Efficient construction of a large nonimmune phage antibody library: The production of high-affinity human single-chain antibodies to protein antigens

TLDR

The authors constructed a 6.7 × 10⁹‑member phage‑displayed human scFv library using improved construction steps and affinity‑selected antibodies against 14 diverse protein antigens. The library yielded high‑affinity panels (average 8.7 scFv per antigen) with sub‑nanomolar affinities, including 220 pM–4 nM binders to ErbB2, and the antibodies proved effective in immunoassays such as staining Chlamydia‑infected cells, confirming the library’s utility for rapid production of functional reagents. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

Abstract

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 × 10 9 members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody–antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia -infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.