Centrifugal Precipitation Chromatography

证明了离心沉淀色谱法在亲和分离中的新应用。该方法使用抗体沉淀分析物蛋白，然后用释放剂解离从柱中洗脱，而抗体稍后从柱内容物中回收。该测试系统使用人血清白蛋白作为抗原（分析物），兔抗人IgG作为抗体。实验开始时，在磷酸盐缓冲液（pH 4.3）中填充40%饱和硫酸铵（AS）的色谱柱（两个通道），然后将样品混合物（0.2 mg HSA +抗HSA， 1 ml蒸馏水）注射到样品通道中，以0.3 ml/min的速度用水洗脱。用紫外检测器在280 nm处对废水进行连续监测，并用分馏收集器将废水收集到试管中。洗脱过量的HSA后，切换流动相，释放溶剂为40-50%含0.5M甘氨酸的AS溶液，经氢氧化铵调节pH为10，使抗原（HSA）与抗体解离。SDS PAGE分析分离后的HSA。该方法成功地用于人血清中HSA的纯化。与传统亲和柱层析法相比，本方法的优点是抗体直接用于目标蛋白的纯化，而不使用固体载体。

A new application of centrifugal precipitation chromatography for affinity separation has been demonstrated. The method uses an antibody to precipitate the analyte protein which is then dissociated by a releasing agent to elute from the column while the antibody is recovered later from the column contents. The test system uses human serum albumin as an antigen (analyte) and a rabbit antihuman IgG as an antibody. The experiment was initiated by filling the column (both channels) with 40% saturated ammonium sulfate (AS) in a phosphate buffer (pH 4.3) followed by injection of the sample mixture ( 0.2 mg of HSA + anti-HSA in one ml of distilled water) into the sample channel which was eluted with water at 0.3 ml/min. The effluent was continuously monitored by a UV detector at 280 nm and collected into test tube using a fraction collector. After an excess amount of HSA was eluted out, the mobile phase was switched with the releaser solvent of 40-50% AS solution containing 0.5M glycine at pH 10 adjusted by ammonium hydroxide to dissociate the antigen (HSA) from the antibody. The fractionated HSA is analyzed by SDS PAGE. The method was successfully applied to purification of HSA from human serum. The present method has an advantage over conventional affinity column chromatography in that the antibody is directly used for purification of the target protein without using the solid support.