AB+AG reactions Detect Identify Quantitate antigen or antibody Disadvantage: Cross reaction -similar or common epitope

Abs Polyclonal: Heterogeneous antibody preparations Recognizes many epitopes on a single antigen Monoclonal: Recognize individual epitoses on an antigen

Antibody-antigen complexes can be detected: Directly Labelling the antibody or the antigen: -enzyme -radioactive -fluorescent dye

Lattice formation

Ag+Ab reactions Precipitation – soluble antibody reacts with soluble antigen Agglutination – particulate antigens bound together by antibody Complement Fixation – antibody binding to antigen triggers activation of complement

Precipitation

Turbidimetry Measures turbidity or cloudiness of a solution by measuring the amount of light PASSING THROUGH the solution. Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness. The more cloudy the solution, the less light can pass through.

Nephelometry Measures SCATTERED light bouncing off antigen- antibody complexes.

Passive Immunodiffusion Reactions in gels Migrate towards each other and where they meet in optimal proportions form a precipitate. Single diffusion, single dimension Single diffusion, double dimension Double diffusion, single dimension Double diffusion, double dimension

Oudin precipitation

Radial immunodiffusion

RADIAL IMMUNODIFFUSION Standard Curve Precipitin Rings A B C a b c Standards Samples

Ouchterlony Gel Diffusion

B and C not identicB and C contain identical Ags + C contain an additional different Ag

Rocket Immunoelectrophoresis Antigen is electrophoresed into gel containing antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

Immunoelectrophoresis

Two-dimensional immunoelectrophoresis. Antigens are separated on the basis of electrophoretic mobility. The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen.

Immunofixation

Agglutination Direct agglutination

Passive Agglutination Employs particles that are coated with antigens, ie, RBCs, polystyrene latex, bentonite or charcoal.

Reverse Passive Agglutination Antibody attached to carrier particle instead of antigen. Serologic Typing of Shigella: Positive Test

Coombs tests Direct Coombs Indirect Coombs

Hemagglutination

Agglutination Inhibition Based on competition between particulate and soluble antigens for limited antibody combining sites. Patient sample added to reagent antibody specific for antigen being tested, if antigen is present it binds to reagent antibody. Reagent particles (latex or RBCs) coated with the same antigen are added, if antigen was present in the sample all reagent antibody binds to it so no antibody is present to react with antigens coating the particles

ELISA Competitive ELISA Noncompetetive ELISA Immunoenzymometric Assay Sandwich or capture assays

Direkt EIA Ag detection

Noncompetetive EIA Ab detection

Competetive EIA

Capture EIA

Sandwich ELISA Used for antigens with multiple epitopes, ie, HCG Antibody to one epitope fluid, antibody to second epitope fixed. Enzyme label used to detect reaction

Complement fixation

Lateral flow

Western blot