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

One of the major obstacles to acquiring catalytic antibodies is that it req uires labor-intensive procedures to select catalytic antibodies from huge r epertories of antibodies. Here, we selected potential catalytic Abs by util izing their affinity towards a short transition-state analog which containe d only the transition-state structural element, and evaluated in detail its efficiency to enrich catalytic Abs. Hybridoma supernatants elicited agains t a phosphonate derivative, the TSA1, were screened by a three-step screeni ng process: step 1, ELISA for TSA1-BSA; step 2, ELISA for the short TSA4; a nd 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 inc reased 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 i s an efficient method to select potential catalytic Abs. Furthermore, we ch aracterized the properties of both the catalytic Abs and the noncatalytic A bs in detail. The catalytic Abs tended to have lower K-d for TSA1 and the s hort TSA2 than noncatalytic Abs. It was also observed that catalytic Abs sh owed clear enantiospecificity toward substrate 6 containing D-phenylalanine while noncatalytic Abs did not. The detailed analysis of kinetic and bindi ng parameters for these antibodies gives us further insight into catalytic antibodies.