can be prolonged.
 Most patients report a nonspecific viral
syndrome or a flu-like illness
 Asymptomatic infections are also common.
 Neutropenia, elevated liver enzymes, and
disseminated intravascular coagulation are
also common.
 Anti-dengue IgM positivity suggests a recent
dengue infection, but IgG positivity may only
indicate infection at an indeterminate time in the
past.
 Recently, IgM and IgG capture ELISAs have been
modified into immunochromographic formats in
which the result of the assay is a color change
visible to the naked eye.
 The most rapid of these gives a diagnosis within 7
minutes.
Dengue IgM & IgG Rapid Strip
Primary dengue:
+ve IgM
-ve IgG result
Secondary infections
+ve IgG
+/- +ve IgM result
 Detection of IgM antibodies to dengue virus by
ELISA is a valuable procedure, particularly in
second and subsequent infections where the
occurrence of complications is high.
 Serum IgM antibodies can be detected from

dengue patients as early as three to five days
after the onset of fever.
 Generally persist for 30 - 90 days, although

detectable levels may be present eight months
post-infection.
.
 Detection of IgM antibodies to dengue virus by
ELISA is a valuable procedure, particularly in
second and subsequent infections where the
occurrence of complications is high.
 Serum IgM antibodies can be detected from

dengue patients as early as three to five days
after the onset of fever
 Generally persist for 30 - 90 days, although

detectable levels may be present eight months
post-infection.
.
 The Flavi virus genera share epitopes inducing
cross-reactive antibodies
 Leading to great difficulty in differentially
diagnosing flavi viral infections.
Next topic is serology of H. pylori

Serology of H. pylori
tests
5.3. Serology of H. pylori
 H. pylori is a gram negative bacilli
 Found in patients with gastritis, peptic ulcers, gastric

adenocarcinoma.
 It is highly motile cork screw shaped organism.
 They produce urase
 Colonize stomach
 Do not ferment carbohydrates

 Cultural growth requires a complex media
 humans , primates and pigs are reservoir host for H.

pylorior
 Infection is more common in people with low socio

economic class in developing countries.
 Transmission is person to person (fecal- oral

transmission)
Gastric inflammation
 H. pylori have multiple factors which contribute to

gastric inflammation.
 Initial colonization of the stomach is facilitated by
blockage of acid production by a bacterial acid
inhibitor protein and neutralization of gastric acids
by ammonia produced by bacterial urase activity.
 The motile h.pylori can then pass through the
gastric mucosa adhere to epithelial cell.
 Localized tissue damage is mediated by urease
byproducts.
 H.pylori also produces factors that stimulate
secretion of interlukein -8 and production of
platelet activating factor that cause hyper
secretion of gastric acid and programmed death
of gastric
 Histologic examination = gastric biopsy specimen

stained with iemsa stain , hematoxylin –eosin or
Gram stain , specificity 100%
 Urease test = rapid way to detect h.pylori

100%specificity
 Culture = insensitive method unless multiple

biopsy is used
 Serology = h.pylori stimulates humoral immune

system as a result of continuous exposure
 The antibody titer remains for long period of time
 The titer measured is not associated with the
severity of disease or response to treatment
 Is important to document exposure to the
bacteria either for epidemiological studies or
initial evaluation of a symptomatic patiens
Serology
 Serological diagnosis of H.pylori can be made by
detection of antibodies in blood and /or fecal
antigen
 Serological tests for detection of antibodies has
false positive rate &it doesn’t distinguish between
active and passive infection
Next topic is serology of Hepatitis virus

Serology of hepatitis
tests
5.4. Serology of Hepatitis
virus
Introduction
 Hepatitis is a generic term referring to an

inflammation of liver
 Some viruses primarily infect the liver and are

called hepatotrophic viruses .
 These includes hepatitis A, B, C, D, E,

1. Hepatitis A virus
 RNA virus
 Spread by feco-oral route
 Has an incubation period of approximately one
month
 Doe not cause chronic liver disease
2. Hepatitis B virus
 DNA virus
 Spread by- blood and contaminated needle
- Sexual contact
- perinatally
 It has an incubation period of approximately 3
months
 It is more infectious than HIV

3. Hepatitis C virus
 RNA virus
 account 90% of transfusion associated hepatitis
4. Hepatitis D virus
 Defective virus that replicates in only HBV
infected cells
 RNA virus
 Similar route of transmission with HBV
5. Hepatitis E virus
 RNA virus
 Has feco- oral route of transmission
 causes sever infection in pregnant ladies

Markers for hepatitis B virus

 Hepatitis B surface antigen (HBsAg)
 Detected during incubation period
 Found during active phase of the disease
 It persists for month and years, the

individual is carrier and potentially
infectious.
 Produced in the cytoplasm of infected
hepatocytes
 It is the outer lipoprotein coat (envelope)
Hepatitis B core antigen (HBcAg)
 It is the core of HBV
 It is located in the nuclei of hepatocytes

 It is not detectable in serum
Hepatitis B e antigen (HBeAg)

 It is a minor component of the viron
 It is found during acute infection and then

usually disappear and usually reliable diagnostic
indication of active infection.
Antibodies
Anti – HBsAg
 Appears after disappearance of HBsAg
 Shows past infection and immunity
 It is a measure of recovery from HBV infection
 In carriers, HBsAg and anti –HbsAg does not develop
Anti-HBeAg
 Not frequently observed in patient with
chronic infection
 Patients with anti HBeAg are usually not
infectious because they have low titer of HBV.
Anti -HBcAg
 Is actively infected individuals antibodies to
HBcAg appear in serum after the
appearance of HBsAg before the onset of
symptoms.
 Reliable marker of recent infection
 Absences of IgM anti HBcAg in HBsAg

positive individuals indicates , carrier state
1. Serological test for HBsAg
 Agar gel diffusion
 In gar gel preparation anti HBs is added to the
control well and the patient sera are added to the
peripheral well
 Radial diffusion of reactants will form precipitation
reaction
Advantage
 it demonstrates specificity by the formation of line
of identity
 it distinguishes the sub types of HBsAg by lines of
partial identity and spur
 it is the simplest method
Disadvantage
 it is less sensitive than other methods
 it requires longer time for optimal result (24-
72hours )
2. Reverse passive latex agglutination
 Principle: The latex particle coated with anti HBsAg

is mixed with the patient serum and observed for
the occurrence of agglutination
 If erythrocytes are employed for the procedure.

The processes called reveres passive
hemagglutination.
3. Chromatographic technique
 It is a qualitative detection of HBsAg in serum or

plasma
 The membrane is pre coated with antiHBsAg on the
test line region of the strip
 Serum or plasma migrates up ward by capillary
action to reach the test and control areas and
forms a colored line.
4. ELISA
 Principle – wells of a micro titer plate coated with
mouse monoclonal antibody to HBsAg conjugated
with horse reddish peroxidase .
 If patient’s serum or plasma contains HBsAg, it
will simultaneously bind to both the solid phase
antibody and anti- HBsAg conjugated with horse
reddish peroxidase (HRPO) .
 The substrate for HRPO is O-phenylene diamine
(OPD) which dissolved in H2O2 is then added on
the preparation.
 The presence of HBsAg in patient’s serum is
detected by the color change after the addition of
specific substrate.
Next topic is serology of Infectious Mononucleosis

5.5 INFECTIOUS MONONUCLEOSIS
Introduction
 Epstein-Barr virus was first discovered in 1964 as the
cause of infectious mononucleosis.
 The mode of transmission is not known, but may be
facilitated by saliva exchange.
 This disorder is usually an acute, benign, and self-
limiting lymphoproliferative condition.
 The virus is shed in the throat during the illness and for
up to a year after infection.
 After the initial infection, the virus tends to become
dormant for a prolonged period and can later reactivate
and be shed from the throat again.
Introduction
 The virus is spread by person-to-person contact,

via saliva. In rare instances, the virus has been
transmitted by blood transfusion or
transplacentally.
 In underdeveloped countries, people are exposed
in early childhood where they are less likely to
develop noticeable symptoms.
 In developed countries such as the United States,
the age of first exposure may be delayed to older
childhood and young adult age when symptoms
are more likely to result.
Introduction
 Infectious Mono is recognized more often in high

school and college students.
 The disease usually runs its course in two to four
weeks, although cases may be as brief as a week
or last six to eight weeks.
 After recovery, weakness may continue for
several months.
Epstein-Barr Viral Infection
 Epstein-Barr virus (EBV) is a human herpes DNA

virus.
 It is estimated that 95 percent of the world
population is exposed to the virus.
 In Infectious Mono the virus affects B-
lymphocytes.
 There are two techniques used to identify EBV;
immunofluorescence and complement fixation.
 It is a systematic immune complex disease of soluble and
tissue-fixed antigen involvement characterized by fever,
fatigue, chills, headache, myalgia, skin rash, splenomegaly
and cervical adenopathy.
 EBV infected B-lymphocytes express a variety of “new”
antigens encoded by the virus. Infection with EBV results in
expression of:
1. Viral Capsid Antigen (VCA)
2.
Early Antigen (EA)
3. Nuclear
Antigen (NA)
Each antigen
expression has corresponding antibody responses.
Epstein-Barr Virus (VCA)
 Viral capsid antigen (VCA) is produced by infected

B cells and can be found in the cytoplasm.
 Anti-VCA IgM is usually detectable early in the
course of infection, 4 to 7 days after onset of
signs and symptoms, but it is low in
concentration and disappears within 2 to 4
months.
Epstein-Barr Virus (EA)
 Early antigen (EA) is a complex of two components, early

antigen-diffuse (EA-D), which is found in both the nucleus and
cytoplasm of the B cells, and early antigen-restricted (EA-R),
which is usually found as a mass only in the cytoplasm.
 Anti-EA-D of the IgG type is highly indicative of acute
infection, but it is not detectable in 10% to 20% of patients
with IM. EA-D disappears in about 3 months; however, a rise
in titer is demonstrated during reactivation of a latent EBV
infection.
 Anti-EA-R IgG is not usually found in young adults during the
acute phase. Anti-EA-R IgG appears transiently in the later
convalescent phase. In general, anti-EA-D and anti-EA-R IgG
are not consistent indicators of the disease stage.
Epstein-Barr Virus (EBNA)
 Epstein-Barr nuclear antigen (EBNA) is found in the nucleus
of all EBV-infected cells. Although the synthesis of NA
precedes EA synthesis during the infection of B cells, EBV-
NA does not become available for antibody stimulation until
after the incubation period of Infectious Mono, when
activated T lymphocytes destroy the EBV genome-carrying
B cells. As a result, antibodies to NA are absent or barely
detectable during acute IM.
 Anti-EBNA IgG does not appear until a patient has entered
the convalescent period. EBV-NA antibodies are almost
always present in sera containing IgG antibodies to VCA of
EBV unless the patient is in the early acute phase of IM.
Patients with severe immunologic defects or
immunosuppressive disease may not have EBV-NA
antibodies, even if antibodies to VCA are present.
Epstein-Barr Virus (EBNA)
 Under normal conditions, antibody titers to NA
gradually increase through convalescence and
reach a plateau between 3 and 12 months post
infection. The antibody titer remains at a
moderate, measurable level indefinitely because
of the persistent viral carrier state established
following primary EBV infection.
 Test results of antibodies to EBV-NA should be
evaluated in relationship to patient symptoms,
clinical history, and antibody response patterns to
EBV-VCA and EA to establish a diagnosis.
Signs and Symptoms
 Mononucleosis is characterized by fever, sore throat,

fatigue, malaise, and loss of appetite.
 Patients generally have swelling of the lymph nodes
in the neck and often have an enlarged spleen.
 No treatment, other than rest, is needed in the vast
majority of cases and there is no vaccine available
to prevent IM.
 In children and infants the time of onset is usually vague
and the duration of prodromal symptoms is difficult to
determine.
 Anorexia, sometimes accompanied by nausea and
vomiting, is a common and non-specific early symptom of
this infection.
 The most important and most characteristic symptom of IM
is a sore throat. This usually develops a few days after the
onset of the illness, increases in severity during the first
week, and then rapidly subsides during the next five to
seven days.
 In many young adults sore throat is the first indication of
sickness and in some it is the only major symptom
throughout the entire illness.
 Although anorexia may persist for as long as there is fever,
its intensity and duration are more directly related to the
severity of sore throat and dysphagia.
 Gross tonsillar and pharyngeal edema may cause virtually
complete pharyngeal obstruction with harsh-sounding
breathing and complete inability to swallow either food or
fluids.
 In some patients the soreness of the throat is so severe
that swallowing even a few sips of water is extremely
painful.
 The headaches of early IM are often retro-orbital
in location but have no characteristic features.
 They may be moderately severe for one or two
days but usually they are mild and rarely last for
more than three or four days.
 Ocular symptoms may be in the form of
photophobia, ocular muscle aching or the
awareness of puffiness.
 Lymphadenopathy, disease of the lymph nodes, is
sometimes accidentally discovered or detected during self-
examination following the development of systemic
symptoms.
 In about 3 percent of all cases of IM, the gross cervical
lymphadenopathy imparts a “bull neck” appearance.
 Enlargement of lymph nodes usually begins two or three
days after the onset of the first symptoms and, by the end
of the week, palpable lymphadenopathy is present in 70-80
percent of all patients.
 Jaundice is a moderately important symptom of
infectious mono as 8-10 percent of patients
eventually become visibly jaundiced.
 In most instances, however, it is not noticed
since it consists of only a transient icteric tint to
the sclerae and mucous membranes, lasting for a
few days.
Clinical Manifestations
 Examination of the blood usually shows an
increase in the white blood cells, due to the
appearance of many atypical lymphocytes in the
blood.
 Blood serum in IM often contains an antibody
known as heterophil antibody that agglutinates,
or clumps, the red blood cells of sheep.
 Heterophil antibodies are antibodies that are
stimulated by one antigen and react with an
entirely unrelated surface antigen present on
cells from different mammalian species.
 Heterophil antibody titers rise during the first two or
three weeks with half or more developing a significant
titer during the first week of illness.
 The level of antibody gradually declines and usually
disappears in eight to twelve weeks following the
onset.
 Elevated titers sometimes linger for four to six months
up to a year or more.
 Heterophil antibody most commonly used in the
serological diagnosis of IM is an IgM antibody which
agglutinates sheep red blood cells.
 The original Paul-Bunnell test was a simple titration of
sheep cell agglutinins but this procedure was subsequently
modified in order to distinguish between sheep cell
agglutinins formed in IM and the Forssman-type antibodies
found in normal serum, serum sickness and in certain other
conditions.
 Tissues rich in Forssman antigen (guinea pig kidney)
absorb Forssman antibodies but do not affect the heterophil
antibodies in IM.
 Heterophil antibodies are absorbed by beef cells,
 Forssman hapten is a glycolipid usually associated with a
protein, the determinant being largely carbohydrate and
therefore heat stable.
Davidsohn Differential slide test
 The principle behind the Paul-Bunnell-Davidsohn test is that the

two types of sheep agglutinins are distinguished by titrating
them before and after absorption with guinea pig kidney and ox
cells.
 Patients serum containing antibodies due to IM is added to
guinea pig kidney cells. These antibodies are not absorbed by
the kidney cells. These antibodies then react with Beef (Ox) red
blood cells which causes agglutination and is a positive test for
IM.
 Patients serum containing Forssman antibodies are added to
guinea pig kidney cells. Antibodies are absorbed by the kidney
cells. These antibodies are then allowed to react with Beef red
blood cells which does not cause agglutination. This is a positive
test for Forssman antigens.
Davidsohn Differential
To be considered absorbed there must be greater than a

three tube difference between the presumptive titer and
the differential titer.
Heterophil Antibody Kidney Extract Beef Erythrocyte

------------------------ ------------------ ---------------------
Infectious Mono Not Absorbed Absorbed
Forssman Absorbed Not Absorbed
Serum Sickness Absorbed Absorbed

Davidsohn Differential
Advantages Disadvantages
 When properly performed,  Davidsohn Differential test

this test is specific for is very time consuming
Infectious Mononucleosis and burdensome.
and false-positive results
are rare.
Mono-Plus
Sample Requirements
 Red top tube of blood (Serum)
 Green top tube of blood (Plasma)
 Purple top tube of blood (Plasma)
 CPDA-1 (Plasma)
 Capillary blood from fingertip (Whole Blood)
Principle
 Qualitative detection of IM heterophil antibodies
in human serum, plasma and whole blood using
direct solid-phase immunoassay technology.
 A band of bovine (Ox) erythrocyte extracts are
impregnated in the test membrane.
 If IM-specific heterophil antibody is present in the
sample, it will be captured by the bovine
erythrocyte extracts.
Principle
 The Developer Solution is then added to the sample
well.
 The solution mobilizes the dye conjugated to the
anti-human IgM antibodies.
 The antigen band can be seen in the Test Window
(T) only when the antibody-dye conjugate binds to
the IM-specific heterophil antibody which has been
bound to the bovine erythrocyte extract.
Principle
 The antibody-dye conjugate will bind to another
band located in the Control Window (C) to
generate a colored band regardless of the
presence of IM heterophil antibodies in the
sample.
 The presence of two colored bands or lines, one
in the Test Window (T) and one in the Control
Window (C), indicates a positive test.
 The presence of a colored band in the Control
Window (C) only indicates a negative result.
Procedure
 Step 1
 Pipette 10uL of serum or plasma in the upper
well.
 Step 2
 Add 2-3 drops of Developer solution to the lower
end of the sample well
 Step 3
 Read test results in 8 minutes.
 Strong positive may appear in less than 3
minutes.
 Must wait 8 minutes to report negative result.
 Results are stable 15 minutes after Developer is
added.
Positive Result Negative Result
 A pink-purple horizontal  A pink-purple horizontal

bar in the Test Window (T) bar in the Control Window
and the Control (C). (C).
 No horizontal bar in the
Test Window (T).
Invalid Result
 If no bar appears in the Control Window (C) the
test is invalid.
 A distinct horizontal bar should always appear in
the Control Window (C).
False positive
 IM heterophil has been associated with disease

states such as: Burkitt’s Lymphoma, viral
hepatitis, adenovirus, leukemia, cytomegalovirus,
rheumatoid arthritis and Toxoplasma gondii. EBV-
specific lab diagnosis may be used for persons
with these illnesses.
 Sera of patients with IM react not only with beef
erythrocytes but also other bovine antigens. False
positives have occurred with bovine heart extract
(cardiolipin).
 Although most patients will have detectable
heterophile levels within three weeks of infection,
occasionally a patient with strong clinical signs of
IM may take as long as three months to develop a
detectable level. This can be resolved by taking
additional specimens every few days and
retesting.
 Some segments of the population who contract IM
do not produce measurable levels of heterophil
antibody. Approximately 50% of children under 4
years of age who have IM may test as IM
heterophil negative. EBV-specific laboratory
diagnosis may be helpful in these cases.
Conclusion
 In addition to clinical signs and symptoms, laboratory testing
is necessary to establish or confirm the diagnosis of IM. This
can provide important information for both the diagnosis and
management of EBV-associated disease.
 If the classic signs and symptoms of IM are absent, a
diagnosis of IM is more difficult to make. A definite diagnosis
of IM can be established by serologic antibody testing. The
antibodies present in IM are heterophil and EBV antibodies.
 EBV is widely disseminated. It is estimated that 95% of
world’s population is exposed to the virus, which makes it the
most ubiquitous virus known to man.
 EBV is only a minor problem for immunocompetent persons,
but it can become a major one for immunologically
compromised patients
 After primary exposure a person is considered to be immune
and generally no longer susceptible to overt re-infection.
References
1. Mono-Plus; Wampole Laboratories, Dist.; Cranbury Laboratories; 1999.
2. Infectious Mononucleosis; Robert J. Hoagland; Grune and Stratton Inc.;

New York and London; 1967.
3. Immunology and Serology in Laboratory Medicine; Mary Louise

Turgeon; The C.V. Mosby Company; St Louis; 1990.
4. Infectious Mononucleosis; Sidney Leibowitz, M.D.; Grune and Stratton;

New York; 1953.
Next topic Serology of CRP, RF, hCG tests

Unit Six
Serology of CRP, RF,
hCG tests
Learning objectives
 At the end of this lesson student will able to:

 Describe serologic diagnosis of CRP, RF, and
hCG
 Determine the factors that influences serologic
tests of CRP, RF, and hCG
Out line
6.1 CRP
6.2 RF
6.3 HCG
Introduction
C-reactive protein (CRP)

 CRP is the most widely used indicator of an
acute-phase response in man for the early
indication of infections, inflammation or other
disease associated with tissue injury.
 Normally, the serum concentration of CRP is 0.1
mg/dl or less.
 After injury, rapid production in the liver results
in concentrations as high as 100mg/dL.
 CRP is synthesized only in the liver, and synthesis
is stimulated by IL-6 and IL-1.
 CRP got its name because it was first identified in
the serum of patients with pneumonia because it
precipitated with the C-polysaccharide on the
pneumococcal cell wall.
 Cleavage of CRP by enzymes from neutrophils
produces fragment that promote chemotaxis and
contain the tetrapeptide called tufsin
 the biological effects of CRP are like those of
immunoglobulin, including the ability to
precipitate, to function as opsonin through
binding to macrophages, and to fix complement.
 Levels of CRP (increases 4 to 6 hours after tissue
injury) parallel the course of the inflammatory
responses and return to lower undetectable levels
as the inflammation subsides.
 It can increase as much as 100 fold in
concentration in acute inflammation
 CRP increases fester than ESR in responding to
inflammation, whereas the leukocyte count may
remain with in normal limits despite infection.
 CRP is the method of choice for screening for
inflammatory and malignant organic disease and
monitoring therapy in inflammation.
 Elevations of CRP occurs in nearly 70 disease
state, including:
 septicemia and meningitis in neonates,
 infections in immunosuppresed patients,
 burns complicated by infection,
 serious postoperative infections,
 myocardial infraction,
 malignant tumors, and
 rheumatic disease.
 In general, the CRP is advocated as indicator of;
 bacterial infection in at-risk patients in whom
the clinical assessment of infection is difficult
to make, but a lack of specificity rules out CRP
as a definitive diagnostic tool.
 In clinical practice CRP is particularly useful when
serial measurement are performed.
 The course of the CRP level may be useful for
 monitoring the effect of treatment and
 for detection of post operative complications or
internal infections.
Note: The half life of CRP is 5 to 7 hrs. It falls mach

more rapidly than other acute phase proteins
when patient recovers.
Laboratory tests for C-reactive protein (CRP)
 Rapid latex agglutination test
 Complement fixation
 Fluorescent antibody
 precipitation
 Laser nephelometry
Rapid latex agglutination test
Principle
 The agglutination test is based on the reaction
between patient serum containing CRP as the
antigen and the corresponding antihuman (CRP)
antibody coated to the treated surface participles.
The coated particles enhance the detection of an
agglutination reaction when antigen is present in
the serum.
Other test includes
 Complement fixation, not for routine clinical
laboratory
 Fluorescent antibody
 Used to study binding of CRP to lymphocytes and their
subpopulation
 Used primary as a research tools for localizing CRP in
tissue.
 precipitation
 Tube method
 Gel electrophoresis
 Laser nephelometry
 Sensitive, rapid and reproducible
 Can be used for large number of samples
 In brief, the procedure involves the
measurement of light that is scattered by the
insoluble immune complexes in aliquot
medium containing polyethylene glycol.
6.2. Serology of RF tests
6.3. SEROLOGY OF RF
Rheumatoid Factor (RF)

 Rheumatoid factor belongs to a large family of
anti-globulin usually defined as antibodies with
specificity for antigen determinants on the Fc
fragment of human or certain animal IgG.
 RF have been associated with three major
immunoglobulin classes: IgM, IgG, and IgA. Of
these IgM and IgG are the most common.
 As indicated by its name, RF has particular
application to diagnosis and monitoring of
rheumatoid arthritis. Rheumatoid arthritis (RA) is
a systemic syndrome in which chronic
inflammation of the joints initiated by auto-
antibodies and maintained by cellular
inflammatory mechanisms is the major feature.
 The auto-antibodies are directed to self-
immunoglobulin determinants.
 The formation of immune complexes in the joint
spaces leads to activation of complement and
destructive inflammation.
 The acute phase is followed by a delayed type
hypersensitivity (DTH) type chronic inflammation.
Chronic RA is believed to be driven by
macrophages after initiation by DTH cells (TH1).
 The most common symptoms include a
symmetric arthritis usually involving the small
joint of the hands or feet and knees.
 Rheumatoid factor has been associated with
some bacterial and viral infection (hepatitis and
infectious mononucleosis) and some chronic
infections (tuberculosis, parasitic disease, sub
acute bacterial endocarditis, and cancer).
 Elevated values may also be observed in the
normal elderly population.
Serologic tests
 Tests for RA are designed to detect certain
macroglobulin in the patient's serum that reacts
with normal human IgG or normal animal IgG (i.e.,
rheumatoid factors).
 The majority of tests use particular carriers (i.e.,
erythrocytes, latex and bentonite particles) that
transform the reaction between RF and IgG into
visible aggregation.
 Basically, all the tests are designed to detect
antibody to immunoglobulin.
 However, they are not identical, because
sometimes human and sometimes animal
immunoglobulin is used as the coating for the
particle.
 In some circumstances, the different tests give
different results; therefore, one can postulate
that a number of rheumatoid factors with
different specificities are involved.
 Included among the various serologic tests for
the detection of RF are:
 the latex fixation tests,
 the sheep cell agglutination test,
 the sensitized alligator erythrocyte test,
 bentonite flocculation test and
 the concanvalin A and complement fixation
test for the detection of IgG RA factor
 the latex fixation and the sheep cell
agglutination are the most popular
Serology of hCG tests
Serology of hCG
Outline
 Introduction
 Serology of hCG in Urine
 Urine pregnancy tests
 Factors that affects urine pregnanacy test
 Urine specimen collection
 Method of determining hCG
Learning Objectives
Upon completion of this lecture and exercises the

student will be able to:
 Describe the structure of hCG and the production
of hCG
 Explain the serologic diagnosis of pregnancy
Introduction
 Pregnancy is the period during which a woman

carries a baby with in her body before giving
birth.
 Pregnancy begins with conception-that is, the
fertilization of an egg by a sperm.
 The fertilized egg is called zygote.
Introduction
 During pregnancy, the chorionic membrane of the

placenta produces a hormone called human
chorionic gonadotrophin (hCG), which stimulates
the secretion of progesterone, by the ovary.
 Progesterone maintains the uterus during the
pregnancy and prevents any further release of
eggs from the ovary.
Structure of the hCG
 HCG is a glycoprotein composed of two non-

covalently linked polypeptides:
 alpha () and the
 beta () sub units.
 The individual sub units lack biological activity

but become active when linked to form the intact
complex.
Production of hCG
 Human chorionic gonadotrophin (hCG) is

synthesized in large amounts by the placenta,
and it appears in urine, blood, amniotic fluid and
serum relatively soon after implantation of the
developing embryo.
 The presence of this hormone serves as basis for
pregnancy testing.
Introduction
 Production of hCG increases steadily during the first

trimester, peaking around the 10th week of gestation.
 Levels then fall to less than 10% of the first trimester
levels during the reminder of the pregnancy.
60
55
50
45
40
35
30
25
20
15
10
0 4 8 12 16 20 24 28 32 36 40
Ovulation period
 The urine and serum of pregnant women contain
high concentrations of human chorionic
gonadotrophin (hCG) produced by trophoblast,
which provide the basis of tests for the diagnosis of
pregnancy.
 Specific and sensitive analytical methods for the -
chain sub unit of hCG permit the detection of
pregnancy as early as 8 days after ovulation (1 day
after implantation).
 hCG concentrations climb early in pregnancy,
reaching a maximum by 8 to 10 weeks of
gestation.
Application of pregnancy tests
 Pregnancy tests are important in investigation of

suspected:
 To detect early pregnancy.
 To determine the adequacy of hormone
production in high risk pregnancies (for
example, habitual abortion)
Application of ……cont
 Ectiopic disease: the implantation of the

fertilized egg in the extra uterine space. Example in
the fallopian tube
 In threatened abortion: to conform the abortion
is complete or not. In this case the quantity of hGC is
decreases gradually.
 In the infection of hydated mole: to give
treatments in the way not harm the fetus to check
before mechanical treatment. Example X-ray.
Detection of hCG in urine
 A number of serologic tests have been used in

pregnancy testing; each designed to detect
minute amounts of hCG when it appears in the
urine during the first few weeks of pregnancy.
 The methods most commonly used now are
based on the agglutination inhibition test
 developed by Noto and Miale (1964)
 The amount of hormone excreted in the urine is
almost the same as that found in the blood.
 By about the 8th day after the first missed period
during the pregnancy, the hCG level is about
1000IU/L (1IU/ml), and by the 12th day it is
about 2500IU/L(2.5 IU/ml).
 Laboratory pregnancy tests are based on
detection of rapidly rising levels of hCG in urine.
Specimens
Urine specimen
 An early morning specimen is preferable because
this is the most concentrated and will therefore
contain the highest level of hCG
 The urine must be collected in
 a clean container, which is free from the all traces
of detergent.
 If the specimen cannot be tested immediately it
should be refrigerated at 4c, but for not longer than
48 hr’s.
 Specimens preserved with boric acid are also
suitable for testing.
 If the urine is cloudy it should be flittered or
centrifuged and the supernatant fluid used.
 Specimens that are heavily contaminated, or
contain large amounts of protein or blood, are not
usually suitable for testing.
 There are different types of commercially
produced kits for pregnancy tests and each of
this is with the technique and precaution with
instructions.
Types of urine testing kits
1. Rapid latex slide test of the inhibition
I. Direct agglutination test
II. Indirect or inhibition test
2. Heamagglutination technique (tube tests of the

inhibition type)
Rapid latex slide test of the
inhibition
I. Direct slide agglutination test
(Direct latex slide tests).

 recently introduced techniques,
 more sensitive than the inhibition tests
I. Direct slide agglutination test
Principle
In the direct slide test, the latex reagent consists

of particles coated with anti hCG antibodies. This
reagent is mixed directly with the urine there will
be agglutination, which is visible to the naked eye
if the urine contains the hormone hCG. If not
there is no visible agglutination.
Procedure
 Prepare clean, dry, detergent free slides.
 Take one drop of early morning urine in Pasteur
pipettes and drop on slide.
 Take 1 drop of reagent (anti-hCG antibody).
NB. Don’t use an expired reagent.

 Mix gently; and see for agglutination.
 If hCG is present in the urine, it will combine with
the antibodies and causes agglutination of the latex
particles.
Urine latex reagent Result
(hCG antibody agglutination
Coated particles)
hCG Antigen hCG antigen

is present combines with hCG
antibody on latex
particles
Positive result
 If no hCG is present in the urine, there will be no
agglutination of the latex particles.
Urine latex reagent Result
(hCG antibody No agglutination
Coated particles)
No hCG No hCG antigen

Antigen combines with hCG
antibody on latex
particles
Positive result
 In the direct slide test, therefore, agglutination of
the particles indicates positive tests and no
agglutination indicates a negative test.
ii. Indirect latex slide test
(inhibition latex slide test)
Principle:
 Colored latex or other visible particles coated with
hCG, antibodies to hCG and urine are mixed with
particles. Negative urine results in visible
agglutination; and positive urine results,
presence of hCG in urine inhibits agglutination (or
protein flocculation).
ii. Indirect latex slide test …..cont
Reagents:
 Antiserum containing anti-hCG-antibody
 Latex reagent containing polystyrene particles
coated (sensitized) with the hCG antigen.
Procedure:
 Take clean slides which is free from detergent
 Mix a drop of urine with one drop of antiserum on
slide.
 Add latex reagent
 See for positive or negative test.
 If hCG is present in the urine it will combine with

the anti-hCG antibody. This will leave no antibody
free to combine with the latex hCG and therefore
there will be no agglutination of the latex
particles.
 If hCG is present in the urine it will combine with the

anti-hCG antibody. This will leave no antibody free to
combine with the latex hCG and therefore there will
be no agglutination of the latex particles.
Urine + antiserum hCG + latex reagent hCG Result
antibody Ag coated particles No

agglutination

 If there is no hCG in the urine, the antibody will be

free to combine with the latex hCG and cause
agglutination of the latex particles.
Urine + antiserum hCG + latex reagent hCG Result
Ab Ag coated particles No Agg

Inhibition tube agglutination
test
Principle
 Colored visible particles (RBCs) coated with hCG,
antibodies to hCG, and urine is mixed with
particles. Negative urine results in visible
agglutination; and positive urine results,
(presence of hCG in the urine) inhibits
agglutination (or protein flocculation).
 This technique uses RBCs coated with the hCG
molecules.
 These particles are mixed with urine sample and
then with a solution containing antibodies to hCG
sub units.
 In the absence of urine any hCG, the antibody
reacts with the hCG coated particles and cusses
agglutinations.
 When hCG is present in the urine sample, it will
react with and neutralize the antibody, thus
inhibiting particle agglutination.
Reagent
 Anti-hCG antiserum.
 RBC coated with hCG.

Procedure
 Take clean non-broken and detergent free
tube.
 Add urine into the tube.
 Add anti-hCG antiserum.
 Mix well. If there is hCG there will be reaction.
 Add RBC coated with hCG.
 Mix the contents. Leave at room temperature

(20 to 28oC) for 1 - 2 hours to allow the red
blood cells to settle to the bottom of the tube.
 If hCG is present in the urine it will react with the
antiserum leaving no antibody to agglutinate the
RBC coated with hCG.
 If the urine contains no hCG the anti-hCG, antibody
will react with the hCG on the red cells and causes
their agglutination (Heamagglutination)
 The agglutinates will settle and be seen covering
evenly the bottom of the tube.
Quantitative assays
 There are several types of technique that aids to

measure the levels of hCG in serum or urine
quantitatively or semi quantitatively. These are:
 Radio immunoassay
 Enzyme linked immunosorbant assay (ELISA)
 Radio receptor assay (RRA).
RIA
 The RIAs are typical competitive binding assays
in which the hCG in the sample competates with
hCG labeled with radioactive iodine for binding
sites on a hCG antibody. Both solid phase and
double antibody procedures are available.
Principle
 Radio labeled (radioactive iodine) hCG competes
with sample analyte for binding to anti-hCG.
Increased hCG in sample decreased bound
radioactivity.
ELISA
Principle
Enzyme labeled anti-hCG reacts with sample hCG

bound to solid-phase anti-hCG. Amount of bound
enzyme actively directly proportional to amount
of hCG in sample.
RRA
Principle
Radio labeled hCG competes with sample analyte

for binding to tissue-receptor sites. Increased
hCG in sample decreased bound radioactivity.
Sensitivity and Specificity of PT test
 Sensitivity: a term used to describe the

probability that a laboratory test is positive (that
is greater than the upper limit of normal) in the
presence of disease.
 Sensitivity is defined as true positives divided by
the sum of true positive and false negative
 Specificity is used to describe the probability
that a laboratory test will be negative (that is,
with in the normal range) in the absence of
disease.
 Specificity is defined as true negative divided by
the sum of true negative and false positives.
Factors that affect pregnancy tests
 The time in the pregnancy when the test is
carried out.
 The presence of excessive amounts of protein or
blood in the urine may cause false positive
results.
 Detergent contaminated urine
 Turbidity of the specimens
 Bacterial contamination of the urine may cause
unreliable results.
 Drugs, which may cause false-positive results,
include
Next topic is Some Miscellaneous Techniques and
Monoclonal Antibody Production
Chapter Seven
Some Miscellaneous
Techniques and Monoclonal
Antibody Production
Out line
7.1. Miscellaneous techniques

+ Introduction to flow cytometry
+ Isolation of Lymphocyte Populations
+ Instrument and quality control
Learning Objectives
At the end of this chapter the students should be
able to:
 Describe the uses of isolation of lymphocyte
populations
 Describe and/or perform lymphocytes isolation
procedures (Methods)
 Describe methods of preparation of monoclonal
antibodies
 List uses of monoclonal antibodies
Introduction to flow cytometry
 The fluorescent antibody techniques are
extremely valuable qualitative tools, but they do
not give quantitative data.
 This shortcoming was remedied by development
of the flow cytometer, which was designed to
automate the analysis and separation of cells
stained with fluorescent antibody.
Separation of fluorochrome-labeled
cells with the flow cytometer.
 Every time a cell passes the laser beam, light is
deflected from the detector, and this interruption
of the laser signal is recorded.
 Those cells having a fluorescently tagged
antibody bound to their cell surface antigens are
excited by the laser and emit light that is
recorded by a second detector system located at
a right angle to the laser beam.
 The simplest form of the instrument counts each
cell as it passes the laser beam and records the
level of fluorescence the cell emits; an attached
computer generates plots of the number of cells
as the ordinate and their fluorescence intensity as
the abscissa.
 More sophisticated versions of the instrument are
capable of sorting populations of cells into
different containers according to their
fluorescence profile.
 Use of the instrument to determine which and
how many members of a cell population bind
fluorescently labeled antibodies is called
analysis;
 use of the instrument to place cells having
different patterns of reactivity into different
containers is called cell sorting.
 The flow cytometer has multiple applications to
clinical and research problems. A common clinical
use is to determine the kind and number of white
blood cells in blood samples.
From flow cytometric analysis, one can obtain the
following information:
1. How many cells express the target antigen as an
absolute number and also as a percentage of cells
passing the beam.
 For example, if one uses a fluorescent antibody specific
for an antigen present on all T cells, it would be possible
to determine the percentage of T cells in the total white
blood cell population.
2. Then, using the cell-sorting capabilities of the
flow cytometer, it would be possible to isolate the
T-cell fraction of the leukocyte population.
3. The distribution of cells in a sample population
according to antigen densities as determined by
fluorescence intensity.
 It is thus possible to obtain a measure of the
distribution of antigen density within the
population of cells that possess the antigen.
 This is a powerful feature of the instrument,
since the same type of cell may express
different levels of antigen depending upon its
developmental or physiological state.
4. The size of cells:
 This information is derived from analysis of the light-
scattering properties of members of the cell population
under examination.
 Flow cytometry also makes it possible to analyze
cell populations that have been labeled with two or
even three different fluorescent antibodies.
 flow cytometer is one of the essential tools for the
detection and classification of leukemia now a days
Miscellaneous technique
Isolation of Lymphocyte Populations
Introduction
In studies on humans, peripheral blood

lymphocytes are most readily available source of
cells. Lymphocytes and their specific
subpopulations can be isolated by: Fluorescent
activated cell sorter (FACS), density gradient
separation and resetting.
 Activities for which the lymphocytes can be
separated could be:
 To detect the ability of a given B cell to produce a
given antibody,
 To detect the ability of a given T cell to produce
particular Cytokines,
 To test the ability of a given cell to be stimulated
by a given mitogen.
 The study of human T cells is best performed
using purified cells, since the presence of other
cell types may have indirect effects on T cell
function. However, for any kind of functional
assay on T cell specificity antigen - presenting
cells are necessary.
A. peripheral blood Mononuclear Cells (PBMC)
Isolation
 The mononuclear cell fraction containing

monocytes and lymphocytes is separated from
polymorphonuclear cells and red blood cells by
density gradient centrifugation.
Equipment and reagents
 Suppl. RPMI - 1640 medium
 Fetal calf serum (FCS)
 Heat-inactivated human serum, blood group AB
 Ficoll - Hypaque
 50 and 15ml conical centrifuge tubes
 Temperature controlled centeifuge with GH-3.7-
horizontal rotor
 Trypan blue, haemocytometer
B. Separation of T and Non-T Cells from
Mononuclear Cells
The E - rosetting Technique
 The E-rosetting technique describes a procedure
for separating T cells and non-T cells from a
population of MNCs (e.g. peripheral blood or
synovial fluid MNCs) This method is based on the
ability of human T cells to blnd to sheep
erythrocytes via their CD2 molecule
Neuraminidase treatment of sheep red blood cells
(SRBCs) enhances the binding of SRBCs to T
lymphocytes (weineretal, 1973).
 First neuraminidase treated SRBCs are prepared.
Secondly, SRBCs and MNCs are mixed to form
rosettes (E*, which are then isolated from the non
-resetting population (E-,e. e. B cells and
monocytes) by Ficoll gradient centrifugation.
 In the last step, bound SRBCs are separated from
the rosetted T cells by hypothoc lyses (Gmelling
Meyling and Ballieux, 1977).
Equipment and reagents for E - resetting
 SRBCs (eg. From Biologische Arbeitsgemeinschaft
Hessen. Germany): sterile PBS suppl, RPMI - 1640
FCS, heat inactivated (Life Technologies inc.) Test -
Neuraminidase (Centeon L.L.C., king of Prussia,
PA): Ficoil density 1.09 (Biochrom).
 15-ml conical centrifuge tubes (e.g. Greiner or
Falcon). Temperature controlled centrifuge (eg.
Beckman or Heraeus).
Preparation of Neuraminidase - treated SRBC
 A suspension of DRBCs (2ml) and sterile PBS
(10ml) are placed in a 15ml conical centrifuge tube
and spun at 2000 rpm (900g) for q0min, where
after the PBS supernatant is removed and the cells
are re-suspended in PBS.
 This washing procedure is repeated twice.
 Before treatment with neuraminidase, washed
SRBCs can be stored at 40C for 3 days.
 Part of the dry SRBC pellet (300μl) is incubated
with 4.6ml RPMI - 1640 and 100μl neuraminidaes
in a water bath (370C, 30 min), washed twice
with RPMI - 1640 (2000rpm. 10min), and finally
re-suspended in RPMI - 1640 to a total volume of
5ml. the suspension stored at 40Cuntil use
Rosette formation and Ficoll density gradient
centrifugation
1. MNCs are prepared by standard Ficoll-Hypaque
centrifugation (washed, counted and suspended
in suppl. RPMI - 1640/10% (10x106 cells m1-1).
The neuramindase - treated SRBCs are mixed
with the MNCs (20-30 min, room temperature) to
allow E-rosette formation, where after the
mixture is layered over a Ficol solution (density
1.09) in a 15ml conical centrifuge tube.
The volumes of SRBCs medium and Ficoll used in
this protocol depend on the number of MNCs to
be separated. The tubes are centrifuged for 30
min a 2800 rpm.
2. Remove and decant about 80% of the upper layer
(RPMI - 1640/ 10% HUS) from the centrifuged
suspension. The E-rosette negative (monocytes/
B cell enriched) layer (E) is recovered from the
interface layer with a pipette, transferred to a 15-
ml conical tube, and washed with a pipette,
transferred to a 15ml conical tube, and washed
with suppl, RPMI - 1640/5% FCS.
3. The E-rosette-positive (T cell) pellet (E+) is
suspended in 1ml RPMI - 1640/10% FCS in the
15-ml tube. Cold distilled water (2ml) is added
for hypotonic lysis of SRNBCs and mixed gently.
After a few seconds, add 8ml RPMI - 1640/10%
FCS. Transfer this suspension to a 50 - ml tube
containing 40 ml RPMI - 1640 / 10% FCS and
centrifuge for 10min at 1300rpm.
Separation of T cell subsets
 Purification T-cell populations by indirect Antibody
panning
 T cells expressing particular cell surface markers,
such as the CD4, CD8, αß-TCR or TCR molecules
can be selected by their capacity to bind to a
antibody-coated plastic plates (Wysockl and Sato,
1978).
 For example to purify CD8+ T cells, isolated T
cells (E+ cells) are treated with a mouse anti -
human monoclonal antibody against the CD4
molecule, and then incubated on plastic dishes
that have been coated with an anti-mouse IgG)
antibody.
 The T-cell population that is not CD4 positive (i.e.
the αßTCR+ CD8+ and the TCR+ subpopulations),
and does not therefore bind the mouse anti
human CD4 antibody, will not adhere to the
coated plate. These CD4 cells can be selected
physically from the adherent CD4+ subpopulation.
T-cell population (E+ cells)
 Appropriate monocional antibody (ewg. OKT4 or
OKT8 hybridism supernatant containing anti - CD4
or anti - CD8 antibodies, or commercially available
anti - CD4, anti - CD8 antibody); suppl. ROMI -
1640; FCS, ehat inactivated PBS, sterlle
 Plastic six - well plates (Macroplate Standard.
Greine): 15-ml conical centrifuge tubes (eg.
Falcon); sterile rubber scraper; temperature
controlled centrifuge (e.g. Beckman or Heraeus).
Pitfalls
 The purity of the adherent cell population is
greater than non-adherent population. However,
it must be considered that the function of the
adherent T-cell adherent T cell population may be
altered by the binding of specific antibodies to
surface molecules to be positively selected.
Immunomagnetic Negative Selection of CD4+ T
cells
 The protocol below is another cell separation
techniques mediated by antibody - antigen
reactions T cells (E+ cells) are incubated with
specific monoclonal antibodies to surface
molecules (anti-CD8) to coat unwanted T cells.
Magnetic beads coated with goat anti-mouse IgG
are then applied to the cell suspension in order to
bind the antibody coated cells.
 After binding; the target cells can be recovered
using a strong magnetic field. Negative isolation
is a method by which the CD4+ subset is purified
from the CD8* subset binding to the coated
magnetic beads. Furthermore. In a positive
selection step, the beads can be removed from
the CD8+ target cells by a process of detachment.
 T-Cell population (E' cells)
 Appropriate monoclonal antibody (e.g anti CD8
antibody by ptarmigan) goat anti-mouse IgG coated
magnetic beads (Dynabeads M-450, Dynal Oslo,
Norway).
 Sterile PBS; FCS, heat inactivated coating medium
(Hanks balanced salt solution (HBSS) without Ca2+
Mg2+ or phenol Red, supplemented with 10% FCS
20mM HEPES). Suppl. RPM1-1640 HUS health
inactivated.
 Magnetic separation device (Dynal MPC-1) mixing
device (Dynal MX1. 2 or 3) 15-ml centrifugation
tubes (eg. Falcon) vortex mixer temperature
controlled centrifuge (e.g. Beckman or Heraeus)
Procedure
Rosette Test
 Subsets of T lymphocytes can be identified by
their differing membrane structures called
markers.
 Markers are categorized as antigen and receptors
can be detected by rosette technique
 The E rosette forming cells were assigned to T
cell lineage and the E-rosettes become the
principle marker for identification and
enumeration of human T cells
 The presence of fcreceptors for IgG or IgG or IgM
-T lymphocyte has been correlated with their
functional activity.
 Cells with IgM receptors were shown to provide
help for B cell differentiation to plasma cell,
whereas cells with IgG receptors were reported to
functions as suppressors
E-ROSETTE TEST
 Spontaneous rosette formation with untreated
sheep erythro0cytes was performed with some
modification
 Separate Lymphocytes and adjusted the count to
2.5x106 /ml in PBS.
 Prepare 1% sheep erythrocyte suspension in PBS
after 3x washing in PBS
 Then 50 micro liters of bovine serum albumin will
be liken in tube in which 100ul of iymphocytes
suspension and 100ul of 1% sheep RBC
suspension will be added.
 Then centrifuge for 5 minutes at 1000rpm
 After incubation at 40c for 1hr, 0.1% toludine will
be added and rosette-forming cells will be
counted.
Next topic is Methods of Monoclonal antibody production

Methods of Monoclonal antibody
production
Hybridoma technique
 Large quantities of absolutely pure, specific
immunoglobulin directed against an antigen of
interest can be produced by fusing a normal
plasma cell making the antibody of interest with a
myeloma cell with the capacity for prolonged
growth in tissue culture.
 The resulting mixed cell is called hybridoma
 The first stage in making a hybridoma is to
generate-
 antibody producing plasma cells
 This is done by immunizing a mouse against the

antigen of inertest and
 repeating the process several times to
ensure that it mounts a good response.
 Two to four days after administration of antigen,
the mouse's spleen is removed and broken up to
form a cell suspension.
 Spleen cells are fused with a myeloma cell line by
the addition of plyethylene glycol (PEG) which
promotes membrane fusion.
 These spleen cells are suspended in culture
medium together with a special mouse myeloma
cell line.
 It is usual to use myeloma cells that don't secrete
immunoglobulin's since this simplifies purification
later on.
 Only a small proportion of the cells fuse
successfully.
 The fusion mixture is then set up in culture with
medium containing 'HAT'.
 HAT is a mixture of hypoxanthine, aminopterin,
and thymidine.
 There are two biosynthetic pathways by which
cells can produce nucleotides and hence nucleic
acids.
 The myeloma cells are selected so that they lack
the enzyme hypoxanthine phosphoribsyl
transferase and as a result can not utilize
hypoxanthine in the culture medium to produce
inosin, a pryimidine precursor.
 They are obliged to utilize an alternative
biosynthetic pathway involving thymidine.But the
aminopterin in the culture is a drug that prevents
myloma cells from making their own thymidine.
 Since the myeloma cells can not use
hypoxanthine and the aminopetrine stops them
from using the alternative synthetic pathway,
they can not make nucleic acids and will soon die.
 Hybrids made from a myeloma and a normal cell
will grow;
 they possess hypoxanthine phosphoribosyl
transferase and
 can therefore use the hypoxanthine and
thymidine in the culture medium and
survive.
 The spleen cells die in culture naturally after
1-2 weeks.
 Any wells containing growing cells are tested
production of the desired antibodies (using RIA or
ELISAs) and if positive, the cultures are cloned by
plating out so that there is only one cell in each
well.
 This produce a clone of cells derived from a single
progenitor, which is both immortal and producer
of monoclonal antibody
Next topic is Recombinant DNA technique

Recombinant DNA technique
Recombinant DNA techniques

 Attempts are also being made to replace
altogether the hybridoma method by recombinant
DNA techniques.
 One such attempt focuses on the gene segments
that specify the Fab of an immunoglobulin
molecule, the VH CH1 and VLCL.
 These segments can be amplified by PCR from
many different mRNA (cDNA) molecules
expressed in a population of cells undergoing an
immune response, the amplified segments' are
inserted into a suitable vector, cloned and paired
randomly (always one, VH CH1 with VLCL, in a
suitable vector) and the pairs translated into
proteins (Fabs).
 Screening of this combinatorial library of
antibodies with labeled antigen then identifies
these combinations that bind this antigen.
 The identified VHCH1-VLCL pairs are placed in to an

exprsion vector, either bacterial or mammalian,
and used to produce large quantities of
antibodies with selected specificity.
Uses of monoclonal Antibodies
 The greatest impact of Mabs in immunology has
been on the analysis of cell membrane antigens.
 Because Mabs have a single specificity compared
to the range of antibody molecules present in the
serum, Mabs have multiple clinical applications
including
 Identifying and quantifying hormones
 Typing tissue and blood
 Identifying infectious agents
 Identifying clusters of differentiation for the
classification and follow-up therapy of leukemia's
and lymphomas
 Identifying tumor antigens and auto antibodies
 Immunotherapy
Next topic is Quality assurance and safety in serology

Quality assurance
and
safety in serology
Quality assurance and safety in
serology
Quality assurance
 Quality assurance is employed in serology
laboratory to support error free performance and
to ensure the highest quality of patient care.
 Important factors in routine quality assurance
program include evaluation of reagents,
equipments and personnel.
 Quality control of reagents commercial reagents in

serology laboratory which are employed to perform
either antigen/ antibody detection, must meet the
required specificity and potency each reagents on
each day use must be inspected visually for color,
cloudiness and
Quality control of the equipments
 Instruments and equipments used in serology

laboratories such as centrifuges and water
baths must be properly maintained and
monitored to ensure that they are working
properly.
 Check the speed and the actual revolution of

a centrifuge. A water bath temperature should
be constantly monitored using a
thermometer.
Quality control of personnel
 Though it is difficult to control the
maintenance of high personnel standard it is
one of the most important factors of quality
assurance program.
 Evaluate the person’s employment in the

laboratory for competency, dedication, trust
and ability to work in successful conditions. It
is also essential to maintain competencies of
personnel by participating in continuing
education activities.
Stages of quality assurance
 Pre analytical stage quality assurance-
associated with the steps before the actual
analysis of the sample. It includes the
following activities
 Checking the request for the following details
 patient name , age , sex , ID number
 specimen type , investigation required
 clinical note ,summarizing the patients

illness
 Collection and transportation of specimen
 Specimen collection
 Selection of appropriate test kit /method
Analytical stage of quality assurance
 This stage is also known as quality control and
associated with the real analysis of patient
sample .
 In order to assure quality of test result one toll
is standard operating procedure.
 It is a document that describes how to perform
various and routine operations in the
laboratory .
 it is also a step by step instruction that the
technician/technologist consult on daily basis to
complete their task.
 There are pre analytical, analytical and post
analytical SOP.
 Standard operating procedure covering
analytical stage encompasses
 Detailed procedure how to perform a test
 Safe handling of patient sample
 Preparation and use of quality control/
positive and negative control sample
 Preparation of antigenic /antibody suspension
in the laboratory
 Reading and interpretation of serological tests
 Cleaning and quality control of equipments
and materials used in serology laboratory.
 Safe working practices and procedures.
 Disposal of tested samples and cleaning of
used materials in the procedure
Post analytical stage of quality assurance
 This is associated with the procedures which are

carried out after performing the test, the
analytical stage.
 To carry out this sage of quality control the SOPs
need to include: reporting of the result and
verifying and interpreting results.
Laboratory safety in serology laboratory
1. Pipetting by mouth is prohibited.
2. Eating, drinking, smoking, storing food and
applying cosmetics is not permitted.
3. Labels must not be licked; materials must not be
placed in the mouth.
4. The testing site must be kept neat, clean and free
of materials that are not pertinent to the work
being done.
5. Work surfaces must be decontaminated after any
spill of potentially dangerous material and at the
end of each working day.
6. Needles must not be recapped. Sharps such
as needles and lancets must not be reused.
They must be disposed of in a special waste/
sharp container.
7. Before taking a finger-prick sample the finger
must be decontaminated. After the sample
has been obtained the wound must be
covered with a plaster.
8. Staff members must wash their hands after
handling infectious materials and before
leaving the laboratory or testing area.
9. Potentially contaminated and ordinary office
waste must be kept in separate and clearly
labeled waste containers.
10. All potentially contaminated materials and
specimens must be decontaminate before
disposal or cleaning for reuse.
11. Only persons who have been advised of the
potential hazards are allowed to enter
testing areas.
12. Doors must be kept closed when testing is in
progress. Children must be excluded from
testing areas
THANK YOU
ALL!
Stay Safe!!

Immunofluorescence Tests: Direct and Indirect

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Immunofluorescence Tests: Direct and Indirect

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Immunofluorescence tests

 There are two type of fluorescent antibody tests

A. Direct fluorescent antibody tests

+ The fluorochrome used is usually

 A tissue or smear containing the organism

 To identify bacteria when the numbers are very

 It may also be used to detect viruses growing in

B. Indirect Fluorescent Antibody Test

 In the IFAT, unlabelled antibody combines with

 To detect antibodies in a patient's

 After allowing time for the antigen and antibody to

 A fluorescent labeled anti-species globulin is added and

 The preparation is examined by fluorescence

 In this test, a known antigen is placed on the slide

 The preparation is then washed and fluorescent-

S.A. Berson and

Rosalyn Yalow, in 1960 to determine levels of

• Radioactive isotopes are molecules with

• carcinoembryonic and α- feto protein

• Liquid phase Assay

• Solid phase Assay

 The sample, labeled antigen and the specific

 After completion of incubation with the

 Developed with the objective of solving the

 Reading assignment (Read

 Named in E.M. Southern, who 1st described it in 1975

 Used to detect specific DNA sequences

 Used in clinical lab to detect gene mutations known to

 Used to detect carriers of the fragile X syndrome and

• Very similar with southern blotting

• Used to detect RNA

• Not used in clinical laboratory mostly

 because compared with other serological tests,

 require no special equipment

 usually less expensive.

 The fundamental and most commonly used reaction in

 Direct agglutination of bacterial antigen with its

E.g. the slide agglutination of salmonella,

To identify bacteria from cultures are difficult to

To check auto-agglutination (false agglutination)

 Specific antibody or known antigen is attached to inert

 When the known antigen or antibody combines with its

iii. Micro titration agglutination tests

 A precipitation reaction may be defined as

 In addition to these two substances,

 are used to detect and quantify antibodies in

 Some tests have a low sensitivity.

 Tube precipitation test

 Gel diffusion tests

 Counter immunoelectrophoresis tests

 When only the antigen or antibody diffuse, with

 Antigen and antibody diffuse towards each other

 Elek gel technique

 In general, complement fixation tests (CFT) are

best performed in reference laboratories where

facilities exist for the careful standardization and

control of reagents, which these tests require.

 Standard amount of complement is added

 Erythrocytes coated with Abs is added

 Amount of erythrocyte lysis is determined

 Tertiary binding tests measure the

1. Tizard. Immunology an introduction,4th