Quatitation of Antibody-Antigen Interactions

Quatitation of Antibody-Antigen Interactions - Scatchard...

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Quantization of Antigen-Antibody Interaction Affinity: The strength of interaction between a single epitope and antigen binding site. Avidity: The strength of binding of antigen-antibody binding when multiple epitopes on an antigen interact with multiple binding sites of an antibody. Affinity Equilibrium Constant (Ka) : The ratio of the forward (k1) to the reverse (k1-) rate constant in an antibody-antigen reaction. Dissociation Equilibrium Constant (Kd): 1/Ka; the ligand concentration at which ½ of the antibody is binding ligand at equilibrium is close to the Kd. Kd = [Ab][Ag] [Ab-Ag] Ag + Ab Ag-Ab k1 k2 Kd (dissociation equilibrium constant) = 1/Ka (units are moles/liter) Analysis of Kd and Ka Values Strong binders => high Ka and a low Kd Weak binders => low Ka and a high Kd Kd < 10^-7 moles/liter are indicative of good binders (esp. nanomolar range) Experimentally Measuring Affinity Data from equilibrium dialysis can be analyzed using
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Unformatted text preview: Scatchard Plot : r= bound ligand / total antibody c= free ligand n= number of binding sites per antibody molecule Slope = -Ka X-intercept = n r/c = Ka(n) –Ka(r) Note: this only works if the antibody is homogeneous: all antigen binding sites identical, e.g. myeloma protein or a monoclonal antibody. Polyclonal antibodies will yield a curve due to different Ka’s for different isoforms. (2) Surface Plasmon Resonance Resonance units are proportional to the degree of binding of soluble ligand to the immobilized receptor. (or soluble antibody to immobilized antigen, as shown here ) - Determining the amount of binding at equilibrium with different known concentrations of receptor (antibody) and ligand (protein antigen) allows you to calculate equilibrium constants (Ka, Kd). -Rate of dissociation and association (k off , k on ) can also be calculated (1) Equilibrium Dialysis...
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This note was uploaded on 05/06/2008 for the course MCB 150 taught by Professor Coscey during the Spring '08 term at Berkeley.

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