Alkaline phosphatase (ALPase), concentrated in the membranes of matrix vesicles, is believed to play a role in initial calcification. To further purify, characterize, and identify this
enzyme in tissue, a
monoclonal antibody was developed against the ALPase of isolated fetal calf matrix vesicles. Splenic lymphocytes derived from mice immunized with
Sepharose 6B-purified fetal calf matrix vesicle ALPase were fused with mouse
plasmacytoma cells (line X63-Ag-8.653) using standard hybridoma technology. Hyperimmune sera and hybridoma culture supernatants were screened for the presence of specific antibody using a newly developed double-
immunosorbent assay in which putative antibody is added to microtiter plate wells precoated with affinity-purified rabbit antimouse
immunoglobulin. After incubation and washing, partially purified fetal calf matrix vesicle ALPase is added to each well. The
enzyme adheres only to wells that contain specific anti-ALPase antibody. These wells are identified by adding the
enzyme substrate
p-nitrophenyl phosphate and reading the wells in a plate-reading spectrophotometer at 405 nm. A hybridoma-producing specific antibody was subsequently cloned and grown as ascities-producing
tumors in
pristane-primed mice. Ouchterlony analysis indicated that the cell line secretes an
immunoglobulin of
IgG1 class. This antibody reacts specifically with ALPase derived from calf matrix vesicles and cross-reacts with ALPase of bovine kidney, liver, and placental origin and human bone but does not cross-react with bovine intestinal ALPase or ALPase derived from matrix vesicles isolated from rachitic rat growth plate cartilage.