A simple method for determining K(A)s of both low and high affinity IgG antibodies.
A rapid and convenient method for measuring affinity constants (K(A)) of IgG antibody-hapten complexes is described. A key advantage of this method is its suitability for quantification of both low and high affinity interactions. A comparison is made of the K(A)s of the low affinity anti-phosphocholine (PC) antibody T15 (K(A) = 2.9 x 10(5) M(-1)) and five heavy chain complementarity determining region 2 (HCDR2) mutant antibodies expressed as IgG2b transfectants. As a demonstration of the general applicability of the technique, colchicine binding to the high affinity monoclonal IgG2a antibody C44 (K(A) = 1.5 x 10(9) M(-1)) is measured also; thus the assay is applicable over a four-log range of affinities. The assay, based upon use of fixed Staphylococcus aureus Cowan I strain cells as an adsorbent for antibody-radiolabeled antigen complexes, is conducted over a range of hapten concentrations at constant antibody concentration. The K(A) is obtained by Scatchard analysis.
O'Hare
T
T
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201, USA. oharet@ohsu.edu
Rittenberg
M B
MB
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex / Q000276:immunology; D003078:Colchicine / Q000276:immunology; D006241:Haptens / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology*; D009154:Mutation; D011863:Radioimmunoassay / Q000379:methods; D013205:Staphylococcal Protein A / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D006241:Haptens; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D003078:Colchicine