An investigation of the use of urease-antibody conjugates in enzyme immunoassays.

The development of urease (E.C.3.5.1.5) as a label for enzyme immunoassay (EIA) procedures is described and the use of such conjugates illustrated with examples. Urease catalyzes the hydrolysis of urea to carbon dioxide and ammonia. The production of ammonia may be detected readily by a pH shift which we have found best indicated by the vivid colour change (yellow to purple) of bromocresol purple incorporated in the substrate solution. This enzyme-substrate system offers a number of important advantages. The substrate in aqueous solution is stable, titration end points are sharp and readily visible and the enzyme is not inhibited by sodium azide. Thus, test reagents may be prepared with this preservative and stored ready to use. Urease of high specific activity is commercially available and because it does not occur in mammalian tissues, it is suitable for use in EIA tests to detect cell-associated antigens and their antibodies. Finally, the enzyme reaction may be stopped by the addition of organomercurial preservatives, thus allowing storage of developed tests for later examination.