Characterization of the early primary antibody response to the 4-hydroxy-3-iodo-5-nitrophenylactate (NIP) and the 2,4-dimitrophenyl (DNP) determinants.

Rabbits, rats and mice were injected intraperitoneally with 4-hydroxy-3-iodo-5-nitrophenylacetate (NIP)-chicken globulin. Antibodies to this hapten were assayed in gradient centrifuged fractions or gel filtration fractions of their sera by neutralization of NIP-phage. The 19S antibodies appeared a few days after immunization. Sometimes 7S antibodies appeared simultaneously or after a lag of a few days. Rabbits were injected into the hind footpads with 2,4-dinitrophenyl (DMP)—bovine γ-globulin in complete Freund's adjuvant. Passive haemagglutination, antigen-binding, radioimmunoelectrophoresis and passive cutaneous anaphylactic reactions were applied to the assay of antibody in sera and in ultracentrifugal, electrophoretic and chromatographic fractions of these sera. The γM and γG antibodies appeared simultaneously in the blood on the 5th or 6th day after immunization. The γM antibodies were detected most readily by haemagglutination, but also by radioimmunoelectrophoresis. The γG antibodies were detected on the 5th or 6th day by antigen-binding, radioimmunoelectrophoresis and passive cutaneous anaphylaxis and only later by haemagglutination. The data are discussed with regard to methods for demonstrating the different classes of antibody to haptens, to the organ and cellular origins of antibodies and to the validity of the postulated sequential synthesis of γM and γG antibodies.