Sheets MD , Amersdorfer P , Finnern R , Sargent P , Lindquist E , Schier R , Hemingsen G , Wong C , Gerhart JC , Marks JD , Lindqvist E .

GM15203-02 NIGMS NIH HHS , R01 GM19363 NIGMS NIH HHS

	Figure 1 Schematic outline of the approach used for library construction. A library of VH and genes was generated from rearranged human V-genes and cloned into the plasmid pCITE3A. The VL genes used for scFv assembly were derived from a previously constructed scFv library contained in the plasmid pHEN1 (12). The vector containing the VL repertoire also contained the scFv linker DNA 5′ to the VL genes. Primers for reamplification of the V-gene repertoires were derived from sequences several hundred bp 5′ (the VH genes) or 3′ (the VL genes) of the scFv gene cloning sites. This approach facilitated the efficiency of PCR assembling a new scFv repertoire and increasing the efficiency of cutting assembled scFv genes with restriction enzymes. (A) VH and linker-VL gene repertoires were generated by PCR from the plasmid DNA of the separate libraries. The VH genes were amplified by using a plasmid specific primer  and an equimolar mixture of HuJH primers  . The linker DNA and VL genes were amplified by using a plasmid specific primer  and an equimolar mixture of RHuJH primers   . The RHuJH primers are complementary to the HuJH primers. (B) The VH and linker DNA-VL gene repertoires were PCR assembled into a scFv gene repertoire. (C) The assembled scFv gene repertoire was cut with the restriction enzymes NcoI and NotI and cloned into the plasmid pHEN1 (17) for phage display.
	Figure 2 V-gene usage and VH CDR3 length of unselected and antigen-specific scFv. The VH and VL genes were sequenced and the germ-line gene was assigned based on homology to a database (VBASE) of germ-line V-genes compiled by Tomlinson et al. (25). Specific VH, Vκ, and Vλ genes are listed on the ordinate, with the VH, Vκ, or Vλ germ-line gene family indicated below. Only V-genes in unselected or selected clones are listed.
	Figure 3 Specificity of anti-Botulinum neurotoxin scFv. Representative scFv (2H6, 3D1, 3B12, and 3C8) isolated respectively from selections on BoNT serotypes A, B, C, and E were studied. Specificity was determined by ELISA.
	Figure 4 Staining of HeLa cells infected with C. trachomatis with the scFv 2A10. The scFv specifically stains C. trachomatis elementary bodies (c) within infected HeLa cells but does not stain uninfected cells. n = nucleus.

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