- Molecular Cloning and Characterization of a Single-Chain Variable Fragment Antibody Specific for Benzoylecgonine Expressed in Escherichia coli
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Kenichiro Mori , Youn Uck Kim
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J. Microbiol. 2008;46(5):571-578. Published online October 31, 2008
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DOI: https://doi.org/10.1007/s12275-008-0123-1
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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.
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Citations
Citations to this article as recorded by  - Cytoplasmic production of soluble and functional single-chain Fv-Fc fusion protein in Escherichia coli
Hiroyuki Sonoda, Yoichi Kumada, Tomohisa Katsuda, Hideki Yamaji
Biochemical Engineering Journal.2011; 53(3): 253. CrossRef - Expression of Antibody Fragments with a ControlledN-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis
Andreas Loos, Bart Van Droogenbroeck, Stefan Hillmer, Josephine Grass, Martin Pabst, Alexandra Castilho, Renate Kunert, Mifang Liang, Elsa Arcalis, David G. Robinson, Ann Depicker, Herta Steinkellner
Plant Physiology.2011; 155(4): 2036. CrossRef
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