title: Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression.
abstract: The detection of methamphetamine and other chemically related illicit drugs relies extensively on immunoassays. Here we report the cloning and affinity maturation of an anti-methamphetamine antibody which is being employed in the current commercial assays. An anti-methamphetamine scFv was cloned from hybridoma cells, expressed in bacteria and its affinity towards methamphetamine and N-ethylamphetamine (ethamphetamine) was determined by Surface Plasmon Resonance (SPR). The anti-methamphetamine scFv gene was subjected to random mutagenesis by error prone PCR and variants with improved affinity were isolated from the resulting library by a novel screening methodology termed Anchored Periplasmic Expression (APEx) [Harvey, B.R., Georgiou, G., Hayhurst, A., Jeong, K.J., Iverson, B.L., Rogers, G.K. (2004). Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc. Natl. Acad. Sci. U. S. A. 101, 9193.]. The isolated clones exhibited improved affinity to these illicit drugs, yet maintained low cross-reactivity to over-the-counter drugs. In addition, all clones displayed improved expression characteristics in Escherichia coli. The affinity improved scFv antibodies are thus likely to be useful in methamphetamine class immunodiagnostics.
authors.affiliation: Institute for Cellular and Molecular Biology, University of Texas, Austin, TX, USA.
authors.forename: Barrett R
authors.initials: BR
authors.lastname: Harvey
authors.forename: Armen B
authors.initials: AB
authors.lastname: Shanafelt
authors.forename: Irina
authors.initials: I
authors.lastname: Baburina
authors.forename: Raymond
authors.initials: R
authors.lastname: Hui
authors.forename: Steve
authors.initials: S
authors.lastname: Vitone
authors.forename: Brent L
authors.initials: BL
authors.lastname: Iverson
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity / Q000235:genetics; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D008694:Methamphetamine / Q000032:analysis / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019151:Peptide Library; D019897:Periplasm / Q000276:immunology; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D008694:Methamphetamine