Molecular cloning and characterization of a single-chain variable fragment antibody specific for benzoylecgonine expressed in Escherichia coli.
Benzoylecgonine is a major metabolite of cocaine. We generated hybridoma cells (C1303) producing anti-benzoylecgonine monoclonal antibody (mAb) with a single-chain variable fragment (scFv) and an antigen-binding 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, V(H)) - linker - (light chain variable region, V(L))] and HL-Fc, respectively. A 45 base-pair-long sequence encoding (Gly(4)-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.
Mori
Kenichiro
K
Department of Microbiology and Immunochemistry Asahikawa Medical College, Asahikawa 078-8510, Japan.
Kim
Youn Uck
YU
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D003001:Cloning, Molecular; D003042:Cocaine / Q000031:analogs & derivatives* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data
D000906:Antibodies; D007135:Immunoglobulin Variable Region; C005618:benzoylecgonine; D003042:Cocaine