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 K d 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.Keywords: catalytic antibody; transition-state analogue (TSA); short TSA; MRL/lpr; enantiospecificity.Based on`transition-state theory' advocated by Pauling [1], Jencks [2] predicted that antibodies elicited against stable mimics of transition states would catalyze the corresponding reactions. This hypothesis has proved fruitful when combined with hybridoma technology [3] that allows production of monoclonal antibodies with catalytic activities [4±7]. However, the entire process is time-consuming and labor intensive, especially the screening of catalytic antibodies from a huge repertoire of antibodies, most of which are noncatalytic. It has been, therefore, a long-awaited goal to develop an easy and efficient screening method for catalytic Abs.Because of the inefficiency of conventional screening methods that utilize binding affinity of antibodies toward transition-state analogue, Tawfik et al. [8] developed an alternative method, catELISA', which allows screening of catalytic activities directly in hybridoma growth medium. In this method, immobilized substrates on ELISA plates are converted by hybridoma supernatants to end product, which is detected by ELISA using antiproduct antibodies. Although this method allows screening of more clones than conventional methods, many critical conditions need to be satisfied as follows: (a) immobilized substrates must be stable; (b) appropriate anti-product antibodies with no cross-reactivity with the substrate(s) are required; (c) conditions must be optimized; and (d) the assay should detect only antibodyspecific reaction, no activity resulting from any enzymes in t...

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