Agglutination 1

Agglutination The interaction between antibody and a particulate antigen results in visible clumping called agglutination. Particulate antigen include: bacteria, white blood cells, red blood cells, latex particles Antibodies that produce such reactions are called agglutinins.

Agglutinin and Agglutinogen An agglutinin is an antibody that interacts with antigen on the surface of particles such as erythrocytes, bacteria, or latex particles to cause their agglutination in an aqueous environment containing electrolyte. An agglutinogen is an antigen on the surface of particles such as red blood cells that react with the antibody known as agglutinin to produce agglutination. The most widely known agglutinogens are those of the ABO and related blood group systems.

Agglutination is the basis for multiple serological reactions including: blood grouping, diagnosis of infectious diseases and noninfectious diseases

Phases of Agglutination Agglutination is a two-Phase reaction that results in the formation of a stable lattice network Primary Phase (Sensitization) Ab reacts with a single antigenic determinant on the surface of Ag. Secondary Phase (Lattice formation) Ab must be able to bridge the gap between particles so that at least one Fab portion is attached to an antigenic determinant on each of two adjacent particles, is dependent on environmental conditions and the relative concentrations of antigen and antibody.

This represents what occurs during stage one of agglutination: Sensitization Antibody molecules attach to their corresponding Antigenic site (epitope) on the red blood cell membrane. There is no visible clumping.

This represents what occurs during stage 2 of agglutination: Lattice Formation Antibody molecules crosslink RBCs forming a lattice that results in visible clumping or agglutination.

Direct Agglutination Agglutination reactions where the antigens are found naturally on a particle are known as Direct Agglutination. Slide Agglutination: The test is performed in a slide. A suspension of bacteria is prepared and is added to a drop of standarised antiserum. Clumping of bacteria is the positive reaction. Clumping occurs instantly in a positive result. Eg: 1) Identification of bacterial strains such as Salmonella, Vibrio etc. 2) Blood grouping.

b) Tube Agglutination: The test is performed in a tube. In these test, the patient’s serum is diluted in a series of tubes and bacterial antigens specific for the suspected disease are added to it. Antigen and antibody reactions are demonstrated by visible clumps of agglutination. It is a standard method used for the quantitative estimation of antibodies in the serum.

Ag-Ab complex

Passive Agglutination It employs carrier particles that are coated with soluble antigens. When antibody instead of antigens is adsorbed on the carrier particle for detection of antigens, it is called reverse passive agglutination. Earlier only RBCs were the major carrier particles used for the coating of antigens. Now a variety of particles like polystyrene latex, bentonite and charcoal are used for this purpose. The use of synthetic beads or particles provide the advantage of consistency, uniformity and stability.

Types of Passive Agglutination 2) Hemagglutination test 1) Latex Agglutination test 2) Hemagglutination test 3) Coagglutination test Hemagglutination test: RBCs are used as carrier particles. RBCs of sheep, human, chick etc are commonly used in this test. When RBCs are coated with antigen to detect antibodies in the serum, the test is called indirect hemagglutination test (IHA). IHA is used for the dignosis of many parasitic diseases including amoebiasis, hydatid disease. When antibodies are attached to the RBCs to detect microbial antigen, it is known as reverse passive hemeagglutination (RPHA)

Radioimmunoassay (RIA) RIA was first described in 1960. It was used to measure the amount of endogenous plasma insulin by Solomon Berson and Rosalyn Yalow. RIA works on the principle of competitive binding. In this method, unlabeled antigen competes with radiolabelled antigen for binding to the antibody. When mixtures of radiolabeled and unlabeled antigen are incubated with the corresponding antibody, the amount of free radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the mixture. The radioactivity can be counted for finding out the concentration of unlabeled antigen present.

Uses: RIA can be used to detect even nanograms of antigen in the serum Can be used for the quantitation of hormones, drugs and various viral antigens Disadvantages: Cost of the equipment and reagents are expensive Short shelf-life of raidolabeled compounds Problems associated with the disposal of radioactive waste.