以抗体竞争结合抗原测定单抗亲和力常数的研究

Measurement of affinity constant of monoclonal antibody by competitive antibody binding to antigen

目的在Friguent方法基础上建立更加稳定可靠的基于抗原/抗体竞争结合原理的单抗亲和力常数(Kd)检测方法。方法以抗体对抗原进行竞争结合,通过双抗体夹心法检测在一定抗体浓度竞争下抗原的结合率计算Kd值。结果用改良方法在两次不同条件下检测抗IL-8单抗的Kd值分别为:2.61×10-12mol/L和2.39×10-12mol/L,而使用Friguent方法在两次不同条件检测抗IL-8单抗的Kd值分别为:5.57×10-10mol/L和1.41×10-10mol/L。结论相对于Friguent方法,本研究计算出的Kd值更能反映抗原/抗体结合的真实情况,而且在不同实验条件下检测结果重现性强。

Objective To develop a more reliable and stable method for determining monoclonal antibody （mAb） affinity constant based on competitive antibody/antigen binding. Methods The Kd value was calculated based on the relationship between the binding proportion of the antigenand the original concentration of the mAb that competed for the binding site of the antigen. Results The Kd values measured with this improved method under two different conditions were 2.61× 10^-12 mol/L and 2.39× 10^12 mol/L, and those with Friguent method in these two conditions were 5.57 × 10^-10 mol/L and 1.41 × 10^-10 mol/L, respectively. Conclusion Compared with Friguent method, the Kd values measured with this improved method are closer to the actual value, and the measurement results under different experiment conditions are more stable.