A simple radioimmunoassay of human placental alkaline phosphatase (Regan isoenzyme) using specific antibody polymers.
Rabbit antiserum against highly purified high-molecular-weight B-variant of human placental alkaline phosphatase (M.W. 200,000) was rendered monospecific by absorption with polymerized pooled male serum proteins; the absorbed antiserum was then polymerized with ethyl chloroformate and used in radioimmunoassay as a stable solid-phase immunoabsorbent. Homogeneous preparation of the enzyme, with a specific activity of 477 mumoles phenol per mg per min, was also obtained by absorbing the chromatographically purified enzyme with polymerized rabbit antiserum directed to whole human serum proteins; the pure enzyme was then labeled with 125-I as the tracer retaining at least 80% of its antigenicity. Only a minute quantity of the polymerized antibody particles is required for each assay in admixture with the labeled and unlabeled enzyme. By adding a small amount of starch-gel particles before low-speed centrifugation, complete phase separation was achieved. The radioimmunoassay could detect 0.4 to 0.8 ng enzyme protein per tube, which is comparable to the sensitivity achieved by enzymic assays. However, radioimmunoassay is advantageous over the enzymic assay in being direct, specific (no interference by the nonplacental-type alkaline posphatase), and capable of detecting both catalytically active and inactive forms of the enzyme. Native variants of placental-type alkaline phosphatase including Regan isoenzyme and Nagao isoenzyme (D-phenotype of normal placental alkaline phosphatase), could thus be directly determined by this procedure in the clinical specimens.
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000097:blood; D000818:Animals; D000918:Antibody Specificity; D001202:Ascitic Fluid / Q000201:enzymology; D001798:Blood Proteins / Q000276:immunology; D002642:Chick Embryo; D004796:Clinical Enzyme Tests; D004305:Dose-Response Relationship, Drug; D005260:Female; D007106:Immune Sera; D007527:Isoenzymes / Q000032:analysis*; D008970:Molecular Weight; D010051:Ovarian Neoplasms / Q000201:enzymology; D010920:Placenta / Q000201:enzymology*; D011108:Polymers; D011247:Pregnancy; D011817:Rabbits / Q000276:immunology; D011863:Radioimmunoassay / Q000379:methods*
D001798:Blood Proteins; D007106:Immune Sera; D007527:Isoenzymes; D011108:Polymers; D000469:Alkaline Phosphatase