Simple method for selecting catalytic monoclonal antibodies that exhibit turnover and specificity.
Monoclonal antibodies were raised against a mono-p-nitrophenyl phosphonate ester to elicit catalytic antibodies capable of hydrolyzing the analogous p-nitrophenyl ester or carbonate. Potential catalytic antibody producing clones were selected, by use of a competitive inhibition assay, on the basis of their affinity for a "short" transition-state analogue, a truncated hapten which maximizes the relative contribution of the transition-state structural elements to binding. Of 30-40 clones that would have been examined on the basis of hapten binding alone, 7 were selected and 4 of these catalyzed the hydrolysis of the relevant p-nitrophenyl ester. This competitive inhibition technique represents a general approach for selecting potential catalytic antibodies and significantly increases the probability of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (kcat/kuncat approximately 10(4)) and substrate specificity for the hydrolysis of the analogous ester and, for three of the antibodies, of the analogous carbonate. The antibodies displayed turnover, an essential feature of enzymes. Evidence that catalysis occurred at the antibody combining sites was provided by the identity of the binding and the catalysis-inhibition specificity patterns.
Tawfik
D S
DS
Department of Pharmaceutical Chemistry, Faculty of Medicine, Hebrew University, Jerusalem, Israel.
Zemel
R R
RR
Arad-Yellin
R
R
Green
B S
BS
Eshhar
Z
Z
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002384:Catalysis; D006241:Haptens; D006868:Hydrolysis; D008968:Molecular Conformation; D009943:Organophosphorus Compounds / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D006241:Haptens; D009943:Organophosphorus Compounds