Assay of anti-hapten antibody with the aid of hapten-coupled bacteriophage

Abstract

Treatment of bacteriophage with 3-iodo-4-hydroxy-5-nitrophenyl acetic acid chloride (NIP) in aqueous medium killed a proportion of the phage but the survivors were made susceptible to inactivation 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 γ-globulin and was neutralized by a sheep antiserum against electrophoretically purified rabbit γ-globulin. The inactivation was strongly inhibited by the hapten and its derivatives. As little as 0.001 micromole per millilitre of NIP—ε-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 per cent of the phage became inactivated. When incubated with phage for 6 hours concentrations of antiserum as low as 10^-7 (1.9×10^-5 μg/ml) or less caused measurable inactivation of the phage. The threshold quantity of antibody was estimated to be less than 10^-5 μg.