Development of hybridomas secreting monoclonal antibodies to the chicken intestinal 1 alpha,25-dihydroxyvitamin D3 receptor.

Four hybridomas that secrete monoclonal antibodies to the chicken intestinal cytoplasmic 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been obtained. Splenic lymphocytes, derived from two male Lewis rats expressing serum antireceptor activity after repeated immunization with a partially purified preparation of this protein, were fused with two nonsecreting murine myeloma cell lines, SP2/0-Ag14 and P3-X63-Ag8.653. Viable hybrids were screened for anti-chicken intestinal 1,25-(OH)2D3 receptor activity by incubation of hybrid media with receptor-hormone complex; this was followed by immunoprecipitation with rabbit anti-rat IgG. Of 1,724 hybridomas assayed by this technique, 4 were positive (2 derived from each animal) for the secretion of an antireceptor immunoglobulin molecule. After cloning by limiting dilution, the cell lines (designated SP2/0-4A5, P3-8C8, SP2/0-8D3, and SP2/0-9A7) were expanded into suspension culture. Antibody-induced alterations in the sedimentation pattern of the native 1,25-(OH)2D3 receptor, coupled with Ouchterlony double-diffusion techniques, indicate that SP2/0-4A5 secretes an IgG2a, SP2/0-9A7 produces an IgG2b, and P3-8C8 secretes an IgG. In contrast, SP2/0-8D3 was found to synthesize an IgM. The monoclonal antibodies react with both occupied and unoccupied chicken intestinal receptor and nuclear receptors, and they crossreact with 1,25-(OH)2D3 receptors from a wide variety of tissue and cultured cell types, including human 1,25-(OH)2D3 receptors. These immunological reagents should prove valuable in the elucidation of the molecular action of 1,25-(OH)2D3.