酶联免疫吸附试验在脊髓灰质炎病毒型内鉴别中的应用

Application of ELISA in Intratypic Differentiation of Poliovirus Isolates from AFP Cases

从 2 0 0 1年 10月开始 ,中国疾病预防控制中心病毒病预防控制所国家脊髓灰质炎 (脊灰 )实验室 ,增加使用酶联免疫吸附试验 (ELISA)对从急性弛缓性麻痹 (AFP)病例粪便标本中分离到的脊灰病毒进行型内鉴别。 2 0 0 2年6 0 7株单型脊灰病毒的ELISA试验结果是 :5 73株为疫苗相似株 (SL) ,3株为非疫苗相似株 (NSL) ,2 9株为双反应株 (DRV) ,2株为阴性反应株 (NRV)。将ELISA试验结果与逆转录 聚合酶链反应 限制性内切酶片段长度多态性分析 (RT PCR RFLP)结果进行比较 ,发现仅 2株ELISA试验结果为DRV的脊灰病毒 ,PCR结果为变异株。同样 ,在 33株PCR结果为变异株的脊灰病毒中 ,仅有 2株ELISA试验结果为DRV。由于两种方法的原理不同 ,它们得出的结论并无很高的一致性。将ELISA试验结果为NSL和DRV、PCR结果为变异株的毒株进行了脊灰病毒全VP1区核苷酸序列分析 ,在ELISA试验结果为DRV和NSL的脊灰病毒的全VP1区氨基酸序列分析结果中 ,仅有Ⅰ型脊灰病毒的氨基酸变异位点在已知的SabinⅠ型抗原决定簇位点 1(site 1)内 ,Ⅱ型和Ⅲ型的变异位点均不在其中 ,这一矛盾的结果说明 ,可能在VP1区以外其它编码区的抗原决定簇起到主要作用 ,或者试剂盒本身的检测系统存在某些需要改进的地方。在ELISA试验过程中 。

ELISA was introduced into National Laboratory for Poliomyelitis for intratypic differentiation(ITD)of poliovirus isolates of AFP cases from Sep 2001 607 single-type poliovirus isolates in 2002 were analyzed using ELISA method,The result indicated that 573 of 607 were Sabin like(SL) viruses,3 were non Sabin like(NSL)viruses,29 were double reaction viruses(DRV),and 2 were non reaction viruses(NRV) Comparing with the results of reverse transcription polymerase chain reaction restriction fragment length polymorphism(PCR RFLP),only 2 DRVs corresponded to the mutant results of PCR RFLP,in the same way,in all 33 mutants of PCR RFLP,only 2 DRVs by ELISA Because of the different principles of the two methods,there was not much consistency between these 2 methods Whole VP1 gene sequencing of the isolates,contaning all NSL,DRV of ELISA or mutants of RT PCR RFLP,was done Through sequencing results of DRV and NSL isolates,the gene mutation correspondly locating in the antigenic epitope of SabinⅠ(siteⅠ)was found only in type 1 viruses,not in type Ⅱ and Ⅲ,these contradictory results showed that other epitope site beyond VP1 gene may play the dominating role,or this system of ELISA has limitation itself Also,type Ⅲ ELISA kit has the problem that is unsolved,and this causes the inaccurate results It seems that the kit should be improved using Chinese polio vaccine type Ⅲ antigen One vaccine-derived poliovirus(VDPV)was detected by both the methods ELISA method is one of the effective methods for ITD,in post eradication era of China,it is necessary to do ITD using 2 methods simultaneously