Production of monoclonal antibodies against gastric parietal cell antigens

Two mice DBA/1 were each immunized with a single injection of one million enriched parietal cells in the hind foot pads. Monoclonal antibodies to be used as research tools in studies on regulatory mechanisms in gastric parietal cells were obtained after fusion of mouse myeloma cells (SP2) with cells from the popliteal lymph nodes of the mice. Twelve hybridomas produced antibodies reactive with structures only present in parietal cells as assessed by immunohistochemistry of oxyntic mucosa sections. Three hybridomas were subcloned and the antibodies produced by them, designated as PC4, PC8, and PC117, were characterized. In an enzyme-linked immunosorbent assay, all antibodies reacted with H,K-ATPase-containing vesicles. The antibody PC8 recognized a 94 kDa protein after immunoblotting of H,K-ATPase-containing vesicles and all antibodies precipitated a 94 kDa protein from [125I]H,K-ATPase-containing vesicles. The antibodies PC4 and PC117 recognized extracellular structures with a polarized distribution in viable, purified parietal cells. The results suggest that the structure recognized by all three antibodies is the alpha-subunit of the H,K-ATPase. The antibodies produced by another hybridoma, PC43, recognized a structure present in parietal and surface epithelial cells of the oxyntic mucosa. In an enzyme-linked immunosorbent assay, they reacted with a high-activity carbonic anhydrase which had been affinity-purified from pig oxyntic mucosa and they recognized a 30 kDa protein after immunoblotting. Thus, monoclonal antibodies against both intracellular and extracellular parietal cell structures were obtained after immunization with a small number of parietal cells.