Measurement of functional affinity of humanized anti-HBsAg Fab using non-competitive ELISA method / 解放军医学杂志

ABSTRACT

Objective To assess functional affinity of humanized anti-HBsAg Fab in order to provide theoretical basis for the radioimmunotherapy of liver cancer bearing HBV. Methods With cognizance of the advantages of solid phase method in time consumption and convenience, we assessed the functional affinity of engineered anti-HBsAg Fab with non-competitive ELISA method. After a series of trials affirming the best concentration and the best time of HBsAg to cover the plate and the proper binding time of Fab to HBsAg to reach an equilibrium, we plotted the OD standard curve of the binding reaction of Fab and HBsAg using four grades of concentration of HBsAg and a series of consistency of Fab. We got the consistency of Fab at OD50% through the standard curve, and then calculated the affinity by Law of Mass Action. Results The affinity of anti-HBsAg Fab is between 10 7 -10 8 M -1 , which was only smaller by 10 times than that of anti-HBsAg IgG. Conclusion The Fab which we constructed in our lab, can strongly bind to the target antigen, thus providing a theoretical basis for its clinical use.