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.

Chloroplast-mediated activation of plant immune signalling in Arabidopsis.

Chloroplasts have a critical role in plant immunity as a site for the production for salicylic acid and jasmonic acid, important mediators of plant immunity. However, the molecular link between chloroplasts and the cytoplasmic-nuclear immune system remains largely unknown. Here we show that pathogen-associated molecular pattern (PAMP) signals are quickly relayed to chloroplasts and evoke specific Ca(2+) signatures in the stroma. We further demonstrate that a chloroplast-localized protein, named calcium-sensing receptor (CAS), is involved in stromal Ca(2+) transients and responsible for both PAMP-induced basal resistance and R gene-mediated hypersensitive cell death. CAS acts upstream of salicylic acid accumulation. Transcriptome analysis demonstrates that CAS is involved in PAMP-induced expression of defence genes and suppression of chloroplast gene expression possibly through (1)O(2)-mediated retrograde signalling, allowing chloroplast-mediated transcriptional reprogramming during plant immune responses. The present study reveals a previously unknown chloroplast-mediated signalling pathway linking chloroplasts to cytoplasmic-nuclear immune responses.