CN103760347B - 一种检测人巨细胞病毒pp65抗原的elisa方法 - Google Patents
一种检测人巨细胞病毒pp65抗原的elisa方法 Download PDFInfo
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Abstract
本发明涉及一种检测人巨细胞病毒(HCMV)PP65抗原的ELISA方法，它是一种检测HCMV活动性感染指标的临床检测方法。本发明主要是解决现有HCMV活动性感染检测方法存在的敏感性或特异性低、费时费力、操作繁琐和使用不便的问题。本发明采用的技术方案是：一种检测人巨细胞病毒PP65抗原的ELISA方法，其包括下列步骤：1)制备检测试剂盒：所述检测试剂盒包括：包被有大鼠抗重组PP65322-561多克隆抗体(pAb)的酶标板、兔抗重组PP65322-561pAb、标准品、HRP-羊抗兔IgG、PBST洗涤液、底物显色液、终止液和封板膜；2)制备待测标本；3)检测人巨细胞病毒PP65抗原。本发明具有较好的特异性、敏感性、稳定性和可重复性，易于规模化和标准化操作等优点。
Description
技术领域
本发明涉及一种检测人巨细胞病毒(HCMV)PP65抗原的ELISA方法，它是一种检测HCMV活动性感染指标的临床检测方法。
背景技术
人巨细胞病毒(humancytomegalovirus，HCMV)属疱疹病毒科β属双链DNA病毒。HCMV可通过口腔、生殖道、胎盘、授乳以及输血或器官移植等多途径传播。人群感染HCMV的显著特点是感染率高，正常成人HCMV抗体阳性率为76.7％－95.8％，我国也是HCMV感染高度流行国家之一。然而正常人初次感染此病毒多无明显症状，病毒也不能彻底清除，而是潜伏体内。当机体免疫功能低下时，潜伏病毒开始复制并大量增殖，继发活动性感染并表现多种临床症状。临床活动性HCMV感染多见于孕妇，新生儿、输血患者、恶性肿瘤和艾滋病患者以及器官移植等使用免疫抑制剂的患者。孕妇活动性感染可通过胎盘、产道或授乳传给胎儿，导致流产，死胎，胎儿形态畸形，器官功能障碍。先天感染的新生儿出生时可伴有黄疸、瘀斑、脉络膜视网膜炎、小头畸形等，而无症状的部分新生儿，随着发育才逐渐被发现听觉、视力低下，智力发育迟缓，运动障碍等。在恶性肿瘤、艾滋病、器官移植等免疫功能减弱或受抑制的患者HCMV的活动性感染常并发间质性肺炎、视网膜炎、食管炎、结肠炎和脑膜脑炎等多器官脏器损害的各种临床综合征，是患者致死或移植术失败的重要原因。因此，早期快速检测HCMV活动性感染以便及时抗病毒药物治疗或及时终止妊娠是降低HCMV危害的重要措施之一。
目前，临床检测HCMV活动性感染常用的检测方法是：①ELISA检测HCMV特异抗体IgM的方法：优点是简便、快速、廉价、易于标准化和规模化操作。但因IgM产生早而消失快、仅感染早期可检测到，所以IgM检测HCMV活动性感染敏感性低，而且由于免疫功能低下者不能充分产生IgM，所以特异性也不够理想。②FQ-PCR检测HCMV核酸的方法：通过定量HCMVDNA判断是否为活动性感染，目前已代替因敏感性过高而被淘汰的普通PCR，尽管如此，由于定量标准不统一，需要配套的特殊设备，敏感性高而特异性较低等因素，FQ-PCR也不是最完美的检测方法。③荧光免疫组化或酶免疫组化检测HCMVPP65抗原的方法：PP65在不同HCMV病毒株中高度保守。HCMV一旦复制出被膜磷蛋白PP65就说明病毒完成复制、产生大量子代病毒并将导致临床疾病的发作，即HCMV的活动性感染。PP65在被膜蛋白中占95％以上，在受感染者外周白细胞中检出率极高，所以PP65是检测HCMV活动性感染既敏感又特异的指标。但是PP65抗原主要存在于白细胞核内，检测PP65抗原需要将白细胞固定在玻片上进行荧光免疫组化或酶免疫组化，显微镜下观察计数PP65抗原阳性细胞，不仅需要配备荧光显微镜或者配套输出设备和有经验的镜检人员，而且操作繁琐，每个样本需要一个玻片固定检测，难以规模化检测。为此，国内个别医院已将荧光免疫组化法改进为流式细胞术检测，但是仍然离不开流式细胞仪和有经验流式细胞术操作分析人员，难以普及到基层医院，也难以规模化批量检测。
发明内容
本发明的目的是解决现有HCMV活动性感染检测方法存在的敏感性或特异性低、费时费力、操作繁琐和使用不便的问题，提供一种简便、快速、准确、费用低廉、易于普及和标准化的检测人巨细胞病毒PP65抗原的ELISA方法。
为解决上述问题，本发明采用的技术方案是：
一种检测人巨细胞病毒PP65抗原的ELISA方法，其包括下列步骤：
1)制备检测试剂盒
所述检测试剂盒包括：包被有大鼠抗重组PP65322-561多克隆抗体(pAb)的酶标板、兔抗重组PP65322-561pAb、标准品、HRP-羊抗兔IgG、PBST洗涤液、底物显色液、终止液和封板膜；
①所述大鼠抗重组PP65pAb的酶标板的制备：
a)大鼠抗重组PP65pAb的制备：
免疫前大鼠断尾采血，分离血清，作为免疫血清效价检测的阴性对照；取70-170μg重组PP65抗原与等体积弗氏完全佐剂混合，振荡混匀制成乳化剂，皮下及皮内多位点免疫大鼠；免疫后14-21天，取70-130μg重组PP65抗原与等体积弗氏不完全佐剂混合，振荡混匀制成乳化剂，第二次皮下及皮内多位点免疫大鼠；第二次免疫后21-30天，与第二次方法一样再次加强免疫；第三次免疫后第10天，采血分离血清，ELISA检测抗体效价，证明免疫成功后，心脏采血获取免疫血清，饱和硫酸铵沉淀法纯化免疫血清，得到所需大鼠抗重组PP65的pAb；
b)包被酶标板：
包被液稀释大鼠抗重组PP65pAb为1︰2000，每孔50μl加入酶标板中，4℃包被过夜；弃去孔内的包被液，PBST洗2次，拍干，加封闭液每孔100μl，置37℃封闭2h，PBST洗3次，拍干；
②所述兔抗重组PP65pAb的制备：
免疫前兔耳缘静脉采血，分离血清，作为免疫血清效价检测的阴性对照；取100-300μg重组PP65抗原与等体积弗氏完全佐剂混合，振荡混匀制成乳化剂，皮下及皮内多位点免疫兔子；免疫后14-28天，取90-170μg重组PP65抗原与等体积弗氏不完全佐剂混合，振荡混匀制成乳化剂，第二次皮下及皮内多位点免疫兔子；第二次免疫后28-42天，与第二次方法一样再次加强免疫；第三次免疫后第10-20天，采血分离血清，ELISA检测抗体效价，证明免疫成功后，心脏采血获取免疫血清，饱和硫酸铵沉淀法纯化免疫血清，得到所需兔抗重组HCMVPP65的pAb；
③所述标准品为封闭液对倍稀释重组PP65抗原从640ng/ml至10ng/ml共7个标准浓度梯度，即640ng/ml、320ng/ml、160ng/ml、80ng/ml、40ng/ml、20ng/ml和10ng/ml；
2)制备待测标本
取待测新鲜抗凝血2-3ml，溶解破坏红细胞，150-250μl生理盐水悬浮白细胞，或调白细胞浓度为105/ml左右，冰浴环境下超声裂解白细胞释放核内抗原，或反复冻融裂解法释放核内抗原，离心取上清即为制备的待测标本；
3)检测人巨细胞病毒PP65抗原
a)将标准品各浓度PP65抗原和待测标本每孔50μl加入酶标板中，37℃孵育lh，PBST洗涤5次，拍干；
b)加兔抗重组PP65pAb，每孔50μl，37℃孵育1h，PBST洗涤5次，拍干；
c)加HRP-羊抗兔IgG，每孔50μl，37℃孵育1h，PBST洗涤5次，拍干；
d)加底物显色液，每孔100μl，室温避光显色15min；
e)加终止液，每孔50μl，酶标仪检测并记录OD450值，根据标准曲线，计算待测样本的PP65抗原含量。
所述大鼠抗重组PP65pAb和兔抗重组PP65pAb还能用针对重组PP65322-561抗原的任意两种多克隆抗体搭配或者与单克隆抗体搭配。
所述HRP-羊抗兔IgG还可以是碱性磷酸酶或生物素标记的羊抗兔IgG。
多克隆抗体(pAb)包含针对多种抗原表位的抗体成份，检测抗原的敏感性较单克隆抗体高，但容易发生非特异交叉反应，本发明采用多次反复免疫动物以获取高效价pAb(1:400000的兔pAb和1:20000的大鼠pAb)，并用免疫动物可能接触到的环境微生物(大肠杆菌、变形杆菌、葡萄球菌、链球菌)抗原与所得pAb反应，克服了其容易出现交叉反应的缺点。通过比较兔和大鼠pAb包被酶标板的检出特异性，选择了大鼠pAb为包被抗体，兔pAb为检测一抗。通过对单纯疱疹病毒Ⅰ型和Ⅱ型感染患者的白细胞抗原的检测，排除了本发明非特异结合单纯疱疹病毒抗原的可能，更肯定了试剂的特异性。
将101份临床疑似HCMV感染患者的血清分别用FQ-PCR试剂(中山医科大学达安基因中心)和IgM试剂(意大利DIA.PRO公司)检测，同时用本发明检测患者外周白细胞PP65抗原。三种检测方法检出的阳性例数分别是26例、18例和25例。检测结果的比较分别见表1和表2。
表1FQ-PCR检测HCMV-DNA与本发明检测HCMV-PP65抗原检测结果比较
本发明与HCMV-DNA(FQ-PCR)检测试剂的一致率为97.0％，两种试剂的检测结果无差异(P>0.05)。本发明相对于HCMV-DNA(FQ-PCR)试剂的敏感度为92.3％，特异度98.7％。但是HCMV-DNA(FQ-PCR)检测试剂存在着敏感性过高的问题，所以本发明实际敏感性和特异性可能更好。
表2.2ELISA检测HCMV-IgM与本发明检测HCMV-PP65抗原检测结果比较
本发明与进口IgM检测试剂的一致率为91.1％。本发明的检出阳性数为25例高于HCMV-IgM(ELISA)试剂的18例，由于HCMV-IgM(ELISA)试剂存在公认的敏感性低的问题，不适合做相对敏感性和特异性的比较。
本发明批内变异系数介于3.9-6.1％之间，批间介于4.2-6.7％之间，均小于常规10％的限制，说明检测体系稳定，有良好的重复性。本发明对PP65抗原的最低检出限是5ng/ml。
此外，利用本发明比较了1例使用免疫抑制剂的肾移植患者更昔洛韦治疗前与治疗2周后的HCMVPP65抗原的量，由194ng降低到15ng，证明了本发明具有监测药物疗效的作用。
因此，与背景技术相比，本发明具有较好的特异性、敏感性、稳定性和可重复性，不仅能够经济简便地早期快速检测HCMV活动性感染，而且易于普及基层医院，易于规模化和标准化操作，并能通过对PP65抗原的定量，判断疾病的预后和进展以及抗病毒药物的疗效。
附图说明
附图为本发明的间接双抗体夹心ELISA的标准曲线图。
具体实施方式
本实施例中的一种检测人巨细胞病毒PP65抗原的ELISA方法，其步骤如下：
1)制备检测试剂盒
所述检测试剂盒包括：包被有SD大鼠抗重组PP65322-561多克隆抗体(pAb)的酶标板、新西兰大耳白兔抗重组PP65322-561pAb、标准品、HRP-羊抗兔IgG、PBST洗涤液、底物显色液、终止液和封板膜；上述抗重组PP65322-561pAb中的PP65322-561以下简称重组PP65；
①所述SD大鼠抗重组PP65pAb的酶标板的制备：
a)SD大鼠抗重组PP65pAb的制备：
免疫前SD大鼠断尾采血约0.5ml，37℃放置30min，再转入4℃待血块完全收缩，3000rmp离心10min，分离血清，56℃水浴30min灭活补体，-20℃保存，作为免疫血清效价检测的阴性对照；取200μg/ml的重组PP65抗原150μg(0.75ml)与等体积弗氏完全佐剂混合，吸入5ml注射器中，振荡混匀制成乳化剂，背部皮下及皮内免疫SD大鼠，皮下注射乳化抗原300μl/位点，皮内注射70μl/位点，每只SD大鼠皮内及皮下各注射4个位点；第15天取100μg重组PP65抗原与等体积弗氏不完全佐剂混合，制成乳化剂后，第二次皮下及皮内各4个位点免疫SD大鼠，皮下注射乳化抗原200μl/位点，皮内注射40μl/位点；第二次免疫后28天，采用与第二次免疫相同的方法再次加强免疫；第三次免疫后第10天，采血分离血清，ELISA检测抗体效价，效价为1︰20000，证明免疫成功，心脏采血获取免疫血清，饱和硫酸铵沉淀法纯化免疫血清，得到所需SD大鼠抗重组PP65的pAb；
b)包被酶标板：
包被液稀释SD大鼠抗重组PP65pAb为1︰2000，每孔50μl加入酶标板中，4℃包被过夜；弃去孔内的包被液，PBST洗2次，每次放置30秒，拍干，加封闭液每孔100μl，置37℃封闭2h，PBST洗3次，每次放置30秒，拍干；
②所述新西兰大耳白兔抗重组PP65pAb的制备：
免疫前兔耳缘静脉采血1ml，37℃放置30min，再转入4℃放置待其血块完全收缩，3000rmp离心10min，分离血清，再用56℃水浴30min灭活补体，置于-20℃保存，作为免疫血清效价检测的阴性对照；取200μg/ml的重组PP65抗原1ml(200μg)与等体积弗氏完全佐剂混合，吸入5ml的容器中，振荡混匀制成乳化剂，皮下及皮内多位点背部免疫已被皮的新西兰大耳白兔，皮下注射400μl/位点，皮内注射50μl/位点，每只新西兰大耳白兔背部皮内及皮下各注射4个位点；第15天取150μg重组PP65抗原与等体积弗氏不完全佐剂混合，制成乳化剂，第二次背部皮下及皮内各4个位点免疫新西兰大耳白兔，第二次免疫后29天，采用与第二次免疫相同的方法再次加强免疫；第三次免疫后第15天，采血分离血清，ELISA检测抗体效价，效价为1︰400000，证明免疫成功，心脏采血获取免疫血清，饱和硫酸铵沉淀法纯化免疫血清，得到所需新西兰大耳白兔抗重组HCMVPP65的pAb；
③所述标准品为封闭液对倍稀释重组PP65抗原从640ng/ml至10ng/ml共7个标准浓度梯度，即640ng/ml、320ng/ml、160ng/ml、80ng/ml、40ng/ml、20ng/ml和10ng/ml；
④所述PBST洗涤液的配方为：0.15MKH2PO40.2g，Na2HPO4·12H2O2.9g，NaCl8.0g，KCl0.2g，0.05％Tween-200.5ml，加蒸馏水至40ml；
⑤所述底物显色液的配方为：
底物显色液是在临用前由底物液A和底物缓冲液B各一半混合而成。底物液A主要成份是四甲基联苯胺(TMB)：TMB20mg，无水乙醇10ml，加蒸馏水至100ml；底物缓冲液B主要成份是PH5.0的磷酸柠檬酸：0.2MNa2HPO425.7ml，0.1M柠檬酸24.3ml，加蒸馏水至100ml；
⑥所述终止液的配方为：
终止液主要成份是2MH2SO4：蒸馏水178.3ml，逐滴加入98％的浓硫酸21.7ml；
⑦所述封板膜为与酶标板的板面大小一致的透明塑料膜；
⑧建立双抗夹心ELISA标准曲线：
a)将PBST用蒸馏水稀释25倍，混匀，用于洗涤；
b)加样：封闭液对倍稀释重组PP65抗原从640ng/ml至10ng/ml共7个标准浓度梯度，即640ng/ml、320ng/ml、160ng/ml、80ng/ml、40ng/ml、20ng/ml和10ng/ml，每孔50μl分别加样，每个样品重复做两个复孔；同时设立封闭液阴性对照2孔，空白对照1孔，37℃孵育lh，PBST洗涤5次，每次放置30秒，拍干；
c)加一抗：每孔加1︰20000的新西兰大耳白兔抗重组PP65pAb50μl，37℃孵育1h，PBST洗涤5次，每次放置30秒，拍干；
d)加酶标抗体：每孔加HRP-羊抗兔IgG50μl，37℃孵育1h，PBST洗涤5次，每次放置30秒，拍干；
e)每孔加底物显色液100μl，室温避光显色15min；
f)加终止液：每孔加终止液50μl，读板，记录OD450值，建立标准曲线，见附图；
2)制备待测标本
取待测新鲜抗凝血2ml，溶解破坏红细胞，200μl生理盐水悬浮白细胞，冰浴环境下超声裂解释放白细胞核内抗原，离心取上清即为制备的待测标本，其具体的制备过程为：
①取待测新鲜抗凝血2ml于15ml离心管中；
②加蒸馏水至15ml，上下翻转混匀10次，静置5min，常温3000rpm离心15min，彻底弃上清；
③加蒸馏水2ml悬浮细胞；
④重复步骤②和③，直至彻底破坏红细胞；
⑤加生理盐水200μl悬浮白细胞，转移白细胞至1.5mlEP管；
⑥在冰浴环境下超声裂解提取白细胞核内抗原，每超声2s间隔2s，共超声99次；
⑦4℃下3000rpm离心15min，转移上清液至EP管即为制备的待测标本；
3)检测人巨细胞病毒PP65抗原
a)将标准品各浓度PP65抗原和待测标本每孔50μl加入酶标板中，37℃孵育lh，PBST洗涤5次，每次放置30秒，拍干；
b)加兔抗重组PP65pAb，每孔50μl，37℃孵育1h，PBST洗涤5次，每次放置30秒，拍干；
c)加HRP-羊抗兔IgG，每孔50μl，37℃孵育1h，PBST洗涤5次，每次放置30秒，拍干；
d)加底物显色液，每孔100μl，室温避光显色15min；
e)加终止液，每孔50μl，以空白孔调零，酶标仪检测450nm波长处吸光度值，即OD450值，根据标准曲线，计算待测样本的PP65抗原含量。
上述实施例中所述的SD大鼠抗重组PP65pAb和新西兰大耳白兔抗重组PP65pAb还能用针对重组PP65322-561抗原的任意两种多克隆抗体搭配或者与单克隆抗体搭配。
上述实施例中所述HRP-羊抗兔IgG还可以是碱性磷酸酶或生物素标记的羊抗兔IgG。
本发明的保护范围不受以上实施例具体数值的限制。
Claims (1)
1.一种非诊断目的检测人巨细胞病毒PP65抗原的ELISA方法，其特征是：包括下列步骤：
1)制备检测试剂盒
所述检测试剂盒包括：包被有大鼠抗重组PP65322-561多克隆抗体(pAb)的酶标板、兔抗重组PP65322-561pAb、标准品、HRP-羊抗兔IgG、PBST洗涤液、底物显色液、终止液和封板膜；
①所述包被有大鼠抗重组PP65322-561pAb的酶标板的制备：
a)大鼠抗重组PP65322-561pAb的制备：
免疫前大鼠断尾采血，分离血清，作为免疫血清效价检测的阴性对照；取70-170μg重组PP65322-561抗原与等体积弗氏完全佐剂混合，振荡混匀制成乳化剂，皮下及皮内多位点免疫大鼠；免疫后14-21天，取70-130μg重组PP65322-561抗原与等体积弗氏不完全佐剂混合，振荡混匀制成乳化剂，第二次皮下及皮内多位点免疫大鼠；第二次免疫后21-30天，与第二次方法一样再次加强免疫；第三次免疫后第10天，采血分离血清，ELISA检测抗体效价，证明免疫成功后，心脏采血获取免疫血清，饱和硫酸铵沉淀法纯化免疫血清，得到所需大鼠抗重组PP65322-561的pAb；
b)包被酶标板：
包被液稀释大鼠抗重组PP65322-561pAb为1︰2000，每孔50μl加入酶标板中，4℃包被过夜；弃去孔内的包被液，PBST洗2次，拍干，加封闭液每孔100μl，置37℃封闭2h，PBST洗3次，拍干；
②所述兔抗重组PP65322-561pAb的制备：
免疫前兔耳缘静脉采血，分离血清，作为免疫血清效价检测的阴性对照；取100-300μg重组PP65322-561抗原与等体积弗氏完全佐剂混合，振荡混匀制成乳化剂，皮下及皮内多位点免疫兔子；免疫后14-28天，取90-170μg重组PP65322-561抗原与等体积弗氏不完全佐剂混合，振荡混匀制成乳化剂，第二次皮下及皮内多位点免疫兔子；第二次免疫后28-42天，与第二次方法一样再次加强免疫；第三次免疫后第10-20天，采血分离血清，ELISA检测抗体效价，证明免疫成功后，心脏采血获取免疫血清，饱和硫酸铵沉淀法纯化免疫血清，得到所需兔抗重组HCMVPP65322-561的pAb；
③所述标准品为封闭液对倍稀释重组PP65322-561抗原从640ng/ml至10ng/ml共7个标准浓度梯度，即640ng/ml、320ng/ml、160ng/ml、80ng/ml、40ng/ml、20ng/ml和10ng/ml；
2)制备待测标本
取待测新鲜抗凝血2-3ml，溶解破坏红细胞，150-250μl生理盐水悬浮白细胞，调白细胞浓度为105/ml左右，冰浴环境下超声裂解白细胞释放核内抗原，或采用反复冻融裂解法释放核内抗原，离心取上清即为制备的待测标本；
3)检测人巨细胞病毒PP65抗原
a)将标准品各浓度PP65322-561抗原和待测标本每孔50μl加入酶标板中，37℃孵育lh，PBST洗涤5次，拍干；
b)加兔抗重组PP65322-561pAb，每孔50μl，37℃孵育1h，PBST洗涤5次，拍干；
c)加HRP-羊抗兔IgG，每孔50μl，37℃孵育1h，PBST洗涤5次，拍干；
d)加底物显色液，每孔100μl，室温避光显色15min；
e)加终止液，每孔50μl，酶标仪检测并记录OD450值，根据标准曲线，计算待测样本的PP65抗原含量。
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| CN103276008A (zh) * | 2013-04-25 | 2013-09-04 | 湖南师范大学 | HCMV pp65原核表达重组菌株及其构建方法 |
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| CN1651918A (zh) * | 2005-01-08 | 2005-08-10 | 王明丽 | 重组抗原pp65包被酶标反应板的制法及ELISA检测试剂盒 |
| CN101261272A (zh) * | 2007-03-09 | 2008-09-10 | 天津市秀鹏生物技术开发有限公司 | Hcmvpp65抗原血症间接免疫荧光法检测试剂盒 |
| CN103276008A (zh) * | 2013-04-25 | 2013-09-04 | 湖南师范大学 | HCMV pp65原核表达重组菌株及其构建方法 |
Non-Patent Citations (3)
| Title |
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| Comparison of Seven Enzyme Immunoassay Systems for Measurement of Cytomegalovirus;ROBERT H. YOLKEN 等;《JOURNAL OF CLINICAL MICROBIOLOGY》;19800630;第11卷(第6期);第546页左栏第2段-第548页右栏倒数第4段 * |
| HCMV pp65在原核细胞中的表达、纯化及其抗血清的制备;王兴满 等;《安徽医科大学学报》;20101231;第45卷(第6期);1.7兔抗HCMVpp65蛋白血清的制备 * |
| 人巨细胞病毒及功能鉴定PP65322~56l片段的原核表达及功能鉴定;李娜 等;《国际病毒学杂志》;20100228;第17卷(第1期);摘要，第1页左栏第1段 * |
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