Synthesis of ligand-specific phage-display ScFv against the herbicide picloram by direct cloning from hyperimmunized mouse

Abstract

Immunoglobulin genes were directly isolated from the splenocytes of a BALB/C mouse hyperimmunized with the auxinic herbicide picloram conjugated to bovine serum albumin. Variable light and heavy domain DNA were joined to produce single-chain Fv (scFv) DNA, which was cloned into phage vector fd-tet-GIIID to display multiple copies of scFv on the filamentous phage minor coat protein gIIIp. The phage-display scFv library (10(4) clones) was selected against picloram conjugated to ovalbumin. After five rounds of panning, individual clones were analyzed. ScFv with different affinities to picloram (IC(50) values ranging from 20 ppb to 10 ppm) were detected in the final enriched pool. The increased avidity of the phage vector enhanced the selection (i.e., panning) of multiple picloram-specific recombinant antibodies. Stringent selection was required to isolate the clones with the highest affinity. Nucleotide sequence analysis of six isolated clones revealed that all of the V(L) belonged to the V kappa 9A family joined to J kappa 2 segments. All of the V(H) belonged to the V(H)()7183 family and joined to two different J segments (i.e., J(H)()2 or J(H)()4). Different from the immune response to large molecular weight molecules (MW > 10,000 Da), which requires both VDJ segment rearrangement and somatic hypermutations, production of high-affinity antibodies to picloram, a small ligand having a formula weight of 241.5 Da, predominantly requires somatic hypermutations.