Antibody Fragments for On-Site Testing of Cannabinoids Generated via in Vitro Affinity Maturation

Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate dev...

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Bibliographic Details
Published in:Biological & pharmaceutical bulletin 2017/02/01, Vol.40(2), pp.174-181
Main Authors: Morita, Izumi, Oyama, Hiroyuki, Yasuo, Mayumi, Matsuda, Kazuhisa, Katagi, Kengo, Ito, Aya, Tatsuda, Hiroka, Tanaka, Hiroyuki, Morimoto, Satoshi, Kobayashi, Norihiro
Format: Article
Language:eng ; jpn
Subjects:
Affinity
affinity maturation
Amino Acid Sequence
Animals
Antibodies, Monoclonal - chemistry
Antibodies, Monoclonal - metabolism
antibody
Antibody Affinity - physiology
cannabinoid
Cannabinoids
Cannabinoids - chemistry
Cannabinoids - metabolism
Cannabis
Complementarity
Complementarity-determining region
Complementarity-determining region 2
Dose-Response Relationship, Drug
Enforcement
Enzyme-linked immunosorbent assay
Fab
Female
Fusion protein
Immunoglobulin Fragments - chemistry
Immunoglobulin Fragments - genetics
Immunoglobulin Fragments - metabolism
Maturation
Mice
Mice, Inbred BALB C
Molecular Docking Simulation - methods
Monoclonal antibodies
N-Terminus
Panning
Phages
Protein Structure, Secondary
single-chain Fv fragment
Substance Abuse Detection - methods
Tetrahydrocannabinol
THC metabolite
Urine
Δ9-tetrahydrocannabinol (THC)
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Summary:Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ9-tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with Ka 6.2×107 M−1 (as Fab fragment) was established by the hybridoma technique. Then, a “wild-type” scFv (wt-scFv) with Ka, 1.1×107 M−1 was prepared by bacterial expression of a fusion gene combining the VH and VL genes for Ab-THC#33. Subsequently, random point mutations in VH and VL were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (Ka 1.1×108 M−1) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the VH-complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose–response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.
ISSN:0918-6158
1347-5215
DOI:10.1248/bpb.b16-00669