Characterization of monoclonal antibodies to oat phytochrome by competitive radioimmunoassays and comparative immunoblots of phytochrome peptides.

A library of 50 hybridomas which make antibodies to oat phytochrome was produced from the fusion of spleen cells from immunized Balb/c mice with P3x63Ag8 myeloma cells. Hybridomas were selected in a medium containing hypoxanthine, aminopterin, and thymidine, and specific hybridomas were screened for production of antibodies to phytochrome using a solid-phase enzyme-linked immunoadsorbent assay which was antigen specific. Positive cultures were cloned three times by limit dilution to assure monoclonal growth and stability. Specificity toward phytochrome was established by Western blot analysis and immunoprecipitation. Epitope specificity of nine monoclonal antibodies was determined by competitive inhibition radioimmunoassays and/or comparative immunoblots of tryptic peptides of phytochrome.