Abstract
Single chain antibody (scfv NC6.8) was generated from the IgG (2b,k) parent mAb cell line NC6.8, a mAb that binds a super-potent trisubstituted guanidino sweetener, N-(p-cyanophenyl)-N'(diphenylmethyl)guanidine acetic acid. The plasmid construct was cloned from the VH and VL MRNA isolated from the parent mAb cell line. Expression was induced in transformed E. coli and scfv was localized in inclusion bodies. ScFv was isolated and solubilized in 6 M Gdn-HCI. Three different methods of affinity purification were compared: 1) anti c-myc mAb for a c-myc-scfv NC6.8 fusion construct, 2) metal chelation for a histidine hexamer-scfv NC6.8 fusion construct, and 3) polyclonal anti-scfv NC 6.8 antibody, for either of the above fusion constructs. The polyclonal sheep antibody to the scfv was prepared using the NC6.8 Fab as an antigen. This antisera was then absorbed to remove polyclonal anti-lg(CH1+CL) activity prior to affinity purification with a NC6.8 IgG column. Antic-myc mabs (9ElO and CT14 cell lines) that react with residues 408439 of the c-myc peptide were grown as ascites; purified IgG was coupled to Affi-gel-10 resin. Identification of purified scfv was accomplished by immunoblotting and ELISA. Analysis using circular dichroism revealed a predominantly D-character of the pure scfv. Determinations of total yield, purity, and activity were compared using different affinity purification techniques.
Jackson, Michael Gary (1996). Comparative affinity purification of single chain antibody NC6.8. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1996 -THESIS -J336.