重组痘苗病毒艾滋病疫苗细胞免疫水平ELISPOT检测方法的建立  

Development of ELISPOT for Cellular Immunity of Recombinant Tiantan Vaccinia Virus-based AIDS Vaccine

作　　者：李媛媛[1] 凌媛[1] 马志新[1] 徐静[1] 

机构地区：[1]北京生物制品研究所第三研究室,北京100024

出　　处：《中国生物制品学杂志》2011年第12期1496-1499,共4页Chinese Journal of Biologicals

基　　金：艾滋病和病毒性肝炎等重大传染病防治专项(2008-ZX10001-010)

摘　　要：目的建立检测重组痘苗病毒(Recombinant Tiantan vaccinia,rTV)艾滋病疫苗细胞免疫水平的ELISPOT方法。方法 rTV艾滋病疫苗经皮内注射BALB/c小鼠,免疫后1～6周,采集小鼠脾细胞,ELISPOT法检测小鼠细胞免疫水平,确定最佳检测时间,并对免疫剂量、肽库刺激浓度进行优化。应用ELISPOT方法对3批rTV疫苗成品进行检测,验证该方法的适用性;按现行检定标准对同批疫苗进行重复检测,验证该方法的重复性;应用ELISPOT和ICS方法同时对rTV疫苗进行检测,验证该方法与ICS法的相关性。结果 rTV艾滋病疫苗最佳免疫剂量为50μl/只,免疫后4周,混合多肽库刺激终浓度为4μg/ml,淋巴细胞分泌IFNγ的水平较高;ELISPOT法适用性和重复性较好,且与ICS法具有较好的相关性(r=0.767,P<0.05)。结论已成功建立了检测重组痘苗病毒艾滋病疫苗细胞免疫水平的ELISPOT方法。Objective To develop an ELISPOT method for cellular immunity of recombinant Tiantan vaccinia（rTV） virus-based AIDS vaccine.Methods BALB / c mice were injected i.d.with rTV virus-based AIDS vaccine,and their splenocytes were collected 1 ～ 6 weeks later and determined for cellular immunity by ELISPOT,based on which the time for determination,dosage of vaccine and peptide concentration for stimulation were optimized.The developed ELISPOT method was verified for applicability by determination of three batches of final products of rTV virus-based AIDS vaccine,for reproducibility by repeat determination of the vaccine of the same batch,and for relationship to ICS methods by determination of vaccine by the two methods simultaneously.Results The optimal dosage of rTV virus-based AIDS vaccine for immunization was 50 μl / mouse.The final peptide concentration for stimulation 4 weeks after immunization was 4 μg / ml.The IFNγ secreted by the lymphocytes of immunized mice reached a high level.The developed ELISPOT method showed high applicability and reproducibility as well as close relationship to ICS method（r = 0.767,P 0.05）.Conclusion An ELISPOT method for cellular immunity of rTV virus-based AIDS vaccine was successfully developed.

关 键 词：痘苗病毒 艾滋病疫苗 细胞免疫 ELISPOT 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]