Production of monoclonal antibodies against high Mr avenin polypeptides

Abstract

Endosperm proteins extracted from oat (cv. Rodney) were separated by SDS-PAGE, electrophoretically blotted on to nitrocellulose, and the polypeptides in the M r 50–60k range were injected into BALB/c mice at 3-week intervals until high antibody titres were obtained. Spleen cells from the immunized mice were fused with P3X63-Ag8.653 myeloma cells, and hybridomas were selected in a medium containing hypoxanthine, aminopterin, and thymidine. Hybridomas were screened for production of antibodies against oat endosperm proteins using a solid-phase enzyme-linked immunosorbent assay, which was antigen-specific. Positive cultures were cloned at least three times by limiting dilution to ensure monoclonal growth and stability. Two clones, 6C2 and 7H10, produced antibodies that bound to high M r wheat gluten, barley, oat and maize endosperm polypeptides but to different epitopes. Clone 6C2 bound predominantly to four oat polypeptides having M r s of 56·6, 51·3, 48·0 and 40·0 k, two of which varied amongst oat cultivars.