Highly sensitive solid-phase immunoenzymometric assay for placental and placental-like alkaline phosphatases with a monoclonal antibody and monodisperse polymer particles.
We have used a mouse monoclonal antibody (H7) to placental alkaline phosphatase (PLAP, EC 3.1.3.1) in developing an immunoenzymometric assay for PLAP and PLAP-like enzymes. The antibody is bound to sheep anti-mouse IgG (Ab2) covalently coupled to tosylated shell-and-core light (1.07 g/cm3) monodisperse polymer particles. Adding the H7-Ab2-polymer particle suspension to a PLAP-containing sample gives maximal binding of the antigen within 10 min. PLAP and PLAP-like enzymes remain active and bound to the solid-phase throughout all assay manipulations, and can thus be saved for future testing. In testing the enzymes for inhibition by L-Phe, L-Phe-Gly-Gly, L-Leu, and L-homoarginine, the effect of all the inhibitors is fully reversible. The assay is highly versatile, and its sensitivity (routinely 0.05 micrograms/L) can be increased 1000-fold by adjusting the sample volume and incubation time (sample volume is irrelevant between 50 microL and 5 mL). We have measured the basal activities of PLAP in men and women and, by using enzyme inhibitors, have characterized it as corresponding to the PLAP-like phenotypes described in normal human testis.
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000097:blood*; D000596:Amino Acids / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D002384:Catalysis; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008863:Microspheres; D009842:Oligopeptides / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D012016:Reference Values
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D009842:Oligopeptides; C043963:phenylalanyl-glycyl-glycine; D000469:Alkaline Phosphatase