激动样活性抗α_1-肾上腺素受体胞外第二肽段抗体的制备  

Preparation of the antibodies against alpha 1-adrenergic receptor with exciting actions

作　　者：周子华[1] 奇琦 廖玉华[1] 王彬[1] 李留东[1] 魏芬[1] 王敏[1] 魏宇淼[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心内科,湖北省武汉市430022 [2]天门市第四人民医院内科,湖北省天门市431700

出　　处：《中国临床康复》2006年第44期51-53,I0002,共4页Chinese Journal of Clinical Rehabilitation

摘　　要：目的:制备提纯抗α1-肾上腺素受体抗体,并对其生物学活性进行鉴定。方法:实验于2003-02/12在华中科技大学同济医学院附属协和医院完成。①选取清洁级1月龄雄性Wistar大鼠6只,取4只用于抗α1-肾上腺素受体抗体的制备。将合成肽用戊二醛与牛血清白蛋白偶联,透析除去未结合的多肽,与等体积的弗氏佐剂混匀后于0,2,4,6,8周对大鼠进行免疫处理,4只/次。另2只作为未免疫对照。免疫前后鼠尾采血并用ELISA法检测抗体的滴度,α1-肾上腺素受体抗原的包被浓度50mg/L,血清稀释度从1∶40开始倍比稀释,辣根过氧化物酶酶标抗体稀释度为1∶2000。用阳性血清与阴性血清的吸光度之比来判定,比值≥2.1为阳性。免疫结束后采血,离心取上清,与等体积的生理盐水混合后逐滴加入饱和硫酸铵至浓度达50%,于磷酸盐缓冲液中透析。②以合成的α1-肾上腺素受体胞外第二肽段多肽为抗原,采用免疫亲和层析的方法,纯化用合成肽免疫大鼠产生的抗α1-肾上腺素受体多克隆抗体,用Bradford对抗体进行定量,十二烷基硫酸钠-聚丙烯凝胶电泳检测抗体纯度,以ELISA法、免疫组化、免疫印迹方法进行抗体免疫活性分析,用激光共聚焦测量细胞内游离钙浓度变化以检测抗体的激动样活性。结果:实验选取清洁级1月龄雄性Wistar大鼠6只,全部进入结果分析。①抗α1-肾上腺素受体抗体的产生和纯度:抗α1-肾上腺素受体抗体在大鼠免疫后2周开始产生,8周后达到很高滴度。提纯的抗体聚丙烯酰胺凝胶电泳法鉴定有较高的纯度。②纯化的抗α1-肾上腺素受体抗体的免疫学活性:抗体浓度为0.1g/L时滴度约1∶2560,并可结合培养平滑肌细胞及心肌组织上的α1-肾上腺素受体,具有较好的反应原性。③纯化的抗α1-肾上腺素受体抗体的生物学活性:抗α1-肾上腺素受体抗体可显著升高分离的成年大鼠心肌细胞游离钙离子�AIM： To prepare and identify the purified antibodies against α1-adrenergic receptor with exciting actions, and evaluate their biological activity. METHODS： The expertments were completed in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology from February to December in 2003. （1）Six Wistar male rats of one month old and cleaning grade were adopted in the experiment, and 4 of them were selected to prepare the antibodies against α1-adrenergic receptor. The synthesized peptide was coupled with bovine serum albumin （BSA） by glutaral to dialyse the uncombined polypeptide and then mix with equal volume of Freund adjuvant at the 0, 2^nd, 4^th, 6^thand 8^th weeks for immune managements in rats, 4 rats one time. Another 2 rats were taken as immune controls. The blood samples from rat tails were used to detect the titre of antibodies by ELISA method. The coating concentration was set as 50 mg/L for α1-adrenergic receptor antigen, and the serum was diluted doubly at 1：40 while horse-radish peroxidase （HRP） antibody was diluted at 1：2 000. Positive was defined as the absorbance （A） ratio of positive serum and negative serum≥2.1. Blood sampling and centrifugation were conducted to fetch supernatant liquid, which was mixed with euqla saline and added with saturation ammonium sulfate by drops to reach the 50% concentration and dialyse in phosphate buffer solution. （2）Using the synthesized peptides of second extracellular loop of the adrenergic receptor as antigen, the polyclonal antibodies to α1-adrenergic receptor produced by immunizing the rats, were purified using the method of affinity chromatography, and were cncentrated using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunity of the purified antibodies was analyzed by Bradford method, ELISA, western blotting, and immunohistochemistry respectively, and the exciting actions were identified by the change of free Ca^2＋ ion in the isolated adult Wistar rats cardiomyocytes, which we

关 键 词：抗体 受体 肾上腺素能α1 免疫活性 

分 类 号：R730.51[医药卫生—肿瘤]