Problems of indirect immunologic assay of specific enzyme activity

The estimation of specific activity of variant enzymes is important for understanding the mechanism of inherited enzyme deficiency. Direct determination of specific enzyme activity is often difficult. Indirect immunologic methods, i.e., the quantitative neutralization method and the electroimmunoassay method using specific antiserum, have been used for estimating the specific activity of variant enzymes. The validity of these indirect methods was examined with the use of normal and variant human glucose 6-phosphate dehydrogenase (G6PD), and three different anti-G6PD sera. The specific activities of purified normal, partially denatured normal, partially purified normal, and partially purified preparations of four different G6PD variants were estimated by the indirect methods. The values estimated by the neutralization method and that obtained by the electroimmunoassay method were substantially different, and different antisera gave different values for the given G6PD preparations. The values estimated by the indirect methods do not reconcile with the specific enzyme activity determined by the direct determination. Therefore, care should be taken in deducing the specific enzyme activity from the immunologic assay.