| Immunoglobulin genes were directly isolated from splenocytes of mice hyperimmunized with the auxinic herbicide picloram conjugated to bovine serum albumin. Variable light and variable heavy domain DNA were joined to produce scFv DNA and cloned into the phage vector fd-tet-GIIID to display multiple copies of scFv on the filamentous phage minor coat protein pIII. The phage-display scFv library was selected against picloram conjugated to ovalbumin. After 5 rounds of panning, scFv with different affinities to picloram (IC50 values ranging from 20 ppb to 10 ppm) were detected in the final enriched pool. Picloram-specific variable fragments (VHH) of heavy chain antibodies (HCAb) were also selected from a naive-llama library using ribosome-display technology. The use of a naive library may reduce the time and resources required for hyperimmunization. Furthermore, only one library is required for isolating antibodies to an unlimited number of target compounds. A cDNA library of VHHs was constructed from lymphocytes of non-immunized llama and engineered to display the antibody fragments as ribosome complexes, joining both the genotype (mRNA) and the phenotype (nascent VHH). After six cycles of panning, two sequences (3-1D2 and 3-1F6) were enriched. The KD values, measured by surface plasmon resonance, were 3 and 254 muM, respectively. Three point mutations causing three independent amino acid changes were enriched in the DNA of both clones and which may have conferred binding advantages to the selected VHHs. Ribosome-display technology may also be efficient in molecular evolution of VHHs. Its ability to accommodate a diverse library (1015 members) is advantageous to the construction of a mutant library with multiple target randomization. Consequently, an experiment to improve the affinity and specificity of a V HH using ribosome-display was conducted. A ribosome display library of the mutants was constructed. Nine different "hotspots" in the CDR2 and CDR3 of a parathyroid hormone (PTH)-specific VHH were randomized. After three rounds of selection, one of the clones analysed had a 30-fold increase in affinity and the cross-reactivity to streptavidin observed in the parent clone was eliminated. The results demonstrated that phage-display and ribosome-display are efficient tools with which to isolate recombinant antibody fragments from different DNA sources. Furthermore, ribosome-display can be used in directed molecular evolution to alter protein structure and function. |
