Three Distinct Phases of Antigen/Antibody Reactions

Primary Phenomenon – Sensitization

Secondary Phenomenon – Lattice formation

Tertiary Phenomenon – Detected by affect on tissues or cells.

ANTIGEN- ANTIBODY REACTIONS

Antigens and antibodies combine with each other specifically and in an observable manner

Serological reactions : Antigen –antibody reactions in vitro

Uses :- In vivo1. Basis of antibody mediated immunity in infectious

diseases2. Tissue injury in hypersensitivity and autoimmune

diseases

In Vitro :- 1. Diagnosis of infections in laboratory

2. In epidemiological surveys

3. Detection of non infectious agents - enzymes 4. Detection and quantification of either Ag or Ab

Antigen-Antibody Binding Affinity

Avidity

Law of Mass Action

Affinity Antibody affinity is the strength of the reaction

between a single antigenic determinant and a single combining site on the antibody.

It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site .

Affinity is the equilibrium constant that describes the Ag-Ab reaction as illustrated in Figure . Most antibodies have a high affinity for their antigens.

Affinity

Avidity Avidity is a measure of the overall strength of binding

of an antigen with many antigenic determinants and multivalent antibodies.

Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies.

Avidity is influenced by both the valence of the antibody and the valence of the antigen.

Avidity is more than the sum of the individual affinities.

Avidity

Law of Mass Action Governs the reversibility of the antigen-antibody

reaction.

Reversible reaction, visible reaction occurs when the rate of binding exceeds the rate of dissociation.

Precipitation Curve Prozone – antibody excess, many antibodies coat

all antigen sites- results in false negative

Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative

Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

Precipitation Curve

Precipitation Curve

Precipitation When a Soluble Ag combines with its Ab in presence of

electrolytes at an opt temp and pH resulting in precipitate.

When a Soluble Ag combines with its Ab in presence of electrolytes at an opt temp and pH resulting in floccules which remain suspended in the medium.

e.g. VDRL test

Flocculation

Passive Immunodiffusion

Reactions in gels

Migrate towards each other and where they meet in optimal proportions form a precipitate.

Four Methodologies

Single diffusion, single dimension

Single diffusion, double dimension

Double diffusion, single dimension

Double diffusion, double dimension

Solution of antibody is carefully layered on top of a solution of antigen, such that there is no mixing between the two.

With time at the interface where the two layers meet, antigen-antibody complexes form a visible precipitate.

Single Diffusion, Single Dimension

Single Diffusion, Single Dimension

Single Diffusion, Two DimensionThe Ab is incorporated in agar gel.

Above which is placed a column of plain agar.

The Ag is poured on the top of this plain agar.

The Ag & Ab move toward each other and a ppt’n band is formed in the region of plain agar where they meet in optimum conc.

The Ab incorporated in agar gel on the slide or Petri dish.

The wells are cut on the surface of gel.

Ag is added to the wells.

The Ag diffuses radially and ring shaped band of ppt’n are formed.

The diameter of the ring is directly proportional to the conc of Ag.

Radial Immunodiffusion

Radial Immunodiffusion

Double Diffusion, Two Dimension Holes punched in agar.

Known antibody or antigen added to center well.

Known sample added to outer well.

Unknown sample added to outer well next to unknown sample.

Wait for bands to form.

The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity.

FIGURE 2:If a solution with antigens X and Y is placed in well 1, a solution with antigen X only is placed in well 2, and antiserum containing antibodies specific for both X and Y is placed in well 3, a reaction similar to that appearing in Fig. 2 will occur. Notice that there is a spur reaction towards the XY well. This indicates that the two antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. Such a reaction with spur formation indicates partial identity

If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines as in Fig. 3

Determine which interpretation fits for samples 1, 2 and 3.

Electrophoretic Techniques

Immunodiffusion can be combined with electrical current to speed things up.

Rocket Immunoelectrophoresis Antigen is electrophoresed into gel containing

antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

Rocket Electrophoresis

Immunoelectrophoresis

Immunoelectrophoresis

Two-dimensional immunoelectrophoresis. Antigens are separated on the basis of electrophoretic mobility. The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen.

AGGLUTINATION A particulate Ag combines with its Ab in presence of

electrolytes at an opt temp and pH resulting in visible clumping of particles

More sensitive than precipitation for detecting Abs

1. Slide Agglutination : Blood grouping, Bacterial typing

2. Tube Agglutination : quantitative method for AbsEg : Widal test, Cold agglutination test

3. Passive Agglutination

Passive Agglutination If we attach soluble Ag to the surface of carrier particle

then ppt’n reaction is converted into agglutination.

e.g:- Latex particle, bentonite, RBC etc.

Some important tests are

o C-Reactive protein test – CRP test

o Rheumatoid Arthritis factor – RA test

o Anti-Streptolysin O test – ASO test

o Bacterial Typing etc

ELISA An absorbing material specific for one of the components

of the reaction i.e Ag or Ab

The Ag or Ab coated on Microtitre wells.

Based on detection of Ag-Ab complex by enzyme substrate reaction.

Reaction further read by ELISA reader spectrophotometrically.

Direct ELISA

Indirect ELISA

Immunoflourescence Immunofluorescence is the labeling of antibodies

or antigens with fluorescent dyes.

Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.

Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.

• There are two ways of doing IF staining

– Direct immunofluorescence

– Indirect immunofluorescence

1. Direct immunofluorescence

• Ag is fixed on the slide

• Fluorescein labeled Ab’s are layered over it

• Slide is washed to remove unattached Ab’s

• Examined under UV light in an fluorescent microscope

• The site where the Ab attaches to its specific Ag will show apple green fluorescence

• Use: Direct detection of Pathogens or their Ag’s in tissues or in pathological samples

Direct immunofluorescence

2. Indirect immunofluorescence:

Indirect test is a double-layer technique

The unlabelled antibody is applied directly to the tissue substrate

Treated with a fluorochrome-conjugated anti-immunoglobulin serum

Advantage over direct IFBecause several fluorescent anti-

immunoglobulins can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test.

It is also more time-efficient since it is only one signal labelled reagent, the anti-immunoglobulin, is prepared during the lengthy conjugation process

RIA

Purpose is to determine the concentration of an antigen in solution

Competitive binding assay

Originally developed by Yalow and Berson in 1960 for insulin

RIAReagentsTracer: labeled antigen

Antibody

Standards: Known concentrations of unlabeled antigen

Unknown samples

Antibody

Labeled Antigen

Labeled Antigen + Sample

•Separate bound from free:

•Antibody labeled tubes can be simply decanted

•Liquid-phase antibodies need to be precipitated•Use a second antibody•PEG •Centrifugation

B%

concentration

Count gamma emission

Counts per minute (CPM) for each tube

A sample containing a higher concentration of the unknown antigen will have a lower CPM