Development and cross-reactive properties of monoclonal antibodies to bovine matrix vesicle alkaline phosphatase.
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.
D000469:Alkaline Phosphatase / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001857:Bone Matrix / Q000201:enzymology* / Q000276:immunology; D002417:Cattle; D003429:Cross Reactions; D007158:Immunologic Techniques; D007164:Immunosorbents; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats
D000911:Antibodies, Monoclonal; D007164:Immunosorbents; D000469:Alkaline Phosphatase