Characterization of the selected phagemid transformed-HB2151 E. coli clones. (A) Indirect ELISA results showing the binding of HuScFv in lysates of 17 huscfv positive E. coli clones to rM2 and BSA control. HuScFv of 10 clones (no. 2, 5, 9, 13, 14, 19, 20, 23, 27 and 29) bound specifically to the rM2. (B) RFLP of huscfv sequences of the 10 ELISA positive clones showed 6 different DNA patterns. Lanes 1–6: RFLP of clones 2, 5, 14, 19, 23 and 27, respectively. (C) Western blot patterns of HuScFv-2, -19, -23 and -27 that bound to rM2; HuScFv-5 and -14 did not bind to the rM2 (data not shown). Lane M: protein molecular weight standard; lane 1: positive control (rM2 blotted strip was probed with mouse PAb to rM2); lane 2: negative control (rM2 blot was probed with lysate of normal E. coli); lanes 3–6, rM2 blotted strips probed with HuScFv-2, -19, -23, and -27, respectively. The antigen-antibody reactive bands were revealed by using mouse anti-E tag-enzyme conjugate and substrate. (D) Western blot results for determining the binding of HuScFv of clones 2, 19, 23 and 27 to nM2. Lane M: protein molecular weight standard; lane 1: positive control which the SDS-PAGE separated rM2 purified from the E. coli lysate was probed with mouse PAb to rM2; lane 2: positive control which SDS-PAGE separated H5N1 virus lysate containing nM2 was probed with mouse PAb to rM2; lanes 3–6, nM2 blotted strips probed with HuScFv of clones no. 2, 19, 23, and 27, respectively; the antigen-antibody reactive bands were detected by mouse anti-6x His tag; lane 7: negative control which the nM2 blotted strip was probed with lysate of normal E. coli. Numbers at the left of (C) and (D) are relative molecular masses (Mr) of proteins.