利用棋盘格实验直接筛选酶免疫测定的最佳包被抗原和抗体浓度

以两个单克隆抗体及其相应的包被抗原为例,研究了棋盘格实验中包被抗原与抗体浓度组合对免疫检测中0孔吸光度值A0、IC50值和标准曲线斜率的影响.结果发现,A0和IC50值随包被抗原与抗体稀释倍数的增加而降低;斜率随抗体稀释倍数增加而降低,而随包被抗原的稀释倍数增加表现出先增加后降低的趋势.在同一抗体浓度和不同包被抗原浓度的组合中,斜率最大值出现在使板吸附达到饱和的最大稀释倍数的包被抗原浓度附近.综合实验结果确认从棋盘格实验中直接筛选最佳包被抗原、抗体浓度组合的标准是:选择使板吸附达到饱和的最大包被抗原稀释倍数为最佳包被抗原浓度,在不同抗体浓度与已选择的最佳包被抗原浓度组合中使0孔吸光度值达1.0左右的抗体稀释倍数为最佳抗体浓度.

Systematic Optimization of Antibody and Coating Antigen Concentrations in Enzyme Linked Immunosorbent Assay Checkerboard Assay

Two monoclonal antibodies and their corresponding coating antigens were used to investigate the influence of both concentration of coating antigen and dilution of antibody on the absorbance of negative well(A0),IC50 value and the slope of standard curve.The results showed that the A0 and IC50values decreased as both the concentrations of antibody and immobilized coating antigen decreased.The slope of the standard curve also declined as the antibody concentration decreased.However,the slope first increased as the coating antigen concentration decreased to reach to a maximum point,and then declined as coating antigen was further diluted.The maximal value of the slope nearly appeared at the minimal coating antigen concentration which allowed forming a saturated monolayer of coating antigen on the microplate surface.The results confirmed the criterion of direct selecting the optimal combination of the coating antigen and antibody via the checkerboard titration.The optimal coating antigen concentration was the maximum dilution which allowed forming a saturated coating antigen on the microplate surface,and the optimal antibody concentration was the dilution which can give an A0 value of approximate 1.0 at the optimal concentration of the selected coating antigen.