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

Abstract

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 K_a 6.2×10⁷ M^-1 (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with K_a, 1.1×10⁷ M^-1 was prepared by bacterial expression of a fusion gene combining the V_H and V_L genes for Ab-THC#33. Subsequently, random point mutations in V_H and V_L 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 (K_a 1.1×10⁸ M^-1) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the V_H-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.