Screening the 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, then the good binders are tested for their catalytic activity. In order to simplify this selection process, a competitive ELISA assay has been developed that allows direct screening of the antibodies on the basis of their catalytic activity. In this assay, the product of the catalyzed reaction binds to an immobilized anti-product antibody in competition with a peroxidase-product conjugate. The screening assay has been developed for the antibody catalyzed hydrolysis of esters of p-aminophenylacetic acid and has been tested on the PLE4-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 fmoles of enzyme. In the standard conditions for the screening of hybridomas obtained from a fusion, the competitive ELISA assay allows detection of catalytic species with values of kcat ** 5 x 10-7 mol l-1 s-1 and kcat/kuncat ** 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 Competitive Immunoassay for the Detection of Esterolytic Activity of Antibodies and Enzymes
BENEDETTI, FABIO;BERTI, FEDERICO;FLEGO, MASSIMILIANO;
1998-01-01
Abstract
Screening the 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, then the good binders are tested for their catalytic activity. In order to simplify this selection process, a competitive ELISA assay has been developed that allows direct screening of the antibodies on the basis of their catalytic activity. In this assay, the product of the catalyzed reaction binds to an immobilized anti-product antibody in competition with a peroxidase-product conjugate. The screening assay has been developed for the antibody catalyzed hydrolysis of esters of p-aminophenylacetic acid and has been tested on the PLE4-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 fmoles of enzyme. In the standard conditions for the screening of hybridomas obtained from a fusion, the competitive ELISA assay allows detection of catalytic species with values of kcat ** 5 x 10-7 mol l-1 s-1 and kcat/kuncat ** 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.Pubblicazioni consigliate
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