人血浆β淀粉样蛋白42 ELISA的建立及其临床初步应用

Development of an ELISA method for the determination of human plasma amyloid protein β42 and study of its preliminary clinical application

目的建立人血浆中β淀粉样蛋白42（Aβ42）酶联免疫吸附试验（ELISA），并初步观察其对诊断阿尔茨海默病（AD）的应用价值。方法用鼠抗Aβ42片段（1～28氨基酸残基）单克隆抗体包被微孔板，用生物素标记的兔抗Aβ42片段（40～42氨基酸残基）为标记抗体，应用生物素-亲和素系统（ABS）进行放大，采用棋盘滴定方法确定最佳实验条件，用Aβ42标准品建立ELISA标准曲线，分别检测不同痴呆程度AD患者及健康人血浆中Aβ水平。结果本研究建立的方法测定范围为20—500pg／mL，最低检出量20pg／mL，批内和批间变异系数（c叻分别为3．8％和7．9％。轻度AD患者血浆中AB42水平为（146．4±9．7）pg／mL，明显高于健康对照组[（95．I±7．2）pg／mL]，中晚期AD患者血浆Aβ42浓度明显下降[（99．4±17．6）pg／mL]，其水平降至与健康对照组差异无统计学意义。结论建立的人血浆Aβ42ELISA可作为AD早期诊断的有效方法，可在临床推广应用。

Objective To develop an enzyme-linked immunosorbent assay （ELISA） of human plasma amyloid protein β42 （ Aβ42 ） and study its preliminary clinical application in diagnosis of Alzheimer＇ s disease （ AD ）. Methods The microplate was coated with the mouse anti-Aβ42 fragments （1-28 amino acids） monoclonal antibody. The biotin-labeled rabbit anti-Aβ42 fragments （40-42 specific amino acids）antibody was adopted as labeling antibody. The sensitivity was amplified by avidin -biotin -system （ABS）. The best reaction condition was decided by the board titration method . The standard curve of ELISA was established using Aβ42 standard product. The plasma Aβ42 level was detected in AD patients with different degree of dementia and in healthy people respectively. Results It was found that the measurement range of this method was 20-500 pg/mL, and the minimal detectable limit was 20 pg/mL, and the intra and inter-coefficients of variation （CV） was 3.8% and 7.9%. The plasma Aβ42 level of the mild AD patients was （ 146.4 ± 9.7 ） pg/ mL. It was significantly higher than the healthy group [ （ 95.1 ± 7.2 ） pg/mL] ; The plasma Aβ42 level in the moderate or severe AD groups [ （ 99.4 ± 17. 6 ） pg/mL ] declined markedly, there was no significant difference eompared with healthy control group. Conclusions The developed ELISA method for the determination of human plasma Aβ42 may become an effective method in early diagnosis of AD, and it could be useful in the clinical application.