k [AbH] Keq = = k' [Ab] [H]

Transcription

1 Dr. Paul C. Montgomery August 2, 2001 Page 1 (of 6) I. Antibody Interactions with Simple Haptens hapten - small organic molecule that becomes part of an antigenic determinant when coupled to a carrier molecule example: DNP NO 2 NO 2 - important because it allowed physical chemists to define a series of important parameters related to Ag/Ab interactions - allows a more precise understanding of the more complex interaction - most simplistic of interactions affinity - mathematical expression defining the strength of interaction between an antibody and a ligand (hapten) Ab + H k k' AbH k [AbH] Keq = = k' [Ab] [H] derive a series of working equations which allows measurement of affinity (expressed as the average intrinsic association constant or Ko) heterogeneity - in this context refers to amino acid diversity within the combining site heterogeniety index - # describing variation in affinities valence - # of sites reacting with Ag on an Ab molecule IgG(2), IgA(2 or 4), IgM(10), IgD(2), determined from Keq expression avidity vs affinity avidity = combining power (valence + affinity) specificity - ability of an antibody to preferentially react with the inducing stimulus example DNP TNP Anti DNP Ab Ko Ko Anti TNP Ab Ko Ko resolving power at level of NO 2 groupings

2 Dr. Paul C. Montgomery August 2, 2001 Page 2 (of 6) ANTIGEN-ANTIBODY INTERACTIONS I (Lecture 5) Benjamini: Chapters 5 II. Antibody Interactions with Soluble Macromolecules begins to approach what's going on biologically examples Anti-A1b (Ab) + Alb (protein) Ab Ag ppt Anti-PS (Ab) + PS Ab Ag ppt A. Quantitative Precipitin Reaction Lattice Diagram (for above QP curve)

3 Dr. Paul C. Montgomery August 2, 2001 Page 3 (of 6) B. Precipitin Reactions in Gels Way to visualize QP Rx note: ppt is not a physical barrier lines represent equivalence zones shows relatedness or differences in Ags 3 basic pptn patterns Detailed analysis of partial identity Induction of antibodies 1. anti-dnp-bsa (DNP is much smaller than site) Ab will always see DNP and BSA 2. anti-bsa (DNP does not cover entire BSA molecule) it is possible to have Ab to BSA only

4 Dr. Paul C. Montgomery August 2, 2001 Page 4 (of 6) PRECIPITIN REACTION IN GELS - PARTIAL IDENTITY 1 = BSA a = DNP X = non recognized carrier (necessary for precipitin Rx only) 1a = BSA-DNP Xa= HSA-DNP Radial Immunodiffusion (adaptation of above reaction) Method employed to quantitate antigens [Benjamini p 97]

5 Dr. Paul C. Montgomery August 2, 2001 Page 5 (of 6) C. Immunoelectrophoresis - separates components based on charge - visualization using antisera - after separation, stepwise: Relationship of immunoelectrophoretic pattern to free boundary electrophoresis (serum)

6 Dr. Paul C. Montgomery August 2, 2001 Page 6 (of 6 ANTIGEN-ANTIBODY INTERACTIONS I (Lecture 5) Benjamini: Chapter III. Other Interactions A. Agglutintion - particulate antigens red blood cells (RBC), bacteria B. Neutralization - toxins inability to function viruses inability to attach C. Complement Mediated - Ab fixes complement cascade can lead to lysis of target cell agglutination in detail RBC agglutination/rbc typing Latex agglutination/rf determination RBC agglutination (electron micrograph) ABO system is one of major histocompatibility systems in humans Ags expressed on RBC surface Ags can also be free in secretions such as saliva Major Blood Groups Determinants A polysac backbone - N acetylgalactosamine B " - galactose O " AB " - both A & B Type Lacks Isoantibodies* (in circulation) O AB anti AB A B anti-b B A anti A AB nothing no isoantibodies O universal donor AB universal acceptor other blood groups including Rh factor *isoantibodies/isohemagglutinins - usually IgM; clump RBC from mismatched donors Erythroblastosis Fetalis