ANTIBODY FRAGMENTS AS INHIBITORS OF JAPANESE RADISH ACID-PHOSPHATASE

V-H (heavy-chain variable region) and V-L (light-chain variable region ) genes were amplified by PCR from hybridomas producing MAb-11 and MAb -18 which inhibited Japanese radish acid phosphatase. Nucleotide seque ncing of the V genes demonstrates that the MAbs contained similar V-H and identical V-L domains. Initially, the V-H and V-L genes were expre ssed in Escherichia coli as single-chain Fv (ScFv) fragments, Fragment s ScFv-11 and ScFv-11, named for MAb-11 and MAb-18, respectively, inhi bited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases , including some phosphomonoesterases and phosphodiesterases from diff erent sources. ScFv-18 also inhibited acid phosphatase from a differen t origin, but stimulated the activity of alkaline phosphatase from cal f intestine, The PCR-amplified V-H and V-L genes were subsequently exp ressed separately in Escherichia coli as fusion products with glutathi one S-transferase. The fusion proteins had little effect on Japanese r adish acid phosphatase. Furthermore, a large number of recombinant ScF v fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, includin g inhibition, stimulation, and none. Among them, an ScFv fragment, des ignated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18.