SPECIFICITY AND BINDING-AFFINITY OF AN ANTI-CROTOXIN COMBINATORIAL ANTIBODY SELECTED FROM A PHAGE-DISPLAYED LIBRARY

A crotoxin-specific, monoclonal, high-affinity, single-chain antibody variable region (scFv) was generated by combinatorial methods using Ph armacia's Recombinant Phage Antibody System. A high-affinity clone, de signated A10G, was selected, and its DNA sequence was determined. Prot ein A10G showed high reaction specificity, with only the closely relat ed rattlesnake neurotoxins, concolor toxin and Mojave toxin, showing c ross-reactivity out of eleven group II phospholipase A(2)s (PLA(2)s) s creened. No group I PLA(2)s cross-reacted in enzyme-linked immunosorbe nt assays. The gene coding for AIOG was subcloned into an expression v ector, and the resulting expressed nonfusion protein, designated A10GP E, was renatured and purified to apparent homogeneity. Dissociation co nstants of A10G with intact crotoxin and crotoxin basic subunit were d etermined to be 7 x 10(-10) and 6.8 x 10(-9) M, respectively. When A10 GPE was preincubated with either the basic subunit or intact crotoxin at molar ratios of up to 5:1, no inhibition of phospholipase activity was observed. Expressed protein, however, could partially neutralize t he lethality of Mojave toxin, a crotoxin homolog, in mice.