阿尔茨海默病尿神经丝蛋白检测方法的建立及其临床意义

Detection of urine neural thread protein for diagnosis of Alzheimer disease and its clinical significance

目的研制检测尿中AD7C—NTP诊断试剂盒，并分析评价其在诊断AD中的临床应用价值。方法固相法合成具有免疫原件的AD7C—NTP抗原决定簇多肽片段，通过免疫动物、制备抗体、配对筛选，以小鼠抗AD7C—NTP抗体作为包被抗体，以生物素标记兔抗AD7C—NTP抗体和辣根过氧化物酶标记亲和素建立ELISA检测尿AD7C—NTP的方法；并用以检测和分析121例AD患者与118名同龄健康人清晨中段尿液中AD7C—NTP的水平差异。结果经过鉴定，小鼠抗AD7C—NTP抗体的ELISA检测效价为1：8000，兔抗AD7C—NTP抗体的ELISA检测效价为1：32000；WB检测人脑标本中抗AD7C—NTP抗体的相对分子质量为41000。自建ELISA检测AD7C—NTP的灵敏度为0．2μg／L，线性范围为0～10μg/L，正常参考值1．5μg/L，平均回收率为100．2％，批内CV为3．8％和4．5％，批间CV为7．6％和6．8％。AD组尿AD7C—NTP[2．25（0．43—8．62）μg/L]高于健康对照组[0．82（0．47～2．77）μg/L，P〈0．01]，AD组和健康对照组阳性率分别为89．3％和15．3％，AD7C—NTP检测的敏感度为89．3％、特异度为84．7％。结论用自行设计合成的多肽片段免疫动物，成功制备了抗AD7C—NTP抗体，初步建立的尿AD7C—NTP的ELISA检测方法精密度和灵敏度高，有可成为临床诊断AD的辅助手段。

Objective To develop urine AD7C-NTP diagnostic kit, analyze and evaluate its application value on AD. Methods Immunogenicity AD7C-NTP peptide fragments had synthesized by solidphase methods. The animals immunized to prepare antibodies. After matching screening, mouse antibody was uesed as coating antibody, biotin-labeled rabbit antibody was used as testing antibody, and horseradish peroxidase was labeled with avidin. The urine AD7C-NTP ELISA detective method was established. The AD7C-NTP levels in morning urine samples of 121 AD patients and 118 age-matched controls were collected. Results AD7C-NTP antibodies were identified. Mouse anti-AD7C-NTP antibody titer in ELISA was 1：8 000, and rabbit anti-AD7C-NTP antibody titer in ELISA was 1：32 000; WB was uesd to detect human brain specimens and there was a single band with molecular weight of 41 000. The lowst detection limit of ELISA methodology was 0. 5 μg/L. The linear range was 0-10 μg/L, normal reference value ≤1.5 μg/L, the average recovery rate was 100. 2%. The intra and inter of CV were 3.8% , 4. 5% , 7. 6% , 6. 8% respectively. The AD7C-NTP levels [2. 25（0. 43-8. 62） μg/L] of urine in AD group was higher than those in contorl group [0. 82（0.47-2. 77） μg/L, P 〈 0. 01 ]. The positive rates in AD group and control group were 89. 3% and 15.3% respectively. The sensitivity was 89. 3% and specificity was 84. 7%. Conclusions The animals are immunized with the self-designed synthetic peptide fragment to prepare AD7C-NTP antibodies successfully. The established ELISA method for detection of urine AD7C-NTP with high sensitivity, and precision can be used as an assistant examination in clinical diagnosis of AD.