A primary enzyme-antibody binding test has been developed to study antigenic differences between normal and mutant enzymes which cannot be distinguished by routine immunoprecipitation techniques. The test depends on enzyme substrate specificity and does not require monospecific antibody nor pure antigen, one of which is needed for most other primary biding tests. Goat antiserum to normal human arylsulfatase A was coupled to sepharose 4B using cyanogen bromide. The mutant enzyme protein from patients with metachromatic leukodystrophy or normal enzyme protein was assayed by its ability to saturate the Sepharose bound antibody and thus block the subsequent binding of a standard normal enzyme preparation. Enzyme activity was then measured by hydrolysis of the substrate, nitrocatechol sulfate. Significant binding differences between normal and mutant enzyme were detected using this method. These antigenic differences were not detectable using immunodiffusion or immunoelectrophoresis. The method also made it possible to compare the binding of enzymes from different species. Monkey enzyme, and, to a lesser extent, dog enzyme, was found to be related to the human enzyme. The primary binding test was also useful in quantitating normal enzyme protein during purification. The determination of enzyme specific activity based on enzyme protein rather than total protein is now possible in the carrier state. The primary enzyme immunoassay (PEIA) suitably modified may be extended to study qualitative and quantitative enzyme or inhibitor protein deficiencies in other diseases caused by inborn errors of metabolism.
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