Immunology LectureRobert J. Boackle, Ph.D. Antigen-Antibody Reactions Specific Objectives: THE STUDENT SHOULD BE ABLE TO 1. Discuss immunoglobulin variability (ie. the variable region) 2. Describe bonds between the variable region and the antigenic determinant 3. Define antibody affinity and antibody avidity 4. Describe a precipitin curve and discuss lattice formation involving proteins verses carbohydrate antigens and be able to define "zone of equivalence". 5. Understand immunodiffusion in agar gels. (identity, nonidentity and partial identity) 6. Have a conceptual understanding of immunoelectrophoresis, Fluorescent antibody techniques and ELISA (enzyme-linked immunoassay) 7. Define "agglutination" and understand the functional differences between monomeric Ab (ie. IgG) and polymeric Ab (ie. IgM and S-IgA)

Definitions: 1. The "antibody affinity" of an antibody-antigen reaction is related to the strength of attractiveness between an antibody (Fab region) and its antigenic determinant. 2. The "antibody avidity" is the total strength of binding of the Fab regions of the population of antibodies evoked to an antigen, and involves the reaction with all the antigenic determinates. Thus it is the total strength of the binding of antibodies to antigens. 3. Immune Complex = Antigen-Antibody Complex [the size depends on the ratio of antigen to antibody]. Also the student should be prepared to answer and discuss the following: 1. List and describe the possible bonds between the immunoglobulin variable region and an antigenic determinant. Then draw and explain a precipitin curve and "lattice formation" involving protein antigens and polyclonal Ab. 2. What is meant by "hypervariable regions" on immunoglobulins? How do B cell clones differ in regard to the hypervariable regions of the immunoglobulins on their surface? At the level of the gene, explain what is believed to account for these clonal diversities. 3. Can two different classes of immunoglobulins have identical variable regions? In your answer include a discussion of the switch mechanism.

ANTIGENIC DETERMINANTS INTERACT WITH SPECIFIC ANTIBODY

CH2 CH3 IgG has a Valence of 2 TWO Identical ANTIGEN BINDING SITES

Movement at the Hinge Region CH2 CH3 IgG Surface of an Antigen i.e. bacterial cell surface

Non-Covalent Interactions Ball in glove fit Antigenic Determinant VL VH

- Gene rearrangements and Mutational Hot Spots Charge-Charge Interactions Hydrophobic Interactions - And good fit ! + - VLVL VHVH +

Y Antibody Affinity VLVL VHVH +

Antigenic determinant 1 Antigenic determinant 2 Antigenic determinant 3 Antigenic determinant 4 PROTEIN ANTIGEN

Y Y Y MUST HAVE POLYCLONAL ANTIBODY and at least two different antigenic determinants TO CROSS-LINK PROTEIN ANTIGENS Immune Complexes

Y Y Y Y ANTIBODY EXCESS NO CROSSLINKS NO Precipitate Y Y Y Y Y Y Y Y Excess Antibody

Y Excess Antigen = Not enough Cross-links to cause a Precipitation

Y Y Y More cross-links, and higher individual affinities = higher AVIDITY of the Immune Complexes Y Y Y

Amount of Precipitate Ab CONC ANTIBODY EXCESS ANTIGEN EXCESS ZONE of Equivalence No Soluble Ag or Ab

Repeating Antigenic Determinants e.g. PEPTIDOGYCAN

CHO ANTIGENS may cross-link with MONOCLONAL Ab Y Y Y Y

AntigenAntibody DOUBLE DIFFUSION Immune Complex

Antigen Antibody Antigen Immune Complexes Zone of Equivalence

Rabbit Serum as antigens 1:4 1:20 Goat anti-rabbit serum (Antibodies to rabbit serum)

Non-Identity Antigen #1 Antigen #2

No Shared Antigenic Determinants Antigen #1 Antigen #2

OUCHTERLONY ANALYSIS Diffusion of Antigens and Polyclonal Antibodies Antigen 1 (Molecule #1) Antibodies to both antigens The same Animal was injected with antigen 1 and with antigen 2 Antigen 2 (Molecule #2) Non-Identity

OUCHTERLONY ANALYSIS Antigen 3 is a part of antigen 4 Antibody Antigen 4 Partial - Identity Remember that Protein Antigens have different antigenic determinants Also remember that this antibody is a multi- clonal antibody such as an anti-serum to an antigenic preparation This animal was only injected with Antigen #4

OUCHTERLONY ANALYSIS Antigen 3 Antibody Antigen 4 Partial - Identity

Antigen 3 Antibodies polyclonal antibody Antigen 4 Partial - Identity Antibodies to determinants c and d are only on Antigen 3 and they pass by antigen 4

OUCHTERLONY ANALYSIS Antigen 5 Antibody Antigen 6 is Antigen 5 Identity These two Antigens are the Same Molecule No spikes were formed because: Antigenic determinants on Antigen 5 captured all the antibodies to Antigen 6 and antigenic determinants on Antigen 6 captured all the antibodies to Antigen 5

Antigens on Cells or on Tissue Sections UV Light Fluorescence

Fluorescence Double layer Sandwich UV Light Antigens

Ag Peroxidase Enzyme is permanently attached to the Antibody Probe Microtiter ELISA Antigens are immobilized to the plastic surface of a Microtiter Plate Enzyme Linked Immuno-Sorbant Assay ELISA Ag Substrate that turns from clear to green

Ag Peroxidase Enzyme is permanently attached to the Antibody Probe Microtiter ELISA Antigens are immobilized to the plastic surface of a Microtiter Plate Enzyme Linked Immuno-Sorbant Assay ELISA Ag Substrate that turns from clear to green

Capture ELISA -- using pre-immobilized mouse monoclonal Ab to capture the Specific Antigen and a second Probe monoclonal Antibody against a different antigenic determinant Ag

Agglutination Y Y Y IgM >>IgG