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

摘要

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性的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ADTC-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1:8 000,兔抗AD7C-NTP抗体的ELISA检测效价为1:32 000;WB检测人脑标本中抗AD7C-NTP抗体的相对分子质量为41 000.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 wag used as testing antibody,and horseradish peroxidase was labeied 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 ELISAwas 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 0.5μg/L The linear range was 0-10μg/L,normal reference value ≤1.5μg/J,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.