CN116840489A - 户尘螨过敏原特异性IgE抗体检测试剂盒及其应用 - Google Patents
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Abstract
本发明涉及生物检测技术领域,尤其涉及户尘螨过敏原特异性IgE抗体检测试剂盒及其应用。本发明对户尘螨提取物进行预处理以及生物素化修饰制备了户尘螨过敏原上清溶液,结合磁微粒混悬液和鼠抗人IgE抗体溶液能够对少量的血清/血浆样本进行快速简单、灵敏度高、特异性好、全自动、全定量的检测。检测时间短,检测成本低,能够广泛应用。
Description
技术领域
本发明涉及生物检测技术领域,尤其涉及户尘螨过敏原特异性IgE抗体检测试剂盒及其应用。
背景技术
人体免疫***在反复接触生活环境中正常无害的物质后发生的过度反应,引起过敏反应的物质通常被称作过敏原,是引起过敏性疾病的罪魁祸首。其发生机制是:过敏原进入机体后,诱导B细胞产生产生特异性IgE(Specific Immununoglobulin E,sIgE),sIgE与肥大细胞或嗜碱性粒细胞表面受体结合使机体处于致敏状态。当相同的过敏原再次进入致敏的机体,与sIgE抗体结合,就会引发细胞膜的一系列生物化学反应,释放出许多活性介质,如组胺、蛋白水解酶、肝素、趋化因子等;从而产生过敏反应,过敏原的检测的重要性也再次体现。
户尘螨过敏原作为一种重要的吸入过敏原,是引起室内过敏性疾病的重要因素,有文献研究表明,60%的成年哮喘患者对尘螨过敏,儿童中更甚,可达到80%。体外定量检测人体血清或血浆中的户尘螨过敏原特异性IgE抗体,可以辅助诊断户尘螨过敏,同时sIgE的定量检测可以评估疾病风险;对疾病进行早期诊断,提前规避风险;为脱敏治疗过程提供用药指导。
目前定量检测人体血清或血浆中的户尘螨过敏原特异性IgE抗体的体外诊断试剂,在中国批准上市的进口产品有1家,国内产品有4家。反应原理均是基于抗原抗体反应的免疫学检测原理。标杆产品户尘螨d1过敏原特异性IgE抗体检测试剂盒(荧光免疫法)是通过共价结合方式包被于ImmunoCAP上的过敏原可与患者标本中的特异性IgE发生反应,酶催化增强化学发光底物产生荧光,荧光强度越高,表明患者血清中的特异性IgE浓度越高。但其价格昂贵,耗时较长约2个小时。因此开发出一种特异性好,灵敏度高,价格低廉,且耗时短的试剂盒对于户尘螨的临床检测具有重要的意义。
发明内容
有鉴于此,本发明要解决的技术问题在于提供户尘螨过敏原特异性IgE抗体检测试剂盒及其应用。本发明提供了对户尘螨提取物进行预处理和生物素化修饰后,利用磁微粒化学发光法检测户尘螨过敏原特异性IgE抗体的试剂盒,以及该试剂盒在临床检测中的应用。
本发明提供了一种试剂组合,其包括磁微粒混悬液、户尘螨过敏原溶液和/或鼠抗人IgE抗体溶液;其中,所述户尘螨过敏原溶液为离心预处理后经过生物化修饰并稀释的上清液。
本发明对户尘螨过敏原溶液进行离心预处理后发现,沉淀中缺少户尘螨的主要致敏蛋白Der p 1和Der p 2,而上清液中的这两种蛋白含量较多,且纯度较高。说明对户尘螨过敏原溶液进行离心处理后能够去除原提取物中的杂质和干扰物,有效降低蛋白背景,纯化主要致敏蛋白,且该预处理步骤操作简单,耗时短。
在本发明所述的试剂组合中,所述户尘螨过敏原溶液离心预处理的步骤包括:所述户尘螨过敏原溶液离心预处理的步骤包括:将户尘螨过敏原提取物以去离子水稀释至2~5mg/mL后,静置2~5h,然后80000~13000g离心3~10min。作为优选,户尘螨过敏原提取物以去离子水稀释至0.2-2mg/mL。
进一步的,所述户尘螨过敏原溶液中的户尘螨过敏原修饰有生物素;修饰的步骤包括:将户尘螨过敏原溶液与生物素化试剂混合,2~8℃震荡反应0.5~3小时后,去除游离生物素,获得生物素化修饰的户尘螨过敏原。
所述户尘螨过敏原与生物素化试剂的摩尔比为1:5~1:20,优选为1:10;其中,在所述生物素化修饰后的稀释采用的缓冲液为:含有1wt%~5wt%牛血清白蛋白、0.1wt%~0.5wt%防腐剂、0.1wt%~0.5wt%Triton100的0.05MTris-NaCl缓冲液,pH值为7~8。其中,所述防腐剂为Proclin 300。
本发明所述的试剂组合中,所述磁微粒混悬液中磁珠的含量为0.5~3mg/mL,优选为1~2mg/mL;所述磁微粒混悬液中,磁微粒包被有链霉亲和素;所述磁微粒包被链霉亲和素的步骤包括将磁微粒用0.05M三羟甲基氨基甲烷盐酸盐洗涤4次后,重悬在含有人血清蛋白的三羟甲基氨基甲烷-氯化钠缓冲液中;这些缓冲液的配合,对于提高试剂盒检测的灵敏度和准确性起到良好的作用,优于其他缓冲液的效果。
所述鼠抗人IgE抗体溶液的浓度为0.1~1.5μg/mL,优选为1.0μg/mL;所述鼠抗人IgE抗体标记有辣根过氧化物酶。
本发明对户尘螨天然提取物的上清液、沉淀以及混合物进行生物素化标记后,对160例样本进行检测发现户尘螨天然提取物上清液的检测结果更准确,与过敏原诊断“金标准”的ImmunoCAP试剂盒的检测结果的阳性符合率均高于户尘螨天然提取物沉淀和混合物的检测结果。说明将户尘螨天然提取物的上清做为过敏原进行检测,能够提高试剂盒的灵敏度。
本发明提供了所述试剂组合在制备检测户尘螨过敏原特异性IgE抗体的试剂盒中的应用。
本发明还提供了户尘螨过敏原特异性IgE抗体定量检测试剂盒,其包括本发明所述的试剂组合以及可接受的助剂。
进一步的,本发明所述的试剂盒还包括鲁米诺试剂、过氧化物试剂和/或校准品。
本发明还提供了一种检测户尘螨过敏原特异性IgE抗体的方法,其包括利用本发明所述的试剂组合或所述的试剂盒对样本进行检测;所述样本为离心预处理后的上清液。
具体的,在本发明的实施例中,所述检测方法包括以下步骤:
1)将20μL校准品或待测标本加至反应杯中;
2)再将20μL链霉亲和素包被磁微粒悬浮液和20μL生物素化的户尘螨过敏原溶液加至反应杯中,37±0.5℃温育15min;
2)温育结束后,磁分离去上清、洗涤液洗涤3次;
3)加入60μL辣根过氧化物酶标记的鼠抗人IgE抗体溶液,37±0.5℃温育15min;
4)温育结束后,磁分离去上清、洗涤液洗涤3次;
5)最后加入50μL底物A液(含鲁米诺试剂)和50μL底物B液(含过氧化物试剂),震荡混匀测定化学发光强度(RLU);
6)选择适当的曲线拟合方式计算样本中户尘螨过敏原特异性IgE的浓度值(IU/mL)。
本发明提供的方法可为诊断目的的,也可为非诊断目的的。例如,包括对人体或动物体,或者离体的来自于人体或动物体的样品进行的以诊断为目的的检测方法;也包括对环境样品或者模拟样品进行的以科研或其他非诊断目的检测方法。
本发明提供的对天然抗原提取物进行离心预处理获取提取物上清,使用提取物上清进行生物素化修饰的方法,提高了抗原载量和抗原纯度,去除了原提取物中的杂质和干扰物,有效提高了试剂盒的阳性检出率即灵敏度。本发明提供的试剂盒适用于磁微粒化学发光仪器平台,具有全自动化检测,检测时间短、检测成本低的优点。
附图说明
图1示离心预处理对户尘螨过敏原粗提物致敏蛋白含量的影响。
具体实施方式
本发明提供了户尘螨过敏原特异性IgE抗体检测试剂盒及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1户尘螨过敏原溶液的预处理
用纯化水溶液稀释户尘螨天然提取物冻干粉(购于郑州伊美诺生物技术有限公司),室温平衡2-5h后,进行高速离心,转速为80000~13000g,离心3-10min。取离心预处理后的提取物上清、提取物沉淀、及离心前的提取物混合物,使用SDS-PAGE电泳确定蛋白含量情况(由郑州伊美诺公司完成),结果如图1所示。
SDS-PAGE蛋白电泳结果显示,户尘螨天然过敏原提取物稀释后离心预处理后,提取物沉淀中缺少户尘螨的主要致敏蛋白Der p1和Der p2;提取物上清相对提取物混合物来看,蛋白条带较为明显清晰且背景较为干净。
实施例2试剂盒中的试剂制备
1、生物素化-户尘螨过敏原提取物溶液制备步骤如下:
(1)、户尘螨过敏原与生物素化试剂的摩尔比为1:10混合;户尘螨过敏原标记浓度为1mg/mL。生物素化试剂为赛默飞(Thermo Scientific)公司的Sulfo-NHS-LC-biotin试剂。
(2)、2~8℃震荡反应0.5-3小时后,使用生物素去除柱去除未反应的生物素,获得的液体为生物素化-户尘螨过敏原提取液。
(3)、生物素化-户尘螨过敏原提取液用含1%~5%牛血清白蛋白(Bovinealbumin,BSA)、0.1%-0.5%防腐剂(Proclin 300)、0.1%-0.5%聚乙二醇单辛基苯基醚(Triton100)的0.05M Tris-Nacl缓冲液(pH=7~8)配制成10ug/mL溶液,即得生物素化-户尘螨过敏原提取物溶液。
2、链霉亲和素包被磁微粒悬浮液的制备步骤如下:
(1)、用0.05M三羟甲基氨基甲烷盐酸盐(Tris-HCl)缓冲液(pH=7~8)重悬磁微粒原液,反复洗涤4次(5-10min/次),磁分离。
(2)、将磁微粒重悬于含1-5%人血清白蛋白(Albumin Human,HSA)的0.05M三羟甲基氨基甲烷-氯化钠(Tris-Nacl)缓冲液(pH=7~8),即得到浓度为1-2mg/mL的链霉亲和素包被磁微粒悬浮液。
3、辣根过氧化物酶标记鼠抗人IgE抗体溶液制备步骤如下:
将HRP标记的鼠抗人IgE抗体用含1~5%BSA、0.1-0.5%酪蛋白(Casein)、0.1-0.5%Proclin 300、0.1-0.5% Triton100的0.05M Tris-Nacl缓冲液(pH=7~8)配制成0.1-1.5μg/mL溶液,即得辣根过氧化物酶标记的鼠抗人IgE抗体溶液。
实施例3检测户尘螨过敏原提取物上清、提取物沉淀、提取物混合物
使用户尘螨过敏原提取物上清、提取物沉淀、提取物混合物分别进行生物素化标记,获得3种生物素化-户尘螨过敏原提取物溶液,使用本发明所述试剂盒与过敏原诊断“金标准”的ImmunoCAP试剂盒平行对160例样本进行检测,利用安图生物全自动化学发光测定仪A2000Plus检测,步骤如下:
1)首先将20uL特异性IgE抗体检测试剂盒(磁微粒化学发光法)中校准品或待测标本加至反应杯中。
2)再将20uL链霉亲和素包被磁微粒悬浮液和20uL生物素化的户尘螨过敏原溶液加至反应杯中,37±0.5℃温育15min;
2)温育结束后,磁分离去上清、洗涤液洗涤3次;
3)然后加入60uL辣根过氧化物酶标记的鼠抗人IgE抗体溶液,37±0.5℃温育15min;
4)温育结束后,磁分离去上清、洗涤液洗涤3次;
5)最后加入50μL底物A液(含鲁米诺试剂)和50μL底物B液(含过氧化物试剂),震荡混匀测定化学发光强度(RLU);
6)选择适当的曲线拟合方式,本试剂盒推荐采用五参数(lin/log)拟合标准曲线,以特异性IgE抗体检测试剂盒(磁微粒化学发光法)中校准品的浓度值为横坐标,其对应化学发光信号值为纵坐标,建立定标曲线,根据待测标本的化学发光信号值计算待测标本中户尘螨过敏原特异性IgE的浓度值(IU/mL)。
检测结果如下表:
表1
结果显示:对户尘螨天然提取物离心预处理步骤,获取户尘螨天然提取物上清作为户尘螨过敏原再进行生物素化修饰,能够提高试剂盒的阳性检出率(灵敏度)。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.试剂组合,其特征在于,包括磁微粒混悬液、户尘螨过敏原溶液和/或鼠抗人IgE抗体溶液;
其中,所述户尘螨过敏原溶液为离心预处理后经过生物素化修饰并稀释的上清液。
2.根据权利要求1所述的试剂组合,其特征在于,所述户尘螨过敏原溶液离心预处理的步骤包括:将户尘螨过敏原提取物以去离子水稀释至2~5mg/mL后,静置2~5h,然后80000~13000g离心3~10min。
3.根据权利要求1所述的试剂组合,其特征在于,所述生物素化修饰的步骤包括:将户尘螨过敏原溶液与生物素化试剂混合,2~8℃震荡反应0.5~3小时后,去除游离生物素,获得生物素化修饰的户尘螨过敏原。
4.根据权利要求1所述的试剂组合,其特征在于,在所述生物素化修饰后的稀释采用的缓冲液为:含有1wt%~5wt%牛血清白蛋白、0.1wt%~0.5wt%防腐剂、0.1wt%~0.5wt%Triton100的0.05M Tris-NaCl缓冲液,pH值为7~8。
5.根据权利要求1~4任一项所述的试剂组合,其特征在于,所述磁微粒混悬液中磁微粒的含量为0.5~3mg/mL;
所述磁微粒混悬液中,磁微粒包被有链霉亲和素。
6.根据权利要求1~5任一项所述的试剂组合,其特征在于,所述鼠抗人IgE抗体溶液的浓度为0.1~1.5μg/mL;
所述鼠抗人IgE抗体标记有辣根过氧化物酶。
7.权利要求1~6任一项所述试剂组合在制备检测户尘螨过敏原特异性IgE抗体的试剂盒中的应用。
8.户尘螨过敏原特异性IgE抗体定量检测试剂盒,其特征在于,包括权利要求1~6任一项所述的试剂组合以及可接受的助剂。
9.根据权利要求8所述的试剂盒,其特征在于,还包括鲁米诺试剂和/或过氧化物试剂。
10.一种非诊断目的的检测户尘螨过敏原特异性IgE抗体的方法,其特征在于,包括利用权利要求1~6任一项所述的试剂组合或权利要求8或9所述的试剂盒对样本进行检测;所述样本为离心预处理后的上清液。
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