二步超离心法分离纯化人血浆Apo B100及兔抗人Apo B100抗血清制备

Preparation of Apo B100 Rabbit Anti-Human Antiserum

建立一种从人血浆中分离纯化Apo B100的方法,并制备兔抗人Apo B100抗血清。将血浆用溴化钾调成1.019g/mL的密度液进行差分超速离心,将分离产物在溴化钾不连续密度梯度液(1.006～1.210g/mL)中进行等密度超速离心。用G-100葡聚糖凝胶柱纯化Apo B100,以ELISA双抗体夹心法检测浓度,真空冷冻干燥。取冻干粉与弗氏佐剂混合后免疫新西兰白兔,制备兔抗人Apo B100抗血清,利用双向免疫扩散检测抗原纯度与抗血清效价。结果是一步超离法分离的Apo B100最高浓度为0.91μg/mL;二步超离法分离的Apo B100最高浓度为1.40μg/mL,对应的抗血清效价为1∶64。结果表明二步超离心法提高了人血浆Apo B100的分离效果。

To investigate a separation and purification method of Apo B100 from human serum and prepare rabbit anti-human Apo B100 antiserum. Plasma is fractionated in the presence of KBr at a density of 1. 019 g/mL by differential ultracentrifugation. Liquid discontinuous density gradient （1. 006 g/mL-1. 210 g/mL） is prepared with separated product by KBr density medium to further ultracentrifugation. The target protein is purificated with Sephadex G-100 column, then double-antibody sandwich ELISA is used to determine the titer. The obtained product is vacuum freeze-dried after dialysis. The freeze-dried powder mixed with Freund＇s adjuvant is immuned to New Zealand White Rabbit to obtain rabbit anti-hu- man Apo B100 antiserum. Purity and valence of the antiserum are detected by double immunodiffusion. The results indicate that the concentration of Apo B100 from one step ultracentrifugation is 0. 91 μg/mL, and the concentration of Apo B100 from two step ultracentrifugation is 1.40μg/mL and the titer of the antiserum is 1 ： 64. The two step ultracentrifugation improves the separation effect of Apo B100 in human Plasma.