Detection of antibody display phage without clearing of bacterial culture.

Filamentous phage display has become a powerful method for the discovery of affinity-binding reagents (6). Linear and constrained peptides, antibody fragments (scFvs and Fabs) and some alternative binding domains have all been displayed on phage particles by fusing to one of the phage coat proteins. Although several phage proteins (derived from gVIII, gVI, gVII and gIX) have all been used as fusion partners for display of recombinant proteins, gIII is most widely used for the display of antibody fragments. Phagemids that contain a phage origin of replication, an antibiotic resistance marker and the gene encoding the antibody/gIII fusion protein are readily constructed with conventional molecular biology techniques. Through largescale ligation, transformation and recombination strategies, large libraries of 108–1011 different recombinants are now being generated for use in affinity selection strategies (1,4,5). Once a library of potential binding agents for phage display is generated, individual phage with the capacity to bind to a chosen protein target must be isolated from an enormous excess of non-binding phage. Typically, a target antigen is immobilized on a solid support and incubated with an aliquot of the phage library. Phage particles that remain attached to the antigen after removal of the unbound fraction can be eluted by pH denaturation or enzymatic cleavage and amplified by reinfection of exponentially growing E. coli. Phage that are generated following this amplification are used as the input in the subsequent cycle of affinity selection (panning). After several rounds of panning, E. coli are reinfected with the enriched phage, and individual clones are isolated. A helper phage (e.g., M13KO7) is used to rescue the phagemid into an infectious phage particle displaying the selected binding domain. These individual phage stocks are screened with ELISA to find the clones with the desired binding properties. Traditionally, overnight cultures of phage-producing bacteria are centrifuged to pellet the bacteria, and the phage supernatants are used in the ELISA (2). Alternatively, phage can be purified and concentrated from cleared supernatants by polyethylene glycol precipitation. Although performing this procedure on a small scale may seem reasonable, developing high-throughput screening procedures requires the investigator to eliminate costly, timeconsuming or unnecessary steps. In our laboratory, centrifugation of cultures limited the throughput in screening isolated clones using phage ELISA. We have generated phage-displaying scFvs or Fabs by PCR amplification of cDNA corresponding to the heavy and light chain variable regions from the HP6002, HP6025 and HP6054 hybridomas (3); cells, obtained from ATCC (Manassas, VA, USA) included CRL-1788, CRL-1775 and CRL-1763, respectively. Assembled scFvs or Fabs were digested with SfiI and NotI, subcloned into the pCANTAB5E vector (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and transformed into TG1 or XL1-Blue-competent E. coli. Individual clones capable of specific binding to the target antigen were isolated by conventional methods and then used to explore alternatives to centrifu-