Journal of Microbiology

Journal Article

Molecular Cloning and Characterization of a Single-Chain Variable Fragment Antibody Specific for Benzoylecgonine Expressed in Escherichia coli: Kenichiro Mori , Youn Uck Kim; J. Microbiol. 2008;46(5):571-578. Published online October 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0123-1

Abstract: Benzoylecgonine is a major metabolite of cocaine. We generated hybridoma cells (C1303) producing antibenzoylecgonine monoclonal antibody (mAb) with a single-chain variable fragment (scFv) and an antigenbinding domain from the C1303 cells. Genes encoding an scFv antibody and constant region (Fc) were amplified from a cDNA library of C1303 cells using PCR. The two frameworks built for scFv and scFv-Fc consisted of HL [(heavy chain variable region, VH) - linker - (light chain variable region, VL)] and HL-Fc, respectively. A 45 base-pair-long sequence encoding (Gly4-Ser)3 was used as the linker, and the mouse IgG1 constant region sequence (225 amino acids) was used as the Fc domain. These two types of recombinant Abs were determined to be 750 bp in length (which corresponds to a 30 kDa protein) in the HL and 1,432 bp in length (which corresponds to a 65 kDa protein) in the HL-Fc, respectively. The parental Ab and HL-Fc affinities against benzoylecgonine were measured by ELISA and found to be nearly equal to the Ab concentration. We were also able to measure HL affinity using an agarose diffusion assay (Ouchterlony test). The affinity of the recombinant single-chain antibody against benzoylecgonine was sufficiently comparable to that of the parent antibodies to be used for the immunodetection of specific drug compounds or the detoxification of drug abusers by immunotherapy.

Generation of Isotype Switch Variants form Hybridoma cells Producing anti-Streptococcus penumoniae 6B Polysaccharide Antibody: Kim, Ji Hye , Ryu, Eun Ja , Park, Moon Kook; J. Microbiol. 1999;37(3):180-184.

Abstract: Hybridoma cells producing IgM anti-pneuococcal 6B polysaccharide antibodies were induced to switch to IgG-producing cells in vitro by treating with acridine orange. Treating 0.5 ㎍/ml of acridine orange for 24 hours generated maximal number of variant cells. The maximal isotype switch frequency was 3×10^-5, which is about 30-fold higher than the frequency of spontaneous switching. Resulting IgG-producing variants were enriched by sib selection and ELISA spot assay. Two IgG3-producing variant cells were finally cloned by limiting dilution. The variant cells produced similar amounts of antibodies as their parental cells did. The two switched antibodies had similar reactivity to pneumococcal 6B polysaccharide. When compared to their parental IgM antibodies, the switched IgG3 than that of IgM antibody. The antibodies will be useful as essential tools for comparative study of the role of heavy chain isotypes in protection against Streptococcus pneumoniae.

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