Immunoassay of pig and human gastric proton pump

Monoclonal antibodies against the K+-dependent adenosine triphosphatase (ATPase) responsible for acid secretion in the pig gastric mucosa were generated by hybridoma technology. Two of these antibodies, shown to bind selectively to subunits of the ATPase and to label intracellular membranes of pig and rabbit parietal cells, were used to develop a sensitive (less than 1 pmol) immunoassay for the ATPase. Enzyme samples were adsorbed to the wells of polystyrene microtitration plates and then incubated sequentially with monoclonal antibody, antimouse immunoglobulin G coupled to alkaline phosphatase, and p-nitrophenyl phosphate. Standard curves relating the absorbance of the wells at 410 nm to log10 micrograms ATPase were fitted by a three-parameter logistic, with a useful assay range of 0.05-10 micrograms ATPase. The immunoassay allows measurement of proton-pumping ATPase levels in human gastric biopsy specimens and may therefore be useful in studies of gastric mucosal function.