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 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 Ka6.2×107M-1(as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with Ka, 1.1×107M-1was prepared by bacterial expression of a fusion gene combining the VHand VLgenes for Ab-THC#33. Subsequently, random point mutations in VHand VLwere 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 (Ka1.1×108M-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.
Kobe Pharmaceutical University.
Izumi
I
Morita
Hiroyuki
H
Oyama
Mayumi
M
Yasuo
Kazuhisa
K
Matsuda
Kengo
K
Katagi
Aya
A
Ito
Hiroka
H
Tatsuda
Hiroyuki
H
Tanaka
Satoshi
S
Morimoto
Norihiro
N
Kobayashi
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000915:Antibody Affinity / Q000502:physiology*; D002186:Cannabinoids / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D062105:Molecular Docking Simulation / Q000379:methods*; D017433:Protein Structure, Secondary; D015813:Substance Abuse Detection / Q000379:methods
D000911:Antibodies, Monoclonal; D002186:Cannabinoids; D007128:Immunoglobulin Fragments