CN104749372B - H9n2流感病毒血凝素蛋白elisa试剂盒 - Google Patents
H9n2流感病毒血凝素蛋白elisa试剂盒 Download PDFInfo
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Abstract
本发明公开了一种甲型H9N2流感病毒血凝素蛋白双抗体夹心ELISA试剂盒。该试剂盒包含有包被单克隆抗体的固相载体，辣根过氧化物酶标记的兔多克隆抗体、H9N2血凝素蛋白标准品、样品稀释液、洗涤液、底物显色液和反应终止液，不但灵敏度好，可以对H9N2流感病毒血凝素蛋白进行定量检测，而且特异性识别甲型H9N2流感病毒，与甲型流感病毒的其它主要亚型包括H1N1，H2N2，H3N2，H5N1和H7N7以及乙型流感病毒的血凝素蛋白无交叉反应。试剂盒操作简单，能够同时快速检测大批样本，既可以用于支持H9N2流感病毒的基础研究，同时，对于开展流感病毒的流行病学研究具有重要意义。
Description
技术领域
本发明属于免疫学领域，具体涉及H9N2流感病毒血凝素蛋白特异性单克隆抗体制备，以及一种定量H9N2流感病毒血凝素蛋白双抗夹心ELISA检测试剂盒。
背景技术
禽流感是一种由禽流感病毒引起的疾病，所表现的临床症状随病毒亚型不同而异。至今已发现的甲型流感病毒的血凝素16个亚型(H1～H16)和神经氨酸酶9个亚型(N1～N9)均能从禽中分离到，但它们并不是都能引起禽流感。目前发现能感染人的禽流感病毒主要有：H5N1、H9N2、H7N7、H7N2、H7N3和H7N9亚型。
H9N2是一种禽流感病毒，属于正黏病毒科A型流感病毒属。1966年HoMee从患温和呼吸道病的火鸡中分离到第一株H9N2亚型禽流感病毒，之后H9N2亚型禽流感在世界范围内迅速流行，尤其是1994～1999年在世界范围内造成了巨大的经济损失。近几年，对我国部分省市鸡场进行的禽流感血清学调查中发现，221个禽流感阳性鸡群中H9亚型阳性鸡群占阳性群总数的93.67％，证实了H9N2亚型禽流感在我国广泛存在，是当前禽流感流行的主要亚型。研究表明，近年来H9N2亚型部分流行株的致病力明显变异，尤其对肉仔鸡，死亡率可达到30％以上；对产蛋高峰期的蛋鸡，其产蛋下降幅度大，恢复困难，因此对养禽业有巨大危害。
H9N2病毒在人类身上发现比较罕见，可以感染人但一般不致人死亡，主要传染源为禽类，在人群中不易传播。1998年广东省的流感监测系统在韶关、汕头市分别发现4例和5例H9N2禽流感病毒感染病例，为全球首次发现人H9N2感染病例。以前认为禽流感和人是有种属屏障的，但近几年的禽流感感染人事件表明，这种屏障正在减弱或消失看，而且禽流感不仅在鸡、鸭、鹅、候鸟等禽类上发生，目前已经开始波及到猪、老虎、海狮等哺乳类动物，因此潜在危害性不断加大。
H9N2禽流感病毒亚型毒性虽然弱于H5N1亚型，人群发病率低，但近年来，随着传染源愈来愈多，感染谱逐渐扩大，以及非典型性禽流感流行增多，给禽流感的消灭和控制带来了巨大困难，H9N2发生变异导致致病性增加和人际传播，引发严重流感疫情的可能性一直存在，因此人类在高度警惕H5N1的同时，也不能忽视H9N2的威胁。
综上所述，H9N2不但严重危害养殖业的发展，其公共卫生意义也日渐显著。对H9N2的检测，在减少世界经济损失和提高人类卫生健康方面，都具有深远的意义。目前H9N2还是以基于核酸检测的定量PCR方法为主，这种方法对环境，样品以及仪器要求较高，需要专业人员操作，不易推广。另外，现有的基于ELISA原理的H9N2检测试剂，缺点是无法进行定量检测，而且针对不同流感病毒亚型的检测特异性差，因此亟需一种操作简单，灵敏度好，特异性高的定量检测试剂。
发明内容
本发明的目的是提供一种检测灵敏度高、准确性强、低成本的H9N2流感病毒血凝素蛋白双抗夹心法检测试剂盒。
本发明所提供的检测试剂盒包括包含有包被单克隆抗体的固相载体，酶标检测多克隆抗体以及蛋白标准品、样品稀释液、洗涤液、底物显色液和反应终止液。
其中所述包被单克隆抗体为鼠单克隆抗体，其轻链和重链氨基酸序列分别为SEQID NO：1和SEQ ID NO：2；所述酶标检测多克隆抗体为辣根过氧化物酶(Horseradishperoxidase，HRP)标记的兔多克隆抗体；所述蛋白标准品为重组H9N2流感病毒血凝素蛋白。
所述鼠单克隆抗体采用重组H9N2流感病毒血凝素蛋白免疫动物，然后选择高血清效价小鼠通过经典杂交瘤技术制备获得；所述酶标兔多克隆抗体采用H9N2流感病毒血凝素蛋白免疫动物，通过蛋白A纯化和抗原亲和纯化技术制备纯化的多克隆抗体，然后按照常规方法利用HRP标记抗体获得。
本发明的技术方案为优选具有高灵敏度、高特异性的单克隆抗体与酶标多克隆抗体配对组合，将单克隆抗体作为包被抗体吸附于固相载体上，包被抗体可以特异性的捕获样本中的H9N2流感病毒血凝素蛋白以及蛋白标准品，加入酶标检测抗体后，形成包被抗体、抗原、检测抗体复合物，相应底物显色后终止，读取样本吸光值，通过与标准曲线比较即可得出样本中H9N2流感血凝素蛋白的含量。
血凝素蛋白是流感病毒感染宿主细胞的关键功能蛋白，同时，也是流感治疗性药物及疫苗开发的重要靶点，本发明采用双抗夹心法，能够定性或定量检测样本中的甲型H9N2流感血凝素蛋白水平，检验方法方便易行，检测灵敏度和准确度高、特异性强、能够同时快速检测大批样本，作为试剂盒关键组成部分的包被和检测抗体均为自主研发，因此成本低，可靠性强，易溯源，预计将在H9N2的相关基础和临床研究中发挥重要作用。
附图说明
图1.H9N2流感病毒血凝素蛋白ELISA试剂盒标准曲线图
具体实施方式
以下结合具体实施例对本发明做进一步的描述和说明。
实施例1H9N2流感血凝素蛋白ELISA试剂盒的组分制备
1.小鼠单克隆抗体的制备：
1)动物免疫
采用Balb/c小鼠作为免疫动物，以义翘神州生物技术有限公司生产的重组H9N2流感血凝素蛋白(货号：11229-V08H)为免疫原，免疫剂量为每次每只小鼠免疫50μg的蛋白。首次免疫时将免疫原与等量的完全弗氏佐剂制成乳化剂，腹部皮下多点注射，间隔2～3周后取相同剂量免疫原与等量不完全弗氏佐剂制成乳化剂，加强免疫两次，三次免疫后使用间接ELISA法测定血清效价，血清效价达到1∶16000以后，选择效价最高的小鼠腹腔加强免疫一次，4天后取脾细胞进行细胞融合。
2)细胞融合和克隆化
取小鼠脾脏，研磨过滤后离心获得单个脾细胞悬液，细胞计数后，按5∶1或10∶1的比例与处于对数生长期的SP2/0小鼠骨髓瘤细胞混合，采用聚乙二醇(PEG)法进行细胞融合，铺板，通过选择培养基的作用，骨髓瘤细胞和脾细胞等未融合或未有效融合的细胞将无法生长，而有效融合的杂交瘤细胞将在培养孔内生长、增殖，并分泌抗体。三次换液后，融合后第9-12天取细胞培养上清，以重组H9N2流感血凝素蛋白作为包被抗原，利用间接ELISA法测定上清液，筛选阳性孔，并通过有限稀释法对阳性细胞进行克隆化培养，直至得到稳定分泌特异性抗体的单克隆杂交瘤细胞株。以上细胞融合和克隆化方法均为免疫学单克隆抗体技术中的常用经典方法。
3)单克隆抗体的生产与纯化
选取杂交瘤细胞株，利用培养瓶或生物反应器进行细胞培养，收集细胞培养上清，利用常规蛋白A亲和层析柱进行纯化，获得的抗体通过SDS-PAGE电泳和间接ELISA法鉴定抗体纯度和特异性后分装，于-20℃低温保存备用。
4)单克隆抗体的序列测定
杂交瘤细胞长期保存可能由于多次传代后不稳定以及污染问题导致阳性克隆丢失，为解决上述问题，在本发明过程中，利用分子生物学技术分别提取了阳性克隆的抗体轻链和重链基因，进行了序列测定，包含有抗体基因的质粒可以在-20℃条件下长期稳定保存，同时，根据抗体基因序列，本专业领域内的技术人员可以按照常规分子生物学方法克隆表达获得相同的单克隆抗体。
抗体基因提取具体方法如下：收集生长状态良好的杂交瘤细胞，按BBI公司的classical total RNA isolation kit说明书的操作方案提取杂交瘤细胞总RNA，通过电泳检测质量，UV测定浓度。按BBI公司的MMLV first strand cDNA synthesis kit说明书的操作方案将mRNA反转录为cDNA，-20℃冻存备用。反转录反应体系为：11μl RNA(2.7μg)，5×reaction buffer4μl，RNase inhibitor(20U/μl)1μl，dNTP mix(10mmol/L)2μl，M-MuLVreverse transcriptase1μl，总反应体积为20μl。根据参考文献设计引物，以cDNA为模板分别PCR获得抗体的轻链和重链片段，反应体系为：2.0μl10×pyrobest buffer，1.6μl2.5mMdNTP，1.4μl10μM引物对，0.4μl杂交瘤细胞cDNA，0.2μl5U/μl Pyrobest DNA Polymerase，反应体系为20μl。扩增条件：变性94℃4min；变性94℃1min，退火58℃1min，延伸72℃1min，30个循环；延伸72℃5min。将轻链和重链片段插入到pcDNA3T载体上，构建pcDNA3-anti-H9N2-L和pcDNA3-anti-H9N2-H载体。将载体转化大肠杆菌，挑取阳性克隆，进行测序鉴定，分析测序结果，获得正确的轻链和重链氨基酸序列。测定结果及序列分析显示该抗体的轻链氨基酸序列为SEQ ID NO：1；重链氨基酸序列为SEQ ID NO：2，详见序列表。具体实验操作参考本领域技术人员熟知的【萨幕布鲁克J等，《分子克隆试验指南》，北京科学出版社】。
2.兔多克隆抗体的制备：
选取新西兰兔作为免疫动物，以重组H9N2流感血凝素蛋白为免疫原进行免疫，免疫剂量为每只兔子每次免疫500μg的蛋白。首次免疫将免疫原与等量的完全弗氏佐剂制成乳化剂，颈背部皮下多点注射，间隔2～3周取相同剂量免疫原与等量不完全弗氏佐剂制成乳化剂，加强免疫，共免疫4-5次，间接ELISA法测定血清效价达到1∶25000以后，心脏取血，通过常规蛋白A亲和层析柱和血凝素蛋白抗原亲和柱两步纯化后得到纯化的多克隆抗体，分装，于-20℃低温保存用于酶标抗体的制备。其中血凝素蛋白抗原亲和纯化具体步骤如下：
a)称0.7g溴化氰活化的琼脂糖凝胶(GE公司)，用1mM HCl溶胀，然后用1mM HCl洗三次，备用；
b)取2mg的血凝素蛋白利用超滤方法将蛋白缓冲液置换成0.1M NaHCO3，pH8.3，并控制体积为1～2ml；
c)将蛋白溶液加入到步骤a)洗涤好的活化琼脂糖凝胶中，室温摇4h；
d)用pH8.0，0.1M的Tris缓冲液封闭未反应基团；
e)装重力柱，柱床体积约2ml，用PBS冲洗，并平衡；
取蛋白A纯化的多抗过柱，用PBS冲洗未结合抗体，用pH3.0柠檬酸缓冲液洗脱特异结合的抗体，用Tris缓冲液中和洗脱液到pH7.0-7.5。
3.酶标抗体的制备：
a)称取5mg的HRP溶解于0.5mL蒸馏水中；
b)加入0.5mL新配的0.1M的NaIO4溶液，室温下避光搅拌20分钟；
c)将上述溶液装入透析袋中，1mM的醋酸钠缓冲液(pH4.4)透析4℃过夜；
d)取5mg亲和纯化的多克隆抗体，加入到1mL0.01M碳酸盐缓冲液中，备用；
e)向c)液中加入0.2M碳酸盐缓冲液(pH9.5)，使pH升高到9.0～9.5，然后立即加入d)液中，室温避光轻轻搅拌2小时；
f)加0.2mL现配的4mg/mL NaBH4液，混匀，于4℃放置2小时；
将上述液装入透析袋中，于pH7.4，0.15M的PBS透析，4℃过夜。
实施例2H9N2流感血凝素蛋白ELISA试剂盒的组建
组建的ELISA试剂盒，包含以下试剂：
a)小鼠单克隆包被抗体；
b)HRP标记的兔多克隆抗体；
c)H9N2流感血凝素蛋白标准品；
d)包被缓冲液：pH9.6，0.05mol/L的碳酸盐缓冲液；
e)封闭液：含有2％牛血清白蛋白的Tris缓冲液；
f)样品稀释液：含有0.1％牛血清白蛋白的磷酸盐缓冲液；
g)洗涤液：含有0.1％吐温的磷酸盐缓冲液；
h)底物显色液：由显色液A和显色液B组成，显色液A为过氧化氢或过氧化脲，显色液B为四甲基联苯；
i)终止液：2mol/L的硫酸
实施例3H9N2流感血凝素蛋白ELISA试剂盒的制备
1.利用正交试验摸索酶联免疫试剂盒的最佳抗体组合及工作浓度
按照紫外分光光度计法，测定抗体及抗原的浓度。采用正交试验方法，摸索最佳抗体组合以及最佳抗体使用浓度，将不同抗H9N2流感血凝素蛋白单克隆抗体稀释至浓度为4μg/ml、2μg/ml、1μg/ml，重组血凝素蛋白浓度稀释至1000pg/ml、100pg/ml、0pg/ml，HRP标记的兔多克隆抗体稀释浓度至4μg/ml、2μg/ml、1μg/ml、0.5μg/ml。综合考虑空白孔背景及阳性实验孔的光吸收值，优选出一株鼠单克隆抗体作为包被抗体，并确认其最佳工作浓度为2μg/ml，HRP标记多克隆抗体的工作浓度为1μg/ml。
2.试剂盒的批量制备
1)包被酶标板：用碳酸盐包被缓冲液(称取3.18g Na2CO3，5.86g NaHCO3，1gNa2N3，定容至2L的去离子水中，调pH值为9.6)，将抗H9N2流感血凝素蛋白单克隆抗体稀释至浓度为2μg/ml，100μL/孔，包被酶标板，4℃孵育过夜，用洗涤液洗板1次，200μL/孔。
2)封闭：每孔加入300μL封闭液封闭非特异性结合位点，室温孵育1小时；然后用洗涤液洗板2次，200μL/孔；拍干后用真空包装机包装，4℃保存备用。
3)蛋白标准品的制备：用样品稀释液稀释H9N2流感血凝素蛋白，分装后冻干，4℃保存备用。
4)酶标抗体的制备：将亲和纯化后的兔多克隆抗体用HRP标记，加入50％甘油，分装后-20℃保存备用。
实施例4ELISA试剂盒包被抗体检测限测定
a)将H9N2流感血凝素蛋白用碳酸盐包被缓冲液稀释成，1.56ng/mL，0.78ng/mL，0.39ng/mL，0.195ng/mL，0.0975ng/mL，0.049ng/mL，0.0245ng/mL，100μl/孔，包被酶标板，4℃孵育过夜，用洗涤液洗板1次，200μL/孔；
b)每孔加入300μL封闭液封闭非特异性结合位点，室温孵育1小时；然后用洗涤液洗板2次，200μL/孔；
c)将抗H9N2流感血凝素蛋白鼠单克隆抗体用样本稀释液稀释至1ug/ml，加入预先包被和封闭好的酶标板，200μL/孔，室温孵育1小时；然后用洗涤液洗板3次，200μL/孔；
d)加入HRP标记的抗小鼠IgG二抗，200μL/孔，室温孵育1小时；然后用洗涤液洗板3次，200μL/孔；
e)加入200μL新鲜配置的TMB底物，室温避光显色20分钟，加入50μL2mol/L硫酸终止反应，酶标仪450nm波长下读值。
f)以各样本组吸光值的平均值减去3倍标准差的数值与空白对照组(用不含有重组蛋白的包被缓冲液包被酶标板，其它步骤同实验组)吸光值平均值加上3倍标准差的数值相比，取样本组数值大于空白对照组数值条件下的最低蛋白包被浓度为抗体的检测限。结果显示抗体的检测限能够达到0.195ng/ml(表1)，说明抗体有较高的检测灵敏度。
表1包被抗体检测限测定
| OD-1 | OD-2 | OD-3 | OD-4 | 平均值 | 标准差SD | 平均值+3sD | 平均值-3SD | 检测限(ng/ml) | |
| 空白对照 | 0.043 | 0.041 | 0.042 | 0.042 | 0.001 | 0.045 | |||
| 0.0245ng/mL | 0.042 | 0.042 | 0.041 | 0.042 | 0.042 | 0.001 | 0.039 | ||
| 0.049ng/mL | 0.041 | 0.038 | 0.042 | 0.041 | 0.041 | 0.002 | 0.035 | ||
| 0.0975ng/mL | 0.045 | 0.044 | 0.047 | 0.05 | 0.047 | 0.003 | 0.038 | ||
| 0.195ng/mL | 0.053 | 0.052 | 0.056 | 0.059 | 0.055 | 0.003 | 0.046 | 0.195 | |
| 0.39ng/mL | 0.069 | 0.069 | 0.067 | 0.065 | 0.068 | 0.002 | 0.062 | ||
| 0.78ng/mL | 0.094 | 0.096 | 0.094 | 0.094 | 0.095 | 0.001 | 0.092 | ||
| 1.56ng/mL | 0.175 | 0.176 | 0.165 | 0.167 | 0.171 | 0.006 | 0.153 |
实施例5H9N2流感血凝素蛋白ELISA试剂盒的特异性测定
将不同流感病毒亚型的重组血凝素蛋白稀释至50ng/ml，用H9N2流感血凝素蛋白ELISA试剂盒测试，结果显示试剂盒特异性的识别甲型H9N2流感病毒(A/Hong Kong/1073/1999)的血凝素蛋白，与其它甲型流感病毒的主要亚型包括H1N1，H1N2，H3N2，H5N1和H7N7检测毒株的血凝素蛋白以及乙型流感病毒(Influenza B/Florida/4/2006)的血凝素蛋白无交叉反应。证明试剂盒特异性好，可以用于支持H9N2流感的流行病学研究，而且对于流感病毒的基础研究也具有重要的实际应用价值。
表2H9N2流感血凝素蛋白ELISA试剂盒的特异性测定
| 血凝素蛋白的亚型与毒株 | OD450nm值 |
| Influenza B/Florida/4/2006 | 0.007 |
| H1N1(A/Brisbane/59/2007) | 0.01 |
| H2N2(A/Canada/720/2005) | 0.003 |
| H3N2(A/Brisbane/10/2007) | 0.016 |
| H5N1(A/Anhui/1/2005) | 0.011 |
| H5N1(A/bar-headed goose/Qinghai/14/2008) | 0.011 |
| H5N1(A/Vietnam/1194/2004) | 0.003 |
| H5N1(A/Indonesia/5/2005) | 0.012 |
| H5N1(A/turkey/Turkey/1/2005) | 0.007 |
| H7N7(A/Netherlands/219/03) | 0.003 |
| H9N2(A/Hong Kong/1073/1999) | 1.692 |
实施例6H9N2流感血凝素蛋白ELISA试剂盒的检测步骤
1.加样
1)取出包被好的酶标板及冻干的标准品，加1ml样品稀释液将标准品溶解。室温下放置20分钟。将标准品从20ng/ml起，做2倍的倍比稀释，共7个点，将稀释液各取100μl加入96孔酶标板中。
2)取待测样本，100μl/孔加入反应孔中，室温下孵育2小时；
3)洗涤液洗板3次，200μL/孔，拍干酶标板。
2.加入检测抗体
将HRP标记抗体用样品稀释液稀释至1μg/mL，加入反应孔中，100μL/孔，室温下孵育1小时，洗涤液洗板3次，200μL/孔，拍干酶标板。
3.显色
1)加入200μL新鲜配制的底物显色液，室温反应20分钟，然后加入50μL终止液终止反应；
2)酶标仪450nm波长下读取吸光值。
4.标准曲线的建立
以标准品的浓度为横坐标，OD值为纵坐标建立标准曲线(图1)，根据测得的样品OD值可计算获得样品中的H9N2血凝素蛋白的含量。
Claims (5)
1.一种检测H9N2流感病毒血凝素蛋白的ELISA试剂盒，其包括：
1)包被H9N2流感病毒血凝素蛋白单克隆抗体的酶标板；
2)酶标记的H9N2流感病毒血凝素蛋白多克隆抗体；
其中，用于包被酶标板的H9N2流感病毒血凝素蛋白单克隆抗体的轻链和重链氨基酸序列分别为SEQ ID NO：1和SEQ ID NO：2；所述酶标记的H9N2流感病毒血凝素蛋白多克隆抗体为辣根过氧化物酶标记的兔多克隆抗体。
2.如权利要求1所述的试剂盒，其特征在于，还包括以下试剂：H9N2血凝素蛋白标准品、样品稀释液、洗涤液、底物显色液和反应终止液，其中，所述H9N2血凝素蛋白标准品为重组表达的血凝素蛋白；所述样品稀释液为含有0.1％牛血清白蛋白的磷酸盐缓冲液；所述洗涤液为含有0.1％吐温的磷酸盐缓冲液；所述底物显色液由显色液A和显色液B组成，显色液A为过氧化氢或过氧化脲，显色液B为四甲基联苯胺；所述反应终止液为2mol/L的硫酸。
3.如权利要求1所述的试剂盒，其特征在于，辣根过氧化物酶标记的抗H9N2流感病毒血凝素蛋白兔多克隆抗体的制备过程如下：用重组H9N2流感病毒血凝素蛋白免疫新西兰兔0.5mg/只/次，皮下多点免疫，间隔2-3周，共免疫四次，心脏取血，经蛋白A和H9N2血凝素蛋白抗原亲和纯化后得到纯化的多克隆抗体，再用辣根过氧化物酶进行标记获得辣根过氧化物酶标记的抗H9N2流感病毒血凝素蛋白兔多克隆抗体。
4.如权利要求2所述的试剂盒，其特征在于，采用重组H9N2流感病毒血凝素蛋白作为标准品，对H9N2流感病毒血凝素蛋白进行定量检测。
5.如权利要求1所述的试剂盒，其特征在于，特异性的识别甲型H9N2流感病毒血凝素蛋白，与甲型流感病毒的其它主要亚型以及乙型流感病毒的血凝素蛋白无交叉反应，其中，甲型流感病毒的其它主要亚型包括H1N1，H2N2，H3N2，H5N1和H7N7。
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