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Serology

AGGLUTINATION TESTS and IMMUNOASSYS


Methods for Ag-Ab
detection
Agglutination
Precipitation
Hemagglutination and Hemagglutination
inhibition
Viral neutralization test
Radio-immunoassays
ELISA
Immunofluorescence
Agglutination tests
Antibodies can agglutinate
multivalent particulate antigens,
such as Red Blood Cells (RBCs) or
bacteria
Some viruses also have the ability to
agglutinate with RBCs.
This behavior is called agglutination.
Serological tests based on
agglutination are usually more
sensitive than those based on
precipitation
Examples
Glass Agglutination Test
Tube Agglutination Test
Passive Agglutination
Test
Microscopic
Agglutination Test
DIRECT AGGLUTINATION
- Test patient serum against large,
cellular antigens to screen for
the presence of antibodies.
Antigen is naturally present on
the surface of the cells.
In this case, the Ag-Ab reaction
forms an agglutination, which is
directly visible.
DIRECT
AGGLUTINATION
The particle antigen may be a
bacterium.
e.g.: Serotyping of E. coli,
Salmonella using a specific
antiserum
The particle antigen may be a
parasite.
e.g.: Serodiagnosis of
Toxoplasmosis
The particle antigen may be a
DIRECT
AGGLUTINATION
These
reactions can
be performed
on slides (rapid
tests) or on
microliter
plates or tubes
for Antibody
titration if
Direct agglutination
Principle
combination of an insoluble
particulate antigen with its
soluble antibody
forms antigen-antibody
complex
particles clump/agglutinate Ag-Ab complex
used for antigen detection
Examples
bacterial agglutination
tests for sero-typing and
sero-grouping e.g., Vibrio
cholerae, Salmonella spp
Positive Negative
Glass Agglutination Test
Tube Agglutination
Test
Also known as the standard agglutination
test or serum agglutination test (SAT)
Test serum is diluted in a series of tubes
(doubling dilutions)
Constant defined amount of antigen is
then added to each tube and tubes
incubated for ~20h @37C
Particular antigen clumps at the bottom of
the test tube
Test is read at 50% agglutination
Quantitative
Widal Testing
Tube Agglutination Test
Tube
Agglutination
TestAgglutination No agglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 1/40


Passive Agglutination
An agglutination reaction that
employs particles that are coated
with antigens or antibody not
normally found in the cell surfaces
Particle carriers include:
Red blood cells
Polystyrene latex
Haemagglutination

RBC
Viral

Haemagglutination
Some viruses contain proteins which bind
to erythrocytes (red blood cells) causing
them to clump together
Viral
Hemagglutination

the attachment of viral particles by their


receptor sites to more than 1 cell.
As more and more cells become attached in
this manner agglutination becomes visible
Readings The

results
Titer: The maximum dilution that
gives visible agglutination.
The end point: is the well with the
lowest concentration of the virus where
there is haemagglutination
2 4 8 16 32 64 128 256 512 1024 2048
4096

The HA titer of this virus in this row is 256


or 28
(1:256 dilution contains (1 HA unit) (one
Hemagglutination test: method
1:8

1:2 1:2 1:2 1:2 1:2


virus

serial dilution

8 16 32 64 128 256
mix with red
blood cells

side view

top view

Titer = 32 HA units/ml

One HA unit :minimum amount of virus that


causes complete agglutination of RBCs
What is Antibody
Titer
Is the
lowest
concentratio
n of
antibodies
against a
particular
antigen. Figure 18.6
Latex Agglutination
Antibody molecules can be bound to
each latex beads
It will increase the potential number
of exposed antigen-binding sites.
When an antigen is present in test
specimen, it may bind to the latex
bead thus forming visible cross-
linked aggregates.
Latex particles can be coated with
antigen (pregnancy testing, rubella
Complement fixation Test
The complement fixation test (CFT)
was extensively used in syphilis
serology after being introduced by
Wasserman in 1909.
Whole complement system is made
up of nine proteins: C1 to C9
Complement proteins are heat labile
and are destroyed by heating at 56C
for 20 30 minutes.
Complement binds to Ag-Ab complex
Principle
Complement takes part in many of the
immunological reactions. It gets absorbed during
the combination of antigens and antibody.
This property of antigenantibody complex to fix
the complement is used in complement fixation test
for the identification of specific antibodies.
The hemolytic system containing sheep
erythrocytes (RBC) and its corresponding antibody
(Amboceptor) is used as an indicator which shows
the utilization or availability of the complement.
If the complement is fixed then there will be no
lysis of sheep erythrocytes, thus denoting a
positive test.
If the complement is available then there will be
hemolysis which is a property of complement,
denoting a negative test.
Components of CFT
Test System
Antigen: It may be soluble or particulate.
Antibody: Human serum (May or may not contain
Antibody towards specific Antigen)
Complement: It is pooled serum obtained from 4 to
5 guinea pigs.
Indicator System (Hemolytic system)
Erythrocytes: Sheep RBC
Amboceptor (Hemolysins): Rabbit antibody to
sheep red cells prepared by inoculating sheep
erythrocytes into rabbit under standard
immunization protocol.
Additional:
Radio-immunoassays
Principle
Radioactively labelled-antibody (or antigen) competes with the
patients unlabeled antibody (or antigen) for binding sites on a
known amount of antigen (or antibody)
Reduction in radioactivity of the antigen-patient antibody
complex compared with control test is used to quantify the
amount of patient antibody / antibody bound
Limited use due to the problems with handling radioisotope

Example
HBsAg

Response
Thyroid function test

Antibody
Radio Immuno Assay
Radio-immunoassays:
Performance, applications
Advantages
highly sensitive
can be used for detection of small quantities
quantification possible

Limitations
expensive
requires isotopes

Time taken
1 day
Labeling
Enzyme-linked Immuno-
technique
Sorbant assay (ELISA)
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
LISA

Micro-plate reader

Positive result

96-well micro-plate
Types of ELISA (Ag Abs
Labeling
technique
tests)
Competitive
Antigen or antibody are
labeled with enzyme and
allowed to compete with
unlabeled ones (in patient
serum) for binding to the
same target
Hydrolysis signal from Ag-
Ab complex (enzyme-
labeled) is measured
Antigen or antibody in
serum is then calculated
No need to remove the
excess/unbound Ag or Ab
from the reaction plate or
tubes)
Labeling
Types of ELISA used in the detection of
antigens and antibodies technique
Non-competitive
must remove excess/unbound Ag
or Ab before every step of reactions
Direct ELISA
Indirect ELISA
Sandwich ELISA
Ab Capture ELISA (similar to
sandwich ELISA but in 1st step,
anti-Ig (M or G) is coated
on the plate
Then antibodies in patient serum
are allowed to capture in next step
ELISA:
Performance, applications
Advantages
Automated, inexpensive
Objective
Small quantities required
Class specific antibodies measurable
Limitations
Expensive initial investment
Variable sensitivity / specificity of variable
tests
Cross contamination
Time taken - 1 day
Labeling
Immuno-fluorescence
technique
Principle
Use fluorescein
isothiocyanate labeled-
immunoglobulin to detect
antigens or antibodies
according to test systems
Requires a fluorescent Cell infected with Dengue virus
microscope
Examples
Herpes virus IgM
Dengue virus
Rabies virus
Scrub and murine typhus

V. Cholerae
Immunofluorescence Helps in
Diagnosis of Various Diseases
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.