SHRI SHANKRACHARYA COLLEGE OF

NSG.

HUDCO, BHILAI

SEMINAR PRESENTATION

ON

Antigen-Antibody Reaction

Presented By:- Guided By:-

Mrs. Biji RameshLecturer SSCN

SESSIO

N2013-1

4

micro

TYPES OF ANTIGEN ANTIBODY REACTION

Precipitation reactionAgglutination reaction Complement fixation testImmunofluorescenceImmunoblottingNeutralization test

ANTIGENS

An antigen has been defined as any substance which, when introduced parenterally into the body, stimulates the production of an antibody with which it reacts specifically and in an observable manner.

ANTIBODIES

Towards the close of the nineteenth century, the humoral basis of immunity was established by the demonstration that following the introduction of an antigen into an animal, certain substances called antibodies.

GENERAL FEATURES OF ANTIGEN ANTIBODY REACTIONS:-

Antigen-antibody reactions have the following general characteristics:

The reaction is specific, an antigen combing only with its homologous antibody and vice versa.

Entire molecules react and not fragments. When an antigenic determinant present in a large molecule or on a ‘carrier’ particle reacts with its antibody, whole molecules or particles are agglutinated.

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

4. The combination occurs at the surface. Therefore, it is the surface antigen that are immunologically relevant.

5. The combination is firm but reversible. The firmness of the union is influenced by the affinity and avidity of the reaction.

6. Both antigens and antibodies participate in the formation of agglutinates or precipitates.

7. Antigens and antibodies can combine in varying proportions, unlike chemicals with fixed valencies.

IMPORTANT PARAMETERS OF SEROLOGICAL TEST:-

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

SPECIFICITY:- 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:- When 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 precipitate.

MECHANISM OF PRECIPITATION:- According to Marrac (1934) multivalent antigens combine with bivalent antibodies in varying proportions, depending on the antigen-antibody ratio in the reacting mixture.

Applications of precipitation reaction

Types of precipitation test:-

1. Ring test- This, the simplest type of precipitation test, consists of layering the antigen solution over a column of antiserum in a narrow tube, a precipitate forms at the junction of the two liquids.

2. Slide test- when a drop each of the antigen and antiserum are placed on a slide and mixed by shaking, floccules appear. The VDRL test for syphilis is an example of slide flocculation.

3. TUBE TEST- A quantitative tube flocculation test is employed for the standardization of toxins and toxoids. Serial dilutions of the toxin/toxoids are added to the tubes containing a fixed quantity of the antitoxin.

4. IMMUNODIFFUTION (PRECIPITATION IN

GEL)- There are several advantages in allowing precipitation to occur in a gel rather than in a liquid medium. The reaction is visible as a distinct band of precipitation, which is stable and can be stained for preservation, if necessary. As each antigen-antibody reaction gives rise to a line of precipitation, the number of different antigens in the reacting mixture can be readily observed.

AGGLUTINATION REACTION

When a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated.

Agglutination is more sensitive than precipitation for the detection of he antibodies.

Applications of agglutination reaction

SLIDE AGGLUTINATION- When a drop of appropriate antiserum is added to a smooth, uniform suspension of a particulate antigen in a drop of saline on a slide or tile, agglutination takes place.

TUBE AGGLUTINATION- When a fixed volume of a particulate antigen suspension is added to an equal volume of serial dilution of an antiserum in test tubes, the agglutination titre of the serum can be estimated.

THE ANTIGLOBULIN (COOMBS) TEST- The antiglobulin test was devised by coombs, mourant and Race (1945) for the detection of the anti-Rh antibodies that do not agglutinate Rh positive erythrocytes in saline.

PASSIVE AGGLUTINATION TEST- By attaching soluble antigens to the surface of carrier particles, it is possible to convert precipitation test into agglutination test. which are more convenient and more and more sensitive for detection of antibodies. Such tests are known as passive agglutination test.

COMPLEMENT FIXATION TEST (CFT)

The ability of activated complement to lyse RBCs is made use of in complement fixation test for detection of antibodies or antigens. This reaction is not visible. An indicator system is used to detect the fixation of complement. This indicator system consists of sheep erythrocytes coated with amboceptor (rabbit antibody to sheep RBCs).

V. IMP

PROCEDURE

The first step consists of adding antigen to serial dilutions of patients serum followed by complement. It is

then incubated for one hour.

In the second step, the indicator system s added to the mixture and is again

incubated at 37 degree Celsius for one hour after which haemolysis is observe.

If patients serum contains antibody, complement will be utilized in the

first step. When indicator system is added in the 2nd step, there will be

no lysis of RBCs (as complement was used up in 1st step).

Therefore, no haemolysis indicates presence of antibodies in the patients

serum and indicate a positive complement fixation test. Presence of

haemolysis indicate a negative complement fixation test.

e.g.- Wassermann reaction formerly used to detect

antibodies in syphilis

Neutralization TESTS

VIRUS NEUTRALISATION TESTS: Neutralization of bacteriphages can be demonstrated by the plaque inhibition test. When bacteriophages are seeded in appropriate dilution on lawn culture of susceptible bacteria, plaques of lysis are produced. Specific antiphage serum inhibits plaques information. Neutralisation of animal viruses can be demonstrated in three systems- animals, eggs and tissue culture.

TOXIN NEUTRALISATION: Toxin neutralisation can be tested in vivo or in vitro. Neutralization tests in animals consists of injecting toxin-antitoxin mixture and estimating the least amount of antitoxin that prevent death or disease in animals.

PRESESNTED BY:- PRAFULL PAL