Enantioselective Monoclonal Antibodies for Detecting Ketamine to Crack Down on Illicit Use.

Ketamine (KT) is a chiral anesthetic agent, (R)- and (S)-enantiomers of which differ in their pharmacological properties. KT has become one of the most commonly used illicit drugs in the world, thus, rapid and feasible on-site testing is required to crack down on the illicit use. Although immunochemical approach with specific antibodies is promising for this purpose, in practice anti-KT antibodies are difficult to obtain. We here disclose generation of monoclonal antibodies against KT. Mice were immunized with either (a) commercially-available or (b) in-house-prepared KT-albumin conjugates. Splenocytes from these mouse groups (a and b) were separately fused with P3/NS1/1-Ag4-1 myeloma cells. After standard screening and cloning, we established 5 hybridoma clones: 2 were derived from group-a mice [generating Ab-KT(a)#2 and #37] and 3 were from group-b mice [generating Ab-KT(b)#9, #13, and #45]. These antibodies exhibited practical performance in competitive enzyme-linked immunosorbent assay systems. When (±)-KT·hydrochloride (HCl) was used as the competitor, dose-response curves showed midpoint values of 30 and 70 ng/assay (a-series antibodies) and 2.0-3.0 ng/assay (b-series antibodies). Remarkably, the a-series antibodies were specific for (S)-KT·HCl, while the b-series antibodies were specific for (R)-KT·HCl. Ab-KT(a)#2 (Ka, 7.5×107 M-1) and Ab-KT(b)#45 (Ka, 7.7×108 M-1) exhibited the highest enantioselectivity for each group, and cross-reactivity with the (R)- and (S)-antipodes was 1.3 and 1.7%, respectively. The hybridomas established here are also valuable as a source of genetic information for the anti-KT antibodies, which is required for progressing to next-generation technologies using genetically engineered antibodies.

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 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.