Monoclonal antibodies against human alkaline phosphatases

Abstract

The alkaline phosphatases (EC 3.1.3. I ) are intrinsic membrane enzymes found in many tissues. Structural and genetic studies divide the isoenzymes of man into three distinct groups: the placental, the intestinal and the hepatic, the renal and the skeletal alkaline phosphatases (Moss, 1982). The placental enzyme is also secreted by certain carcinomas where its appearance in serum can be used as a tumour marker (Haije, 1982; Jeppsson et al., 1983). Placental and intestinal alkaline phosphatase have some common antigenic sites; antisera raised against placental alkaline phosphatase contain antibodies that cross-react with the intestinal enzyme. Liver and bone alkaline phosphatase can be distinguished by their different electrophoretic mobility on charge-separation gels and by their different stability to denaturation with urea or heat. It is thought that these two enzymes are products of the same gene locus and that their different properties are due to tissue-specific modification, including variations in glycosylation. Elevated serum alkaline phosphatase activity in disease is typically due to increased osteoblastic activity in the bone and/or liver disease such as cholestasis. I t is frequently desirable to discover the tissue source of an elevated serum alkaline phosphatase but the current methods of distinguishing between liver and bone alkaline phosphatase are only semiquantitative and unsuitable for routine analysis. Monoclonal antibodies are capable of very high specificity and a monoclonal antiboldy specific for liver or bone alkaline phosphatiise could be used to set u p a rapid. sensitive and specific assay for the isoenzymes of alkaline phosphatase. Six monoclonal antibodies against placental alkaline phosphatase were raised by conventional methods (Galfre & Milstein, 1981). Balb/c mice were immunized with a commercial preparation of placental alkaline phosphatase (15 units/mg, l-2'?{ pure), obtained from Sigma Chemical Co. The culture supernatants were screened for antiplacental alkaline phosphatase antibodies by the enzymeantigen immunoassay of Jemmerson &, Fishman (1982). Epitope analysis by the method of Soos & Siddle (1983) showed that the six monoclonal antibodies could be placed in three epitope groups. Three antibodies all recognized an antigenic site which was also found on adult and foetal intestinal alkaline phosphatase. One ant.ibody recognized a separate antigenic site common to the intestinal and placental alkaline phosphatases. Two antibodies, recognizing the same antigenic site, were spes-ific for placental alkaline phosphatase. Liver alkaline phosphatase was partially purified to 15 units/mg of protein ( 1 -20/, pure) by solubilizing microsomal membranes in the zwitterionic detergent Sulphobetaine 14, followed by DE-52 ion-exchange chromatography and concanavalin A chromatography. Six monoclonal antibodies I07