Efficient screening for catalytic antibodies using a short transition-state analog and detailed characterization of selected antibodies.

Abstract

One of the major obstacles to acquiring catalytic antibodies is that it requires labor-intensive procedures to select catalytic antibodies from huge repertories of antibodies. Here, we selected potential catalytic Abs by utilizing their affinity towards a short transition-state analog which contained only the transition-state structural element, and evaluated in detail its efficiency to enrich catalytic Abs. Hybridoma supernatants elicited against a phosphonate derivative, the TSA1, were screened by a three-step screening process: step 1, ELISA for TSA1-BSA; step 2, ELISA for the short TSA4; and step 3, competitive-inhibition by the short TSA2. Only 22. 8% of positive mAbs from step 1 were found to be catalytic. The rate of catalytic Abs increased to 45.7% using screening steps 1 plus 2, and reached 83.3% using all three screening steps. This clearly suggests that our screening protocol is an efficient method to select potential catalytic Abs. Furthermore, we characterized the properties of both the catalytic Abs and the noncatalytic Abs in detail. The catalytic Abs tended to have lower Kd for TSA1 and the short TSA2 than noncatalytic Abs. It was also observed that catalytic Abs showed clear enantiospecificity toward substrate 6 containing d-phenylalanine while noncatalytic Abs did not. The detailed analysis of kinetic and binding parameters for these antibodies gives us further insight into catalytic antibodies.