Lignification of plant cell walls: impact of genetic manipulation.

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 3 109 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. Antibodies that bind with high specificity and high affinity to a target molecule are essential tools for biological research. These reagents have proven invaluable for: (i) detecting and quantitating levels of gene expression; (ii) determining the subcellular, cellular, and tissue location of gene expression; and (iii) identifying the molecules interacting with a gene product, for example by immunoprecipitation. Numerous new applications for basic research, as well as clinical use, have resulted from the development of recombinant antibodies constructed from Ig variable (V) region genes (1–3). Single-chain Fv antibodies (scFv) have proven particularly useful. scFv consist of the antigen-binding domains of Ig heavy (VH) and light (VL) chain regions connected by a flexible peptide linker (4), all encoded by a single gene. The single gene design of scFv simplifies the construction of fusion proteins such as cancer immmunotoxins (5) and facilitates intracellular expression in eukaryotic cells to achieve phenotypic knockout of antigen function (6–8). The intracellular expression of antibodies is proving to be an effective new strategy for studying the function of specific proteins in vivo where conventional genetic approaches are not feasible. Genome projects have led to an increasing rate of gene discovery and an accelerating need for antibodies to study gene expression and function. Until recently, hybridoma technology, a slow and cumbersome process, was used to produce mAbs for such applications. Separate immunizations are required for each antigen, and the cell fusion process required to generate hybridomas is laborious and inefficient. In addition, production of antibodies to antigens conserved between species is difficult and antibodies from hybridomas are murine and hence immunogenic if used therapeutically. Recent advances using antibody phage display now make it possible to overcome these limitations and generate human mAbs that recognize any desired antigen (1–3, 9). For phage display, the antigen-binding regions of VH and VL genes are cloned and used to construct scFv (or Fab) gene repertoires. A phage antibody library is created by cloning these repertoires as fusion proteins with a minor coat protein of bacteriophage (the gene 3 protein) (10–12). Each resulting phage has a functional antibody protein on its surface and contains the gene encoding the antibody incorporated into the phage genome. Particular phage antibodies that specifically bind to proteins and small molecules can be separated from nonbinding phage antibodies with affinity chromatography techniques (12–15). This strategy requires no immunization, the antibody genes are cloned, and generally the antibody fragments express well in Escherichia coli. The number and affinity of the antibodies generated to a particular antigen is a function of library size and diversity, with larger libraries yielding a greater number of high-affinity antibodies (14, 15). Unfortunately, the construction of large phage-displayed antibody libraries has remained difficult. If such libraries are to be a common tool of life scientists the efficient production of these reagents must become routine, especially because library diversity and utility are lost on library reamplification. In this paper, we describe a strategy to optimize the construction of phage-display antibody libraries. By using this strategy, a very large phage-displayed single-chain antibody library consisting of 6.7 3 109 members was produced. This library then was used to isolate panels of antibodies to 14 different protein antigens. Analysis of antibody–antigen interactions revealed high-affinity binding with Kds for the ErbB2 protein ranging between 220 pM and 4 nM.