CN116284264A - 用于检测1型rhdv抗体的多肽、试剂盒及方法 - Google Patents
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Abstract
本发明公开了一种用于检测1型RHDV抗体的多肽,其氨基酸序列如SEQ ID NO.1所示。所述多肽在制备检测1型RHDV抗体的试剂盒中的应用,在检测1型RHDV抗体中的应用。本发明还公开了一种检测1型RHDV抗体的试剂盒,包括用于检测1型RHDV抗体的多肽。所述检测1型RHDV抗体的试剂盒在检测1型RHDV抗体中的应用。本发明的用于检测1型RHDV抗体的多肽,对于1型RHDV抗体的特异性高,安全性好,具有良好的抗原性。本发明的检测1型RHDV抗体的试剂盒,适用于间接ELISA检测法,可对1型RHDV抗体水平作出评价,具有很高的特异性和敏感性,适合批量样品的检测。
Description
技术领域
本发明涉及用于检测1型RHDV抗体的多肽、试剂盒及方法,属于抗体检测技术领域。
背景技术
兔病毒性出血症(viral haemorrhagic disease of rabbits)又名兔出血性肺炎、兔出血症或兔瘟,是由兔出血症病毒(rabbit haemorrhagic disease virus,RHDV)感染所致的兔的一种急性、败血性、高度接触传染性、致死性和以全身实质器官出血为主要特征的传染病。很多国家及地区均报道有该病原的存在,给养兔业带来严重的经济损失。目前国内发生高致死率的RHDV已有两血清型,其中已有针对1型RHDV的疫苗。
目前国内主要通过血凝抑制试验来进行RHDV抗体检测,RHDV只和人O型红血球发生凝集反应,与兔或其他哺乳动物红血球均无凝集反应;但是该方法中人O型血球获取不易,且公共卫生条件很难达到。因此,如何评价1型RHDV疫苗的免疫效果是亟需解决的问题。
酶联免疫吸附试验(ELISA)在某种程度上比血凝抑制敏感性更高,更易于标准化,利用RHDV全病毒或基因工程技术表达的VP60蛋白建立RHDV的ELISA抗体检测方法已有报道,然而这种包被抗原蛋白存在较多的抗原表位,在实际检测过程中难以与2型RHDV抗体相区别,不适于1型RHDV疫苗免疫抗体的评估。单一的抗原表位多肽是建立ELISA检测抗体的最佳包被抗原,可实现1型RHDV抗体的特异性检测,为兔出血症病毒的疫苗免疫评估提供技术支撑。
发明内容
针对上述现有技术,本发明提供了一种用于检测1型RHDV抗体的多肽、试剂盒及方法。
本发明是通过以下技术方案实现的:
一种用于检测1型RHDV抗体的多肽,命名为多肽E22,其氨基酸序列为:AENSSASVATAGIGG,如SEQ ID NO.1所示。
所述用于检测1型RHDV抗体的多肽在制备检测1型RHDV抗体的试剂盒中的应用,在检测1型RHDV抗体中的应用。
一种检测1型RHDV抗体的试剂盒,包括上述用于检测1型RHDV抗体的多肽。
进一步地,所述试剂盒包括多肽E22包被的ELISA板。
进一步地,所述多肽E22包被的ELISA板是通过以下方法制备得到的:将多肽E22加入包被液中,浓度为10μg/mL,按100μL/孔剂量加入至ELISA反应板中,于37℃下作用2小时,并于4℃包被10~16小时,拍干后用1%的BSA(牛血清白蛋白)溶液于37℃封闭2小时,用PBST洗涤,拍干,干燥,即得,装入含干燥剂的包装袋保存,备用。
进一步地,所述包被液选自碳酸盐缓冲液,优选用pH为9.6的0.05mol/L的碳酸盐缓冲液。
进一步地,所述试剂盒还包括样品稀释液、10×浓缩洗涤液、酶结合物工作液、显色液、终止液、阳性对照和阴性对照。
进一步地,所述试剂盒是由以下组分组成的:多肽E22包被的ELISA板,5块;样品稀释液,400mL;10×浓缩洗涤液,400mL;酶结合物工作液,100mL;显色液,200mL;终止液,100mL;阳性对照,2mL;阴性对照,2mL。
进一步地,所述样品稀释液为PBST,即含0.05%吐温-20的pH 7.4的0.01mol/L的磷酸盐缓冲液。
进一步地,所述10×浓缩洗涤液为含0.5%吐温-20的pH 7.4的0.1mol/L的磷酸盐缓冲液。
进一步地,所述酶结合物工作液为HRP-羊抗兔IgG(辣根过氧化物酶标记的羊抗兔IgG)。
进一步地,所述显色液为四甲基联苯胺(TMB)溶液和柠檬酸-磷酸盐缓冲液的混合液,二者体积比为1:1,四甲基联苯胺溶液的浓度为0.2mg/mL,柠檬酸-磷酸盐缓冲液中含有浓度为0.5‰的过氧化氢尿素。
进一步地,所述终止液为浓度0.31%的氢氟酸溶液。
进一步,所述阳性对照为经筛选获得的RHDV阳性血清,其OD630nm≥1.0,并含有1000U/mL的青链霉素。
进一步地,所述阴性对照为经筛选获得的阴性血清,其OD630nm≤0.243,并含有1000U/mL的青链霉素。
所述检测1型RHDV抗体的试剂盒在检测1型RHDV抗体中的应用。
进一步地,具体应用时,所述检测1型RHDV抗体的试剂盒的使用方法为:将待检血清用样品稀释液按1:200的比例进行稀释,随后按100μL/孔剂量加入至多肽E22包被的ELISA板中,同时设阴性对照及阳性对照,于37℃孵育1小时;反应结束后,弃去反应孔中的液体,每孔加洗涤液350μL,洗涤3~5次,拍干;随后每孔加100μL的酶结合物工作液,于37℃孵育30分钟;随后加洗涤液洗涤3~5次拍干;并依次加入100μL显色液,于37℃避光孵育15分钟,并加100μL终止液终止反应,用酶标仪在630nm波长下测定各孔吸光度A值,读值计算并判定结果,判定标准为:当待检样本OD630nm值与阴性对照OD630nm值的比值(P/N)大于或等于2.1,且待检样本OD630nm值大于0.243,判为阳性。
一种检测1型RHDV抗体的方法,同上。
本发明的用于检测1型RHDV抗体的多肽E22,对于1型RHDV抗体的特异性高,安全性好,只与1型RHDV阳性血清特异结合,不与2型RHDV阳性血清和其它病原的阳性血清发生交叉反应,具有良好的抗原性,使得本发明的检测试剂盒具有很高的特异性和敏感性。
本发明的检测1型RHDV抗体的试剂盒,适用于间接ELISA检测法,可对1型RHDV抗体水平作出评价,适合批量样品的检测,操作简便、快速。
本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。
附图说明
图1:HI抗体检测的结果示意图。
图2:本发明的ELISA检测试剂盒的抗体检测结果示意图。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而,本发明的范围并不限于下述实施例。本领域技术人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。
下述实施例中所涉及的仪器、试剂、材料,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料,可通过正规商业途径获得。下述实施例中所涉及的实验方法、检测方法,若无特别说明,均为现有技术中已有的常规实验方法、检测方法。
本发明的多肽E22,为人工合成的多肽,可以按照本领域技术人员所熟知的多肽合成的方法进行合成,如多肽的固相合成。
实验抗原多肽的筛选
RHDV的VP60蛋白是病毒的主要结构蛋白,本发明基于免疫学的线性抗原表位原理,根据VP60蛋白序列N端130个氨基酸序列,从蛋白序列的第一个氨基酸开始人工合成含15个氨基酸的多肽,每间隔2个氨基酸合成一条,共合成了62条多肽。利用1型RHDV阳性血清进行ELISA筛选,具体如下:
1.用碳酸盐缓冲液将合成肽(预先用1mL DMSO稀释)稀释为50μg/ml,每孔100μL包被。放湿盒37℃作用1h后4℃过夜,PBST洗涤4次。加1%BSA溶液,每孔300μl。放湿盒,37℃封闭2h,PBST洗涤5次。
2.用PBST以1:200稀释1型RHDV阳性血清(HI效价为6log2)和SPF兔阴性血清,每孔100μl。37℃作用1h。PBST洗涤5次。
3.加入羊抗兔的二抗,每孔100μl。37℃作用30min,PBST洗涤5次。
4.加底物(TMB)每孔100μl。避光作用15min。
5.加入100μl终止液,每孔100μl。于OD 630nm酶标仪读数。每条多肽重复3孔,结果见表1。
由表1可知,E22多肽具有很好的抗原性和免疫原性,明显优于其它61条多肽。
表1
实施例1封闭液的选择
将多肽E22先用DMSO溶解,然后用碳酸盐缓冲液稀释至蛋白浓度为50μg/mL,每孔100μL包被。放湿盒37℃作用1h后4℃过夜,PBST洗涤4次。加封闭液(如表1所示),每孔300μl,37℃封闭2h,然后PBST洗涤5次。用PBST以1:200稀释1型RHDV阳性血清和SPF兔阴性血清,每孔100μl,37℃作用1h。PBST洗涤5次。加入羊抗兔的二抗(1:2000稀释),每孔100μl。37℃作用30min,PBST洗涤5次。加底物(TMB)每孔100μl。避光作用15min。加入终止液,每孔100μl。于OD 630nm酶标仪读数。结果见表2。
表2 封闭液的选择
经过对5种封闭液进行的ELISA实验(4次重复),1%BSA溶液效果最佳。
实施例2多肽E22的特异性验证
将多肽E22先用DMSO溶解,然后用碳酸盐缓冲液稀释至蛋白浓度为10μg/mL,每孔100μL包被。放湿盒37℃作用1h后4℃过夜,PBST洗涤4次。加封闭液(1%BSA溶液),每孔300μl,3个重复。放湿盒,37℃封闭2h,PBST洗涤5次。用PBST以1:200稀释1型RHDV阳性血清(HI效价为5log2)、2型RHDV阳性血清(HI效价为5log2)和SPF兔阴性血清,每孔100μl,37℃作用1h。PBST洗涤5次。加入羊抗兔的二抗(1:2000稀释),每孔100μl。37℃作用30min,PBST洗涤5次。加底物(TMB)每孔100μl。避光作用15min。加入终止液,每孔100μl。于OD 630nm酶标仪读数。结果见表3。
表3 多肽的特异性验证
表2所示结果表明,多肽E22只能与1型RHDV阳性血清发生特异性反应,而与2型RHDV阳性血清无交叉反应。
实施例3试剂盒成分配制
试剂盒是由以下组分组成的:多肽E22包被的ELISA板,5块;样品稀释液,400mL;10×浓缩洗涤液,400mL;酶结合物工作液,100mL;显色液,200mL;终止液,100mL;阳性对照,2mL;阴性对照,2mL。
所述样品稀释液为PBST,即含0.05%吐温-20的pH 7.4的0.01mol/L的磷酸盐缓冲液,配制方法为:取KH2PO4 0.2g,NaHPO4·12H2O 2.9g,NaCl 8g,定容至1000mL,再加0.5mL吐温-20。
所述10×浓缩洗涤液为含0.5%吐温-20的pH 7.4的0.1mol/L的磷酸盐缓冲液,配制方法为:取KH2PO4 2g,NaHPO4·12H2O 29g,NaCl 80g,定容至1000mL,再加5mL吐温-20。
所述酶结合物工作液为HRP-羊抗兔IgG(辣根过氧化物酶标记的羊抗兔IgG)。
所述显色液为四甲基联苯胺溶液和柠檬酸-磷酸盐缓冲液的混合液,二者体积比为1:1,四甲基联苯胺溶液的浓度为0.2mg/mL,柠檬酸-磷酸盐缓冲液中含有浓度为0.5‰的过氧化氢尿素,配制方法为:称取200mg四甲基联苯胺,用100mL无水乙醇溶解后,以双蒸水定容至1000mL;称取21g柠檬酸(C6H8O7·H2O),28.2g无水磷酸氢二钠(Na2HPO4),6.4mL的0.75%过氧化氢尿素,双蒸水定容至1000mL,调pH值至5.0;控制二者的体积比为1:1。
所述终止液为浓度0.31%的氢氟酸溶液:取氢氟酸0.31mL,双蒸水定容至100mL。
所述阳性对照为经筛选获得的RHDV阳性血清:将制备的阳性血清用样品稀释液作1:300稀释(其OD630nm≥1.0),加入1000U/mL的青链霉素,无菌过滤,作为试剂盒的阳性对照。
所述阴性对照为经筛选获得的阴性血清:将筛选获得的阴性血清(OD630nm≤0.25),加入1000U/mL的青链霉素,无菌过滤,作为试剂盒的阴性对照。
实施例4间接ELISA反应条件的确定
本实施例采用方阵试验确定多肽抗原和血清最佳工作浓度。用包被缓冲液将多肽E22分别稀释成1μg/mL、5μg/mL和10μg/mL等,包被ELISA反应板,100μL/孔;抗1型RHDV的阳性血清和阴性血清用样品稀释液分别作1:100、1:200和1:300稀释;进行间接ELISA测定;加入显色液显色,加入终止液终止反应;并测定光波长630nm的OD值,结果如表4所示。
取阳性血清OD630nm1.0左右,阴性血清OD630nm0.2左右,且阳性血清OD630nm/阴性血清OD630nm即P/N值大于2.1的抗原浓度和血清稀释度为最佳工作浓度,结果表明血清最佳稀释度为1:200,多肽最佳包被浓度为10μg/mL。
表4 多肽抗原和血清最佳工作浓度的确定(OD630nm值)
将收集的36份SPF兔血清(RHDV抗体阴性),在最佳工作条件下进行间接ELISA测定,以确定兔血清在无RHDV感染时其吸收值范围因此,确定以待检样本OD630nm值与阴性OD630nm值的比值(P/N)大于或等于2.1,且待检样本OD630nm值大于0.243判为阳性,如见表5所示。
表5 RHDV抗体临界值的确定
实施例5 1型RHDV多肽间接ELISA抗体检测反应板的制备
将多肽E22先用DMSO溶解,然后用pH为9.6的0.05mol/L的碳酸盐缓冲液稀释至蛋白浓度为10μg/mL,按100μL/孔剂量加入至ELISA反应板中,于37℃下作用2小时,并于4℃包被14小时,拍干后用1%的BSA溶液于37℃封闭2小时,用PBST洗涤5次,拍干,干燥,即得,装入含干燥剂的包装袋保存,备用。
实施例6ELISA抗体检测试剂盒的使用
步骤如下:
(1)将待检血清用样品稀释液作1:200稀释,按100μL/孔加入抗体检测板中,同时设阴性血清对照、阳性血清对照,37℃孵育1h;
(2)弃去反应孔中的液体,每孔加洗涤液(PBST)350μL,洗涤5次,拍干;
(3)每孔加100μL的酶结合物工作液,37℃孵育30min;
(4)重复上述步骤(2);
(5)加入100μL显色液,37℃避光孵育15min;
(6)加100μL终止液,用酶标仪在630nm波长下测定各孔吸光度A值,读值计算并判定结果。
实施例7特异性试验
交叉试验:用E22包被抗原建立的间接ELISA抗体检测试剂盒分别检测兔轮状病毒、兔魏氏梭菌、兔巴氏杆菌等阳性血清及阴性血清,每样本4个重复,进行交叉反应性测定,检测结果显示只有1型兔出血症病毒阳性血清为阳性结果,其余均为阴性。
实施例8敏感性试验
将1型RHDV阳性血清(HI效价为7log2)和阴性血清分别作1:50~1:3200稀释,其余条件按最佳反应条件进行ELISA检测。结果表明,1型RHDV阳性血清稀释到1:800时,其OD630nm仍高于临界值0.243且P/N大于2.1(如表6所示),表明该ELISA检测试剂盒具有较高的敏感性。
表6 敏感性实验
实施例9重复性试验
分别用两次包被的酶标板,检测5份1型RHDV阳性血清和5份阴性血清,每个样品重复检测5次,测定其变异系数CV%(CV=S.D./X×100%,S.D.:标准差,X:算术平均值)。结果表明变异系数最大为3.96%,最小为0.46%。10份血清变异系数都较小,具有较好的重复性。
实施例10临床应用
在兔场随机选择7只6周龄健康兔,用商品化的1型兔瘟灭活疫苗进行免疫,于免疫前、免疫后7d、14d、21d、28d、35d、42d分别采集血清进行ELISA抗体和血凝抑制试验(HI)抗体检测。HI方法为将25μl生理盐水加入96孔板中,每行第1孔加入25μl免疫后阳性血清,加入25μl已稀释至4单位的RHDV抗原,室温静止30min,最后每孔加入25μl的1%人“O”型红细胞。将7次采集的7只兔血清抗体分别计算几何平均值,统计分析ELISA检测值与HI滴度的相关系数为0.90,结果如图1、图2所示,本发明的检测试剂盒适宜于1型兔出血症病毒疫苗抗体检测。
给本领域技术人员提供上述实施例,以完全公开和描述如何实施和使用所主张的实施方案,而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。
Claims (10)
1.一种用于检测1型RHDV抗体的多肽,其特征在于:其氨基酸序列为AENSSASVATAGIGG,如SEQ ID NO.1所示。
2.权利要求1所述的用于检测1型RHDV抗体的多肽在制备检测1型RHDV抗体的试剂盒中的应用,或在检测1型RHDV抗体中的应用。
3.一种检测1型RHDV抗体的试剂盒,其特征在于:包括权利要求1所述的用于检测1型RHDV抗体的多肽。
4.根据权利要求3所述的检测1型RHDV抗体的试剂盒,其特征在于:所述试剂盒包括多肽E22包被的ELISA板。
5.根据权利要求4所述的检测1型RHDV抗体的试剂盒,其特征在于,所述多肽E22包被的ELISA板是通过以下方法制备得到的:将多肽E22加入包被液中,浓度为10μg/mL,按100μL/孔剂量加入至ELISA反应板中,于37℃下作用2小时,并于4℃包被10~16小时,拍干后用1%的BSA溶液于37℃封闭2小时,用PBST洗涤,拍干,干燥,即得。
6.根据权利要求3所述的检测1型RHDV抗体的试剂盒,其特征在于:所述试剂盒还包括样品稀释液、10×浓缩洗涤液、酶结合物工作液、显色液、终止液、阳性对照和阴性对照。
7.根据权利要求6所述的检测1型RHDV抗体的试剂盒,其特征在于,所述试剂盒是由以下组分组成的:多肽E22包被的ELISA板,5块;样品稀释液,400mL;10×浓缩洗涤液,400mL;酶结合物工作液,100mL;显色液,200mL;终止液,100mL;阳性对照,2mL;阴性对照,2mL;
所述包被液选自pH为9.6的0.05mol/L的碳酸盐缓冲液;
所述样品稀释液为PBST;
所述10×浓缩洗涤液为含0.5%吐温-20的pH 7.4的0.1mol/L的磷酸盐缓冲液;
所述酶结合物工作液为HRP-羊抗兔IgG;
所述显色液为四甲基联苯胺溶液和柠檬酸-磷酸盐缓冲液的混合液,二者体积比为1:1,四甲基联苯胺溶液的浓度为0.2mg/mL,柠檬酸-磷酸盐缓冲液中含有浓度为0.5‰的过氧化氢尿素;
所述终止液为浓度0.31%的氢氟酸溶液;
所述阳性对照为经筛选获得的RHDV阳性血清,其OD630nm≥1.0,并含有1000U/mL的青链霉素;
所述阴性对照为经筛选获得的阴性血清,其OD630nm≤0.243,并含有1000U/mL的青链霉素。
8.权利要求3~7中任一项所述的检测1型RHDV抗体的试剂盒在检测1型RHDV抗体中的应用。
9.根据权利要求8所述的应用,其特征在于:具体应用时,将待检血清用样品稀释液按1:200的比例进行稀释,随后按100μL/孔剂量加入至多肽E22包被的ELISA板中,同时设阴性对照及阳性对照,于37℃孵育1小时;反应结束后,弃去反应孔中的液体,每孔加洗涤液350μL,洗涤3~5次,拍干;随后每孔加100μL的酶结合物工作液,于37℃孵育30分钟;随后加洗涤液洗涤3~5次拍干;并依次加入100μL显色液,于37℃避光孵育15分钟,并加100μL终止液终止反应,用酶标仪在630nm波长下测定各孔吸光度A值,读值计算并判定结果,判定标准为:当待检样本OD630nm值与阴性对照OD630nm值的比值大于或等于2.1,且待检样本OD630nm值大于0.243,判为阳性。
10.一种检测1型RHDV抗体的方法,其特征在于:将待检血清用样品稀释液按1:200的比例进行稀释,随后按100μL/孔剂量加入至多肽E22包被的ELISA板中,同时设阴性对照及阳性对照,于37℃孵育1小时;反应结束后,弃去反应孔中的液体,每孔加洗涤液350μL,洗涤3~5次,拍干;随后每孔加100μL的酶结合物工作液,于37℃孵育30分钟;随后加洗涤液洗涤3~5次拍干;并依次加入100μL显色液,于37℃避光孵育15分钟,并加50μL终止液终止反应,用酶标仪在630nm波长下测定各孔吸光度A值,读值计算并判定结果,判定标准为:当待检样本OD630nm值与阴性对照OD630nm值的比值大于或等于2.1,且待检样本OD630nm值大于0.243,判为阳性;
所述多肽E22的氨基酸序列如SEQ ID NO.1所示。
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