Serological techniques

and their applications

PRESENTED BY,
REKHA K.R.
PALB4142
DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
Serology & Immunology
SEROLOGY
◦ The scientific study of blood sera and their effects.
◦ Subdivision of immunology concerned with in-vitro Ag-Ab
reaction.
◦ Concerned with the laboratory study of the activities of the
components of serum that contribute to immunity.
 Classification of Antigen-
Antibody Interactions
1. Primary serological tests: (Marker techniques) e.g.
– Enzyme linked immuno sorbent assay (ELISA)
– Immuno fluorescent antibody technique (IFAT)
– Radio immuno assay (RIA)

2. Secondary serological tests: e.g.

– Agglutination tests
– Complement fixation tests (CFT)
– Precipitation tests
– Serum neutralization tests (SNT)
– Toxin-antitoxin test

3. Tertiary serological test: e.g.

– Determination of the protective value of an anti serum in an animal.
Enzyme-Linked Immuno
Sorbent Assay (ELISA)
 ELISA, is a biochemical technique used mainly in serology to detect
the presence of an antibody / antigen in a sample.
 ELISA is an unknown amount of antigen is affixed to a surface, and
then a specific antibody is washed over the surface so that it can bind
to the antigen
In the case of fluorescence ELISA, when light is shown upon the
sample, any antigen/antibody complexes will fluoresce so that the
amount of antigen in the sample can be measured.
Enzyme-linked Immuno-Sorbant assay
Labeling
(ELISA) technique
Principle

◦ use of enzyme-labeled immunoglobulin to detect antigens or antibodies

◦ signals are developed by the action of hydrolyzing enzyme on chromogenic
substrate
◦ optical density measured by micro-plate reader.

Examples
◦ Hepatitis A (Anti-HAV-IgM, anti-HAV Iggy)
To detect Antibody (indirect
ELISA):
To detect Antigen (sandwich ELISA):
Immuno fluorescent antibody
technique (IFAT)
 Immunofluorescence is a technique used for light microscope with a
fluorescent microscope and is used primarily on microbiological
samples.
 This technique uses the specificity of antibodies to their antigen to
target fluorescent dyes to specific biomolecules targets within a cell and
therefore allows visualization of the distribution of the target molecule
through the sample .
 It makes use of fluorophores to visualize the location of the antibodies.
Immuno fluorescent antibody
technique (IFAT)
Immunofluorescence testing
uses fluorescent Ab either directly or indirectly to visualize cells or cell
aggregates that have reacted with the FAbs

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 Labeling
Immuno-fluorescence
Principle
technique
◦ Use fluorescein isothiocyanate
labeled-immunoglobulin to detect
antigens or antibodies according to
test systems
◦ Requires a fluorescent microscope
Examples Cell infected with Dengue virus
◦ Herpes virus IgM
◦ Dengue virus
◦ Rabies virus
◦ Scrub and murine typhus

V. Cholerae
Types of immuno- Labeling
fluorescence technique

Direct immuno-fluorescence Steps Direct FA Indirect FA Sandwich FA

◦ Used to detect antigen 1st

Indirect and sandwich

immuno-fluorescence 2nd

◦ Antigen detection
◦ Antibody detection 3rd

Legend
Ag= =FITC-conjAnti-Ig

Ab=
=FITC-conjugated Ab

4th
Immuno-fluorescence:
Performance, applications

Advantages
Sensitive and specific
Can be used for discrepant analysis
Limitations
Expensive (Reagents and equipment)
Subjective
Cross reactivity
Non-specific immuno-fluorescence

Time taken
few minutes to few hours
RADIO IMMUNO ASSAY
It is a very sensitive in vitro assay technique used to measure
concentrations of antigens by use of antibodies . As such, it can be seen
as the inverse of a radio binding assay, which quantifies an antibody by
use of corresponding antigens.
In this method a known quantity of an antigen is made radioactive ,
frequently by labeling it with gamma radioactive isotopes of Iodine ,
attached to tyrosine . This radio labelled antigen is then mixed with a
known amount of antibody for that antigen .
As a result the two specifically bind to one another.
Radio Immuno Assay
Agglutination tests
Agglutination
When the antigen is particulate, the reaction of an antibody with the
antigen can be detected by agglutination (clumping) of the antigen. The
general term agglutinin is used to describe antibodies that agglutinate
particulate antigens.

a. Qualitative agglutination test

Agglutination tests can be used in a qualitative manner to assay for the
presence of an antigen or an antibody. The antibody is mixed with the
particulate antigen and a positive test is indicated by the agglutination of
the particulate antigen.
Qualitative agglutination test
Radioimmunoassay (RIA)

• Involves the separation of a protein (from a mixture)

using the specificity of antibody - antigen binding and
quantify it using radioactivity

•The technique was introduced in 1960 by Berson

and Yalow as an assay for the concentration of
insulin in plasma.

•Here radioactive materials are not administered

to the individual but are used as reagents.
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b. Quantitative agglutination test
Agglutination tests can also be used to measure the level of antibodies
to particulate antigens. In this test, serial dilutions are made of a sample
to be tested for antibody and then a fixed number of cells or bacteria or
other such particulate antigen is added.
Then the maximum dilution that gives agglutination is determined. The
maximum dilution that gives visible agglutination is called the titer. The
results are reported as the reciprocal of the maximal dilution that gives
visible agglutination.
Quantitative agglutination test
Passive hemagglutination:
The agglutination test only works with particulate antigens. This is
called passive hemagglutination.
The test is performed just like the agglutination test. Applications include
detection of antibodies to soluble antigens and detection of antibodies to
viral antigens.
APPLICATIONS OF AGGLUTINATION TEST
ABO blood grouping
Rh blood grouping
Widal test for typhoid
Weil felix test for typhus
Coomb’s test for the identification of anti Rh antibodies
Brucella agglutination test for brucellosis
Leptospira agglutionarion for leptospirosis
Cold agglutination test for pneumonia, malaria and
trypanosomiasis
Haemagglutination inhibition test for the diagnosis of certain
viral and parasitic diseases
ABO blood grouping and Rh
typing
To the drop of blood sample anti-serum A and to another drop
anti-serum B is added. If the blood sample is clumped with
antiserum A it is GROUP A
If clump formation occur with anti-serum B then its GROUP B
Clumping with both anti- sera then it is GROUP AB
No clumping its GROUP O
For Rh typing, a drop of blood sample is mixed with a drop of
anti-Rh serum
if clumping occurs its Rh+ve
If there is no clumping then its Rh-ve
Complement fixation test
The complement fixation test is an immunological test looking for
evidence of infection. It tests for the presence of either specific antibody
or specific antigen in a sample's serum.
 It uses sheep red blood cells (sRBC), anti-sRBC antibody and
complement, plus specific antigen (if looking for antibody in serum) or
specific antibody (if looking for antigen in serum).
If either the antibody or antigen is present in the sample's serum, then
the complement is completely utilized, so the sRBCs are not lysed. But
if the antibody (or antigen) is not present, then the complement is not
used up, so it binds anti-sRBC antibody, and the sRBCs are lysed.
The Wassermann test is one form of complement fixation test.
Applications of
RIA
 Analysis of hormones, vitamins,metabolites, diagnostic markers
◦ Eg. ACTH, FSH, T3, T4, Glucagon, Insulin, Testosterone, vitamin B12, prostaglandins,
glucocorticoids,

 Therapeutic drug monitoring:

◦ Barbiturates, morphine, digoxin,

 Diagnostic procedures for detecting infection

◦ HIV, Hepatitis A, B etc

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 Precipitation Tests

◦ One of the easiest of serological tests

◦ Relies on fact that antigens and antibody mixed in the
proper proportion form large macromolecular
complexes called precipitates
◦ Correct proportions are important to create
precipitation
◦ Two techniques determine optimal antibody and
antigen concentrations
◦ Immunodiffusion
◦ Immunelectrophoresis
 Precipitation tests
Radial Immunodiffusion
In radial immunodiffusion antibody is incorporated into the agar gel as it is poured
and different dilutions of the antigen are placed in holes punched into the agar. As
the antigen diffuses into the gel, it reacts with the antibody and when the equivalence
point is reached a ring of precipitation is formed.

Immunoelectrophoresis
In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched
out of an agar gel and the antigens are electrophoresed so that the antigen are
separated according to their charge. After electrophoresis, a trough is cut in the gel
and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are
produced in the equivalence zone when an antigen/antibody reaction occurs.
Countercurrent electrophoresis
In this test the antigen and antibody are placed in wells punched out of
an agar gel and the antigen and antibody are electrophoresed into each
other where they form a precipitation line.
Serum Neutralization Test
Serum neutralization test is a test in which the sample serum, which
may contain a neutralizing antibody and the microorganism of interest ,
is either placed in the cell culture or injected into the host organism so as
to evaluate levels of protective antibodies present within the serum.
Serum neutralization test is used to quantify the titre of neutralizing
antibody for a virus.
Currently it is considered to be the ‘Gold standard’ for detecting and
measuring antibodies that can neutralize the viruses that cause many
diseases . It has higher sensitivity than other tests like agglutination test
and many commercial Enzyme immunoassay without the compromising
specificity .
Toxin antitoxin test
A toxin antitoxin system is a set of two or more closely linked genes
that together encode both a protein ‘poison’ and a corresponding
antidote.
When these systems are contained on plasmids they ensure that only
the daughter cells that inherit the plasmids survive after the cell
division . If the plasmid is absent in a daughter cell, the unstable
antitoxin is degraded and the stable toxic protein kills the new cell. This
is known as post segregational killing . Toxin antitoxin systems are
widely distributed in prokaryotes, and the organisms often have them in
multiple copies .
Toxin antitoxin assays have been developed to characterize toxin
potency. Antitoxin is used in conjunction with a toxin to find the amount
needed to neutralize a set amount of toxin.
Uses
 Cases requiring increased sensitivity and specificity of
identification
 Cases requiring faster report turn around time
 Confirmation of culture
 Identification of organisms that are non-viable or cannot be
cultured
 Identification of fastidious, slow growing organisms
 Identification of organisms that are dangerous to culture
 Identification of organisms in small numbers or in small
volume specimens
Uses
 Monitoring of disease progression or initiation or
modification of therapy
 Drug susceptibility testing
 Differentiation of anti genically similar organisms
 Molecular epidemiology and infection control.
 Disease diagnosis by characterization of genetic materials
without direct identification of infectious agent
 Determination of virulence of antimicrobial resistance genes