All sequences were unique, indicating that diversity of the scFv-PhoA library was maintained throughout the assay

All sequences were unique, indicating that diversity of the scFv-PhoA library was maintained throughout the assay. antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers DM4 a higher success rate than previous methods based on scFv libraries, andmost importantlyit enables high-throughput procedures. Keywords: antibody, single-chain Fv (scFv), alkaline phosphatase, bacteria, which are grown on a hydrophilic filter placed on an antigen-coated membrane. Colonies form on the filter, scFv antibodies produced by the colonies diffuse out, they bind to the antigen on the membrane, and their presence is detected by superimposing the spot on the colony. A gene encoding scFv with affinity for the antigen is obtained. The colony assay identifies clones with high reliability by directly observing antibodyCantigen binding, thus resulting in a low false-positive rate. In addition, the method can be easily used to screen libraries with an order of magnitude larger (105~106) than those employed in hybridoma technology (103~104), DM4 resulting in more positive clones over a shorter period. Nevertheless, the size of a colony assay library is much smaller than that afforded by phage display (109~1010) [3]. To obtain monoclonal antibodies with the desired characteristics, the colony assay needs DM4 to be sufficiently efficient to handle larger libraries. Here, we aimed to improve colony assay efficiency by replacing scFv with scFv fused to bacterial alkaline phosphatase (scFv-PhoA). The strategy of Pho-A fusion was successfully applied to produce in MYO7A various eukaryotic molecules, including hormones [14,15] and antibody fragments [16,17,18,19,20]. All these fusion proteins were secreted in periplasm of where they folded correctly, yielding homogeneous, stable, and bifunctional molecules. Fusion of scFv to the N-terminus of bacterial alkaline phosphatase considerably improved performance [21,22]. The periplasmic localization of PhoA-tagged scFv ensures dimerization of the PhoA moiety into its enzymatically active form and the correct folding of scFv via disulfide bond formation [23]. PhoA enables direct enzymatic detection of scFv fusions without the need for a secondary reagent such as an anti-His-tag antibody [24]. Positive clones showing specific binding to the antigen could be detected directly and rapidly, thus strongly improving assay processivity. 2. Results 2.1. Development of the Colony Assay with scFv-PhoA The colony assay procedure based on the scFv-PhoA library is illustrated in Figure 1. The hydrophilic filter and antigen-coated nitrocellulose membrane were placed on the agar plate, and bacteria transformed with the scFv-PhoA library were spread on the filter. Colonies became visible after 14 h of incubation at 30 C, at which point, scFv-PhoA expression was autoinduced. The bacterial colonies produced soluble periplasmic scFv-PhoA fusions, and those showing affinity against the antigen were captured by the antigen immobilized on the membrane. After incubation for 24 h at 30 C, the filter harboring the colonies was transferred to a fresh agar plate and the nitrocellulose membrane was developed to detect antigen-binding scFv-PhoA fusions by chemiluminescence. This was achieved simply by applying the alkaline phosphatase substrate without using additional enzyme-conjugated antibodies and corresponding washing and reaction steps, thus shortening protocol time to about 1/10 of the original. To identify positive colonies, the filter and the membrane were superimposed so that the colonies on the filter and the positive chemiluminescence signals were aligned. The colonies corresponding to the positive signals were then picked and cultured in the medium to identify candidate genes. Open in a separate window Figure 1 Schematic diagram of the procedure describing the colony assay with a single-chain variable fragment fused to the gene [23]. Open in a separate window Figure 2 (a) Partial sequence of pET-NXNN-PhoA shown together with the cloning site and the restriction DM4 sites used in the cloning strategy. (b) Schematic representation of scFv cloned into pET-NXNN-PhoA. Orange square, signal sequence for PhoA DM4 (ssPhoA); blue square, two N-terminal amino acids of PhoA (PhoA (1-2)); dotted rectangle, heavy-chain variable domain (VH); gray square, linker ((G4S)3); striped rectangle, light-chain variable domain (VL); blue.