基于蛋白A-琼脂糖凝胶黄曲霉毒素B_1免疫亲和柱的制备

Preparation of Immunoaffinity Column for AFB_1 with Monoclonal Antibodies Immobilized on Protein A-Sepharose

将初步纯化的抗黄曲霉毒素B1单克隆抗体,定向偶联于蛋白A-琼脂糖凝胶,制备成免疫亲和层析柱。结果显示,辛酸-饱和硫酸铵纯化后的单抗质量浓度为17.5 mg/m L,纯度为45.1%,抗体亚型为Ig G1。取纯化后抗体48μL与1 m L蛋白A凝胶偶联,偶联率达98.1%,高效液相色谱测定其平均相对柱容量为105 ng/0.125 m L凝胶、柱空白为0,不同样品(玉米粉、面粉、花生油、酱油、醋等)平均加标回收率在80%以上。该亲和柱在2~8℃保存12个月后,相对柱容量仍可达72 ng/0.125 m L凝胶。该法制备的亲和柱相对柱容量大,稳定性好,可用于样品中黄曲霉毒素B1分离净化的要求。

The immunoaffinity column(IAC) for aflatoxin B1(AFB1) was prepared by directed coupling of protein A-sepharose with the anti-AFB1 monoclonal antibody which was preliminarily purified by caprylic acid-ammonium sulfate method. The results showed that the concentration, purity and antibody subtype of the purified monoclonal antibody were 17.5 mg/m L, 45.1% and Ig G1, respectively. When coupling 48 μL of purified antibody with protein A-sepharose, and the coupling rate was 98.1%. The average relative column capacity and column blank were 105 ng/0.125 m L gel and 0, respectively. The average recoveries of standard addition in different samples(corn flour, wheat flour, peanut oil, soy sauce) were all above 80%. After being stored at 2–8 ℃ for 12 months, the relative capacity of the IAC was 72 ng/0.125 m L gel. These results show that the anti-AFB1 affinity column prepared by this method has large relative capacity and good stability which meet the requirement for separation and purification of AFB1 from real samples.