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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

Immuno-

diffusion

(Mancini)

The specific antibody to the antigen is incorporated in the

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

Known concentration of antigen is added in successive

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

Immuno-

diffusion

(Ochterlony)

Wells are punched in agarose gel, with a central well

being surrounded by peripheral wells.

The antigen solution is kept in the central well and sera

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-

current

Immuno-

electrophores

is

Like double immunodiffusion, specific antigen and

antibody molecules migrate towards each other and form

a precipitin arc where they meet in equivalent

proportions. However, unlike double immunodiffusion,

the migration takes place under the influence of electric

current and not through passive diffusion.

Immuno-

electrophores

is

An antigen mixture is first resolved into its components

through electrophoresis.

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

Disease

Research

Laboratory

(VDRL) test

Cardiolipin antigen is reacted with regain (antibody) in

heat inactivated patients sera and rotated on a VDRL

rotator at 180 rpm for 4 mins.

A positive reaction shows formation of clumps when

observed under the microscope. A negative reaction

shows presence of uniformly distributed crystals under

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the microscope.

Rapid

Plasma

Reagin (RPR)

test

To obviate the need of a microscope and heat inactivation

of serum, cardiolipin antigen is coated on carbon

particles.

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

Aggluti-

nation

Antigen is coated on latex particles and in presence of

specific antibody present in patient serum, there occurs

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-

tination

Antigen is coated on RBCs and in presence of specific

antibody present in patient serum, there occurs visible

agglutination of the RBCs. Example: Rose Waaler Test

C. Enzyme Linked Immunosorbent Assay:

Method Principle

Indirect

ELISA

Used for antibody detection:

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

ELISA

Used for antigen detection:

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

ELISA

Used for antigen detection:

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

Inhibition Test

This test is used for the detection of antibodies to

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

O (ASO) test

This test detects the presence of antibodies to

Streptolysin O antigen in patient serum. The

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biological activity of Streptolysin O toxin of

Streptococcus pyogenes is to hemolyse RBC

suspensions.

Presence of antibodies to this antigen in clinical

samples inhibits the hemolysin activity. The highest

dilution of patient serum that neutralizes this

hemolysin activity is considered 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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