Quantitation of aspartate aminotransferase isoenzymes by immunologic methods: Use of antibodies directed against the mitochondrial isoenzyme

Abstract

Mitochondrial aspartate aminotransferase (m-AST) was purified to homogeneity from human liver and used to prepare antisera in rabbits, and the antibodies were partially purified. Two convenient immuno-techniques were developed using these antibodies to estimate AST isoenzyme activity in human sera and tissue extracts: (i) A homogeneous immunoinhibition assay requires a 1-h incubation in the presence of anti-m-AST; residual soluble enzyme (s-AST) activity is then determined. Average coefficient of variation (CV) for this assay system was 9.6% for sera with residual s-AST within the normal range and 4.5% for specimens with elevated activities. A small portion (ca. 2%) of m-AST was not inhibited by this technique. (ii) An immunoprecipitation assay requires incubation of the specimen with antibody in the presence of polyethylene glycol; the mixture is then centrifuged, and supernatant s-AST is determined. Average CV for this assay was 6.4% at 22 U/l and 1.2% at 90 U/l of s-AST. Average CV for estimation of 140 U/l of m-AST was 3.6%. Precipitation of m-AST was complete at ≤ 500 U/l. The average contribution of m-AST to total activity in serum was 12% (2.0 ± 1.5 U/l). Isoenzyme patterns after cardiac surgery suggested that the appearance of m-AST in serum is delayed and that it does not merely follow the pattern of total AST activity.