The development and evaluation of a homogeneous immunoassay for the isoenzymes of aspartate aminotransferase.
Purified isoenzymes of aspartate aminotransferase (AST) from human liver (mitochondrial) and erythrocytes (cytoplasmic) were used to elicit antisera in rabbits. Each antiserum was characterized for titer and specificity. Complexes formed upon addition of each isoenzyme to its specific antiserum were demonstrated to be catalytically inactive. Results obtained when either filtration or centrifugation was used for separating the complexes (heterogeneous assay) were comparable to those obtained when the complexes were not separated (homogeneous assay) from the mixture before assay. A quality control system was designed to monitor specificity in addition to the usual parameters. The precision for the inhibition of cytoplasmic (CV 4.8%) and mitochondrial (CV 3.5%) isoenzymes was within that of the enzymatic assay. Several parametric conditions of the enzyme-antibody reactions were examined, and the assay was adapted for semi-automation. The homogeneous assay was evaluated with a series of pseudo specimens containing known mixtures of pure isoenzymes to determine the extent of recovery (99.8%) for a particular isoenzyme in the presence of varying concentrations of the other isoenzyme. In addition, sera from patients having elevated AST concentrations were examined for isoenzyme contributions to total AST activity. A mean recovery of 96% was obtained for these specimens.
D000918:Antibody Specificity; D001219:Aspartate Aminotransferases / Q000032:analysis*; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D007118:Immunoassay / Q000379:methods; D007527:Isoenzymes / Q000032:analysis*
D007106:Immune Sera; D007527:Isoenzymes; D001219:Aspartate Aminotransferases