中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2012年
1期
42-46
,共5页
王华成%罗金刚%刘学军%杨梦心%王英%许英%段朝晖
王華成%囉金剛%劉學軍%楊夢心%王英%許英%段朝暉
왕화성%라금강%류학군%양몽심%왕영%허영%단조휘
阿尔茨海默病%淀粉样β蛋白%肽碎片%酶联免疫吸附测定
阿爾茨海默病%澱粉樣β蛋白%肽碎片%酶聯免疫吸附測定
아이자해묵병%정분양β단백%태쇄편%매련면역흡부측정
Alzheimer disease%Amyloid beta-protein%Peptide fragments%Enzyme-linked immunosorbent assay
目的 建立β淀粉样肽42(β-amyloid peptide 42,Aβ42)酶联免疫吸附测定法(enzymelinked immunosorbent assay,ELISA),并探讨其检测阿尔茨海默病(alzheimer disease,AD)患者血清Aβ42含量及其早期诊断、治疗的临床意义.方法 利用噬菌体抗体库技术制备抗Aβ42的单链抗体,用作包被抗体；并与用Aβ42免疫兔制备的抗Aβ42的多克隆抗体及辣根过氧化物酶标记的羊抗兔IgG建立检测外周血Aβ42的ELISA方法.然后,用其检测120例AD患者和120例血管性痴呆(vascular dementiao,VD)或脑梗死患者、120名健康人血清的Aβ42含量,同时通过重复性、稳定性、回收试验和与国外试剂比对分析进行方法学评价与诊断性能分析.结果 建立的Aβ42 ELISA法检测2份血清标本重复20次的批内变异系数(coefficient of varible,CV)分别为3.6％和3.5％,重复检测20 d的批间CV分别为6.8％和7.1％；回收率为97.2％～103.1％,线性范围为0.050～2μg/L;在37℃放置6d及4℃保存6个月的活性降低均＜12％.与比利时INNOTEST双抗体夹心ELISA β淀粉样肽检测试剂盒平行检测90份标本,自建方法检测Aβ42含量为(0.207±0.039)μg/L,INNOTEST试剂盒为(0.206 ±0.038) μg/L；两种方法有较好的一致性(回归方程为Y=1.011X-0.003,R2=0.979,P＜0.01).ELISA检测AD组血清Aβ42为(0.247 ±0.032) μg/L,VD或脑梗死组为(0.173±0.028) μg/L,健康对照组为(0.172±0.032)μg/L;AD组分别高于VD或脑梗死组和健康对照组(q值分别为18.687、18.907,P均＜0.01).用受试者操作特征(receiver operating characteristic,ROC)曲线确定Aβ42的临界值为0.212 μg/L,正常参考值范围为0～0.212 μg/L时,ELISA Aβ42诊断VD的敏感度为86.7％(104/120),特异度为90.8％(218/240).结论 建立的AD血清Aβ42 ELISA法的敏感度和特异度较高,重复性及稳定性较好,为AD患者的早期诊断和治疗监测提供了新的有效方法.
目的 建立β澱粉樣肽42(β-amyloid peptide 42,Aβ42)酶聯免疫吸附測定法(enzymelinked immunosorbent assay,ELISA),併探討其檢測阿爾茨海默病(alzheimer disease,AD)患者血清Aβ42含量及其早期診斷、治療的臨床意義.方法 利用噬菌體抗體庫技術製備抗Aβ42的單鏈抗體,用作包被抗體；併與用Aβ42免疫兔製備的抗Aβ42的多剋隆抗體及辣根過氧化物酶標記的羊抗兔IgG建立檢測外週血Aβ42的ELISA方法.然後,用其檢測120例AD患者和120例血管性癡呆(vascular dementiao,VD)或腦梗死患者、120名健康人血清的Aβ42含量,同時通過重複性、穩定性、迴收試驗和與國外試劑比對分析進行方法學評價與診斷性能分析.結果 建立的Aβ42 ELISA法檢測2份血清標本重複20次的批內變異繫數(coefficient of varible,CV)分彆為3.6％和3.5％,重複檢測20 d的批間CV分彆為6.8％和7.1％；迴收率為97.2％～103.1％,線性範圍為0.050～2μg/L;在37℃放置6d及4℃保存6箇月的活性降低均＜12％.與比利時INNOTEST雙抗體夾心ELISA β澱粉樣肽檢測試劑盒平行檢測90份標本,自建方法檢測Aβ42含量為(0.207±0.039)μg/L,INNOTEST試劑盒為(0.206 ±0.038) μg/L；兩種方法有較好的一緻性(迴歸方程為Y=1.011X-0.003,R2=0.979,P＜0.01).ELISA檢測AD組血清Aβ42為(0.247 ±0.032) μg/L,VD或腦梗死組為(0.173±0.028) μg/L,健康對照組為(0.172±0.032)μg/L;AD組分彆高于VD或腦梗死組和健康對照組(q值分彆為18.687、18.907,P均＜0.01).用受試者操作特徵(receiver operating characteristic,ROC)麯線確定Aβ42的臨界值為0.212 μg/L,正常參攷值範圍為0～0.212 μg/L時,ELISA Aβ42診斷VD的敏感度為86.7％(104/120),特異度為90.8％(218/240).結論 建立的AD血清Aβ42 ELISA法的敏感度和特異度較高,重複性及穩定性較好,為AD患者的早期診斷和治療鑑測提供瞭新的有效方法.
목적 건립β정분양태42(β-amyloid peptide 42,Aβ42)매련면역흡부측정법(enzymelinked immunosorbent assay,ELISA),병탐토기검측아이자해묵병(alzheimer disease,AD)환자혈청Aβ42함량급기조기진단、치료적림상의의.방법 이용서균체항체고기술제비항Aβ42적단련항체,용작포피항체；병여용Aβ42면역토제비적항Aβ42적다극륭항체급랄근과양화물매표기적양항토IgG건립검측외주혈Aβ42적ELISA방법.연후,용기검측120례AD환자화120례혈관성치태(vascular dementiao,VD)혹뇌경사환자、120명건강인혈청적Aβ42함량,동시통과중복성、은정성、회수시험화여국외시제비대분석진행방법학평개여진단성능분석.결과 건립적Aβ42 ELISA법검측2빈혈청표본중복20차적비내변이계수(coefficient of varible,CV)분별위3.6％화3.5％,중복검측20 d적비간CV분별위6.8％화7.1％；회수솔위97.2％～103.1％,선성범위위0.050～2μg/L;재37℃방치6d급4℃보존6개월적활성강저균＜12％.여비리시INNOTEST쌍항체협심ELISA β정분양태검측시제합평행검측90빈표본,자건방법검측Aβ42함량위(0.207±0.039)μg/L,INNOTEST시제합위(0.206 ±0.038) μg/L；량충방법유교호적일치성(회귀방정위Y=1.011X-0.003,R2=0.979,P＜0.01).ELISA검측AD조혈청Aβ42위(0.247 ±0.032) μg/L,VD혹뇌경사조위(0.173±0.028) μg/L,건강대조조위(0.172±0.032)μg/L;AD조분별고우VD혹뇌경사조화건강대조조(q치분별위18.687、18.907,P균＜0.01).용수시자조작특정(receiver operating characteristic,ROC)곡선학정Aβ42적림계치위0.212 μg/L,정상삼고치범위위0～0.212 μg/L시,ELISA Aβ42진단VD적민감도위86.7％(104/120),특이도위90.8％(218/240).결론 건립적AD혈청Aβ42 ELISA법적민감도화특이도교고,중복성급은정성교호,위AD환자적조기진단화치료감측제공료신적유효방법.
Objective To establish a enzyme-linked immunosorbent assay (ELISA) method for detecting the β-amyloid peptide 42 ( Aβ42 ) and explore its clinical meaning for diagnosis and treatment in the early stages of the alzheimer disease ( AD).Methods Using the Aβ42 single chain variable fragment constructed by phage antibody library display system as coat antibody,associated with the Aβ42 polyclonal antibody acquired by Aβ42 immunized rabbit and HRP labeled goat anti rabbit IgG to establish ELISA method for detecting the Aβ42 in peripheral blood.The method was used it to test the Aβ42 in 120 vascular dementia VD) or cerebral vessel infarction patients and 120 AD patients and 120 controls.The methodology performance were evaluated.Results The inter and intra coefficient of variable (CV) of this self-established ELISA method was 3.6％ and 3.5％,6.8％ and 7.1％ respectively.The recovery rate was 97.2％ -103.1％.The linear range was 0.050 - 2 μg,/L.Its reactivity decreased ＜ 12％ when it was put in both 37 ℃ for 6 days and 4 ℃ for 6 months.Compared with the Belgium INNOTEST reagent by testing 90 samples simultaneously,the results of self-established method was (0.207 ± 0.039 ) μg/L,the results of INNOTEST was (0.206± 0.038 ) μg/L; the regression equation was Y =1.011X - 0.003,R2 =0.979,P ＜0.01.The Aβ42 in blood of AD group was (0.247 ± 0.032 ) μg/L,VD or cerebral vessel infarction group was (0.173 ±0.028) μg/L,control group was (0.172 ±0.032) μg/L.The Aβ42 in AD group was higher than that in the VD or cerebral vessel infarction group and control group (q =18.867,18.907respectively,P ＜ 0.01 ).The cut off value was 0.212 μg/L decided by the receiver operating characteristic (ROC) curve.The reference interval was 0 -0.212 μg/L.The sensitivity of this ELISA method was 86.7％(104/120) and specificity was 90.8％ (218/240).Conclusions The ELISA method for detecting Aβ42 in peripheral blood established by the study is sensitive and specific and has good precision and stability.It could provide a new effective criterion and support for the early diagnosis and treatment of the AD patients.
