Principles of Immunology Antigen-Antibody Interactions 4/25/06

Word/Terms List Agglutinin EIA Equivalence zone FIA Immunodiffusion Immunoelectrophoresis RIA Titer

Affinity =  attractive and repulsive forces Strength of the reaction between a single antigenic determinant and a single Ab combining site Ab Ag High Affinity Ab Ag Low Affinity Affinity =  attractive and repulsive forces

Specificity The ability of an individual antibody combining site to react with only one antigenic determinant. The ability of a population of antibody molecules to react with only one antigen.

Cross Reactivity Cross reactions The ability of an individual Ab combining site to react with more than one antigenic determinant. The ability of a population of Ab molecules to react with more than one Ag Anti-A Ab Ag C Similar epitope Cross reactions Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope

Factors Affecting Measurement of Ag/Ab Reactions Ab excess Ag excess Affinity Avidity Equivalence – Lattice formation Ag:Ab ratio Physical form of Ag

Tests Based on Ag/Ab Reactions All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

Agglutination Tests Lattice Formation

Agglutination/Hemagglutination Definition - tests that have as their endpoint the agglutination of a particulate antigen Agglutinin/hemagglutinin Y +  Qualitative agglutination test Ag or Ab

Agglutination/Hemagglutination Quantitative agglutination test Titer Prozone 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 4 Patient 1 2 3 5 6 7

Agglutination/Hemagglutination Definition Qualitative test Quantitative test 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Applications Blood typing Bacterial infections Fourfold rise in titer Practical considerations Easy Semi-quantitative

Passive Agglutination/Hemagglutination Definition - agglutination test done with a soluble antigen coated onto a particle Y +  Applications Measurement of antibodies to soluble antigens

Agglutination/Hemagglutination Inhibition Definition - test based on the inhibition of agglutination due to competition with a soluble Ag Y +  Prior to Test Y +  Test Patient’s sample

Agglutination/Hemagglutination Inhibition Definition Applications Measurement of soluble Ag Practical considerations Same as agglutination test

Precipitation Tests Lattice Formation

Radial Immunodiffusion Method Ab in gel Ag in a well Ag Ab in gel Ag Concentration Diameter2 Interpretation Diameter of ring is proportional to the concentration Quantitative Ig levels

Immunoelectrophoresis Method Ags are separated by electrophoresis Ag - + Ag Ab Ag Ab Interpretation Precipitin arc represent individual antigens

Immunoelectrophoresis Method Interpretation Qualitative Relative concentration

Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (EIA) Lattice formation not required

Competitive RIA/ELISA for Ag Y +  Prior to Test Labeled Ag Method Determine amount of Ab needed to bind to a known amount of labeled Ag Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor Y +  Test Patient’s sample Labeled Ag

Solid Phase Non-Competitive RIA/ELISA Y Ag Immobilized Ab in Patient’s sample Labeled Anti-Ig Ab detection Immobilize Ag Incubate with sample Add labeled anti-Ig Amount of labeled Ab bound is proportional to amount of Ab in the sample

Solid Phase Non-Competitive RIA/ELISA Y Ag Immobilized Ag in Patient’s sample Labeled Ab Ag detection Immobilize Ab Incubate with sample Add labeled antibody Amount of labeled Ab bound is proportional to the amount of Ag in the sample

Tests for Cell Associated Antigens Lattice formation not required

Immunofluorescence Direct Y Ab to tissue Ag is labeled with fluorochrome Ag Y Fluorochrome Labeled Ab Tissue Section

Immunofluorescence Y Qualitative to Semi-Quantitative Indirect Ab to tissue Ag is unlabeled Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab Qualitative to Semi-Quantitative

Assays Based on Complement Lattice formation not required

Y Complement Fixation Ag No Ag Ag mixed with test serum to be assayed for Ab Standard amount of complement is added Erythrocytes coated with Abs is added Amount of erythrocyte lysis is determined Ag No Ag Ag Y Patient’s serum Ag Y