CAJ | 학술논문

目的在获得具有中和活性的针对Ⅰ型登革病毒(dengue virus serotype Ⅰ,DENV-1)的抗体基础上,评价已建立的非结构蛋白1(nonstructural protein 1,NSI)抗原捕获酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分析抗体中和活性的可行性,为中和抗体的筛选和疫苗效果的评价提供一个更为快速、简便的检测方法.方法使用重组DENV-1包膜蛋白Ⅲ区(envelopeprotein domainⅢ,EDⅢ)免疫5只BALB/c小鼠制备单克隆抗体(简称单抗),采用间接ELISA、间接免疫荧光法(immunofluorescenee assay,IFA)和免疫印迹法(Western Blot)对单抗进行筛选及鉴定;同时用该重组蛋白免疫1只新西兰兔,制备多克隆抗体血清;以传统噬斑减少中和试验(plaquereduction neutralization test,PRNT)作为参比方法,采用NSI抗原捕获ELISA对所获抗体进行中和活性检测.结果获得4株针对DENV-1 EDⅢ单抗和1份兔多抗血清,且被PRNT试验证明均具有中和活性,四株单抗腹水(1A1、1B3、3D3、9D6)和兔多抗血清中和效价分别为1:1024、1:512、1:256、1:4096和1:4096.使用NS1抗原捕获ELISA法检测不到PRNT中和效价最低的3D3抗体对DENV-1有中和能力,而检测其余3株单抗(1A1、1B3、9D6)和多抗兔血清中和效价均远低于PRNT测定结果,分别为1:32、1:32、1:128和1:128.结论简单、快速的NS1抗原捕获ELISA可用于评价DENV抗体的中和活性,该方法适合于高效价中和抗体的筛选,有望成为评价疫苗效果的方法之一.
목적 재획득구유중화활성적침대Ⅰ형등혁병독(dengue virus serotype Ⅰ,DENV-1)적항체기출상,평개이건립적비결구단백1(nonstructural protein 1,NSI)항원포획매련면역흡부법(enzyme-linked immunosorbent assay,ELISA)분석항체중화활성적가행성,위중화항체적사선화역묘효과적평개제공일개경위쾌속、간편적검측방법.방법 사용중조DENV-1포막단백Ⅲ구(envelopeprotein domainⅢ,EDⅢ)면역5지BALB/c소서제비단극륭항체(간칭단항),채용간접ELISA、간접면역형광법(immunofluorescenee assay,IFA)화면역인적법(Western Blot)대단항진행사선급감정;동시용해중조단백면역1지신서란토,제비다극륭항체혈청;이전통서반감소중화시험(plaquereduction neutralization test,PRNT)작위삼비방법,채용NSI항원포획ELISA대소획항체진행중화활성검측.결과 획득4주침대DENV-1 EDⅢ단항화1빈토다항혈청,차피PRNT시험증명균구유중화활성,사주단항복수(1A1、1B3、3D3、9D6)화토다항혈청중화효개분별위1:1024、1:512、1:256、1:4096화1:4096.사용NS1항원포획ELISA법검측불도PRNT중화효개최저적3D3항체대DENV-1유중화능력,이검측기여3주단항(1A1、1B3、9D6)화다항토혈청중화효개균원저우PRNT측정결과,분별위1:32、1:32、1:128화1:128.결론 간단、쾌속적NS1항원포획ELISA가용우평개DENV항체적중화활성,해방법괄합우고효개중화항체적사선,유망성위평개역묘효과적방법지일.
Objective To produce neutralizing antibodies against envelope protein domain Ⅲ (ED Ⅲ ) of dengue virus serotype Ⅰ ( DENV-1 ) and evaluate the nonstruetural protein 1 ( NSI ) antigen capture enzyme-linked immunnsorbent assay (ELISA) for identification of antibody neutralizing abilities. Methods Five BALB/c mice and one New Zealand Rabbit were immunized with recombinant EDⅢ protein of DENV-1 for the production of hybridomas and hyperimmune sera. Indirect ELISA, immunofluoreseence assay (IFA) and Western Blot analyses were applied to identify specificity of antibodies. Comparing to plaque reduction neutralization test (PRNT) ,the new established DENV-1 specific NSI antigen capture ELISA was used for detecting the neutralizing abilities of these antibody. Results Four strains of monoclonal antibodies (mAbs) named 1A1,1B3,3D3 and 9D6 and one hyperimmune serum of rabbit were obtained ,all of which were approved to have neutralizing abilities to DENV-1 with the PRNT titer of 1: 1024,1 : 512,1:256,1 : 4096 and 1: 4096. MAb 3D3 with the lowest neutralization titer in PRNT had not shown neutralizing ability to DENY-1 in NSI antigen capture ELISA,while MAbs 1A1,1B3 and 9D6 and the rabbit hyperimmune serum eould protect the C6/36 from being infected by DENV-1 with the neutralization titer of 1:32,1:32,1:128 and 1:128 in this assay. Conclusion NSI antigen capture ELISA could be used to identify antibody neutralizing abilities to DENV, it was a faster and more convenient way to screen antibodies with high neutralization fiter and might also be used as one of the methods to evaluate the effects ofvaccines.