Abstract
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 it 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 105 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 109 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.
Original language | English (US) |
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Pages (from-to) | 161-167 |
Number of pages | 7 |
Journal | Journal of Immunological Methods |
Volume | 218 |
Issue number | 1-2 |
DOIs | |
State | Published - Sep 1 1998 |
Keywords
- Affinity constant
- Colchicine
- Phosphocholine
- Protein A
ASJC Scopus subject areas
- Immunology and Allergy
- Immunology