8-羟基脱氧鸟苷高特异性ELISA检测方法的建立及其用于妊娠期糖尿病的对比研究

The establishment of 8 hydroxydeoxyguanosine detection by a high specificity ELISA assay, and the comparative study of its used in gestational diabetes mellitus

目的:建立生物素-亲和素双抗夹心酶联免疫吸附法检测8-羟基脱氧鸟苷(8-OHdG)的方法,对比研究妊娠期糖尿病患者8-OHdG水平。方法:通过偶联形成8-OHdG-mcKLH复合物作为免疫原得到8-OHdG的高特异性配对抗体1D4、3B8,最终建立生物素-亲和素标记的双抗夹心ELISA定量检测8-OHdG的方法。结果:所建立的方法批内批间变异系数均<15%,稳定性良好;回收率为99.2%、100.8%和94.8%。妊娠期糖尿病患者血清8-OHdG水平(7.3±1.3ng/ml)高于对照组(2.2±0.7ng/ml)(P<0.001)。结论:本研究建立的8-OHdG检测方法为研究氧化损伤与疾病关系提供了更为准确、便捷的检测方法。血清8-OHdG水平的异常改变可能与妊娠期糖尿病的发生进程密切联系。

Objective:To establish a method for the detection of 8-OHdG by biotin avidin sandwich antibody ELISA,and to comparison study the level of 8 OHdG levels of patients with gestational diabetes mellitus(GDM).Methods:The 8 OHdG-mcKLH complex was formed by conjugation and used as immunogen to obtain the highly specific paired antibodies 1 D4 and 3 B8 of 8 OHdG.A biotin avidin labeled double antibody sandwich ELISA was developed for the quantitative detection of 8 OHdG.The method was used for detecting serum 8 OHdG levels of patients with GDMand healthy pregnant women.Results:The coefficient of variation intraassay and between the assays was less than 15%,which had good stabilities,high accuracy and precision.The recovery rates were 99.2%,100.8%and 94.8%.Serum 8 OHdG level of patients with GDM was(7.3±1.3)ng/ml,which was significant higher than that[(2.2±0.7)ng/ml]of normal pregnant women(P<0.001).Conclusion:The level of serum 8 OHdG of patients with GDM may be closely related to the occurrence of GDM and the progression of the disease.The method of detecting 8 OHdG in this study have provide more accurate and convenient test method for studying the relationship between oxidative damage and disease.