An immunochemical procedure for determination of mitochondrial aspartate aminotransferase in human serum.
An immunochemical procedure is described for quantitation of mitochondrial aspartate aminotransferase (m-AspAT; EC 2.6.1.1) activity in human serum specimens. Antibodies directed against purified soluble aspartate aminotransferase (s-AspAT) from human erythrocytes were produced in rabbits and partly purified. Antibody sufficient for analyses of > 6000 specimens could be obtained from 15 mL of rabbit antiserum; contaminant AspAT activity of the antibody preparation was < 0.4 U/L. Addition of antibody directly to purified AspAT isoenzymes resulted in inhibition of s-AspAT but had no measurable effect upon m-AspAT. Antibody is incubated with serum in the presence of polyethylene glycol for 60 min at room temperature, then 60 min at 4 degrees C, and centrifuged (7000 x g, 4 degrees C, 15 min). No detectable s-AspAT activity remains in the supernatant fluid; thus m-AspAT activity can be measured directly. Precision, both within-day and day-to-day, was < 1 U/L, or 3.0% of residual m-AspAT activity. The method completely removed 1200 U of purified s-AspAT activity per liter; addition of s-AspAT to serum in increasing concentrations of about 500 U/L had no effect upon the measurement of residual m-AspAT activity. Results of the procedure described showed excellent correlation with those by an alternative procedure involving antibodies directed against m-AspAT. Addition of both anti-s- and anti-m-AspAT antibodies resulted in complete removal of serum AspAT activity. Univalent Fab fragments prepared anti-s-AspAT antibodies were capable of directly inhibiting s-AspAT activity without precipitation. Although a homogeneous immunoinhibition assay was possible, the greater precision of the precipitation assay made it preferable.
D000818:Animals; D001219:Aspartate Aminotransferases / Q000097:blood*; D006801:Humans; D005779:Immunodiffusion; D007527:Isoenzymes / Q000097:blood*; D008928:Mitochondria / Q000201:enzymology*; D011817:Rabbits / Q000276:immunology; D013552:Swine
D007527:Isoenzymes; D001219:Aspartate Aminotransferases