Detection of catalytic monoclonal antibodies.
Several laboratories have now shown that monoclonal antibodies having enzyme-like properties can be generated. The generation of catalytic antibodies makes use of the same basic procedures that have been used for the generation of binding monoclonal antibodies, yet the process involves an additional crucial step: screening for catalytic activity. In this paper we address the unique problems involved in the detection of inefficient catalytic activity that is accompanied by uncatalyzed background reaction. An analysis that allows optimization of assay conditions and estimation of the minimal antibody concentration required to observe catalysis is presented. The results indicate that the structure of the substrate should be optimized to increase its affinity (i.e., decrease its Km) and reduce its concentration to pseudo-first-order conditions (S(O) much less than Km) so that the signal observed in the presence of a catalytic antibody (delta Pcat) is significantly higher than that of the background (delta P(uncat)). Other factors involved in the screening procedures, e.g., sensitivity of the assay, solubility and reactivity of the substrate, and purity of the antibody preparation, are also discussed. The effect of these assay parameters on the ability to detect catalytic activity is demonstrated with p-nitrophenyl ester-hydrolyzing antibodies.
Tawfik
D S
DS
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Green
B S
BS
Eshhar
Z
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000378:metabolism*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004798:Enzymes / Q000378:metabolism*; D006241:Haptens / Q000378:metabolism; D008433:Mathematics; D051379:Mice; D009596:Nitrophenols / Q000276:immunology / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004798:Enzymes; D006241:Haptens; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C023602:O-4-nitrophenyl phenylphosphonate