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目的建立检测谷氨酸脱羧酶抗体(GADA)的新方法,即生物素-链霉亲合素放大ELISA系统(ELISA,BAS ELISA),并进行验证及初步临床应用。方法人脑组织经过破碎、抽提、二次凝胶过滤层析与阴、阳离子交换层析分离蛋白,收集纯化的谷氨酸脱羧酶(GAD);以链霉亲和素(SA)预包被,使GAD生物素化(bio-GAD),探讨检测GADA的最优实验条件,并对BAS ELISA的灵敏度、特异性、准确性及精密性进行验证。然后选择新诊断的成年1型糖尿病患者106例(T1DM组),以健康体检者98例为对照组,抽取空腹外周静脉血3 ml,分离血清冷冻标本,用构建的BASELISA和德国宝灵曼公司Anti-GAD65 ELISA检测血清中的GADA,对比研究并进行统计学分析。结果成功构建了检测GADA的BAS ELISA新方法;在T1DM组中,BAS ELISA方法测得T1DM的GADA阳性率显著高于anti-GAD65 ELISA方法(82.08%vs 52.83%,P<0.01);在对照组中BASELISA方法测得T1DM的GADA阳性率也明显高于anti-GAD65 ELISA方法(10.20%vs 6.12%,P<0.05)。结论利用生物素-链霉亲合素放大系统可以构建检测GADA的ELISA新方法;生物素-链霉亲合素放大ELISA系统在检测GADA方面具有较高的特异性、灵敏度、准确性和精密性,提高了GADA检测的阳性率,降低T1DM的漏检率,为早期检测T1DM提供新方法。
OBJECTIVE: To establish a new method for the detection of glutamic acid decarboxylase antibody (GADA), namely, biotin-streptavidin amplification ELISA system (ELISA, BAS ELISA) and to verify and preliminary clinical application. Methods The human brain tissue was broken, extracted, separated by secondary gel filtration, anion exchange and cation exchange chromatography to collect the purified glutamate decarboxylase (GAD). The streptavidin (SA) The GAD was biotinylated (bio-GAD) to investigate the optimal experimental conditions for the detection of GADA. The sensitivity, specificity, accuracy and precision of the BAS ELISA were also verified. And then select the newly diagnosed adult type 1 diabetes in 106 patients (T1DM group), with 98 healthy subjects as the control group, fasting peripheral blood of 3 ml, frozen serum samples were isolated, with the construction of BASELISA and Germany Bauhiniai company Anti-GAD65 ELISA serum GADA, comparative studies and statistical analysis. Results In the T1DM group, the GADA positive rate of T1DM was significantly higher than that of anti-GAD65 ELISA (82.08% vs 52.83%, P <0.01) in T1DM group. In the control group The GADA positive rate of T1DM measured by BASELISA was also significantly higher than that of anti-GAD65 ELISA (10.20% vs 6.12%, P <0.05). Conclusions A new ELISA method for GADA detection can be constructed using the biotin-streptavidin amplification system. The biotin-streptavidin amplification ELISA system has high specificity, sensitivity, accuracy and precision in detecting GADA , Increased the positive rate of GADA test, reduced the missed detection rate of T1DM, and provided a new method for early detection of T1DM.