乙脑病毒IgG抗体单克隆抗体捕获ELISA检测方法的建立
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摘要
乙型脑炎(Japanese Encephalitis,JE)是由蚊虫传播的人畜共患急性传染病,其病原体是乙型脑炎病毒(Japanese Encephalitis Virus,JEV)。JEV能够引起人类中枢神经系统病变,JE死亡率高,后遗症严重。JE是疫苗可预防性疾病,接种乙脑疫苗以及自然状态下感染JEV后产生的病毒特异性抗体可长时间存在,检测乙脑病毒抗体的方法有中和实验(NI)、间接免疫荧光(IFA)、血凝抑制试验(HIT)和酶联免疫吸附试验(ELISA)等多种。NI是检测中和抗体的金标准,但操作复杂,对人员要求高,且需要操作活病毒,存在生物安全的隐患;IFA敏感性和特异性较好,但对设备要求高,难以在基层开展;HI敏感性较低;比较而言,ELISA具有敏感性、特异性高,操作简单等优点,是重要的抗体检测方法。
目前中国乃至全球都缺乏理想的乙脑病毒IgG抗体ELISA检测试剂,现有试剂的检测结果普遍存在本底值高、假阳性多、试剂间检测结果一致性差等问题,本研究旨在利用乙脑单克隆抗体建立一种乙脑病毒IgG抗体的快速检测方法,以用于人群乙脑病毒IgG抗体的检测。主要研究内容和主要结果如下:
一.标准血清的建立
由于正常人群中乙脑病毒中和抗体主要以IgG抗体形式存在,因此通过检测血清中乙脑病毒中和抗体的水平可以大概反映血清中乙脑病毒IgG抗体的水平。本研究在建立方法前利用中和实验方法检测了456份血清的乙脑病毒中和抗体水平,从中筛选出80份中和抗体水平清晰的血清作为建立方法用的标准血清,其中包括中和抗体滴度小于1∶10的有20份,大于等于1∶10且小于1∶20的有20份,大于等于1∶20且小于1∶40的有20份,大于等于1∶40的有20份,为乙脑捕綢gG抗体ELISA检测方法的建立和评价提供了标准血清。
二.乙脑病毒IgG抗体单克隆抗体捕获ELISA检测方法的建立
1.乙脑病毒抗原的制备
建立ELISA方法检测乙脑病毒IgG抗体首先需要制备乙脑抗原。本研究以乙脑病毒P3株作为抗原制备的毒株,C6/36细胞为扩增细胞系。经扩增和常规处理后,对病毒上清进行灭活处理,灭活上清在相同条件下回传C6/36细胞连续3代未见细胞病变,说明没有感染性病毒存在。为提高抗原的浓度和纯度,采用Millipore小型切向流系统对300mL灭活病毒细胞上清进行浓缩和纯化(除去膜透过性杂质起到纯化作用),得到12mL病毒浓缩液;对浓缩液和滤过液进行JEV的分子生物学和ELISA检测,结果显示浓缩液经10~4倍稀释后PCR扩增仍出现目的条带,滤过液及其10~2倍稀释液PCR扩增均未见条带;ELISA结果显示浓缩液中抗原浓度有20～30倍增加,滤过液检测结果为阴性,显示浓缩效果较好。
2.乙脑病毒单克隆抗体的制备
对实验室保存的乙脑病毒单克隆抗体细胞株JEV07#进行复苏培养,培养至3天后,取上清检测JEV抗体,ELISA和IFA检测结果均为阳性,说明单抗细胞分泌JEV抗体。继续扩增培养,获得足量单克隆抗体细胞时,取上清检测JEV抗体,ELISA检测单抗的OD值为1.397。单抗细胞腹腔注射10只Balb/c小鼠,收获25mL腹水,SP2/0细胞腹腔注射1只Balb/c小鼠,收获1mL腹水。ELISA检测腹水滴度,结果显示SP2/0腹水为阴性,单抗腹水的滴度超过8×10~3。
3.乙脑病毒IgG抗体单克隆抗体捕获ELISA检测方法的建立
本研究的特点是利用乙脑病毒单克隆抗体的特异性和均一性特点,对乙脑抗原进行捕获,起到纯化作用,以减少病毒上清中血清蛋白和细胞组分带来的高本底和假阳性问题。
工作条件的确定:通过优化反应条件建立乙脑病毒IgG抗体单克隆抗体ELISA检测方法。首先通过包被足量人IgG抗体确定1∶80,000为酶标抗体的工作浓度。其次方阵稀释乙脑病毒单克隆抗体和乙脑抗原,固定血清的稀释度1∶100和酶标抗人IgG抗体的稀释度1∶80,000,确定1∶2,000包被单克隆抗体,1∶100稀释抗原,1∶100稀释待测血清,1∶80,000稀释酶标抗人IgG抗体是最优的反应条件,此时,阳、阴性对照的P/N值(23)最高,且阴性对照的OD值(0.049)小于0.1。
灵敏性评价:为了评价方法的敏感性,用新建方法筛选出9份阳性血清,并按OD值大小分为三组:弱阳性组(OD值≤0.4)、中等强度阳性组(0.4＜OD值≤0.8)和强阳性组(OD值＞0.8)。对每份血清1∶200起系列倍比稀释8个滴度并利用新建方法进行测定,结果显示新建方法对弱阳性标本、中等强度阳性标本和强阳性标本检测的最高稀释度分别可达到1∶400、1∶800和1∶12,800,表明该方法检测乙脑病毒IgG抗体的敏感性很高。
稳定性评价:为了评价方法的稳定性,用新建方法筛选出不同抗体滴度的阳性血清标本9份,阴性血清标本3份进行检测。每份标本先后经过4次重复检测,结果表明四次检测结果的判定均一致,并且每份标本检测的OD值变异度不超过6.8%,显示该方法具有良好的稳定性。
三.乙脑病毒IgG抗体单克隆抗体捕获ELISA检测方法的评价
为进一步评价新建方法的实际应用效果,本研究中以IFA为标准,利用第一部分建立的80份标准血清比较了新建立方法与一种国产间接试剂和一种进口试剂的检测性能,此外还探讨了ELISA检测方法与中和实验方法的关系。
三种不同ELISA检测方法的检测性能评价:可靠性评价显示新建立ELISA方法和进口试剂的可靠性高(97.5%),优于国产间接法试剂(93.8%);以IFA为标准,比较三种不同ELISA检测方法的检测性能,结果显示新建立方法与IFA的一致性最高(91.3%),敏感性(95.6%)优于其他两种ELISA试剂检测性能,特异性(85.7%)稍低于进口试剂(100%);进口试剂的特异性高,但敏感性较低;国产间接试剂敏感性和特异性均低于80%。
ELISA检测方法与中和实验的关系探讨:与中和实验的判定结果相比,三种不同ELISA检测方法和中和实验的一致性探讨表明新建立方法与中和实验结果的一致性(80%)高于其他两种试剂;中和实验和新建立方法的检测结果的散点图分析表明两者无线性相关性。
综上所述,本研究利用乙脑单克隆抗体建立了乙脑病毒IgG抗体的ELISA检测方法,检测性能评价表明该方法与IFA一致性最高(91.3%),具有较高的敏感度(95.6%)和特异度(85.7%),优于其他两种试剂。该方法检测效果较好,可以用于人群中乙脑病毒IgG抗体的检测。
Japanese encephalitis(JE) is a zoonosis transmitted by mosquitoes,which pathogen is Japanese encephalitis virus(JEV).JEV causes human central nervous system changes with high mortality rate and severe sequela.JE is a vaccine-preventable disease.After vaccination or natural infection antibodies specific to JEV will occur and last for a long time.There are several methods to detect antibodies to JEV,such as neutralization test(NT),indirect immunofluoresence assay (IFA),hemaglutination inhibition test(HI),enzyme-linked immunosorbent assay (ELISA),and so on.NT is the golden standard for antibody detection but has strict requirement for technologist and has some limitation because of the live virus.IFA is specific and sensitive but has strict requirement for equipment.HI has low sensitivity. Compared with above,ELISA is an important method because of its specific, sensitivity,easy-done.
At present,there is lack of good ELISA reagent to detect IgG antibody to JEV worldwide.Aim of this study is to construct an ELISA detection system which can rapidly detect JEV IgG antibodies and can be used to detect JEV IgG antibodies in the population.Details of the study and results are listed below:
Development of monoclonal antibody capture ELISA for detection of IgG Antibody to Japanese Encephalitis Virus(GAC-ELISA for JEV)
1.Establishment of serum panels for development of detection system
In human bodies neutralization antibodies to JEV mainly exist at the type of IgG, so detection of neutralization antibodies can mainly reflect the level of IgG antibodies.
In this study NT was carried out on 456 serum samples collected in 2006 and 80 serum samples were selected and put into serum panels.According to neutralization antibodies titers,20 were below 1:10,20 were 1:10-1:20,20 were 1:20-1:40,20 were above 1:40.
2.Development of GAC-ELISA for JEV 2.1 Preparation of JEV antigen
In this study P3 strain was amplified in C6/36 cell lines.After routine treatment, virus supernatant was inactivated.The inactivated supernatant was inoculated on C6/36 cell lines and no CPE was observed for 3 sequenced passages which indicated that the virus was fully inactivated.In order to improve the concentration and purification,300mL inactivated supernatant was disposed using Millipore Labscale system and 12mL concentrate was obtained.The filtrate and concentrate was tested with molecular biological method and ELISA.The results showed that the amplification of PrM segment was found in the 10~4 times diluents of concentrate but wasn't found in the filtrate and its diluents.ELISA results showed that the antigen was condensed by 20 to 30 times but no antigen was found in the filtrate.
2.2 Preparation of JEV monoclonal antibody(MAb)
JEV07# was resuscitates.Supernatant was collected 3 days later to test whether it could produce MAb or not.It indicated that JEV07# could produce Mab from the positive ELISA result and positive IFA result.While enough JEV07# cells were obtained the supernatant was tested again,it showed the OD value of was 1.397. JEV07# cells were injected into 10 Balb/c mice.25mL ascites were obtained.SP2/0 cells were injected into 1 Balb/c mouse and 1mL ascites were obtained.ELISA results showed the titer of ascites from JEV07# was higher than 8×10~3 while ascites from SP2/0 were negative.
2.3 Development of GAC-ELISA for JEV
In this study specific and homogeneous JEV MAb was used to capture JEV antigen in order to purify it and lower problems triggered by serum protein and cell components.
Determination of working concentration:The plate was coated with sufficient human IgG,HRP conjugated anti-human IgG was diluted and dilution 1:80,000(at 1.0 of OD value) was determined to be the working concentration.MAb was diluted and coated with coating buffer,treated antigen was diluted with wash buffer and added to the plate,then serum and HRP conjugated anti-human IgG was added with 1:100 dilution and 1:80,000 dilutions respectively.The results showed that 1:2,000 MAb, 1:100 antigen,1:100 serum and 1:80,000 HRP conjugated anti-human IgG was the best working dilution.At these working dilutions max P/N(23) was obtained and the OD value produced by negative control is below 0.1.
Valuation of sensitivity:9 different-OD-value positive samples were detected by GAC-ELISA.Weakly positive samples(OD value≤0.4) were positive at dilution of 1:400,positive samples(0.40.8) was at the highest dilution of 1:12,800.
These results showed sensitivity of the way was high.
Valuation of stability:9 different-OD-value positive samples and 3 negative samples were detected repeatedly.Each sample was detected four times.The positive are still positive and the negative are still negative.CV of each sample was less than 6.8%.
These results showed that the way had good stability.
Evaluation of GAC-ELISA for JEV
To further evaluate the effect of GAC-ELISA,GAC-ELISA,national indirect kit and import kit compared using IFA as the standard.And some discussion of the relationship between ELISA and NT was done.
Performance evaluation of GAC-ELISA,national indirect kit and import kit:
Reliability evaluation of three different IgG ELISA methods showed that GAC-ELISA and import kit had higher reliability(97.5%)than national indirect kit(93.8%);Taking IFA as the standard,performance evaluation showed that GAC-ELISA has higher consistency(91.3%) and higher sensitivity(95.6%) than other two kits and good specificity(85.7%);import kit has 100%specificity but low sensitivity;both specificity and sensitivity of national indirect kit are lower than 80%.
The relationship between ELISA and NT:Analysis of the consistency between ELISA and NT showed that GAC-ELISA had higher consistency(80%) than other two kits;Scatter chart showed there was no linearity between the results GAC-ELISA and NT.
In summary,this study GAC-ELISA was developed using JEV MAb.Performance evaluation showed that the new GAC-ELISA had high consistency(91.3%) with IFA and higher sensitivity(95.6%) and specificity(85.7%) than other two kits. GAC-ELISA can be used as a routine method to detect IgG antibodies to JEV in population.
目前中国乃至全球都缺乏理想的乙脑病毒IgG抗体ELISA检测试剂,现有试剂的检测结果普遍存在本底值高、假阳性多、试剂间检测结果一致性差等问题,本研究旨在利用乙脑单克隆抗体建立一种乙脑病毒IgG抗体的快速检测方法,以用于人群乙脑病毒IgG抗体的检测。主要研究内容和主要结果如下:
一.标准血清的建立
由于正常人群中乙脑病毒中和抗体主要以IgG抗体形式存在,因此通过检测血清中乙脑病毒中和抗体的水平可以大概反映血清中乙脑病毒IgG抗体的水平。本研究在建立方法前利用中和实验方法检测了456份血清的乙脑病毒中和抗体水平,从中筛选出80份中和抗体水平清晰的血清作为建立方法用的标准血清,其中包括中和抗体滴度小于1∶10的有20份,大于等于1∶10且小于1∶20的有20份,大于等于1∶20且小于1∶40的有20份,大于等于1∶40的有20份,为乙脑捕綢gG抗体ELISA检测方法的建立和评价提供了标准血清。
二.乙脑病毒IgG抗体单克隆抗体捕获ELISA检测方法的建立
1.乙脑病毒抗原的制备
建立ELISA方法检测乙脑病毒IgG抗体首先需要制备乙脑抗原。本研究以乙脑病毒P3株作为抗原制备的毒株,C6/36细胞为扩增细胞系。经扩增和常规处理后,对病毒上清进行灭活处理,灭活上清在相同条件下回传C6/36细胞连续3代未见细胞病变,说明没有感染性病毒存在。为提高抗原的浓度和纯度,采用Millipore小型切向流系统对300mL灭活病毒细胞上清进行浓缩和纯化(除去膜透过性杂质起到纯化作用),得到12mL病毒浓缩液;对浓缩液和滤过液进行JEV的分子生物学和ELISA检测,结果显示浓缩液经10~4倍稀释后PCR扩增仍出现目的条带,滤过液及其10~2倍稀释液PCR扩增均未见条带;ELISA结果显示浓缩液中抗原浓度有20～30倍增加,滤过液检测结果为阴性,显示浓缩效果较好。
2.乙脑病毒单克隆抗体的制备
对实验室保存的乙脑病毒单克隆抗体细胞株JEV07#进行复苏培养,培养至3天后,取上清检测JEV抗体,ELISA和IFA检测结果均为阳性,说明单抗细胞分泌JEV抗体。继续扩增培养,获得足量单克隆抗体细胞时,取上清检测JEV抗体,ELISA检测单抗的OD值为1.397。单抗细胞腹腔注射10只Balb/c小鼠,收获25mL腹水,SP2/0细胞腹腔注射1只Balb/c小鼠,收获1mL腹水。ELISA检测腹水滴度,结果显示SP2/0腹水为阴性,单抗腹水的滴度超过8×10~3。
3.乙脑病毒IgG抗体单克隆抗体捕获ELISA检测方法的建立
本研究的特点是利用乙脑病毒单克隆抗体的特异性和均一性特点,对乙脑抗原进行捕获,起到纯化作用,以减少病毒上清中血清蛋白和细胞组分带来的高本底和假阳性问题。
工作条件的确定:通过优化反应条件建立乙脑病毒IgG抗体单克隆抗体ELISA检测方法。首先通过包被足量人IgG抗体确定1∶80,000为酶标抗体的工作浓度。其次方阵稀释乙脑病毒单克隆抗体和乙脑抗原,固定血清的稀释度1∶100和酶标抗人IgG抗体的稀释度1∶80,000,确定1∶2,000包被单克隆抗体,1∶100稀释抗原,1∶100稀释待测血清,1∶80,000稀释酶标抗人IgG抗体是最优的反应条件,此时,阳、阴性对照的P/N值(23)最高,且阴性对照的OD值(0.049)小于0.1。
灵敏性评价:为了评价方法的敏感性,用新建方法筛选出9份阳性血清,并按OD值大小分为三组:弱阳性组(OD值≤0.4)、中等强度阳性组(0.4＜OD值≤0.8)和强阳性组(OD值＞0.8)。对每份血清1∶200起系列倍比稀释8个滴度并利用新建方法进行测定,结果显示新建方法对弱阳性标本、中等强度阳性标本和强阳性标本检测的最高稀释度分别可达到1∶400、1∶800和1∶12,800,表明该方法检测乙脑病毒IgG抗体的敏感性很高。
稳定性评价:为了评价方法的稳定性,用新建方法筛选出不同抗体滴度的阳性血清标本9份,阴性血清标本3份进行检测。每份标本先后经过4次重复检测,结果表明四次检测结果的判定均一致,并且每份标本检测的OD值变异度不超过6.8%,显示该方法具有良好的稳定性。
三.乙脑病毒IgG抗体单克隆抗体捕获ELISA检测方法的评价
为进一步评价新建方法的实际应用效果,本研究中以IFA为标准,利用第一部分建立的80份标准血清比较了新建立方法与一种国产间接试剂和一种进口试剂的检测性能,此外还探讨了ELISA检测方法与中和实验方法的关系。
三种不同ELISA检测方法的检测性能评价:可靠性评价显示新建立ELISA方法和进口试剂的可靠性高(97.5%),优于国产间接法试剂(93.8%);以IFA为标准,比较三种不同ELISA检测方法的检测性能,结果显示新建立方法与IFA的一致性最高(91.3%),敏感性(95.6%)优于其他两种ELISA试剂检测性能,特异性(85.7%)稍低于进口试剂(100%);进口试剂的特异性高,但敏感性较低;国产间接试剂敏感性和特异性均低于80%。
ELISA检测方法与中和实验的关系探讨:与中和实验的判定结果相比,三种不同ELISA检测方法和中和实验的一致性探讨表明新建立方法与中和实验结果的一致性(80%)高于其他两种试剂;中和实验和新建立方法的检测结果的散点图分析表明两者无线性相关性。
综上所述,本研究利用乙脑单克隆抗体建立了乙脑病毒IgG抗体的ELISA检测方法,检测性能评价表明该方法与IFA一致性最高(91.3%),具有较高的敏感度(95.6%)和特异度(85.7%),优于其他两种试剂。该方法检测效果较好,可以用于人群中乙脑病毒IgG抗体的检测。
Japanese encephalitis(JE) is a zoonosis transmitted by mosquitoes,which pathogen is Japanese encephalitis virus(JEV).JEV causes human central nervous system changes with high mortality rate and severe sequela.JE is a vaccine-preventable disease.After vaccination or natural infection antibodies specific to JEV will occur and last for a long time.There are several methods to detect antibodies to JEV,such as neutralization test(NT),indirect immunofluoresence assay (IFA),hemaglutination inhibition test(HI),enzyme-linked immunosorbent assay (ELISA),and so on.NT is the golden standard for antibody detection but has strict requirement for technologist and has some limitation because of the live virus.IFA is specific and sensitive but has strict requirement for equipment.HI has low sensitivity. Compared with above,ELISA is an important method because of its specific, sensitivity,easy-done.
At present,there is lack of good ELISA reagent to detect IgG antibody to JEV worldwide.Aim of this study is to construct an ELISA detection system which can rapidly detect JEV IgG antibodies and can be used to detect JEV IgG antibodies in the population.Details of the study and results are listed below:
Development of monoclonal antibody capture ELISA for detection of IgG Antibody to Japanese Encephalitis Virus(GAC-ELISA for JEV)
1.Establishment of serum panels for development of detection system
In human bodies neutralization antibodies to JEV mainly exist at the type of IgG, so detection of neutralization antibodies can mainly reflect the level of IgG antibodies.
In this study NT was carried out on 456 serum samples collected in 2006 and 80 serum samples were selected and put into serum panels.According to neutralization antibodies titers,20 were below 1:10,20 were 1:10-1:20,20 were 1:20-1:40,20 were above 1:40.
2.Development of GAC-ELISA for JEV 2.1 Preparation of JEV antigen
In this study P3 strain was amplified in C6/36 cell lines.After routine treatment, virus supernatant was inactivated.The inactivated supernatant was inoculated on C6/36 cell lines and no CPE was observed for 3 sequenced passages which indicated that the virus was fully inactivated.In order to improve the concentration and purification,300mL inactivated supernatant was disposed using Millipore Labscale system and 12mL concentrate was obtained.The filtrate and concentrate was tested with molecular biological method and ELISA.The results showed that the amplification of PrM segment was found in the 10~4 times diluents of concentrate but wasn't found in the filtrate and its diluents.ELISA results showed that the antigen was condensed by 20 to 30 times but no antigen was found in the filtrate.
2.2 Preparation of JEV monoclonal antibody(MAb)
JEV07# was resuscitates.Supernatant was collected 3 days later to test whether it could produce MAb or not.It indicated that JEV07# could produce Mab from the positive ELISA result and positive IFA result.While enough JEV07# cells were obtained the supernatant was tested again,it showed the OD value of was 1.397. JEV07# cells were injected into 10 Balb/c mice.25mL ascites were obtained.SP2/0 cells were injected into 1 Balb/c mouse and 1mL ascites were obtained.ELISA results showed the titer of ascites from JEV07# was higher than 8×10~3 while ascites from SP2/0 were negative.
2.3 Development of GAC-ELISA for JEV
In this study specific and homogeneous JEV MAb was used to capture JEV antigen in order to purify it and lower problems triggered by serum protein and cell components.
Determination of working concentration:The plate was coated with sufficient human IgG,HRP conjugated anti-human IgG was diluted and dilution 1:80,000(at 1.0 of OD value) was determined to be the working concentration.MAb was diluted and coated with coating buffer,treated antigen was diluted with wash buffer and added to the plate,then serum and HRP conjugated anti-human IgG was added with 1:100 dilution and 1:80,000 dilutions respectively.The results showed that 1:2,000 MAb, 1:100 antigen,1:100 serum and 1:80,000 HRP conjugated anti-human IgG was the best working dilution.At these working dilutions max P/N(23) was obtained and the OD value produced by negative control is below 0.1.
Valuation of sensitivity:9 different-OD-value positive samples were detected by GAC-ELISA.Weakly positive samples(OD value≤0.4) were positive at dilution of 1:400,positive samples(0.4
These results showed sensitivity of the way was high.
Valuation of stability:9 different-OD-value positive samples and 3 negative samples were detected repeatedly.Each sample was detected four times.The positive are still positive and the negative are still negative.CV of each sample was less than 6.8%.
These results showed that the way had good stability.
Evaluation of GAC-ELISA for JEV
To further evaluate the effect of GAC-ELISA,GAC-ELISA,national indirect kit and import kit compared using IFA as the standard.And some discussion of the relationship between ELISA and NT was done.
Performance evaluation of GAC-ELISA,national indirect kit and import kit:
Reliability evaluation of three different IgG ELISA methods showed that GAC-ELISA and import kit had higher reliability(97.5%)than national indirect kit(93.8%);Taking IFA as the standard,performance evaluation showed that GAC-ELISA has higher consistency(91.3%) and higher sensitivity(95.6%) than other two kits and good specificity(85.7%);import kit has 100%specificity but low sensitivity;both specificity and sensitivity of national indirect kit are lower than 80%.
The relationship between ELISA and NT:Analysis of the consistency between ELISA and NT showed that GAC-ELISA had higher consistency(80%) than other two kits;Scatter chart showed there was no linearity between the results GAC-ELISA and NT.
In summary,this study GAC-ELISA was developed using JEV MAb.Performance evaluation showed that the new GAC-ELISA had high consistency(91.3%) with IFA and higher sensitivity(95.6%) and specificity(85.7%) than other two kits. GAC-ELISA can be used as a routine method to detect IgG antibodies to JEV in population.
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