title: Determination of the relative affinities of antibody fragments expressed in Escherichia coli by enzyme-linked immunosorbent assay.
abstract: We have previously utilized an M13 phage expression system and codon-based mutagenesis to increase the avidity of the tumor-specific antibody, BR96, 65-fold (Yelton et al., 1995, J. Immunol. 155, 1994-2004). Mutants with improved affinity were identified by screening phage-expressed antibodies on a carcinoma cell line. In this study we describe a more broadly applicable assay which permits rapid and quantitative comparison of affinities of related antibodies produced in an M13 phage expression system. BR96 variants displaying a range of affinities were expressed as soluble antibody fragments (Fabs) in the periplasmic space of bacteria and isolated from small-scale cultures grown in a 96-well format, yielding between 142 ng and 1.06 microg of Fab. Although the small-scale cultures expressed variable levels of Fab, the lower quantities were sufficient to saturate microtiter plates coated with a limiting amount of anti-human Fab antibody, resulting in the capture of uniform quantities of the Fab variants. The relative affinities of the variants were then compared by assessing binding to biotinylated antigen followed by detection with streptavidin-alkaline phosphatase conjugates. This approach permitted the direct comparison of the relative affinities of large numbers of antibody variants in a single step without multiple antibody dilutions. The assay is readily adaptable for screening phage-expressed antibody libraries against any biotinylated target and does not require purified antigen. For instance, the biotinylated BR96 antigen utilized in these studies was from a total cell extract prepared by labeling the surface of live tumor cells followed by detergent extraction. Thus, this approach may be applicable to the screening of antibody libraries against other cell surface antigens, such as transmembrane receptors, which are difficult to purify.
authors.affiliation: Ixsys, Inc., 3550 Dunhill Street, San Diego, California 92121, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Watkins
authors.forename: G
authors.initials: G
authors.lastname: Beuerlein
authors.forename: G
authors.initials: G
authors.lastname: Pecht
authors.forename: P R
authors.initials: PR
authors.lastname: McFadden
authors.forename: S M
authors.initials: SM
authors.lastname: Glaser
authors.forename: W D
authors.initials: WD
authors.lastname: Huse
mesh_terms: D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000912:Antibodies, Neoplasm / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000032:analysis / Q000276:immunology*; D000954:Antigens, Surface / Q000032:analysis; D017104:Bacteriophage M13; D001710:Biotin; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D020113:Phenolphthalein; D010635:Phenolphthaleins; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D019809:Streptavidin; D014407:Tumor Cells, Cultured
chemical_list: D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D010635:Phenolphthaleins; D011994:Recombinant Proteins; D020113:Phenolphthalein; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase