ANTIGEN-ANTIBODY REACTIONS

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

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


USES: In vivo: It forms the basis of immunity against infectious diseases. It may lead to tissue injury in some hypersensitivity reactions and autoimmune diseases.

In vitro:
  

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

CHARACTERISTICS


The reaction is specific, the specificity not absolute, cross reactions may occur. Entire molecule react and not fragments. There is no denaturation of the antigen or antibody during the reaction. The combination occurs at the surface. The combination is firm but reversible.

Influenced by Affinity and Avidity of the reaction AFFINITY: Refers to the intensity of attraction between the antigen and antibody molecules. AVIDITY: It is the strength of the bond after the formation of antigen and antibody complex.

Both antigen and antibody participates in the formation of the agglutinates and precipitates. Antigens and antibodies can combine in varying proportions.

REACTIONS OCCUR IN 3 STAGES:

PRIMARY STAGE:  Initial interaction without any visible effects  Rapid  Reversible reaction SECONDARY STAGE: 1.Follows primary stage, leading to demonstrable events such as  Precipitation  Agglutination  Lysis of cells  Killing of live antigens  Neutralization of toxins  Fixation of complement  Immobilization of motile organisms  Enhancement of phagocytosis

2.Antibodies are designated by the reactions  Antibody causing agglutination as Agglutinin (Antigen called as Agglutinogen)  Antibody causing precipitation as Precipitin (Antigen called as Precipitinogen) TERTIARY STAGE: AntigenAntigen-antibody reactions lead to neutralization or destruction of injurious antigens or to tissue damage.

COMPARATIVE EFFICIENCY OF THE IMMMUNOGLOBULIN CLASSES IN DIFFERENT SEROLOGICAL REACTIONS: REACTION Precipitation Agglutination IgG Strong Weak IgM Weak Strong Weak Strong IgA Variable Moderate Negative Negative

Complement fixation Strong Lysis Weak

MEASUREMENT OF ANTIGEN AND ANTIBODY

 

Measurement as units or titre The antibody titre of a serum is the highest dilution of the serum which shows an observable reaction with the antigen in the particular test. SENSITIVITY: Refers to the ability of the test to detect even very minute quantities of antigen or antibody. SPECIFICITY: Refers to the ability of the test to detect reactions between homologous antigens and antibodies only and with no other.

SEROLOGICAL REACTIONS:
PRECIPITATION REACTION:


Take place in liquid media or in gels (Agar, Agarose, Polyacrylamide). When a soluble antigen combines with its antibody in the presence of electrolytes (NaCl) at a suitable temperature and pH, the antigen-antibody complex forms an insoluble antigenprecipitate. If the precipitate remain suspended as floccules, the reaction is known as 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:


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: - Carried out as either a qualitative or quantitative test. - Sensitive - Forensic application Identification of blood and seminal stains Food adulterants RING TEST: Layering antigen solution On column of antiserum in a narrow tube Precipitate forms at the junction Example: Ascolis thermoprecipitin test Grouping of Streptococci by the Lancefield technique

SLIDE TEST: Drop of antigen and antibody on a slide Mixed by shaking Floccules appear Example: VDRL Test TUBE TEST: Quantitative tube flocculation test

For standardization of toxins and toxoids

Example: Kahn test IMMUNODIFFUSION (Precipitation in gel) Advantages: - Reaction is visible - Different antigens in the reacting mixture can be readily observed - Indicates identity, cross-reaction and nonidentity between different crossantigens

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 (RADIAL IMMUNODIFFUSION) Antiserum incorporated in agar gel poured on a flat surface Antigen is added to the wells Ag diffuses radially and forms ring shaped bands of precipitationprecipitation-Halo Diameter of the halo concentration of antigen

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

RADIAL IMMUNODIFFUSION

4.

DOUBLE DIFFUSION IN TWO DIMENSIONS (OUCHTERLONY PROCEDURE) Agar gel is poured on a slide Wells are cut Antiserum placed in the central well Antigens in the surrounding wells If two adjacent antigens are identical Line of precipitate fuse If unrelated Partial identity Lines cross each other Spur formation

Example: Eleks gel test

DOUBLE DIFFUSION IN TWO DIMENSIONS

IMMUNOELECTROPHORESIS: Electrophoresis followed by immunodiffusion. Agar or agarose gel on a slide Ag well and Ab trough cut on it Test serum Ag well Electrophoresed Antibody Diffusion Precipitin lines Trough For 18 24 hours

Photographed, stained and preserved

Uses: Testing for normal and abnormal serum and proteins in urine.

IMMUNOELECTROPHORESIS

ELECTROIMMUNODIFFUSION: Development of precipitin lines speeded up by electrically driving the Ag and Ab. COUNTERELECTROPHORESIS (CIE): Simultaneous electrophoresis of the Ag and Ab in opposite directions Precipitation Uses: - Detection of alphafetoprotein in serum - Detection of antigens of Cryptococcus and Meningococus in CSF.

COUNTERELECTROPHORESIS (CIE)

ROCKET ELECTROPHORESIS: Antiserum is incorporated in agarose Ag in increasing concentrations is placed in wells Ag is electrophoresed into the Ab containing agarose Pattern of immunoprecipitaiton resembles o rocket.

ROCKET ELECTROPHORESIS

Agglutination Reaction:Reaction:Temp, pH

Particulate Antigen + Antibody

Clumped or Electrolytes agglutinated.

More sensitive than precipitation for the detection of antibodies. Applications of Agglutination Reaction:Reaction:Slide Agglutination:Agglutination:Drop of antiserum Particulate Antigen in a drop of saline Positive: USES:USES:Clumping of the particles & Cleaning of the drop.

Identification of bacterial isolates. Blood grouping & cross matching.

Tube agglutination:agglutination:Fixed volume of a particulate antigen Serial dilutions of antiserum in test tubes Agglutination titre of the serum USES:USES:estimated.

Diagnosis of Typhoid widal test Brucellosis Typhus fever weil felix reaction

The antiglobulin (coombs) test:test:Devised by Coombs, Mourant & Race (1945) Detection of anti Rh antibodies. PRINICIPLE:PRINICIPLE:Sera containing incomplete anti Rh antibodies Mixed with Rh positive red cells Ab globulin coats the surface of the RBC No agglutination. When such RBC are treated with a rabbit antiserum. Cells agglutinated.

Direct Coombs test Indirect USES:USES:-

- Sensitisation occurs in vivo

- Haemolytic disease of newborn - Sensitisation performed in vitro.

For the detection of anti Rh antibodies for demonstrating any type of incomplete or non agglutinating antibody. Example:Example:Brucellosis.

COOMBS TEST

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

Carrier particles:
Red cells, Latex particles or bentonite Ex:Ex:Detection of ASO,CRP,RA factor,HCG. Rose waaler test:- For RA factor test:-

Direct Immunofluorescence: Indirect -Fluorescent dyes fluorescein isothiocyanate Lissamine rhodaminerhodamineRhodamine Auramine etc., Detect antigens. Antibodies tagged with fluorescent dyes Detection of unknown antigen Detection of bacteria,virus, other antigens in blood, CSF, Urine, faeces, tissue Diagnosis of rabies. Disadvantage:Disadvantage:Specific fluorescent labelled Ab required. For each antigen. Uses:

Direct:Direct:Principle:

Direct Immunofluorescence

Indirect:Indirect:-

Detection of antibody Known Antigen Unknown Antibody (serum) Ab present binds with antigen

Fluorescein tagged Ab to human globulin is added Fluorescence occurs Advantage: Single antihuman globulin fluorescent Ab to any Ag.

Indirect Immunofluorescence

Radio immunoassay (R I A) - Quantitation of hormones, drugs, hepatitis B surface Ag, Ig E & viral Antigens.
Antigen (Test)

Antibody
Antigen (known Radio labelled)

- Concentration of test Ag calculated using the reference curve.

ELISA:
- Detection of antigens and antibodies. - Principle of ELISA is same as that of IF, but an enzyme is used. - The test can be done in polystyrene tubes (Macro ELISA) or Polyvinyl Microtiter plates (Micro ELISA)

SANDWICH ELISA: (Antigen detection)

wells coated with specific antibody. Specimen added. Antigen present binds to coated antibody To detect this Ag Ab reaction, Ab conjugated with an enzyme added. Conjugated Ab binds to Ag Substrate added Positive result:- Colour production result:Read by spectrtophotometer or ELISA reader.

SANDWICH ELISA: (Antigen detection)

wells coated with specific antibody. Specimen added. Antigen present binds to coated antibody To detect this Ag Ab reaction, Ab conjugated with an enzyme added. Conjugated Ab binds to Ag Substrate added Positive result:- Colour production result:Read by spectrtophotometer or ELISA reader.

Indirect ELISA: (Ab detection)

Wells coated with antigen Sera added Ab present binds to coated antigen To detect, goat antihuman Ig conjugated with an enzyme added Substrate added Colour production

Substrate


Enzyme
Horse radish peroxidase Alkaline phosphatase

O phenyl diamine Dihydrochloride P nitrophenyl Phosphate

Competitive ELISA:
  

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

Wells coated with HIV Antigen Sera added and incubated Antibodies present- Ag-Ab reaction present- AgEnzyme labelled specific HIV Ab added No Ag left for these Ab to act, These Ab remain free and washed during washing Substrate added No enzyme to act Positive reaction: No colour reaction:

ELISA

CASSETTE OR CYLINDER ELISA

Specific Type 1 and 2 Ag immobilized on the nitrocellulose membrane Serum added Positive serum Ab bind to appropriate Ag Washing Remove unbound Ab, conjugate added Washing - Remove unbound conjugate, substrate added Positive result coloured spot

ADVANTAGES:
  

Testing one or few samples of sera at a time Test is rapid (10 mins) For 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:
Immunoferritin Test:- To detect antigen. Test:Ferritin (electron dense substance) conjugated Ab + Ag Visualized under electron microscope

Immunoelectronmicroscopy:
Ag mixed with specific Ab Electron microscope Clumps seen

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

Immunoenzyme Test: (To detect Antigen)

Enzymes conjugated with Antibodies Tissue sections treated with peroxidase labelled antisera. Reaction visualized under electron microscope

Immunoblotting: Western blotting: (To detect proteins) Proteins electrophoretically separated in a gel. Transferred to a nitrocellulose paper. Reacted with test sera (Ab) and enzyme conjugated anti human Ig Substrate added Colour produced Detection of DNA Southern Blotting. Detection of RNA Northern Blotting

Immunoblotting

Thank You