COMMON ANTIGEN- ANTIBODY REACTIONS

A. Precipitation Reaction:
Precipitation may be defined as a method of antigen-antibody reaction in
which a soluble antigen reacts specifically with a soluble antibody to
produce a visible band/arc, when they are combined in equivalent
proportions and under optimum conditions of temperature, pH and
electrolyte concentration.
Some of the important forms of precipitation reaction are as follows:
Method

Principle

Single Radial

The specific antibody to the antigen is incorporated in the

Immuno-

agarose gel and a series of wells are punched in the gel.

diffusion

Known concentration of antigen is added in successive

(Mancini)

wells and the unknown antigen sample is added in a

separate well.
On overnight incubation, a precipitin ring is produced
around the antigen well.
The diameter of each ring is directly proportional to the
antigen concentration in the well. Hence, the unknown
concentration of antigen can be calculated from the
diameter of precipitin ring around the well.

Double

Wells are punched in agarose gel, with a central well

Immuno-

being surrounded by peripheral wells.

diffusion

The antigen solution is kept in the central well and sera

(Ochterlony)

from different patients in the different peripheral wells.

If any sera contains antibody to the antigen in the
central well, a precipitin arc would appear between the
two wells.
The relatedness between the antibody reactivities in the
sera placed in the neighbouring wells is reflected as
follows:
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Margin of the arcs: complete identity

Spur formation: partial identity

Crossing of the arcs: complete non-identity

Counter-

Like

double

immunodiffusion,

current

antibody molecules migrate towards each other and form

Immuno-

electrophores

proportions. However, unlike double immunodiffusion,

is

the migration takes place under the influence of electric

precipitin

arc

where

they

specific

meet

antigen

in

and

equivalent

current and not through passive diffusion.

Immuno-

An antigen mixture is first resolved into its components

electrophores

through electrophoresis.

is

Rectangular troughs are then cut in the agar, parallel to

the direction of electrophoretic migration and loaded with
patients serum.
A

precipitin

arcs

are

produced

between

the

electrophoresis lane and the trough depending on the

particular antigenic components to which the patients
serum has specific antibody.
Flocculation Reaction: This is a type of precipitation reaction, in which
soluble antigen reacts specifically with a soluble antibody to produce
complexes, which

being

too

light

to

precipitate

down,

float

as

floccules/Clumps.
The VDRL/ RPR test is an example of slide flocculation reaction.
Method

Principle

Venereal

Cardiolipin antigen is reacted with regain (antibody) in

Disease

heat inactivated patients sera and rotated on a VDRL

Research

rotator at 180 rpm for 4 mins.

Laboratory

A positive reaction shows formation of clumps when

(VDRL) test

observed under the microscope. A negative reaction

shows presence of uniformly distributed crystals under
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the microscope.
Rapid

To obviate the need of a microscope and heat inactivation

Plasma

of serum, cardiolipin antigen is coated on carbon

Reagin (RPR) particles.

test

A positive reaction shows visible black clumps and a

negative reaction shows uniformly distributed discrete
black crystals.

B. Agglutination Reaction:
This is an antigen- antibody reaction in which a particulate antigen
reacts with its specific antibody, in the presence of electrolytes at a
suitable temperature and pH, to produce clumping or agglutination of
the

particles.

Agglutination

occurs

optimally

when

antigen

and

antibodies react in equivalent proportions.

Some of the important forms of agglutination reaction are as follows:
Method

Principle

Widal Test

Patient sera is taken in dilutions in a series of test tubes,

arranged in 4 rows.
A specific antigen is added to all the tubes in each row, viz.
H antigens of Salmonella Typhi, Salmonella Paratyphi A and
Salmonella Paratyphi B and the shared O antigen.
The tubes are incubated at 37C for 12-18 hours.
In presence of specific antibody the H antigen produces
large, loose, fluffy clumps, whereas the O antigen produces
compact, chalky, granular disc.
The highest dilution showing such appearance is taken as
the agglutinating titer.

Latex

Antigen is coated on latex particles and in presence of

Aggluti-

specific antibody present in patient serum, there occurs

nation

visible agglutination of the latex particles. Example: Latex

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agglutination kits are available for detection of Rheumatoid

Factor, CRP, ASO, etc.
Hemagglu- Antigen is coated on RBCs and in presence of specific
tination

antibody present in patient serum, there occurs visible

agglutination of the RBCs. Example: Rose Waaler Test

C. Enzyme Linked Immunosorbent Assay:

Method

Principle

Indirect

Used for antibody detection:

ELISA

The specific antigen is coated on the well. The well is

washed to remove unbound antigen. Patient serum is
added to the well and specific antibodies, if present in the
sample, bind to the coated antigen. Unbound antibodies
are removed by washing. Anti- human antibody molecules,
conjugated with enzymes like Horse Radish Peroxidase
(HRP) or Alkaline Phosphatase (ALP), are added to the well
and bind to the specific antibodies present in the patient
serum. After washing to remove unbound conjugate, a
chromogenic substrate (OPD for HRP/ PNPP for ALP) is
added to generate a coloured product.

Sandwich

Used for antigen detection:

ELISA

The specific antibody is coated on the well. The well is

washed to remove unbound antibody molecules. Patient
sample is added to the well and specific antigen, if present
in the sample, bind to the coated antibody. Unbound
antigens are removed by washing. Detector antibody
molecules, specific for the antigen of interest, conjugated
with enzymes like Horse Radish Peroxidase (HRP) or
Alkaline Phosphatase (ALP), are added to the well and bind
to the specific antigen present in the patient sample. After
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washing to remove unbound conjugate, a chromogenic

substrate (OPD for HRP/ PNPP for ALP) is added to
generate a coloured product.
Competitive Used for antigen detection:
ELISA

The patient sample, containing the antigen of interest, is

incubated with its specific antibody. The antigen- antibody
mixture is transferred to an antigen- coated well. The well
is

washed

to

remove

unbound

antigen-

antibody

complexes. Enzyme (HRP or ALP) - conjugated secondary

antibody, specific for the antibody used in the first step, is
added to the well. After washing to remove unbound
conjugate, a chromogenic substrate (OPD for HRP/ PNPP
for ALP) is added. Development of a coloured product
indicates absence of the antigen of interest in the patient
sample.

D. Neutralisation Reaction:
Method

Principle

Hemagglutination This test is used for the detection of antibodies to

Inhibition Test

Hemagglutinating viruses. These viruses, because of

the presence of Hemagglutinin spikes on their
envelope,

cause

hemagglutination

of

RBC

suspensions.
In presence of antibodies to these Hemagglutinin
proteins in patient samples, hemagglutination is
inhibited. The highest dilution of patient serum that
neutralizes

this

hemagglutination

activity

is

considered as the Hemagglutination Inhibition titer.

Anti- Streptolysin This test detects the presence of antibodies to
O (ASO) test

Streptolysin

antigen

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in

patient

serum.

The

biological

activity

Streptococcus

of

pyogenes

Streptolysin
is

to

toxin

hemolyse

of

RBC

suspensions.
Presence of antibodies to this antigen in clinical
samples inhibits the hemolysin activity. The highest
dilution of patient serum that
hemolysin

activity

is

considered

neutralizes this
as

the

Anti-

Streptolysin O titer.

E. Immunochromatography:
These tests are also called lateral- flow tests or strip tests which are
suitable for point-of-care application due to their technical simplicity.
In this format, the clinical sample is added directly to the test strip. If the
analyte of interest is present in the sample, it reacts with specific
antibody molecules immobilized on colloidal gold particles present in the
testing system. The antigen-antibody complex migrates along the test
strip and forms a band at a position in the test strip where a set of
capture antibodies (specific for the analyte of interest) is immobilized.
This band is called the Test band and is indicative of the sample being
positive for the analyte of interest. Further down the strip is immobilized
a second set of capture antibodies, specific for the antibody molecules
coated on the gold particles. Binding of the gold particles to this second
set of capture antibodies produces the Control band which indicates
the proper performance of the test.

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