A Monoclonal Antibody to Intestinal Alkaline Phosphatase Made Against D98/AH-2 (HeLa) Cells

Abstract

A monoclonal antibody (AAP1) to human intestinal alkaline phosphatase (ALP) was produced by immunizing a mouse with D98/AH-2 (HeLa) cells, which produce the enzyme ectopically. The antibody, which did not inhibit enzyme activity using p-nitrophenyl phosphate as the substrate, was of the IgG2A class and did not show complement-dependent cytotoxicity. In trace binding assays, AAP1 bound only to cells that expressed an intestinal-like form of human ALP, including some human intraspecific (D98/AH-2 .times. human lymphocyte or fibroblast) hybrids. Immunoprecipitation of immune complexes from cell-free extracts of D98/AH-2 cells, using protein A containing Staphylococcus aureus and AAP1 antibody, resulted in precipitation of all the ALP activity. The precipitated material had a subunit MW of 80,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In non-denaturing conditions, AAP1 antibody prevented ALP activity migration into the gel when cell-free extracts were made from human adult or fetal intestine, or D98/AH-2 cells. Similarly, AAP1 could be used to precipitate ALP activity from these extracts but not from extracts of human liver, kidney or placenta.