SEROLOGICAL TEST

DR.SYEDA.G.S

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 Parameters of serological tests
 Antigen–antibody reactions in vitro are known as
serological reactions

Sensitivity -True positives/

GENERAL True positives + False negatives
FEATURES Specificity-True negatives/
AG-AB True negatives + False positives
REACTION
 Qualitative assays: Detection of the mere presence of an
antigen or antibody
 Quantitative assays: Estimation of the quantity of an
antigen or antibody

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Ag-Ab reactions
Conventional
Newer techniques
Conventional -
techniques -
 Enzyme linked immunosorbent assay (ELISA) Precipitation
 Precipitation reaction
 Enzyme linked fluorescent assay (ELISA) Agglutination

 Agglutination reactionAssay (IFA)
Immunofluorescence CFT

 Chemiluminescence-linked
Complement fixation test immunoassay (CLIA)
Neutralisation

 Immunohistochemistry
Neutralization test
 Rapid tests-
 Lateral flow assay (Immunochromatographic test)
 Flow through assay
 Western blot
 Immunoassays using electron microscope

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 REACTIONS ON
WHICH ANTIGEN-
ANTIBODY ASSAYS
ARE BASED
A. PRECIPITATION REACTIONS
When a soluble antigen combines with its
antibody in the presence of electrolytes (NaCl) at a
suitable temperature and pH, the antigen–
antibody complex forms an insoluble precipitate

Flocculation: When, instead of sedimenting, the

precipitate remains suspended as floccules, the
reaction is known as flocculation(liquid medium)

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PRECIPITATION REACTIONS
The amount of precipitate formed depends on the amount of antigen and antibody and can be
plotted into three phases
Prozone phenomenon: Excess antibody
Zone of equivalence: Optimum proportions
 Post-zone phenomenon: Excess antigen
Mechanism: Lattice formation with alternating antigen and antibody molecules

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VDRL

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VDRL TILE

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VDRL SIGNIFICANT TITRE IS >1:200

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APPLICATIO  Grouping of streptococci by the Lancefield’s technique
NS OF  The VDRL test for syphilis(RPR ) by slide flocculation

PRECIPITATI  To test toxigenicity in diphtheria bacilli-ELEKS gel precipitation

test)
ON  Forensic application in the identification of blood and seminal
stains
REACTIONS

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 AGGLUTINATION
REACTION
Agglutination: When a particulate antigen is mixed with its
antibody in the presence of electrolytes at a suitable
temperature and pH, the particles become clumped or
agglutinated
◦ Slide agglutination
◦ Tube agglutination

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Table 15.2 Clinical application of agglutination tests to detect antibodies in patients’ serum

AGGLUTINATION REACTION
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 1. Agglutination on a slide
 Identification of bacterial isolates
 Blood grouping and cross-matching
2. Agglutination in tubes
AGGLUTINAT  Serial dilutions of the serum
 Febrile agglutination tests
ION Advantages -More sensitive than precipitation for igM
REACTION detection.

Limitations of agglutination tests

 Agglutination test for brucellosis: Prozone phenomenon
 Incomplete or ‘blocking’ antibodies: False negative results
 Cross-reacting/heterophile antigens: False positive results

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Slide agglutination
Performed to confirm the identification and
serotyping of bacterial colonies grown in
culture.

Method used for blood grouping and cross

matching.

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WIDAL/TIDAL KIT

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TUBE AGGLUTINATION
Antibody titer can be estimated as the highest dilution of the serum which produces a visible
agglutination.
Typhoid fever (Widal test)
Acute brucellosis (Standard agglutination test)
Coombs Antiglobulin test
Heterophile agglutination tests:
Typhus fever (Weil Felix reaction)
Infectious mononucleosis (Paul Bunnell test)
Mycoplasma pneumonia (Cold agglutination test)

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 Significant titre
S.typhi O Ag is 100
S.typhi H Ag is 200

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Microscopic Agglutination
Agglutination test is performed on a microtiter plate.

Result is read under a microscope.

MAT is done for leptospirosis

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Passive agglutination test(Ab
detected):
When a soluble antigen is attached to the surface of carrier particles, it is possible to convert
precipitation tests into agglutination tests

Indirect hemagglutination test (IHA)

LAT-Latex agglutination test

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Passive agglutination
Indirect hemagglutination test (IHA)-
RBC(carrier molecule)s are used not used now a a days.

Latex agglutination test (LAT)-Latex -Polystyrene

latex particles (carrier molecule)adsorbing several types
of antigens.

black color card.

Drop of patient’s serum (containing antibody) is added

to a drop of latex solution coated with the antigen and

the card is rocked. ASO test

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 Significant titre >200

Detects –Antibody to streptolysin toxin of

Streptococcus pyogens

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Reverse Passive Agglutination Test
(for Antigen Detection)
RPHA (Reverse passive hemagglutination assay) -RBCs -hepatitis B surface antigen (HBsAg) –
Latex agglutination test
Carrier-Latex particles
Detection of CRP (C reactive protein), RA (rheumatoid arthritis factor), capsular antigen
detection in CSF (for pneumococcus, meningococcus and Cryptococcus) and streptococcal
grouping.

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CRP Significant titre
>0.6 mg/dl .Acute phase reactant protein—
CRP (Ag detection )
CRP levels invariably rise after major surgery
but fall to normal within 7-10 days.
Absence of this fall is indicative of possible
septic or inflammatory post operative
complications.
Serum CRP measurement also provides useful
information in patients with myocardial infarct
CRPmeasurement thus provides a simple
screening test for organic disorders
Rock the slide -2 minute

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Coagglutination test
Staphylococcus aureus
Used in past for antigen detection test in the past (e.g. Salmonella antigen detection from blood
and urine).
Obsolete at present.

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 COMPLEMEN
T FIXATION
TEST (CFT)
 The ability of the antigen–antibody
complexes to ‘fix’ complement is
made use of in the CFT
 Used for the detection of both
antibodies and antigens
 Requires five reagents—antigen,
antibody, complement, sheep
erythrocytes and amboceptor
(rabbit antibody to sheep red cells)

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COMPLEMENT FIXATION TEST
(CFT)
Applications of complement fixation test
 To detect and quantify an antibody that does not agglutinate or precipitate with its antigen
 To detect low levels of antibody (less than 1 μ gram per millilitre) during early infection
 To screen numbers of viral or bacterial infections to detect IgG antibodies (for some
respiratory virus and Coxiella burnetii antibodies, and occasionally antibodies from CSF)

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ENZYME-  An immunosorbent—an absorbing material specific to
LINKED one of the components of the reaction, i.e., the antigen
or the antibody
IMMUNOSORBE  Enzyme–substrate conjugates
NT ASSAY  Enzyme converts colourless substrates to a coloured
product bound to the antibody
(ELISA)

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TYPES OF ELISA
Indirect ELISA
Competitive ELISA
Sandwich ELISA
Capture ELISA
Cylinder or cassette ELISA
Enzyme-linked immunospot assay (ELISpot)
IgG avidity ELISA

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 Performed on a microtiter plate containing 96 wells,
made up of polystyrene, polyvinyl or polycarbonate
mac ELISA kits are commercially available; contain
all necessary reagents (such as enzyme conjugate,
dilution buffer, substrate/chromogen, etc).

Procedure involves a series of steps done

sequentially; at each step, a reagent is added - then
incubated, followed by washing of the wells
[manually or by an automated ELISA washer.
terial

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DIRECT
Well + Ag (test serum) + primary Ab-Enzyme +
substrate-chromogen → Color change

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INDIRECT
Differs from the direct ELISA in that the
secondary antibody (Ab targeted to Fc
region of any human Ig) is labelled
with enzyme instead of primary
antibody

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Sandwich ELISA
antigen gets sandwiched between a capture
antibody and a detector antibody.

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PRINCIPLE DENGUE NS1 Ag
MICROLISA
Direct Sandwich ELISA
The microwells are coated with Anti-dengue NS1antibodies
The samples are added in the wells
enzyme conjugate (monoclonal anti-dengue NS1 antibodies linked to
Horseradish Peroxidase (HRPO)) is added.
Dengue NS1 (from serum sample) is “trapped” or “sandwiched” between the
antibody and antibody HRPO conjugate..

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Unbound conjugate is then washed off with wash buffer.
Upon addition of the substrate buffer and chromogen, a blue colour
develops.
The intensity of developed blue colour is proportional to the
concentration of dengue NS1 antigen in sample.
To limit the enzyme-substrate reaction, stop solution is added and a
yellow colour develops which is finally read at 450nm
spectrophotometrically

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Competitive ELISA
ELISPOT is currently used in IGRA (interferon gamma assay), for diagnosis of latent
tuberculosis; where the sensitized T cells capable of producing the IFN-γ are measured.
IgG Avidity ELISA

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 Diagnosis of Infectious diseases like
 Hepatitis, EBV, cytomegalovirus IgM/IgG, dengue IgG,
influenza, TORCH panel, hepatitis B, C, etc.
 Rotavirus detection in fecal specimens and enterotoxin
USES OF of E. coli in feces
 Syphilis IgG/IgMH. pylori , IgG and antigen detection
ELISA  Food toxins like chloramphenicol, streptomycin,
penicillin, aflatoxins, etc.
 Food adulterants including E. coli, Campylobacter and
Salmonella antigens
 Human allergen-specific IgE and IgA ELISA

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OTHER IMPORTANT IMMUNOLOGICAL ASSAYS

Chemiluminescence immunoassay (CLIA)

Immunoelectroblot or western blot
Immunochromatographic assay/lateral flow assay
Immunoelectron microscopic assays
Immunofluorescence assay
Immunohistochemical techniques
Flow cytometry

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RAPID TESTS
Point of care (POC) tests, because unlike ELISA and other immunoassays, the Point of Care tests
can be performed independent of laboratory equipment

Two principles of rapid tests are available:

Lateral flow assay

Flow through assay

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 OTHER
IMPORTANT
IMMUNOLOGI
CAL ASSAYS
Fig. 15.8 Lateral flow immunoassay

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Principle of ICT (Antigen
Detection)LATERAL FLOW
The test system consists of a nitrocellulose
Test band: At the test line, the Ag-labeled Ab
membrane (NCM) and an absorbent pad.
complex is immobilized by binding to the monoclonal
Test line-Monoclonal ab coat
Ab in the test line to form a colored band
Control –Anti human Ig
Control band: The free colloidal gold labeled Ab can
move further and binds to the anti-human Ig to form
Absorbent pad –Specific Ab tagged with
a color control band. chromogenic marker is present in sample
window
If the control band is not formed, then the test is
considered invalid irrespective of whether the test
band is formed or not

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HEPACARD
The method uses Anti-HBsAg antibodies conjugated to colloidal gold and
Anti-HBsAg antibodies immobilized on a nitrocellulose strip in a thin line.
The test sample flows laterally through an absorbent pad.
If the sample contains HBsAg, the colloidal gold-antibody conjugate binds to the antigen,
forming an antigen-antibody-colloidal gold complex.
Theis complex then migrates through the NCM .
Complex meets the line of immobilized monoclonal antibody at (Test line) “T”.
The complex is trapped forming an antibody-antigen-antibody collidal gold complex. Pink test
line seen. Ab-AG-AB colloidal gold complex

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pink band at T indicating the sample is reactive for HBsAg. REACTIVE
Absence of a coloured band in the test region indicates a non-reactive test result.
A red procedural control line should always develop at ‘C’ called valid test

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DENGUE NS1AG, IgM ,IgG Test
(ICT/Lateral flow assay

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Flow-through Assay/
Protein A is used for labeling antibody HIV TRI DOT
instead of gold conjugate.
As the patient’s sample passes through
the membrane, HIV antibodies, if present
bind to the immobilized antigens
Test band-Pr.A binds to FC portion of HIV
Ab if present to give pink dot
Control band- can bind any IgG present ,
IgG-protein A complex can further bind to
the antihuman Ig at the control line

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HCV TRI DOT

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WESTERN BLOT TECHNIQUE
Denaturation of proteins with the detergent
sodium dodecyl sulfate (SDS) and the use of an
electric current to pull them through
polyacrylamide gel electrophoresis (PAGE)
1-Separation of Ag fragments by gel
electrophoresis- according to molecular weights
weight
2-Transfer of Ag fragments to NCM
3-Detection of Ab against each fragment by
enzyme immunoassay

NCM-Nitro celluose membrane

Supplemental test
USE-HIV, Hydatid disease

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WBT

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Table 15.3 Summary of common immunological tests performed in a clinical
laboratory

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