STEREOSPECIFIC MONOCLONAL-ANTIBODIES TO NICOTINE AND COTININE AND THEIR USE IN ENZYME-LINKED IMMUNOSORBENT ASSAYS
STEREOSPECIFIC MONOCLONAL-ANTIBODIES TO NICOTINE AND COTININE AND THEIR USE IN ENZYME-LINKED IMMUNOSORBENT ASSAYS
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DOI：
10.1016/0022-1759(86)90077-3
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发表时间：
1986-01-01
影响因子：
2.2
通讯作者：
LANGONE, JJ
中科院分区：
文献类型：
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作者：
BJERCKE, RJ;COOK, G;LANGONE, JJ
已制备出针对烟草生物碱（S）-（-）-尼古丁及其主要代谢产物（S）-（-）-可替宁的立体特异性单克隆抗体（McAb）。通过一种筛选程序选择出9种抗尼古丁和4种抗可替宁的杂交瘤，该筛选程序利用了对尼古丁或可替宁的³H标记的天然异构体进行免疫沉淀。这些杂交瘤在经降植烷预处理的同系BALB/c小鼠的腹水中生长。获得了浓度高达7.5毫克/毫升腹水且结合亲和力通常超过10⁸ M⁻¹的抗体。开发了酶联免疫吸附测定法（ELISA），其中将与多聚-L -赖氨酸共价结合的尼古丁或可替宁衍生物包被在聚氯乙烯微量滴定板的孔上。包被的孔依次在有或无抑制剂的情况下与McAb、兔抗鼠免疫球蛋白一起孵育，然后在加入底物之前与辣根过氧化物酶标记的蛋白A（HRP - SpA）一起孵育。这些抗体具有高度特异性，并且与几种尼古丁代谢产物和其他结构相关化合物的交叉反应极小。在各自的测定中，仅需0.25纳克（S）-（-）-尼古丁和0.12纳克（S）-（-）-可替宁就能使抗体结合产生50%的抑制，并且仅需0.05纳克尼古丁和0.02纳克可替宁就能产生15%的抑制。这些测定比用兔抗血清和HRP - SpA开发的类似ELISA或利用兔抗血清和³H标记配体的常规放射免疫测定法（RIA）灵敏5 - 10倍。通过基于McAb的ELISA和常规RIA对吸烟者和非吸烟者唾液样本中尼古丁（r = 0.967）和可替宁（r = 0.981）水平的测定之间具有良好的相关性。
Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or cotinine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 108 M-1 were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly-L-lysine were coated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5-10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and 3H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) and cotinine (r = 0.981) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.