ANTIGEN-ANTIBODY

REACTIONS

DR.B.V.RAMANA, M.D.

DEFINITION:DEFINITION:

Antigens and antibodies combine with each other Antigens and antibodies combine with each other specifically and in an observable manner.specifically and in an observable manner. These Antigen-Antibody reactions These Antigen-Antibody reactions in vitroin vitro are are known as Serological tests.known as Serological tests.

USES:USES:In vivo:In vivo:

It forms the basis of immunity against infectious It forms the basis of immunity against infectious

diseases.diseases.It may lead to tissue injury in some hypersensitivity It may lead to tissue injury in some hypersensitivity reactions and autoimmune diseases.reactions and autoimmune diseases.

In vitro:In vitro:

For diagnosis of infection.For diagnosis of infection. Helpful for epidemiological studies.Helpful for epidemiological studies. For identification of non-infectious agents such as enzyme.For identification of non-infectious agents such as enzyme. Detection and quantitation of either antigens or antibodies.Detection and quantitation of either antigens or antibodies.

CHARACTERISTICSCHARACTERISTICS The reaction is specific, the specificity not absolute, cross The reaction is specific, the specificity not absolute, cross

reactions may occur.reactions may occur.

Entire molecule react and not fragments.Entire molecule react and not fragments.

There is no denaturation of the antigen or antibody during There is no denaturation of the antigen or antibody during the reaction.the reaction.

The combination occurs at the surface.The combination occurs at the surface.

The combination is firm but reversible.The combination is firm but reversible.

Influenced by Affinity and Avidity of the reactionInfluenced by Affinity and Avidity of the reaction

AFFINITY:AFFINITY: Refers to the intensity of attraction between Refers to the intensity of attraction between the antigen and antibody molecules. the antigen and antibody molecules.

AVIDITY:AVIDITY: It is the strength of the bond after the It is the strength of the bond after the

formation of antigen and antibody complex. formation of antigen and antibody complex.

Both antigen and antibody participates in the formation of Both antigen and antibody participates in the formation of the agglutinates and precipitates. the agglutinates and precipitates.

Antigens and antibodies can combine in varying Antigens and antibodies can combine in varying proportions. proportions.

REACTIONS OCCUR IN 3 STAGES:REACTIONS OCCUR IN 3 STAGES:

PRIMARY STAGE:PRIMARY STAGE: Initial interaction without any visible effectsInitial interaction without any visible effects Rapid Rapid Reversible reaction Reversible reaction

SECONDARY STAGE:SECONDARY STAGE:1.Follows primary stage, leading to demonstrable events 1.Follows primary stage, leading to demonstrable events

such assuch as PrecipitationPrecipitation AgglutinationAgglutination Lysis of cellsLysis of cells Killing of live antigensKilling of live antigens Neutralization of toxinsNeutralization of toxins Fixation of complementFixation of complement Immobilization of motile organismsImmobilization of motile organisms Enhancement of phagocytosisEnhancement of phagocytosis

2.Antibodies are designated by the reactions2.Antibodies are designated by the reactions Antibody causing agglutination as Agglutinin Antibody causing agglutination as Agglutinin

(Antigen called as Agglutinogen)(Antigen called as Agglutinogen) Antibody causing precipitation as Precipitin Antibody causing precipitation as Precipitin

(Antigen called as Precipitinogen)(Antigen called as Precipitinogen)

TERTIARY STAGE:TERTIARY STAGE:

Antigen-antibody reactions lead to neutralization Antigen-antibody reactions lead to neutralization or destruction of injurious antigens or to tissue or destruction of injurious antigens or to tissue damage.damage.

COMPARATIVE EFFICIENCY OF THE COMPARATIVE EFFICIENCY OF THE IMMMUNOGLOBULIN CLASSES IN DIFFERENT IMMMUNOGLOBULIN CLASSES IN DIFFERENT SEROLOGICAL REACTIONS:SEROLOGICAL REACTIONS:

REACTIONREACTION IgG IgG IgM IgM IgAIgA

Precipitation Strong Weak VariablePrecipitation Strong Weak Variable

Agglutination Weak Strong ModerateAgglutination Weak Strong Moderate

Complement fixation Strong Weak NegativeComplement fixation Strong Weak Negative

Lysis Weak Strong NegativeLysis Weak Strong Negative

MEASUREMENT OF ANTIGEN AND ANTIBODYMEASUREMENT OF ANTIGEN AND ANTIBODY Measurement as units or titreMeasurement as units or titre

The antibody titre of a serum is the The antibody titre of a serum is the highest dilutionhighest dilution of the of the serum which shows an observable reaction with the antigen serum which shows an observable reaction with the antigen in the particular test.in the particular test.

SENSITIVITY:SENSITIVITY: Refers to the ability of the test to detect even Refers to the ability of the test to detect even very minute quantities of antigen or antibody.very minute quantities of antigen or antibody.

SPECIFICITY:SPECIFICITY: Refers to the ability of the test to detect Refers to the ability of the test to detect reactions between homologous antigens and antibodies reactions between homologous antigens and antibodies only and with no other. only and with no other.

SEROLOGICAL REACTIONS:SEROLOGICAL REACTIONS:

PRECIPITATION REACTION:PRECIPITATION REACTION: Take place in liquid media or in gels (Agar, Agarose, Take place in liquid media or in gels (Agar, Agarose,

Polyacrylamide).Polyacrylamide).

When a soluble antigen combines with its antibody in the When a soluble antigen combines with its antibody in the presence of electrolytes (NaCl) at a suitable temperature presence of electrolytes (NaCl) at a suitable temperature and pH, the antigen-antibody complex forms an insoluble and pH, the antigen-antibody complex forms an insoluble precipitate.precipitate.

If the precipitate remain suspended as floccules, the If the precipitate remain suspended as floccules, the reaction is known as reaction is known as Flocculation.Flocculation.

If high quantities of antigens are added to the

same amount of antiserum in different tubes,

precipitation will be found to occur most rapidly

and abundantly in one of the middle tubes in

which the antigen and antibody are present in

optimal or equivalent proportions.

PROZONE PHENOMENON:PROZONE PHENOMENON: Sera rich in antibody may sometimes give a false negative

precipitation or agglutination result, unless several dilutions are tested.

MECHANISM OF PRECIPITATION: Marrack proposed the lattice hypothesis Multivalent antigens combine with bivalent antibodies,

precipitation occur only when a large lattice is formed.This occurs in the zone of equivalence.

In the zone of antigen excess, the valencies of the antibody are fully satisfied which results in failure to form a large lattice.

In the zone of antibody excess, the valencies of the antigen are taken up with antibody and results in failure to form a large lattice

APPLICATIONS OF PRECIPITATION REACTION:APPLICATIONS OF PRECIPITATION REACTION:

- Carried out as either a qualitative or quantitative test.- Carried out as either a qualitative or quantitative test.- Sensitive - Sensitive - Forensic application Identification of blood and - Forensic application Identification of blood and

seminal stains Food seminal stains Food adulterants adulterants

RING TEST:RING TEST:Layering antigen solutionLayering antigen solution

On column of antiserum in a narrow tubeOn column of antiserum in a narrow tube

Precipitate forms at the junctionPrecipitate forms at the junction

Example:Example: Ascoli’s thermoprecipitin test Ascoli’s thermoprecipitin test Grouping of Streptococci by the Lancefield technique Grouping of Streptococci by the Lancefield technique

SLIDE TEST:SLIDE TEST:Drop of antigen and antibody on a slideDrop of antigen and antibody on a slide

Mixed by shakingMixed by shaking

Floccules appearFloccules appearExample:Example: VDRL TestVDRL Test

TUBE TEST:TUBE TEST:Quantitative tube flocculation test For standardization of toxins and Quantitative tube flocculation test For standardization of toxins and toxoids toxoidsExample:Example: Kahn test Kahn testIMMUNODIFFUSION (Precipitation in gel)IMMUNODIFFUSION (Precipitation in gel)Advantages:Advantages:- - Reaction is visibleReaction is visible-- Different antigens in the reacting mixture can be readily observed Different antigens in the reacting mixture can be readily observed-- Indicates identity, cross-reaction and nonidentity between different Indicates identity, cross-reaction and nonidentity between different

antigensantigens

1.SINGLE DIFFUSION IN ONE DIMENSION (OUDIN PROCEDURE)

Antibody incorporated in agar gel in a test tube

Antigen solution is layered over it

Antigen diffuses downward

Form a line of precipitation

Number of bands indicates the number of different antigens present

2. DOUBLE DIFFUSION IN ONE DIMENSION (OAKLEY-FULTHORPE PROCEDURE)

Antibody incorporated in gel

A column of plain agar placed above it

Antigen layered on top of agar

Antigen and antibody move towards each other through the intervening column of plain agar

Band of precipitate at optimum proportion

3.SINGLE DIFFUSION IN TWO DIMENSIONS 3.SINGLE DIFFUSION IN TWO DIMENSIONS

(RADIAL(RADIAL IMMUNODIFFUSION)IMMUNODIFFUSION)Antiserum incorporated in agar gel poured on a flat Antiserum incorporated in agar gel poured on a flat

surface surface

Antigen is added to the wells Antigen is added to the wells

Ag diffuses radially and forms ring shaped bands of Ag diffuses radially and forms ring shaped bands of precipitation-Halo precipitation-Halo

Diameter of the halo Diameter of the halo concentration of concentration of antigen antigen

Uses:Uses:Estimation of the immunoglobulin in sera Estimation of the immunoglobulin in sera Screening for antibodies to influenza virusesScreening for antibodies to influenza viruses

RADIALRADIAL IMMUNODIFFUSIONIMMUNODIFFUSION

4.4. DOUBLE DIFFUSION IN TWO DIMENSIONS DOUBLE DIFFUSION IN TWO DIMENSIONS (OUCHTERLONY PROCEDURE)(OUCHTERLONY PROCEDURE)

Agar gel is poured on a slideAgar gel is poured on a slide

Wells are cutWells are cut

Antiserum placed in the central wellAntiserum placed in the central wellAntigens in the surrounding wellsAntigens in the surrounding wells

If two adjacent antigens are If two adjacent antigens are identical identical Line of precipitate fuseLine of precipitate fuse

If unrelated If unrelated Lines cross each otherLines cross each other

Partial identity Partial identity Spur formationSpur formation

Example:Example: Elek’s gel test Elek’s gel test

DOUBLE DIFFUSION IN TWO DOUBLE DIFFUSION IN TWO DIMENSIONSDIMENSIONS

IMMUNOELECTROPHORESIS:IMMUNOELECTROPHORESIS:Electrophoresis followed by immunodiffusion.Electrophoresis followed by immunodiffusion.

Agar or agarose gel on a slide Agar or agarose gel on a slide

Ag well and Ab trough cut on it Ag well and Ab trough cut on it

Test serum Test serum Ag wellAg well

Electrophoresed Electrophoresed

Antibody Antibody Trough Trough

Diffusion Diffusion For 18 – 24 hours For 18 – 24 hours

Precipitin lines Photographed, stained and preservedPrecipitin lines Photographed, stained and preservedUses:Uses: Testing for normal and abnormal serum and proteins in urine. Testing for normal and abnormal serum and proteins in urine.

IMMUNOELECTROPHORESISIMMUNOELECTROPHORESIS

ELECTROIMMUNODIFFUSION:ELECTROIMMUNODIFFUSION:

Development of precipitin lines Development of precipitin lines speeded up by speeded up by electrically driving the Ag and Ab. electrically driving the Ag and Ab.

COUNTERELECTROPHORESIS (CIE):COUNTERELECTROPHORESIS (CIE):Simultaneous electrophoresis of the Ag and Ab in Simultaneous electrophoresis of the Ag and Ab in

opposite opposite directionsdirections

PrecipitationPrecipitationUses:Uses:

- - Detection of alphafetoprotein in serumDetection of alphafetoprotein in serum- - Detection of antigens of Cryptococcus and Detection of antigens of Cryptococcus and

Meningococus in CSF. Meningococus in CSF.

COUNTERELECTROPHORESIS (CIE)COUNTERELECTROPHORESIS (CIE)

ROCKET ELECTROPHORESIS:ROCKET ELECTROPHORESIS:

Antiserum is incorporated in agaroseAntiserum is incorporated in agarose

Ag in increasing concentrations is placed in wells Ag in increasing concentrations is placed in wells

Ag is electrophoresed into the Ab containing agarose Ag is electrophoresed into the Ab containing agarose Pattern of immunoprecipitaiton resembles o rocket. Pattern of immunoprecipitaiton resembles o rocket.

ROCKET ELECTROPHORESISROCKET ELECTROPHORESIS

Agglutination Reaction:-Agglutination Reaction:- Temp, pH Temp, pH

Particulate Antigen + Antibody Particulate Antigen + Antibody Clumped or Clumped or ElectrolytesElectrolytes agglutinated. agglutinated.

-- More sensitive than precipitation for the detection of More sensitive than precipitation for the detection of antibodies. antibodies.

Applications of Agglutination Reaction:-Applications of Agglutination Reaction:-Slide Agglutination:-Slide Agglutination:- Drop of antiserum Drop of antiserum

Particulate Antigen in a drop of saline Particulate Antigen in a drop of saline

Positive:Positive: Clumping of the particles & Clumping of the particles & Cleaning of the drop. Cleaning of the drop.

USES:-USES:--- Identification of bacterial isolates.Identification of bacterial isolates.

-- Blood grouping & cross – matching.Blood grouping & cross – matching.

Tube agglutination:-Tube agglutination:-

Fixed volume of a particulate antigenFixed volume of a particulate antigen

Serial dilutions of antiserum in test tubesSerial dilutions of antiserum in test tubes

Agglutination titre of the serum → estimated. Agglutination titre of the serum → estimated. USES:-USES:- Diagnosis of Typhoid → widal testDiagnosis of Typhoid → widal test

BrucellosisBrucellosisTyphus fever → weil felix reaction Typhus fever → weil felix reaction

The antiglobulin (coombs) test:-The antiglobulin (coombs) test:- -- Devised by Coombs, Mourant Devised by Coombs, Mourant

& Race (1945) & Race (1945) -- Detection of anti – Rh antibodies.Detection of anti – Rh antibodies.

PRINICIPLE:-PRINICIPLE:-

Sera containing incomplete anti – Rh antibodiesSera containing incomplete anti – Rh antibodies

Mixed with Rh positive red cellsMixed with Rh positive red cells

Ab globulin coats the surface of the RBC No Ab globulin coats the surface of the RBC No agglutination. agglutination.

When such RBC are treated with a rabbit antiserum. When such RBC are treated with a rabbit antiserum.

Cells agglutinated. Cells agglutinated.

Direct - Direct - Sensitisation occurs in vivoSensitisation occurs in vivo

Coombs testCoombs test - Haemolytic disease of newborn- Haemolytic disease of newborn

Indirect - Indirect - Sensitisation performed in vitro. Sensitisation performed in vitro.

USES:-USES:--- For the detection of anti – Rh antibodies For the detection of anti – Rh antibodies

for demonstrating any type of incomplete or non for demonstrating any type of incomplete or non agglutinating antibody. agglutinating antibody.

Example:-Example:- Brucellosis. Brucellosis.

COOMBS TESTCOOMBS TEST

Passive Agglutination Test:-Passive Agglutination Test:-

Soluble Antigen + carrier particles Soluble Antigen + carrier particles Particulate Ag. Particulate Ag. (Precipitation Test)(Precipitation Test) (Agglutination test) (Agglutination test)

Carrier particles:Carrier particles:Red cells,Red cells,Latex particles or bentonite Detection of Latex particles or bentonite Detection of

ASO,CRP,RA factor,HCG. ASO,CRP,RA factor,HCG.Ex:-Ex:- Rose waaler test:-Rose waaler test:- For RA factorFor RA factor

Direct Direct Immunofluorescence:Immunofluorescence:

Indirect Indirect -Fluorescent dyes – fluorescein isothiocyanate -Fluorescent dyes – fluorescein isothiocyanate Lissamine rhodamine- Lissamine rhodamine- Rhodamine – Auramine etc., Rhodamine – Auramine etc.,

Direct:-Direct:- Detect antigens. Detect antigens.

Principle:Principle: Antibodies tagged with fluorescent dyes Antibodies tagged with fluorescent dyes

Detection of unknown antigenDetection of unknown antigen

Uses:Uses: Detection of bacteria,virus, other antigens in blood,Detection of bacteria,virus, other antigens in blood,CSF, Urine, faeces, tissue CSF, Urine, faeces, tissue Diagnosis of rabies. Diagnosis of rabies.

Disadvantage:-Disadvantage:- Specific fluorescent labelled Ab required.Specific fluorescent labelled Ab required.For each antigen.For each antigen.

Direct ImmunofluorescenceDirect Immunofluorescence

Indirect:-Indirect:- Detection of antibody Detection of antibody

Known Antigen Known Antigen

Unknown Antibody (serum)Unknown Antibody (serum)

Ab present – binds with antigen Ab present – binds with antigen

Fluorescein tagged Ab to human globulin is addedFluorescein tagged Ab to human globulin is added

Fluorescence occurs Fluorescence occurs Advantage:Advantage: Single antihuman globulin fluorescent Ab to Single antihuman globulin fluorescent Ab to

any Ag. any Ag.

Indirect ImmunofluorescenceIndirect Immunofluorescence

Radio immunoassay (R I A)Radio immunoassay (R I A)

- Quantitation of hormones, drugs, hepatitis B surface Ag,- Quantitation of hormones, drugs, hepatitis B surface Ag,Ig E & viral Antigens. Ig E & viral Antigens.

Antigen (Test)Antigen (Test)

Antibody Antibody Antigen (known Radio labelled) Antigen (known Radio labelled)

- Concentration of test Ag calculated using - Concentration of test Ag calculated using the reference curve. the reference curve.

ELISA:ELISA:- Detection of antigens and antibodies.- Detection of antigens and antibodies.- Principle of ELISA is same as that of IF, but - Principle of ELISA is same as that of IF, but an enzyme is used. an enzyme is used.- The test can be done in polystyrene tubes - The test can be done in polystyrene tubes

(Macro – ELISA) (Macro – ELISA) or Polyvinyl Microtiter plates (Micro – or Polyvinyl Microtiter plates (Micro –

ELISA)ELISA)

SANDWICH ELISA: (Antigen detection)SANDWICH ELISA: (Antigen detection)

wells coated with specific antibody.wells coated with specific antibody.

Specimen added.Specimen added.

Antigen present – binds to coated antibody Antigen present – binds to coated antibody To detect this Ag – Ab reaction, To detect this Ag – Ab reaction, Ab conjugated with an enzyme added. Ab conjugated with an enzyme added.

Conjugated Ab binds to Ag Conjugated Ab binds to Ag

Substrate added Substrate added

Positive result:-Positive result:- Colour production Colour production Read by spectrtophotometer or ELISA reader. Read by spectrtophotometer or ELISA reader.

SANDWICH ELISA: (Antigen detection)SANDWICH ELISA: (Antigen detection)

wells coated with specific antibody.wells coated with specific antibody.

Specimen added.Specimen added.

Antigen present – binds to coated antibody Antigen present – binds to coated antibody To detect this Ag – Ab reaction, To detect this Ag – Ab reaction, Ab conjugated with an enzyme added. Ab conjugated with an enzyme added.

Conjugated Ab binds to Ag Conjugated Ab binds to Ag

Substrate added Substrate added

Positive result:- Colour production Positive result:- Colour production Read by spectrtophotometer or ELISA reader. Read by spectrtophotometer or ELISA reader.

Indirect ELISA: (Ab detection)Indirect ELISA: (Ab detection)

Wells coated with antigenWells coated with antigen

Sera added Sera added

Ab present – binds to coated antigen Ab present – binds to coated antigen

To detect, goat antihuman Ig conjugated with an To detect, goat antihuman Ig conjugated with an

enzyme added enzyme added

Substrate addedSubstrate added

Colour productionColour production

SubstrateSubstrate EnzymeEnzyme O – phenyl diamine O – phenyl diamine

Dihydrochloride Dihydrochloride Horse radish peroxidaseHorse radish peroxidase P – nitrophenyl P – nitrophenyl

PhosphatePhosphate Alkaline phosphatase Alkaline phosphatase

Competitive ELISA: Competitive ELISA:

Detection of HIV Antibodies.Detection of HIV Antibodies. Positive result – No colourPositive result – No colour Negative result – colour. Negative result – colour.

Wells coated with HIV Antigen Wells coated with HIV Antigen

Sera added and incubatedSera added and incubated

Antibodies present- Ag-Ab reactionAntibodies present- Ag-Ab reaction

Enzyme labelled specific HIV Ab addedEnzyme labelled specific HIV Ab added

No Ag left for these Ab to act,No Ag left for these Ab to act,

These Ab remain free and washed during washingThese Ab remain free and washed during washing

Substrate added – No enzyme to actSubstrate added – No enzyme to act

Positive reactionPositive reaction: No colour: No colour

ELISAELISA

CASSETTE OR CYLINDER ELISACASSETTE OR CYLINDER ELISA

Specific Type 1 and 2 Ag immobilized on the Specific Type 1 and 2 Ag immobilized on the

nitrocellulose membrane nitrocellulose membrane

Serum added Serum added

Positive serum – Ab bind to appropriate AgPositive serum – Ab bind to appropriate Ag

Washing – Remove unbound Ab, conjugate added Washing – Remove unbound Ab, conjugate added

Washing - Remove unbound conjugate, substrate Washing - Remove unbound conjugate, substrate added added

Positive result – coloured spotPositive result – coloured spot

ADVANTAGES:ADVANTAGES:

Testing one or few samples of sera at a timeTesting one or few samples of sera at a time Test is rapid (10 mins)Test is rapid (10 mins) For detection of HIV type 1 and 2 AbFor detection of HIV type 1 and 2 Ab

Uses:-

1. Detection of HIV antibodies in serum.

2. Detection of Mycobacterial Ab in TB.

3. Detection of Rotavirus in faeces.

4. Detection of Hepatitis – B markers in serum.

Immunoelectronmicroscopic Tests:Immunoelectronmicroscopic Tests: Immunoferritin Test:-Immunoferritin Test:- To detect antigen. To detect antigen.

Ferritin (electron dense substance) conjugated Ab + Ag Ferritin (electron dense substance) conjugated Ab + Ag

Visualized under electron microscopeVisualized under electron microscope

Immunoelectronmicroscopy:Immunoelectronmicroscopy: Ag mixed with specific Ab Ag mixed with specific Ab

Electron microscope Electron microscope

Clumps seen Clumps seen

USES:-USES:- Diagnosis of Hepatitis – A and viruses causing diarrhea. Diagnosis of Hepatitis – A and viruses causing diarrhea.

Immunoenzyme Test: (To detect Antigen)Immunoenzyme Test: (To detect Antigen)

Enzymes conjugated with Antibodies Enzymes conjugated with Antibodies

Tissue sections treated with peroxidase labelled Tissue sections treated with peroxidase labelled antisera.antisera.

Reaction visualized under electron microscopeReaction visualized under electron microscope

Immunoblotting:Immunoblotting: Western blotting: (To detect proteins) Western blotting: (To detect proteins)

Proteins electrophoretically separated in a gel. Proteins electrophoretically separated in a gel.

Transferred to a nitrocellulose paper. Transferred to a nitrocellulose paper.

Reacted with test sera (Ab) and enzyme conjugated Reacted with test sera (Ab) and enzyme conjugated anti human Iganti human Ig

Substrate added Substrate added

Colour produced Colour produced

Detection of DNA – Southern Blotting.Detection of DNA – Southern Blotting.Detection of RNA – Northern BlottingDetection of RNA – Northern Blotting

ImmunoblottingImmunoblotting