ASSAY OF ANTI-HAPTEN ANTIBODY WITH AID OF HAPTEN-COUPLED BACTERIOPHAGE

Abstract

Treatment of bacteriophage with 3-iodo-4-hydroxy-5-nitro-phenyl acetic acid chloride (NIP) in aqueous medium killed a proportion of the phage but the survivors were made susceptible to in-activation by rabbit immune sera to NIP-chicken globulin conjugate. The serum factor inactivating NIP-phage (T2) was eluted from a Sephadex G-200 column as 7S [gamma]-globulin and was neutralized by a sheep antiserum against electrophoretically purified rabbit [gamma]-globulin. The inactivation was strongly inhibited by the hapten and its derivatives. As little as 0. 001 micromole per millilitre of NIP- E-amino caproic acid was inhibitory. Inactivation of NIP-phage was ascribed to anti-NIP antibody. Inactivation of NIP-phage (T2) in strong anti-NIP sera approximately followed first-order reaction kinetics until 99% of the phage became inactivated. When incubated with phage for 6 hr. concentrations of antiserum as low as 10-7 (1.9x10-5 [mu]g/ml) or less caused measurable inactivation of the phage. The threshold quantity of antibody was estimated to be less than 10-5 [mu]g.