东南大学学报(医学版)
東南大學學報(醫學版)
동남대학학보(의학판)
JOURNAL OF SOUTHEAST UNIVERSITY(MEDICAL SCIENCE EDITION)
2015年
4期
501-506
,共6页
文继月%孟多佳%封晔%田赛%时成波%孟继鸿
文繼月%孟多佳%封曄%田賽%時成波%孟繼鴻
문계월%맹다가%봉엽%전새%시성파%맹계홍
戊型肝炎病毒%疫苗%重组蛋白%单克隆抗体%双抗体夹心法酶联免疫吸附试验
戊型肝炎病毒%疫苗%重組蛋白%單剋隆抗體%雙抗體夾心法酶聯免疫吸附試驗
무형간염병독%역묘%중조단백%단극륭항체%쌍항체협심법매련면역흡부시험
hepatitis E virus%vaccine%recombinant protein%monoclonal antibody%double antibody sandwich enzyme-linked immunosorbent assay
目的:建立双抗体夹心法ELISA检测HEV抗原,用于定量检测戊型肝炎p179疫苗生产过程中初制品、半成品和疫苗成品。方法:将单克隆抗体3G1、5G5、1G10、3A10和4E9分别进行辣根过氧化物酶标记,通过竞争ELISA检测确定最佳抗体配对用于建立双抗体夹心法ELISA,对p179生产过程中所得制品进行抗原定量检测。结果:5种单克隆抗体可以分成两组,分别针对p179疫苗抗原上的两种不同抗原表位,选择3G1为包被抗体、4E9为酶标抗体建立的双抗体夹心法ELISA具有良好的敏感性和特异性,对p179抗原检测的最低浓度可至15.6 ng? ml-1。该方法能够有效检测p179疫苗生产过程中的初制品、半成品和疫苗成品的抗原含量,反映抗原纯化各步骤的得率。结论:建立的双抗体夹心法ELISA可作为p179疫苗生产过程中的抗原检测方法,这有助于定量检测p179疫苗生产过程中的抗原成分。
目的:建立雙抗體夾心法ELISA檢測HEV抗原,用于定量檢測戊型肝炎p179疫苗生產過程中初製品、半成品和疫苗成品。方法:將單剋隆抗體3G1、5G5、1G10、3A10和4E9分彆進行辣根過氧化物酶標記,通過競爭ELISA檢測確定最佳抗體配對用于建立雙抗體夾心法ELISA,對p179生產過程中所得製品進行抗原定量檢測。結果:5種單剋隆抗體可以分成兩組,分彆針對p179疫苗抗原上的兩種不同抗原錶位,選擇3G1為包被抗體、4E9為酶標抗體建立的雙抗體夾心法ELISA具有良好的敏感性和特異性,對p179抗原檢測的最低濃度可至15.6 ng? ml-1。該方法能夠有效檢測p179疫苗生產過程中的初製品、半成品和疫苗成品的抗原含量,反映抗原純化各步驟的得率。結論:建立的雙抗體夾心法ELISA可作為p179疫苗生產過程中的抗原檢測方法,這有助于定量檢測p179疫苗生產過程中的抗原成分。
목적:건립쌍항체협심법ELISA검측HEV항원,용우정량검측무형간염p179역묘생산과정중초제품、반성품화역묘성품。방법:장단극륭항체3G1、5G5、1G10、3A10화4E9분별진행랄근과양화물매표기,통과경쟁ELISA검측학정최가항체배대용우건립쌍항체협심법ELISA,대p179생산과정중소득제품진행항원정량검측。결과:5충단극륭항체가이분성량조,분별침대p179역묘항원상적량충불동항원표위,선택3G1위포피항체、4E9위매표항체건립적쌍항체협심법ELISA구유량호적민감성화특이성,대p179항원검측적최저농도가지15.6 ng? ml-1。해방법능구유효검측p179역묘생산과정중적초제품、반성품화역묘성품적항원함량,반영항원순화각보취적득솔。결론:건립적쌍항체협심법ELISA가작위p179역묘생산과정중적항원검측방법,저유조우정량검측p179역묘생산과정중적항원성분。
Objective:To establish a double antibody sandwich ELISA for detection of hepatitis E virus antigen, and quantitative detection of p179 vaccine in early, semi-finished and finished products during vaccine production process.Methods: McAbs 3G1, 5G5, 1G10, 3A10 and 4E9 were prepared, conjugated with horseradish peroxidase and were used in a competitive inhibitory ELISA.Then, the appropriate paired McAbs were chosen to the establishment of the double antibody sandwich ELISA.Serial dilutions of p179 vaccine were used for the optimization of the double antibody sandwich ELISA and then the in-process p179 vaccine products were detected and quantified with the established assay.Results:Double antibody sandwich ELISA that can detect p179 vaccine during production process was established and optimized.McAbs 3 G1 was selected as a coating antibody and 4 E9 as an enzyme-conjugated antibody.Repeated detection of p179 using the sandwich ELISA has shown appreciable specificity and sensitivity,with a minimum detectable concentration of p179 vaccine up to 15.6 ng? ml -1 .No cross reactivity was observed with both freeze-dried hepatitis A attenuated live vaccine and Hepatitis B virus surface antigen recombinant vaccine.Conclusion: The established double antibody sandwich ELISA can be used for the detection and quantitation of p179 antigen during vaccine production process.