Preparation of Apo B100 Rabbit Anti-Human Antiserum

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-human 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 1.40 μg/mL and the titer of the antiserum is 1∶64.The two step ultracentrifugation improves the separation effect of Apo B100 in human Plasma.