A new simplified method for preparation of a synthetic phage antibody with practically acceptable detection sensitivity on immunoblots

Abstract

We produced a library of phage clones displaying a synthetic repertoire of 1 × 10⁸ scFv antibody fragments by combinatorial mutagenesis of several amino acid residues in complementarity determining region 3 (CDR3) of a single antibody sequence used as a template. Phage antibodies specific to a cerebellum-specific protein, MEGF1/fat2, were successfully isolated from clones in this library, but their affinity in binding to MEGF1/fat2 was too low for use in immunoblotting applications. In an effort to obtain a practically useful phage antibody from these primary phage antibodies, a new simplified method based on PCR using unequally degenerate oligonucleotides was devised and applied. A secondary phage antibody showing 5-fold slower dissociation from the targeted antigen than the parental one was successfully obtained from a mutant library consisting of about 10⁷ clones through only two rounds of panning. This affinity maturation followed by fusion with bacterial alkaline phosphatase improved sensitivity in immunoblot assays, with the detection limit lowered to one nanogram level. The results indicate that this method offers a straightforward route to generate a recombinant phage antibody with practically acceptable immunoblot sensitivity from a medium-size single-pot library, from which specific and high-affinity primary phage antibodies are difficult to isolate directly.