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

Immunoglobulin genes were directly isolated from the splenocytes of a BALB/ C mouse hyperimmunized with the auxinic herbicide picloram conjugated to bo vine serum albumin. Variable light and heavy domain DNA were joined to prod uce single-chain Fv (scFv) DNA, which was cloned into phage vector fd-tet-G IIID 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, ind ividual clones were analyzed. ScFv with different affinities to picloram (I C50 values ranging from 20 ppb to 10 ppm) were detected in the final enrich ed pool. The increased avidity of the phage vector enhanced the selection ( i.e., panning) of multiple picloram-specific recombinant antibodies. String ent selection was required to isolate the clones with the highest affinity. Nucleotide sequence analysis of six isolated clones revealed that all of t he VL belonged to the V kappa 9A family joined to J kappa2 segments. All of the VH belonged to the V(H)7183 family and joined to two different J segme nts (i.e., J(H)2 or J(H)4). Different from the immune response to large mol ecular weight molecules (MW > 10000 Da), which requires both VDJ segment re arrangement and somatic hypermutations, production of high-affinity antibod ies to picloram, a small ligand having a formula weight of 241.5 Da, predom inantly requires somatic hypermutations.