哇巴因抗体制备的方法学研究

目的　探索合适的实验条件 ,制备高度特异性以及高效价的哇巴因抗体 ,促进国内对内源性哇巴因这种皮质类固醇激素的研究。方法　利用外源性哇巴因分别与牛血清白蛋白 (BSA)及卵清蛋白 (OVA)耦联 ,制备抗原免疫家兔 ,根据对兔免疫后动物体内抗体效价连续监测的结果来制定免疫方案 ,制备哇巴因抗血清 ,分别应用硫酸铵分段盐析法和DEAE D52 纤维素阴离子非保留色谱相配伍的方法纯化哇巴因抗体 ,并对该抗体进行特异性、亲和性及纯度测定。结果　所得抗体效价最高达 1∶(1× 1 0 6) ,亲和常数最高为 1 .59× 1 0 8,除了与地高辛存在 9.2 0 %～1 9.80 %的交叉结合反应外 ,与结构类似的其他几种物质 ,如强的松、安体舒通、西地兰、睾酮及地塞米松的交叉结合率均小于 5% ,SDS PAGE结果显示所得抗体为电泳纯。结论　应用本研究中所探索出的实验方法 ,获得了高特异性、高亲和性以及高效价的哇巴因抗体 ,可用于对内源性哇巴因的临床与实验研究。

Research method of production ouabain antibody

Objective To probe suitable conditions for producing high titerand specificity ouabain antibody,and burgeon researching of a new adrenocoriticoid-endogenous ouabain in China.Methods Ouabain coupled with ovalbumin and bovine serum albumin as antigen to immunize rabbits,immunizing plans was based successive assaying ouabain antibody titer of immunized rabbits.Purified ouabain antibody was gained by the fractional precipitation of ammonium suiphate and DEAE-D52 anion cellulose chromatography.Results High titer(as high as 1×105 by ELISA)and high specificity (the smaller 5% cross reaction rate with the substances of simlar structure such as suprednisoe,spironolactone,deslanoside,dexame-thasonum,thstosterone except digoxin.Conclusion Found methods of production anti-ouabain serum,and gained ouabain antibody of high titer,high affinity,high specificity could be used in clinical and experimental research of endogenous ouabain.