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-phosphochol...

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Bibliographic Details
Published in:Journal of immunological methods 1998-09, Vol.218 (1-2), p.161-167
Main Authors: O'Hare, T, Rittenberg, M B
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal - genetics
Antibodies, Monoclonal - immunology
Antibody Affinity
Antigen-Antibody Complex - immunology
Colchicine - immunology
Haptens - immunology
Immunoglobulin G - immunology
Mutation
Radioimmunoassay - methods
Staphylococcal Protein A - immunology
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Summary: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.
ISSN:0022-1759
DOI:10.1016/S0022-1759(98)00130-6