Microtiter plate binding assay for cholinergic compounds utilizing the nicotinic acetylcholine receptor.
Microtiter plate binding assay for cholinergic compounds utilizing the nicotinic acetylcholine receptor.
复制标题
利用烟碱乙酰胆碱受体对胆碱能化合物进行微量滴定板结合测定。
DOI：
10.1021/ac00047a025
复制
发表时间：
1992
影响因子：
7.4
通讯作者：
Rechnitz,GA
中科院分区：
文献类型：
--
作者：
Chen,L;Martin,GB;Rechnitz,GA
本文建立了一种测定烟碱型乙酰胆碱受体胆碱能化合物的受体结合分析法。通过在96孔微量滴定板中进行测定，该方法适用于药物开发中的大规模筛选。与早期方法相比，酶标记的固相提取显著简化了测定方案。该试验基于生物素-BSA在mlcrotHer上的固定，由于抗生物素蛋白-生物素相互作用，mlcrotHer可吸收avldln标记的过氧化物酶。为了进行测定，配体（分析物）和生物素α-银环蛇毒素缀合物（aBgt-blotln）依次结合囊泡结合的烟碱乙酰胆碱受体。这是在试管中进行的，测定I，或在生物素化的mlcrottter孔中进行的，测定II。然后将抗生物素蛋白-HRP加入到该混合物中;游离的aBgt-blotln缀合物和固定化的生物素-BSA竞争抗生物素蛋白位点。吸出测定溶液后，测定结合的酶活性，其与加入的aBgt-blotln的量直接相关。生成胆碱能化合物的剂量-反应曲线和Scatchard图以评估表观结合常数。动力学研究进行了优化的目的。最终测定可在4小时内进行，样品处理最少。
A receptor-based binding assay for the determination of cholinergic compounds of the nicotinic acetylcholine receptor has been developed. By conducting the assay In a 96-well microtiter plate, the method Is suitable for large-scale screening In drug development. Solid-phase extraction of the enzyme label significantly simplifies the assay protocol compared to earlier methods. The assay Is based on Immobilization of biotin-BSA on the mlcrotHer which takes up avldln-labeled peroxidase due to avidin-biotin Interaction. To perform the assay, a ligand (the analyte) and a biotin a-bungarotoxin conjugate (aBgt-blotln) sequentially bind to a vesicle bound nicotinic acetylcholine receptor. ThisIs done either In a test tube, assay I, or In a biotinylated mlcrottter well, assay II. Avidln-HRP Is then added to this mixture; free aBgt-blotln conjugate and Immobilized biotin-BSA compete for the avidin sites. After the assay solution has been aspirated off, bound enzyme activity Is determined which Is directly related to the amount of aBgt-blotln added. Dose-response curves of cholinergic compounds and Scatchard plots were generated to evaluate the apparent binding constants. Kinetic studies were conducted for the purpose of optimization. The final assay can be performed In under 4 h with a minimum of sample handling.