Rabbit IgA, IgM and IgG Antihapten Responses Assayed by Cla Fixation and Transfer

Abstract

The present report describes an adaptation of the C1a fixation transfer test (C1a FTT) for measurement of rabbit antibody in three immunoglobulin classes—IgM, IgG and IgA—without serum fractionation. IgM antibody was measured directly and IgA and IgG antibodies were measured indirectly using specific antisera against rabbit IgG and IgA to permit complement fixation by antibodies in these immunoglobulin classes. The ability of the test to discriminate among antibodies in these classes was demonstrated by immunochemical examination of the anti-antisera and by use of the C1a FTT to show that 1) the effects of the enhancing anti-antisera could be summated, 2) the enhancing ability of the anti-antisera could be specifically inhibited by purified rabbit immunoglobulins, and 3) antibody activity in rabbit serum could be fractionated by DEAE-cellulose gradient chromatography into its immunoglobulin components. The ability of the test to quantitate antibody in each class was examined by assay of serial dilutions of rabbit antiserum by the C1a FTT; the amount of C1a fixed and quantitated was found to be directly proportional to the amount of antibody present. Primary and secondary humoral responses of rabbits to intravenously administered haptenic arsanilic acid were studied using the C1a FTT. In the primary response, IgA antibody reached an early peak together with IgM antibody. IgG antibody had a more gradual rise and peaked later than IgA or IgM; a small second peak of IgA antibody sometimes accompanied the IgG peak. In the secondary response IgA antibody showed an accelerated onset, rising to an earlier but lower peak than in the primary response. The secondary IgM antibody response was similar to that of IgA while IgG antibody reached an earlier, higher peak level than in the primary response.