An improved rapid method for selecting monoclonal antibodies with high catalytic activities.
Monoclonal antibodies were raised against a beta-naphthyl phosphonate hapten (1) to elicit antibodies capable of catalyzing the hydrolysis of beta-naphthyl acetate (3). After cell fusion, potential catalytic antibody-producing hybridomas were selected, by use of a competitive inhibition assay on the basis of the binding activity for a short transition-state analogue (inhibitor 5), followed by use of high-performance liquid chromatography analysis for the hybridoma supernatants to screen the antibodies processing catalytic activities. It was shown that supernatants of 12 wells had high binding activity with inhibitor and of them, 7 had catalytic activities. After cloning by limiting dilution, we got two hybridoma clones producing monoclonal antibodies which catalyzed the hydrolysis of beta-naphthyl acetate. This combination of competitive inhibition assay with high-performance liquid chromatography analysis represents an improved rapid approach for the screening of potential catalytic antibodies and significantly increases the possibility of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (Kcat/K(uncat) approximately 10(6) and specificity.
Yuan
Y R
YR
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China.
Yang
B H
BH
Ji
Y Y
YY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D006241:Haptens / Q000737:chemistry / Q000276:immunology; D006868:Hydrolysis; D008722:Methods; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D006241:Haptens