摘要
目的为从天然化合物中高效筛选具有体液免疫抑制活性的先导化合物,本研究将构建高灵敏且价格低廉的小鼠免疫球蛋白IgG的ELISA检测方法。方法利用山羊抗小鼠IgG的Fc段抗体、生物素标记的山羊抗小鼠IgG的F(ab’)2段抗体、辣根过氧化物酶标记的链霉亲和素构建小鼠免疫球蛋白IgG的ELISA检测方法;通过对包被抗体浓度、检测抗体浓度和链霉亲和素浓度的优化,使检测方法灵敏度达到最高;再利用该检测方法检测体外培养小鼠B细胞上清中IgG浓度验证方法的有效性。结果在山羊抗小鼠IgG的Fc段抗体浓度达到5μg·mL^(-1),生物素标记的山羊抗小鼠IgG的F(ab’)2段抗体浓度达到0.25μg·mL^(-1),辣根过氧化物酶标记的链霉亲和素浓度达到0.5μg·mL^(-1),小鼠免疫球蛋白IgG的ELISA检测方法灵敏度达到最高(0.05ng·mL^(-1)),线性范围在0.1~10ng·mL^(-1);该检测方法可以高灵敏检测体外培养LPS刺激小鼠B细胞分泌的IgG抗体。结论本研究成功构建高灵敏且价格低廉的小鼠免疫球蛋白IgG的ELISA检测方法。
Objective To efficiently and reliably screen natural compounds for lead compounds with immunosuppressive activity,high sensitivity and low-cost enzyme-linked immunosorbent assay for mouse IgG was constructed.Methods Goat antibody specific to Fc fragment of mouse IgG,goat antibody specific to F（ab＇）2fragment of mouse IgG,and horseradish peroxidase-labeled streptavidin（HRP-SA）were used to construct ELISA for mouse IgG.The concentration of capture antibody,detection antibody and HRP-SA were optimized to improve the sensitivity of this assay.Then to validate this method,the assay was used to determine the level of mouse IgG secreted by B cells cultured in vitro.Results While the concentration of capture antibody reached 5μg/ml,the concentration of detection antibody reached0.25μg/ml,and HRP-SA reached 0.5μg/ml,the highest sensitivity was achieved（0.05ng/ml）and the linear range was from 0.1to 10ng/ml.This assay could be used to determine the level of mouse IgG secreted by B cells cultured in vitro with high sensitivity.Conclusion This study successfully constructed high sensitivity and low-cost ELISA for mouse IgG.
出处
《河北北方学院学报（自然科学版）》
2017年第8期12-16,共5页
Journal of Hebei North University:Natural Science Edition
基金
内蒙古医科大学第三临床医学院医学研究基金项目(NO.2014-24)
