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Neutralization of Haptenated Bacteriophage F2 by Anti-Hapten Antibodies: Direct Evidence for a Critical Site

From the Division of Biological Sciences, Cornell University, Ithaca, New York, 14850

Abstract

The neutralization of trinitrophenyl (TNP) derivatives of the small RNA bacteriophage f2 by rabbit anti-DNP antibodies was studied by the plaque assay technique. In agreement with previous work using other hapten-bacteriophage conjugates, we found that whole antibody was much more effective in the neutralization reaction than were corresponding Fab fragments. Measurement of the association constant for whole antibody with heavily substituted phage derivatives (TNP₃₀₅-f2) gave a result (Ka = 3.2 x 10¹⁰) which was 10³ greater than the corresponding value for Fab fragments. This difference was, in part, reflected in a 400-fold decrease in the second order rate constant for the phage-antibody association reaction, in going from G antibody to Fab fragment. Analysis of these results lends further support for a simple steric model of phage neutralization by antibody.

The relative effectiveness of antibody in neutralizing TNP-f2 conjugates was in large measure controlled by the degree of phage modification. For example, a lightly substituted f2 derivative (TNP₃-f2) remained fully viable in the presence of a large excess of antibody, providing direct evidence for the infectivity of antibody-f2 aggregates. A quantitative study of the infectivity of another lightly substituted TNP-f2 conjugate (TNP₃₀-f2) led directly to a model of f2 neutralization, in which an antibody molecule must directly interact with a single critical site on the surface of f2 in order to inactivate the phage. A consideration of the icosahedral structure of f2 suggests that f2 neutralization occurs by attachment of a single antibody molecule at or in the vicinitv of A-protein.

Footnotes

¹ Present address: Department of Pathology, Stanford University, School of Medicine, Stanford, California.

² Reprint requests should be addressed to Dr. L. I. Slobin, Sction of Microbiology, Cornell University, Division of Biological Sciences, Ithaca, New York 14850.