Antigen antibody reaction

These reactions serve several purposes. In the body they form the basis of antibody mediated immunity in infectious diseases or of tissue injury in some types of hypersensitivity reaction and autoimmune diseases. In laboratory it is used for diagnostic purposes. These reactions can be used for detection and quantitation of either antigen or antibody.

Practical Importance
1. Diagnosis of infectious diseases, hyper sensitivity states through identification of either Ag or Ab.
2. Quantitation of Ag or Ab.
3. Classification of bacteria on the basis of antigenic constitution.
4. Detection of adulteration of food stuffs.
5. Detection of hormones and enzymes by antibodies prepared against them.
6. Determination of compatibility between blood cells, tissues and organs of two individuals of the same species.

Antigen antibody reaction 
Occur in 3 stages
Primary stage:
Initial interaction between the two without any visible effect. Reversible reaction. This can be detected by estimating free or bound antigen or antibody separately.

Second Stage :
Leading to demonstrable events
Precipitation, Agglutination , lysis of cells, killing of live antigens, neutralization of toxins, fixation of complement, enhancement of phagocytosis, immobilization of motile organisms.
Tertiary stage :
Neutralization or destruction of injurious Ag. Tissue damage include clinical allergy, other immunological diseases.

General features of antigen-antibody reaction
1. Reaction is specific : Ag combines with homologous Ab
2. Entire molecules react, not fragments.
3. No denaturation of Ag or Ab during reaction.
4. Combination occurs at the surface. So surface Ag are immunologically relevant
5. Combination is firm, but reversible.
6. Both Ag and Ab participate in formation of agglutinate or precipitate.
7. Ag and Ab can combine in varying proportions. Both are multivalent (Different binding sites)

Measurement of Ag and Ab
Measurement may be in terms of mass (mg.or Nitrogen units or titre).The antibody titre of a serum is the highest dilution of serum which gives an observable reaction with the Ag in the particular test.
Important parameters of serological tests are specificity and sensitivity.

Isolation of pure antibodies
This is done by sequential protein fractionating steps include
1) Precipitation of gamma globulins in 30-50% amm. sulphate
2) Gel filtration to obtain molecule of correct size
3) Ion exchange chromatography to isolate molecules which are +vely charged at neutral pH
4) Affinity chromatography

Serological reactions.
1. Precipitation reaction
2. Agglutination reaction
3. Complement fixation tests
4. Neutralization tests
5. Opsonisation
6. Immunofluorescence
7. Radio immuno assay
8. Enzyme immuno assays
9. Immuno electro blot techniques
10. Immuno electron microscopic tests

Precipitation reaction
Based on the ability to precipitate when Ag and Ab combined in proportions at or near equivalence. Soluble Ag combines with its Ab in the presence of electrolytes (Nacl) at a suitable tempt and pH, the Ag-Ab complex form an insoluble precipitate. Precipitation can take place in liquid media or in gels.

To the same amount of antiserum in different tubes an increasing concentration of Ag. are added then the amount of immune complex precipitated rises and then falls. The precipitation curve generated in this have 3 zones.

Ab excess zone – Free ab can be detected in the supernatant
Equivalence zone – Added antigen is sufficient to combine with and precipitated all the Ab ,so neither free Ab nor Ab can be detected in the supernatant
Antigen excess zone – reduced amount of precipitate as excess Ag.
cause solubilisation of Ag-Ab complex.
This is called zone phenomenon

Practical application :
May be carried out as qualitative or quantitative test
Sensitive for detecting Ag as 1 microgram of protein can be detected by precipitation reaction
Forensic application in – identification of blood and seminal stains
Testing for food adulterants
Less sensitive for the detection of Ab

Types
1. Ring test : pouring Ag solution over a column of antiserum, precipitate forms at the junction of both liquids
2. Slide test : Eg. VDRL test for syphilis
3. Tube test

Immuno diffusion (precipitation in gel)
Advantage :
Reaction is visible as distinct band of precipitation, can be stained for preservation.
Each Ag-Ab reaction gives a line of precipitation .The number of different Ag in the reacting mixture can be identified.

Methods:
1.Single diffusion in one dimension
Ab is incorporated in Agar gel in a test tube and Ag solution is layered over it. Ag diffuses downward through agar gel forming a line of precipitation appears to move downward. It dissolves when Ag. concentration increases

2.Double diffusion in one dimension
Ab is incorporated in gel, above which placed a column of plane agar. Ag. Solution is layered on top. Ag and Ab move towards each other through plane agar and form a band of precipitate where they meet at optimum concentration

3.Single diffusion in 2 dimension
Ab incorporated in agar gel poured on a flat surface. Ag is added to the wells cut on the surface of gel. Ag diffuses radially and form ring shaped band of precipitation. The diameter of halo gives the estimate of Ag concentration.
Eg: used for screening sera for Ab to influenza virus.

4. Double diffusion in two dimensions
Widely employed method. Helps to compare different Ag and  antiserum directly. Agar gel is poured on slide ,wells are cut. Anti serum is placed in the central well and different Ag. In the surrounding well. If two adjacent Ag. are identical, the lines of  precipitation formed by them will fuse. If they are unrelated the  lines will cross each other. Eg : Elek test for toxigenicity of  diphtheria bacilli, precipitation occurs when it is toxigenic  Immuno electrophoresis
Involves the electrophoretic separation of a composite Ag into its constituent proteins.

1. Ag are separated in an agar gel by placing an electric charge across it. The gel’s pH is chosen so that +vely charged proteins move to the –ve electrode and vice versa
2. A trough is then cut between wells and filled with the Ab which is left to diffuse.
3. Ag and Ab form precipitation arcs.

Agglutination reaction
When a particulate antigen is mixed with its Ab in the presence of electrolyte at suitable temperature and pH the particles are agglutinated.More sensitive for the detection of Ab. Agglutination occurs optimally when Ag and Ab react in equivalent proportion. zone phenomina can be seen. Monovalent Ab do not cause agglutination, but they combine with Ag.

Application
1.Slide agglutination
A drop of antiserum is added to particulate Ag in a drop of saline on slide.Clumbing occur if the test is +ve and is used for blood grouping and cross matching
2.Tube agglutination
Quantitative method for the measurement of Ab :
Principle; when a fixed volume of a particulate Ag suspension is added to an equal volume of dilution of an antiserum in test tubes. Agglutination titre of the serum can be estimated.(used in typhoid, brucellosis & typhus fever)
eg : Widal test is typhoid
(Flagellar) H Ag and O Ag of typhoid bacilli is used
Use : Measurement of H & O antibodies in patient’s sera. Two tubes are used. Dreyer’s agglutination tube for H agglutination and Felix tube for O agglutination . Equal volumes (.4ml) of dilution of serum and H & O antigen are mixed in corresponding test tubes & incubated in water bath at 370c overnight. Control tubes containing the Ag. and normal saline are set to check autoagglutination. The H agglutination leads to the formation of loose cotton woolly clumps, while O agglutination form a disc like pattern at the bottom of the tube.

Interpretation of results of widal tests.
1. Agglutination titre depend on stage of disease .Agglutinins usually appear by the end of first week. The titre increases steadily till 3rd or 4th week, after which it declines.
2 .Demonstration of a rise in titre of Ab by testing 2 or more serum samples is more diagnostic.
3.Titires 1/100 or more for O agglutinins and 1/200 or more for H agglutinins are significant.
4.Agglutinin may be present on account of prior disease, inapparent infection or immunization. H agglutinin persists longer than O.
5.Cases treated with chloramphenicol may show poor response.

Paul Bunnel test – in IMN
There are agglutinins to sheep erythrocytes in patient’s sera, which are adsorbed by Ox red cells but not by guinea pig kidney extract.
Cold agglutination test in primary atypical pneumonia
patients sera agglutinate human O group erythrocytes at 4o C is reversible at 37oC.
The antiglobulin (Coombs) tests

Principle :
Rh +ve erythrocytes are mixed with incomplete antibody, then Ab coats the cell, cannot produce agglutination. On addition of antiglobulin serum which is complete antibody to immunoglobulin, agglutination takes place.

Types- Direct & indirect
Direct – sensitization of erythrocytes with incomplete Ab takes place in VIVO, as in disease due to Rh incompatibility. When the red cells of erythroblastotic infants are washed free of unattached protein and then mixed with a drop of Coombs serum, agglutination takes place.
In the indirect Coombs test sensitisation of red cells with Ab globulins is performed in vitro, for detection of anti Rh Ab(agglutination takes place). Coomb’s test for detection of incomplete & non agglutinating Ab.

Rose Waaler test – RA
Auto antibody (RA factor) appears in serum, which acts as an Ab to gamma globulin. RA factor is able to agglutinate red cells coated with globulins.
Complement fixation test

Principle :
Ability of Ag – Ab complexes to fix complement .CFT needs five reagents : Ag, Ab, complement, sheep erythrocyte, amboceptors (rabbit Ab to sheep red cell)
eg : Wasserman reaction (in syphilis)
Inactivated patient’s serum is incubated at 37oC for 1 hr with the Wassermann Ag and a fixed amount of guinea pig complement. If the serum contains syphilitc Ab the complement will be utilized in Ag-Ab reaction. If the serum does not contain syphilitc Ab, the complement will be left free.

Then testing for complement in the post incubation period is done by adding sensitized cells (sheep erythrocytes) incubating at 370C for 30mts. Lysis of erythrocyte indicate, complement was not used in the first step, so no syphilitic Ab (negative CFT).

1.Antigen + Test serum(contain Ab) – Complement fixed
+ Complement
+ Hemolytic system – no haemolysis; +ve test
2. Ag + Test serum ( not contain Ab) – Complement not fixed
+ Complement
+ haemolytic system – Haemolysis; –ve test.

Neutralization test
Virus neutralization tests
Neutralization of viruses by their Ab can be demonstrated. Toxin neutralization: eg : Schick test in diphtheria
Based on ability of circulating antitoxin to neutralize the diphtheria toxin given intradermally and indicate immunity or susceptibility to disease. Now it is not used, can be done by neutralization in cell culture.
Intradermal injection of skin test dose of diphtheritic toxin on the forearm. Reading on 1-2 days and 5-7 days later. (Similar injection of inactivated toxoids in other arm)
Results
1.No reaction on either arm indicate negative reaction
2.No reaction in control arm ; circumscribed erythematic reaction on test arm after 1-2 days, persisting for 7 days , positive test.
3.Pseudo reaction in both arm
appearing within 24 hrs fades by 4th day –ve, indicating immunity.
4.Combined reaction in which both areas initially showed pseudo reaction; followed by +ve reaction, denote susceptibility and hypersensitivity.

Immuno fluorescence 
Fluorescence is the property of absorbing light rays of one particular wave length and emitting rays with a different wave length. Fluorescent dye can be conjugated to Ab ,such labeled Ab can be used to locate and identify Ag in tissues. It can be used for the identification of bacteria, viruses or other antigens by direct immuno fluorescence.
eg : detection of rabies virus antigens in brain smears

Disadvantage: 
Separate fluorescent conjugate have to be prepared against each Ag to be tested. This is overcome by indirect immuno fluorescent test by using an antiglobulin fluorescent conjugate eg : Fluorescent treponimal Ab test. (Fluorescent dyes, convert UV light to visible light)
A drop of test serum is placed on a smear of T. pallidum on a slide After incubation the slide is washed off, leaving antibody globulin if present, coated on the Treponima pallidum. Smear is then treated with fluorescent labeled antiserum. Then fluorescent conjugate react with antibody globulin. After washing away all the unbound fluorescent conjugate ,when the slide is examined under UV light treponems will be seen as bright object against a dark back ground., +ve test.
Fluorescent dyes may also be conjugated with complement. Labeled complement can be employed in detection of Ag & Ab

Commonly used fluorescent dyes are
1) fluorescein isothiocyante (blue green fluorescence)
2) Lissamine rhodamine (exhibit orange red fluorescence )

Radio immuno assay (RIA)
Measurement of Ag and Ab by using radio isotopes ‘Binder-ligand assay’ is the term used for these reactions. The substance (Ag) whose concentration is to be determined is called analytic or ligand. The binding protein (ab) binds to ligand is called binder.

Application : Quantitation of hormones, drugs, tumour markers IgE and Viral antigens.
Fixed amount of Ab and radio labeled Ag react in the presence of unlabelled Ag. Both Ag’s compete for the limited binding site on Ab. This is determined by the level of unlabelled (serum) Ag present in reacting system. After the reaction, Ag is separated into free and bound fractions and their radio active count is measured. The concentration of the test Ag is calculated by ratio of bound and total Ag labels.

Enzyme immuno assay
Enzyme labeled conjugates are used for localization of Ag in tissues . So there is no radiation hazard. This includes all assays based on the measurement of enzyme labelled Ag, hapten or Ab.
Two types
Homogenous
Heterogenous
Homogenous: There is no need to separate bound and free fractions
Hetergenous: Requires separation of free and bound fractions by
centrifugation or by absorption on solid surface.
eg : Enzyme linked immunosorbent assay (ELISA)

ELISA
It involves an immunosorbent – absorbing material specific for one of the component of reaction, Ag or Ab
Application :
ELISA is done using 96 well microtitre plates.Detection of Ab by ELISA can be illustrated by the anti HIV antibody test.

Purified inactivated HIV Ag is adsorbed into microassay plate wells. Test serum diluted in buffer is added to the well and incubated at 370C for 30mt. The well is then washed. If the serum contains anti HIV Ab, it will form a stable complex with HIV Ag on the plate.A goat antihuman immunoglobulin Ab, conjugated with peroxidase enzyme is added and incubated for 30 mts. After thorough washing the substrate,
O – phenylene diamine dihydrochloride is added and after 30 mts, the colour that develops is read using a micro assay plate reader.
A simple modification of ELISA is cylinder or cassette ELISA Testing one or a few samples of sera at a time. Result obtains within 10mts (2 hrs in ELISA). This is used for HIV type I & 2 Ab.

Immuno electro blot techniques
eg : Western Blot test : definitive test for HIV infection
HIV proteins separated according to their electrophoretic mobility are blotted into strips of nitro cellulose paper. These strips then made to react with test sera and then with enzyme conjugated anti human globulin. Suitable substrate is then added which produces a prominent colour band when the Specific antibody has reacted with the separated viral protein. The position of the band on the strip indicate the Ag with which the Ab has reacted.

Immuno electron microscopic test:
1.Immuno electron microscopy :
Viral particles mixed with specific antisera one observed under electron microscope. Application : study of viruses . Hepatitis 2.Immunoferritin test :
Ferrtin can be conjugated with Ab and such labeled Ab reacting with Ag can be visualized through electron microscope.
3.Immuno enzyme test
Some stable enzymes such as peroxidase can be conjugated with Ab. Tissue sections carrying corresponding Ag can be visualized under electron microscope.

Reference :
Immunology – Roitt- Brostoff-Male
Basic pathology by Kumar , Cotran, Robbins
Text book of Microbiology by Jayaram Panikkar
Boyd’s text book of pathology