Application of phage display to high throughput antibody generation and characterization

Abstract

High-throughput antibody generation and characterization <p>A phage display library has been constructed containing over 1010 human antibodies, allowing the large-scale generation of antibodies. Over 38,000 recombinant antibodies against 292 antigens were selected, screened and sequenced, and 4,400 resultant unique clones characterized further.</p> AbstractWe have created a high quality phage display library containing over 10 10 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.

Cross-domain inhibition of TACE ectodomain

“…In light of both recent structural advances and prior biochemical observations, we hypothesized that selectively targeting noncatalytic regions of the complete TACE ectodomain would produce a more specific cell-surface inhibitor. To this end, recombinant human TACE ectodomain was biotinylated, checked for wildtype activity, and exposed to a naïve human scFv antibody phagedisplay library (24) for two rounds of solution-phase selections. As previous attempts to directly target metalloprotease catalytic sites have resulted in unwanted cross-reactivity (e.g., TIMPs, ADAM prodomains, and SMIs), we blocked the TACE catalytic cleft with the broad-spectrum metalloprotease inhibitor CT1746 (25) during initial selections (Fig.…”

“…Individual scFv clones were isolated from E. coli periplasm and ELISA screened against immobilized recombinant TACE ectodomain in the absence of CT1746. Comprehensive screening details have been outlined previously (24,26). Following initial screening, selected anti-TACE scFv clones were expressed in 500 mL auto-induction (38) shake flask cultures and periplasmic fractions were purified by IMAC.…”

“…Recombinant human TACE ectodomain (Arg 215 -Arg 651 ) was biotinylated at a 1∶1 ratio using N-succinimidyl biotin (Invitrogen AL-01), checked for wildtype activity in a quenched-fluorescent peptide cleavage assay, and exposed to the naïve human scFv phage-display library of McCafferty (24) in the presence of 50 μM CT1746 (25). Following two rounds of solution-phase selection, the eluted polyclonal scFv population was cloned into pSANG10-3F (37) and transformed into BL21(DE3) RIPL E. coli (Stratagene 230280).…”

Advances in the production of human monoclonal antibodies

“…The large antibody repertoires can be generated using the process of combinatorial infection and in vivo recombination, to display scFv on the surface of the phage. [27][28][29] After bio-panning the phages bound to a range of antigens or haptens, antigen-specific antibody fragments with good affinities can be identified. 27,[29][30][31] To date, several improvements have been made in phage display technology to increase the efficiency of the acquisition of antigenspecific scFv, to augment the affinity of scFv for antigens, and to increase the specificity of scFv.…”

“…It is the next method being successfully used to select antigen-specific variable region genes and to express functional antibody fragments with unique specificity. [22][23][24][25][26][27] To isolate human antibodies, the library of diverse human immunoglobulin-heavy chain variable (VH) gene and lightchain variable (VL) gene segments are prepared by reversetranscription of mRNA from B cells and PCR amplification. The gene encoding single chain variable fragment (scFv) can be created by randomly combining VH and VL gene segments using PCR.…”