Enantioselective Monoclonal Antibodies for Detecting Ketamine to Crack Down on Illicit Use
Enantioselective Monoclonal Antibodies for Detecting Ketamine to Crack Down on Illicit Use
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DOI：
10.1248/bpb.b17-00762
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发表时间：
2018-01-01
影响因子：
2
通讯作者：
Kobayashi, Norihiro
中科院分区：
文献类型：
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作者：
Morita, Izumi;Oyama, Hiroyuki;Kobayashi, Norihiro
氯胺酮(KT)是一种手性麻醉剂，其(R)-和(S)-对映体具有不同的药理性质。KT已成为世界上最常用的非法药物之一，因此，需要快速可行的现场检测来打击非法使用。虽然免疫化学方法与特定的抗体有希望达到这一目的，但在实践中，抗KT抗体是很难获得的。我们在这里公开了一代抗KT的单抗。用(A)市售的或(B)自制的KT-白蛋白结合物免疫小鼠。将小鼠脾细胞分别与P3/NS1/1-Ag4-1骨髓瘤细胞融合。经过标准筛选和克隆，我们建立了5个杂交瘤克隆：2个来自a组小鼠[产生抗体-KT(A)#2和#37个竖条]，3个来自b组[产生抗体-KT(B)#9、#13和#45]。这些抗体在竞争性的酶联免疫吸附检测系统中表现出实用性能。以(+/-)-KT-盐酸盐(HCl)为对照，量效曲线显示中点分别为30和70 ng/次(a系列抗体)和2.0~3.0 ng/次(b系列抗体)。值得注意的是，a系列抗体是针对(S)-KT-HCl的，而b系列抗体是针对(R)-KT.HCl的。AB-KT(A)#2(K-a，7.5×10(7)M(-1))和Ab-KT(B)#45(K(A)，7.7×10(8)m(-1))对映体选择性最高，与(R)和(S)对映体的交叉反应率分别为1.3%和1.7%。这里建立的杂交瘤也是抗KT抗体的遗传信息来源，这是使用基因工程抗体发展下一代技术所必需的。
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 vertical bar 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 (K-a, 7.5x10(7)M(-1)) and Ab-KT(b)#45 (K (a), 7.7x10(8)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.