M受体亚型放射配基结合－免疫沉淀分析方法的建立及考核

利用１％洋地黄皂甙溶解大鼠脑匀浆Ｍ受体，用非选择性配基３Ｈ－ＱＮＢ标记可溶性Ｍ受体，以自制的Ｍ１、Ｍ２亚型抗血清和固相二抗分别特异性地免疫沉淀相应的亚型，分析单一受体亚型。对方法进行了考核，包括受体的溶解效率、抗原抗体反应时间、抗血清稀释度、反应温度等对分析结果的影响。对大鼠脑Ｍ受体的试验表明，用两种亚型抗血清进行的免疫沉淀分析呈典型的单位点饱和曲线，抗体对配基与受体的结合反应无影响。

Establishment of a radioligand binding-immunoprecipitation technique for the assay of subtypes of M-cholinergic receptors

Specific polyclonal antibodies against M1 and M2 subtypes of cholinergic receptors were prepared in this laboratory.The M-cholinoceptor molecules in brain homogenate were solubilized with 1% digitonin and labelled with 3H-QNB prior to immumuprecipitation. One of the specific antibodies was then added to form antigen-antibody complex with the respective subtype and the complex was precipitated by the aid of a second antibody. Thus the receptor-ligand binding parameters of a single subtype were obtained from the radioactivities of the precipitates.Typical single- site saturation curves were obtained for both M1 and M2 subtypes.No influence of the antibodies on receptor-ligand binding was detected.Results of studies on the solubilization of menbrane receptors, the time course of antibody-antigen reaction,the optimal dilution factor and the reaction temperature are also presented in the paper.