Comparison of affinity purification techniques for single chain antibody NC 6.8

M. G. Jackson, T. C. Brodnicki, D. M. Kranz, D. S. Linthicum

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Single chain antibody (scFv NC6.8) was generated from the IgG (2b,jfc) 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 Gn-HCl. Three different methods of affinity purification were compared: 1) anti c-mvc mAb for a c-myc-scFv NC6.8 fusion construct, 2) metal chelation for a histidine hexamerscFv 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-Ig(CH1+CL) activity prior to affinity purification with a NC6.8 IgG column. Anti-c-myc mAbs (9E10 and CT14 cell lines) that react with residues 408-439 of the c-myc peptide were grown as ascites; purified IgG was coupled to Affigel10 resin. Identification of purified scFv was accomplished by immunoblotting and ELIS A. Analysis using circular dichroism revealed a predominantly β-character of the pure scFv. Determinations of yield, purity, and activity were compared usine different purification techniques.

Original languageEnglish (US)
Pages (from-to)A1058
JournalFASEB Journal
Issue number6
StatePublished - Dec 1 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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