HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by DeHeer, D. H. Articles by Edgington, T. S. Search for Related Content PubMed Articles by DeHeer, D. H. Articles by Edgington, T. S.

Relationship Between Antibody Affinity and Hemolytic Plaque Diameter

I. Purified anti-DNP Antibody¹

From the Department of Molecular Immunology, The Research Institute of Scripps Clinic, La Jolla, California 92037

Abstract

The relationship between antibody affinity and hemolytic plaque diameter was explored with immunochemically purified anti-DNP antibody of demonstrated affinity and heterogeneity. Anti-DNP antisera were prepared by immunizing two groups of two rabbits each with 5 and 50 mg of DNP-BGG, respectively. Anti-DNP IgG antibody from each antiserum was purified by ammonium sulfate precipitation, DEAE cellulose and Sephadex G-200 chromatography, and elution from a DNP-aminoethylcellulose immunoabsorbent. The affinity and heterogeneity of each preparation were determined by equilibrium dialysis with ³H-DNP-EACA. Each antibody preparation was analyzed by hemolytic plaque assay with trinitrophenylated sheep erythrocytes in an agarose layer. These assays demonstrated a proportionality between antibody affinity and plaque diameter, with the largest plaques being formed by antibody of high affinity. Incorporation of free DNP-EACA into the assays demonstrated: a) that the highest affinity plaques were neutralized more efficiently than low affinity plaques; b) high affinity antibody resulted in a higher inhibition slope than low affinity antibody; c) inhibition slope was directly proportional to the heterogeneity of antibody affinity; and d) a linear correlation existed between the concentration of competing antigen that neutralized 50% of the antibody (Ab₅₀) and the affinity of the antibody. These results provide a basis for the experimental use of inhibition assays to assess anti-hapten antibody affinity and substantiate the validity of using Ab₅₀ in analyses of relative antibody affinity.

Footnotes

¹ This work was supported by Research Grant AM-18163 from the National Institute of Arthritis, Metabolic and Digestive Diseases. This is Publication No. 1633 from the Immunology Departments of the Research Institute of Scripps Clinic.