Studies of the idiotypic network through the use of a novel hemolytic plaque assay
A minimal requirement in investigations of the behavior of the idiotypic network during immunization is the ability to quantitate both the idiotypic (Ab1) and antiidiotypic (Ab2) responses. Quantitation of Ab2 in serum is complicated by the simultaneous presence of Ab1, so that Ab1-Ab2 immune complexes escape detection. In contrast, immune complexes should not complicate the enumeration of Ab2 producing lymphocytes in a hemolytic plaque assay. This study utilizes a procedure that allows detection of Ab2 producing cells in such an assay. The procedure relies upon the insertion of the appropriate antibody (Ab1) into the membrane of indicator SRBC through a covalently attached dipalmitoyl phosphatidylethanolamine (DPPE) tail. When the Ab2 response following murine immunization with DNP-Ficoll was analyzed using such an assay, peak plaque forming cell (PFC) numbers were found to coincide with peak Ab1 PFC numbers in both the primary and secondary response. In addition, this Ab2 response was found to be T independent. The murine immune response to DNP-HGG demonstrated a peak Ab2 PFC response which followed the peak Ab1 PFC response after both primary and secondary immunization. This Ab2 response was determined to be T dependent. The secondary responses to both DNP-Ficoll and DNP-HGG showed increased levels of Ab2 PFC and decreased levels of Ab1 PFC in comparison to the primary responses to the same antigens, suggesting that immunoregulation may occur within these idiotypic networks. Inhibition studies of these same Ab2 producing cell populations suggest that an increase in average affinity may occur between the primary and secondary responses to DNP-Ficoll in the absence of a change in heterogeneity, and a decrease in heterogeneity may occur between the primary and secondary responses to DNP-HGG in the absence of a change in average affinity. Initial attempts to study Ab2 PFC levels in other antigenic systems using an analagous detection system are also described.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign