Detection of native and recombinant Bacillus macerans cyclodextrin glycosyltransferase using microtiter elisa techniques

Abstract

Direct, sandwich, and competitive ELISA methods were developed to quantify Bacillus macerans cyclodextrin glycosyltransferase (CGTase) and its 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 antibody binding. Sandwich ELISA showed the most sensitive detection range (0.2 ∼ 50 μg ml −1) against CGTase. We improved a competitive ELISA method by coating the microtiter plate with denatured CGTase in 6 m guanidine-HCI 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 CGTase and its thioredoxin fusion protein in the recombinant E. coli. These methods are specifically useful when detecting and quantifying recombinant CGTase proteins in which mutations may have reduced or eliminated enzymatic activity.