DETECTION OF NATIVE AND RECOMBINANT BACILLUS-MACERANS CYCLODEXTRIN GLYCOSYLTRANSFERASE USING MICROTITER ELISA TECHNIQUES

Direct, sandwich, and competitive ELISA methods were developed to quan tify Bacillus macerans cyclodextrin glycosyltransferase (CGTase) and i ts thioredoxin fusion molecule using rabbit polyclonal antibodies. The direct ELISA method was used to demonstrate equivalent molar response of both native CGTase and the thioredoxin-CGTase fusion protein to an tibody binding. Sandwich ELISA showed the most sensitive detection ran ge (0.2 similar to 50 mu g ml(-1) against CGTase. We improved a compet itive ELISA method by coating the microtiter plate with denatured CGTa se in 6 M guanidine-HCl solution, resulting in enhanced rapid response . This competitive ELISA method was specific, precise, and rapid when the method was used to quantify CGTase and monitor production of CGTas e and its thioredoxin fusion protein in the recombinant E. coli. These methods are specifically useful when detecting and quantifying recomb inant CGTase proteins in which mutations may have reduced or eliminate d enzymatic activity. (C) 1998 Elsevier Science Inc.