建立双抗体夹心法ELISA定量检测戊型肝炎p179疫苗

Quantitative detection of the hepatitis E p179 vaccine with a double antibody sandwich eLISA

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。

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： Mc Abs 3G1,5G5,1G10,3A10 and 4E9 were prepared, conjugated with horseradish peroxidase and were used in a competitive inhibitory ELISA. Then,the appropriate paired Mc Abs 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. Mc Abs 3G1 was selected as a coating antibody and 4E9 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.