Engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression.
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.
Harvey
Barrett R
BR
Institute for Cellular and Molecular Biology, University of Texas, Austin, TX, USA.
Shanafelt
Armen B
AB
Baburina
Irina
I
Hui
Raymond
R
Vitone
Steve
S
Iverson
Brent L
BL
Georgiou
George
G
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
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D008694:Methamphetamine