IMMUNOLOGY-SEROLOGY LABORATORY NOTES

SAFETY IN THE IMMUNOLOGY-SEROLOGY LABORATORY

Standard Precautions
- All human blood and other body fluids are treated as potentially infectious for
human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other blood-
borne microorganisms that can cause disease in human beings.
BLOOD is the most important source of HIV, HBV, and other blood borne pathogens in the
occupational setting.

 HBV can be present in extraordinarily high concentrations in blood.

 HIV is usually found in lower concentrations.
 HBV- stable in dried blood and blood products at 25C for up to 7 days.
 HIV retains infectivity for more than 3 days in dried specimen at room temperature
and for > 1 week in an aqueous environment at room temperature.

Both HBV and HIV may be transmitted INDIRECTLY.

 Viral transmission – from contact with inanimate objects, such as work surfaces or
equipment contaminated with infected blood or certain body fluids.
 Viral exposure – virus is transferred to the skin or mucous membranes by hand
contact between a contaminated surface and nonintact skin or mucous membranes.
 The MOT for HBV and HIV are similar, but the potential for transmission in the
occupational setting is greater for HBV than HIV.

Both HBV and HIV may be DIRECTLY transmitted by various portals of entry.
 Percutaneous (parental)- inoculation of blood, plasma, serum, or other body fluids
form accidental needlesticks,
 Contamination of the skin with blood or certain body fluids without overt puncture,
caused scratches, abrasions, burns, weeping, or exudative skin lesions.
 Exposure of mucous membranes to blood or certain body fluids, as the direct result of
pipetting by mouth, splashes, or spattering.
 Direct transmission of virus can result by centrifuge accidents or the improper removal of
rubber stoppers from test tubes, producing droplets.

Most exposures do not result in infection. (the risk varies not only with the type of exposure
but also with the amount of infected blood in the exposure, the length of contact with the
infectious material, and the amount of virus in the patient’ s blood or body fluid or tissue at
exposure.

1|Pag
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY NOTES

SPECIMEN PREPARATION AND PROCESSING

 SERUM- the most frequently encountered specimen in immunological testing.

 Avoid hemolysis, this may produce a false-positive test.
 The blood specimen is allowed to clot at room temperature or at 4C and then centrifuged.
 Fresh, non-heat inactivated serum is usually recommended for testing.
 Serum may be stored between 2C and 8C for up to 72 hours.
 If any additional delay in testing, the serum should be frozen at -20C or below.
 It is important to consider the phase of the disease and the condition of the patient at the
time of specimen collection. This is important in assays in diagnosis of infectious
diseases.
o ACUTE SERUM – sample drawn during the acute phase of the illness- when the
disease is fist discovered or suspected.
o CONVALESCENT SERUM- sample drawn during the convalescent phase,
usually about 2 weeks.

Inactivation of complement
 The process that destroys complement activity.
 Complement is known to interfere with the reactions of:
o Syphilis test and complement components (C1q).
o Agglutinate latex particles and cause a false-positive reaction in passive
agglutination assays.
o May also cause lysis of the indicator cells in hemagglutination assays.
 Complement in body fluids can be INACTIVATED by heating to 56C for 30 minutes.
 When more than 4 hours has elapsed since inactivation, REINACTIVATED by heating
it to 56C for 10 minutes.

SEROLOGICAL TEST PRINCIPLES

TYPES OF IMMUNOLOGIC REACTIONS

TYPE REMARKS EXAMPLES

PRIMARY  NON-VISIBLE combination of 1. Immunofluorescence
REACTION single antibody to single (IF)
antigen site. 2. Radioimmunoassay
 Only involves sensitization. (RIA)
 Requires purified Ags and Abs. 3. Immunoenzymatic
 Tests to detect this reaction are assays (IEA)
technically difficult, complex,

2|Pag
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY NOTES

expensive, may require special

equipment and are time
consuming.
SECONDARY  IN VITRO demonstrable 1. Precipitation in
REACTION antigen-antibody reaction. solution or gel
 Followed by a secondary 2. Direct agglutination
manifestation, forming cross or Hemagglutination
links or lattice formation. 3. Complement fixation
 Methods used to detect these
reactions are quick and easy to
perform, less expensive, less
time consuming and usually do
not require special equipment.
TERTIARY  IN VIVO antigen-antibody 1. BIOLOGICAL
REACTION reaction that is not visible but is reactions such as
detected by the affect of the phagocytosis.
reaction on tissues or cells.

TERMS:
 SENSITIVITY
- Ability of a clinical test to be POSITIVE in the presence of its homologous antigen.
- Represents how much of a given substance is measured; the more sensitive the test, the
smaller the amount of assayed substance that is measured.
 SPECIFICITY
- Ability of a clinical test to be NEGATIVE in the absence of its homologous antigen.
- Represents what is being measured. A highly specific test measures only the
assay substance in question; it does not measure interfering or similar substance.
 CROSS REACTION
- Reaction of an antibody with an antigen (heterologous) other than the one that induced
its formation, reaction may be weaker than that with the inducing (homologous) antigen.
 AFFINITY
- It is the initial force of attraction that an antibody for a specific antigenic epitope or
determinant.
 AVIDITY
- It is the sum of all attractive forces between an antigen and an antibody.
- A measure of the overall stability of an antigen-antibody complex.

3|Pag
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY NOTES

PRIMARY SEROLOGIC REACTIONS

LABELED IMMUNOASSAYS
- Involves antigen and antibody interaction, with either reactants labeled with a marker
so that the amount of binding may be monitored
- use LIGANDS
o substances that complex to another substance.
o Substance to be measured.
- Terminologies:
o Labels- act as marker to detect antigen-antibody reaction
 Examples:
 Fluorescence immunoassay- uses fluorescent substances as labels.
 Radioimmunoassay- uses radioisotopes as labels
 Enzyme immunoassay- uses enzyme as labels
o Labeled species- may be antigen or antibody to which the label is attached
o Solid phase- media such as the well or microplate to which an antigen
or antibody may be attached to
o Heterogenous- requires a step to physically separate from unbound analyte
o Homogenous- no separation step is necessary because enzyme activity
diminishes when binding of antibody and antigen occurs
- Separation Step
o Purpose: provides a simple way to separate bound and free reactants
o Methods used:
 Use of physical means
 Includes: decanting, centrifugation, or filtration, followed by
washing step to remove any remaining unbound analyte
 Use of solid-phase vehicle
 Includes: polystyrene test tubes, microtiter plates, glass or
polystyrene beads, magnetic beads, and cellulose
membranes
- Detection Step
o Depends on the label used
 Radioimmunoassay: counting radioactivity using gamma (solid
scintillation)
 Enzyme immunoassay: change in absorbance in substrate measured
via spectrophotometry
 Fluorescence immunoassay: can be determined via fluorometers,
fluorescent microscope, flow cytometers and spectrofluorometers

4|Pag
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY NOTES

5|Pag
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY NOTES

Enzyme Immunoassay (EIA)

- General Principle: based on binding for specific antibody between labeled and unlabeled
antigens in a patient’s sample
- Can be used qualitatively and quantitatively; heterogenous or homogenous
- Can be used for antigen detection or antibody detection
- Enzyme labels used:
Enzyme Sources
Acetylcholinesterase Electrophorus electicus
Alkaline phosphatase Escherichia coli
Beta-galactosidase Escherichia coli
Glucose oxidase Aspergillus niger
Glucose-6-phosphate dehydrogenase Leuconostoc mesenteroides
Lysozymes Egg white
Maltate dehydrogenase Pig heart
Peroxidase Horseradish

-Involves colorimetric reaction

 DARKER color = HIGHER concentration of analyte
- Forms of Enzyme Immunoassay:
Forms Detects Solid phase Labeled species Concentration
DIRECT Antigens None Ab directed to the Directly
Ag proportional
NON- Indirect Antibodies Antigen AHG Directly
COMPETITIVE proportional
Capture/ Antigen Antibodies Ab directed to the Directly
Sandwich Ag proportional
COMPITITIVE Antigen Antibodies Antigen Inversely
proportional
IMMUNOENZYMTERIC Antigen Unattached Ab directed to the Directly
antigens Ag proportional

6|Pag
Prepared by Dawn Kimberly L. Padua,

METHOD
Enzyme-linked Immunosorbent
assay (ELISA)
Sandwich ELISA or Capture
assay
Enzyme-multiplied Homogenous
immunoassay technique (EMIT)
IMMUNOLOGY-SEROLOGY LABORATORY
DESCRIPTION USES
Heterogenous Used as SCREENING
Non-competitive: Indirect METHOD for HIV
Heterogenous Used to measures Igs,
Non-competitive: Capture hormones, proteins and
detects tumor markers
Used for determination of
low molecular weight
analytes not readily measured
by other methods

Fluorescence immunoassay or Fluorescent Antibody Test (FAT)

- Albert coons (1941)
- Fluorescence in visualized using FLUORESCENCE
microscope. Fluorescent probes used:
 Fluorescein isothiocyanate- green fluorescence
 Rhodamine – red-orange fluorescence
 Phyocyanin- red fluorescence
 Texas red - fluorescence

 DIRECT or SINGLE LAYER (DFA)

- Best suited to antigen (unknown) detection.
- Uses KNOWN FLUORESCEIN-LABELED ANTIBODY.
- (+) fluorescence

 INDIRECT or DOUBLE LAYER (IFA)

- Both UNKNOWN ANTIGEN or ANTIBODY detection.
- Uses KNOWN FLUORESCEIN-LABELED ANTIGLOBULIN.
- (+) fluorescence

6|Page
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

 FLUORESCENCE POLARIZATION IMMUNOASSAY (FPIA)

- Based on the change in polarization of fluorescent light emitted from a labeled
molecule when it is bound by antibody.
- Labeled antigens compete with unlabeled antigen in the patient sample for a
limited number of antibody binding sites.
- Degree of fluorescence polarization is inversely proportional to concentration of
the analyte.

Radioimmunoassay (RIA)
- First type of immunoassay developed.
- Pioneered by Rosalyn Yalow and Berson in the 1959.
- Radioisotope labels used: 𝟏𝟐𝟓𝐈 ; 𝟏𝟑𝟏𝐈 ; 𝐇𝟑 ; 𝐂𝟏𝟒
- Most popular radioisotope: 𝟏𝟐𝟓𝐈 (half-life 60 days)
- Radioactivity (radioactive decay) is measured by GAMMA SCINTILLATION
COUNTER

- Forms of RIA:
 COMPETITIVE RIA
- Uses the principle of competitive binding
- “Displacement/Radioligand Inhibition”
- Analyte being detected competes with a radiolabeled analyte for a limited number
of binding sites
- Amount of label in bound phase is INVERSELY PROPORTIONAL to the amount of
patient antigen
o If patient has a little amount of antigens present, it will make the radioactivity
high. Vice versa.
- Sensitivity of the assay is usually enhanced by adding reagents in the following order:
 First: Antibody
 Second: Unlabeled Antigen
 Third: Labeled Antigen

7|Page
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

 NON-COMPETITIVE RIA
- Sandwich assay
- “Immunoradiometric assay (IRMA)”
- Amount of labeled antibody is DIRECTLY PROPORTIONAL to the amount of patient
analyte

- Modification of RIA:
 RADIOIMMUNOSORBENT TEST (RIST)
- 1st step developed for the measurement of TOTAL SERUM IgE
- Serves as a screening test to determine if more specific allergy testing is indicated
- Competitive RIA (older method)

 RADIOALLERGOSORBENT TEST (RAST)

- 1966
- Measures allergen specific IgE
- Non-competitive solid phase immunoassay (older method)
- Principles of the test remain the same, but newer testing methods involves the use of
enzyme or fluorescent labels rather than radioactivity

 RADIOIMMUNOPRECIPITATION
- Used to determine total IgE
- Employs a soluble 2nd antibody to precipitate the bound antigen (double antibody
technique)
- It is a competitive binding assay

PRIMARY SEROLOGIC REACTIONS

AGGLUTINATION
- Involving the aggregation of particulate (carrier) antigen in the presence of
specific antibody
- Antigen: “Agglutinogen”
- Antibody: “Agglutinin”
- Tests are only semiquantitative but highly sensitive
- Stages of Agglutination:
o 1st: SENSITZATION
 Occurs when ANTIGEN-BINDING SITES of the antibodies become
closely associated with ANTIGENIC DETERMINANTS or EPITOPES
of the antigen
nd
o 2 step: LATTICE FORMATION

8|Page
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

 Occurs when ANTIBODIES on coated cells FORM CROSS-LINKAGES

BETWEEN CELL resulting visible clumping
- Grading of Agglutination Reactions (A- Tube method, B-Slide method)

- Types of Agglutination Reactions:

DIRECT AGGLUTINATION
o ANTIGEN that is NATURALLY attached to the carrier molecule
o Carrier can be RBCs or bacteria
o Example:
 Widal Test – detects antibodies to Salmonella
 Antigens/Reagents used: Salmonella O and H antigens
 Weil-Felix Test – detects antibodies to Rickettsia
 Antigens/Reagent used: OX-2, OX-19 and OX-K
 HEMMAGLUTINATION
 If an agglutination reaction involves red blood cell
 Best example is ABO blood group typing of human red blood cells

9|Page
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

INDIRECT/ PASSIVE AGGLUTINATION

o ANTIGEN that are ARTIFICIALLY attached to the carrier molecule
o Carriers used include: Polyesterene latex, bentonite, beads or charcoal
o Carries are coated with antigen that is NOT normally found on their surfaces
o Used to detect ANTIBODIES
o Example:
 ASO Latex Agglutination Test – detects ASO antibodies
 Reagent: SLO in latex particle

REVERSE PASSIVE
o ANTIBODY that are ARTIFICIALLY attached to the carrier molecule; attachment
is through the Fc region, not the Fab region
o Carriers used include: Polyesterene latex, bentonite, beads or charcoal
o Carries are coated with antibody that is NOT normally found on their surfaces
o Example:
 CRP Latex Agglutination Test – to detects C-reactive protein
 Reagent: Anti-CRP

AGGLUTINATION INHIBITION
o Based on competition between particulate and soluble antigens for limited antibody-
antigen combining sites
o Has two stages:
 1st stage: neutralization of antigen by addition of soluble reagent antibodies
 2nd stage: indicator phase; addition of antigen-coated particles to bind

10 | P a g
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

o Often used when antigen is to be detected on biological fluids

o Results:
 Positive: ABSENCE OF AGGLUTINATION
 Negative: PRESENCE OF AGGLUTINATION
o Example: HCG for pregnancy testing

PRECIPITATION
- Involving the aggregates of soluble antigen in the presence of specific antibody
- Antigen: “Precipitinogen”
- Antibody: “Precipitin”
- Simplest method for detection of antigen-antibody reactions
- Precipitation Reactions
o Precipitation technique by Light Measurement
 NEPHELOMETRY
 involves measurement of light scattering
 quantitation of immunoglobulins
 TURBIDIMETRY
 Involves measurement of light intensity (light blocked)
 Measures cloudiness of solution
o Precipitation by Passive Immunodiffusion Techniques
 Linear immunodiffusion
 Radial immunodiffusion
o Precipitation by Electrophoretic Techniques
 Rocket immunoelectrophoresis
 counter immunoelectrophoresis
 Immunofixation technique

11 | P a g
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

PASSIVE IMMONODIFFUSION
o PRINCIPLE: Soluble Ags and/or Abs diffuse through a gel or other
semisolid media until their concentration reaches optimal proportion and
forms stable immunoprecipitated in a band or line
o Molecule of different sizes tend to diffuse through gels at different rates.
 The higher the MW, the slower the rate of diffusion
o No electrical current is used to speed up the process
o TERMS:
 Single Diffusion
 Only ONE reactant (usually Ag) is moving
 Double Diffusion
 BOTH Ag and Ab are moving through the medium
 Single Dimension
 Reaction in tubes – Ag and Ab migrates up and down
 Double Dimension
 Reaction in petri dish – Ag and Ab diffuse rapidly
 Single Diffusion – Single Dimension
o “OUDIN”
o Earliest gel precipitation technique (started around 1946)
o Antibody (serum) is in agar tube; Antigen is overlain in the top
o Antigen moves through the gel to form precipitin band
o 2 types of precipitin band
 R type
 Rabbit antisera
 Form fuzzy edges
 Not well-defined margin
 H type
 Horse antisera
 Clear or well-defined margin

 Single Diffusion – Double Dimension

o “RADIAL IMMUNODIFFUSION”
o Specimen is placed in wells. Wells are cut from an agar plate
o Agar plate contains antibody
o Positive results are formation of precipitin rings
o Measures immunoglobulin concentration
o Types of RID
 FAHEY AND MCKELVEY
 “Kinetic method”
 Measures after 18 hours

12 | P a g
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

 The diameter is proportional to the log of the concentration

 MANCINI
 “Endpoint method”
 Measurement of 24 hours (for IgG) to 72 hours (for IgM)
 Square of diameter is then directly proportional to the
concentration of the antigen

 Double Diffusion – Single Dimension

o “OAKLEY AND FULTHORPE”
o Modified Oudin test
o Antibody is placed in a tube, overlaid with a neutral agar and overlaid with
an antigen
o The reaction is manifested by the formation of a precipitin bands or line
through the gel at the point of equivalence

 Double Diffusion – Double Dimension

o “OUCHTERLONY”
o Semiquantitative analysis of concentration of antigen or antibody
o Involves the use of agar in which holes or wells are cut
o Antigen and antibody are placed on separate wells and form a precipitin line at
the point of equivalence
o Ag and Ab diffuses and bind to each other, forming a line at the equivalence
zone, perpendicular to the axis between the well
o If the Ag concentration = Ab concentration, the precipitin line forms is equidistant
from the wells
o Results:
 IDENTITY
 Indicated by the presence of a smooth arc
 Antigens are serologically identical but not necessarily
chemically identical
 NON-IDENTITY
 Presence of crossed (or intersecting) lines
 Each of the antigen are recognized by the antibody but they are
not identical to each other
 PARTIAL IDENTITY
 Presence of single spur, pointing towards the simpler antigen
 Antibody recognizes both antigens but these antigens are
only partially identical
 DOUBLE PARTIAL IDENTITY
 Double spurring

13 | P a g
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

 Not a common reaction

ELECTROPHORETIC TECHNIQUE
- “Immunoelectrophoresis”
- Antigens and antibodies diffuse through a semi solid medium and employs the use of
electric current
- In zone electrophoresis, macromolecules are separated on the basis of charge in a
buffer saturated support matrix
o ANTIGEN: Negatively charged particles will migrate to the positive pole (Anode)
o ANTIBODY: Positively charged particles migrate to the negative pole (Cathode)
- General steps in electrophoresis
o Electrophoresis/separation
o Staining
o Densitometry
- One-stage electrophoresis
 Rocket electrophoresis
o “Laurell technique”
o Electrophoretic counterpart of the Oudin test
o Antibody is incorporated in the gel; only the antigen diffuses with the
aid of electric current
o For quantitation of antigens other than immunoglobulins
o Positive result: formation of precipitin rockets (bullet-shaped)
 Counterimmunoelectrophoresis
o Also known as countercurrent immunoelectrophoresis

14 | P a g
Prepared by Dawn Kimberly L. Padua,
 IMMUNOLOGY-SEROLOGY LABORATORY

o For the detection and semiquantitative measure of bacterial or

fungal antigens in body fluids
o Positive result: formation of precipitin line

- Two-stage electrophoresis
o Two-step process:
 1st stage: electrophoretic separation of proteins in the sample
 2nd stage: either passive immunodiffusion or electrophoresis
 Classic Immunoelectrophoresis
o Electrophoresis + immunodiffusion (Antibody is placed through)
o Positive result: precipitin arc
 Immunofixation electrophoresis
o Electrophoresis + immunodiffusion (Antibody is overlain in surface of gel)
o Positive result: precipitin bands
 Crossed (two-dimensional) immunoelectrophoresis
o Electrophoresis + electrophoresis (Antibody is incorporated in the 2nd gel)
o Positive results: precipitin rockets

References:
Clinical Immunology and Serology: A Laboratory Perspective by: Christine Stevens, 3rd edition
Immunology & Serology in Laboratory Medicine by: Mary Louise Turgeon, 5th edition
A Concise Review of Clinical Laboratory Science by: Joel Hubbard, 2nd edition
Quick Review Cards for Medical Laboratory Science Cards by: Valeri Polansky
ACTS Immunology and Serology Review Lecture Notes from Ms. Judea Marie Policarpio,
RMT, IMT (ASCPi)
Immunology and Serology Lecture Notes from Dr. Ruchelle Rayco-Turqueza

15 | P a g
Prepared by Dawn Kimberly L. Padua,