Electrophoresis of enzyme--monoclonal antibody complexes: studies of human placental alkaline phosphatase polymorphism.

Abstract

Enzyme-monoclonal antibody complexes formed between six different monoclonal antibodies and the six phenotypes of human placental alkaline phosphatase [orthophosphoricmonoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] that represent the homozygous and heterozygous combinations of the three common alleles have been examined by electrophoresis in starch, acrylamide, and agarose gels. Since the complexes formed retain full enzyme activity, they could be detected after gel electrophoresis by an enzyme stain. Distinctive electrophoretic patterns were obtained with each monoclonal antibody. Differential binding of certain of the antibodies with the products of different alleles produces clear discrimination of various homozygous and heterozygous phenotypes. This discrimination parallels the results previously obtained by using a quantitative binding radioimmunoassay. The results show that this general method should prove useful in screening hybridoma fluids for the presence of monoclonal antibodies to specific enzymes; in the detection of allelic variation, even where this is not expressed by electrophoretic differences among the uncomplexed enzymes; and in discriminating between homozygotes and heterozygotes. It could also prove to be a useful tool in the elucidation of the molecular structures of enzyme-monoclonal antibody complexes.

Applications of Monoclonal Antibodies in Enzyme Genetics

“…The same was found in many cases for the B isozymes. With certain antibodies somewhat increased mobility of the B isozymes was observed, however, apparently because the retardation due to increased size was overrid den in these cases by an increase in net negative charge (16). In acrylamide gel electrophoresis, both the A and B isozymes were invariably retarded.…”

“…In addition, the antibodies were nonprecipitating. These properties make it possible to investigate the relative mobilities of the antibody/enzyme complexes compared with the un complexed enzymes in gel electrophoresis by staining for alkaline phosphatase activity (16). The mobilities of the complexes in starch and polyacrylamide gels depend both on molecular sizes and on net charge.…”

Differentiation of immunochemically related enzymes in different primate species by monoclonal antibodies

“…In the electrophoretic system, binding of a particular antibody to a particular ALP is demonstrated by the disappearance of the zone of activity corresponding to that seen in the absence of antibody and the appearance of slower migrating zones corresponding to the enzyme/antibody complexes [5]. With the control human placental ALP type 1, this effect was observed with all 18 antibodies.…”

Evidence for two isozymes of leukocyte alkaline phosphatase in leukemic leukocytes

“…Incubation at 37°C of a cell sonicate containing both isozymes does not alter the ratio of the two isozymes, nor does altering the time of sonication. Digestion of cells with Bromelain [35] does not alter the relative concentration of the two isozymes, nor does digestion of the preparation with neuraminidase (A. Yang and D. Miller, unpublished data). This suggests that the molecular weight differences are not due to altered glycoprotein characteristics of the two isozymes, but rather to a distinct molecular structure.…”