Screening of a large number of clones produced in a fusion is often the bottleneck in the isolation of catalytic antibodies. The usual approach requires two steps: clones are first selected for their high affinity to the antigen and then the good binders for their catalytic activity. Following this way, one looks for catalysis within a set of antibodies previoulsy selected for a different and not necessarily related property. To overcame this problem, Tawfik and Green developed the catELISA technique, which consist essentially in a non competitive ELISA test for the reaction product: plates are coated with a reagent conjugate which is transformed into product in the presence of a catalytic species.a

The non competitive catELISA

The competitive catELISA

This tecnique allows a fast and direct selection of catalytic clones. Nevertheless it can be used only in a qualitative way, and the reactiviyt of the immobilized substrate can be very different from its behaviour in solution. We have thus developed a competitive catELISA: in this assay the product of the catalyzed reaction binds to an immobilized anti-product antibody in competition with a peroxidase-product conjugate.b

The screening assay has been developed for the antibody catalyzed hydrolysis of esters of p-aminophenylacetic acid and has been tested on the porcine liver esterase catalyzed hydrolysis of the same substrates. This test allows the detection of product formation at the nanomolar level, while, in a typical assay, the catalytic activity of PLE can be traced down to 200 fmol of enzyme. Under standard conditions for the screening of hybridomas obtained from a fusion, the competitive catELISA allows the detection of catalytic species with values of k_cat > 5 x 10^-7 mol l^-1 s^-1 and k_cat/k_uncat > 50.

While the assay has been designed for the selection of catalytic antibodies, other potential applications of this methodology are in the screening of libraries of engineered and designed enzymes and, in general, in the quantitative measurement of enzyme activity.

a. D.S. Tawfik, B.S. Green, Z. Eshar, Anal. Biochem. 1992, 202, 35.

b. F. Benedetti, F. Berti, M. Flego, M. Resmini, E. Bastiani, Anal. Biochem., 1998, 256, 67.