磁珠离子交换吸附法纯化兔血清中多克隆抗体的研究

Ion-Exchange Purification of Polyclonal Antibody from Rabbit Serum Using Magnetic Beads

以羧基化的超顺磁纳米磁珠作为离子交换吸附介质,建立一种新型的非层析离子交换吸附方法,用于对兔血清中抗副溶血弧菌多克隆抗体进行纯化。通过优化吸附、洗脱缓冲液pH值、盐离子浓度以及洗涤液Tween-20体积分数,确立磁珠离子交换吸附纯化条件,并将该纯化方法和辛酸-硫酸铵法、亲和层析法进行比较。结果表明:血清中的多克隆抗体能够在pH6.0的磷酸盐缓冲液(0.01mol/L)中被磁珠完全吸附,经过含0.1%Tween-20的洗涤液两次洗涤,能够达到较好的洗涤效果,用含0.3mol/L NaCl的磷酸盐缓冲液(pH8.0)可以将吸附的抗体洗脱;经该法纯化后,多抗纯度为82.6%,回收率为58.0%,效价为1/64000;每克磁珠可纯化10mL的血清,整个过程只需要1h。该方法和辛酸-硫酸铵法、亲和层析法相比有明显的优势,因此具有较好的应用前景。

A novel non-chromatographic ion-exchange adsorption technique was developed to purify polyclonal antibody against Vibrio parahaemolyticus from rabbit serum using carboxylated superparamagnetic nanobeads.pH and NaCl concentration of adsorption and desorption buffers and Tween-20 concentration in washing buffer were studied,and the developed method was compared with caprylic acid-ammonium sulfate precipitation and SPA affinity chromatography.The results showed that the polyclonal antibody could be completely adsorbed by magnetic beads in 0.01 mol/L phosphate buffer（pH 6.0） and desorbed in 0.01 mol/L phosphate buffer（pH 8.0） containing 0.3 mol/L NaCl,and unadsorbed proteins were removed by washing twice with washing buffer containing 0.1% Tween-20.After the purification,the polyclonal IgG purity was 82.6%,the recovery yield 58.0%,and the titer 1/64000.In total,10 mL of rabbit serum could be treated by 1 g of magnetic beads,and the whole process could be finished within 1 h.The method has obvious advantages compared with caprylic acid-ammonium sulfate precipitation and SPA affinity chromatography,thereby presenting a promising application prospect.