CN116626307A - 一种IgA抗体检测试剂盒及检测方法 - Google Patents
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Abstract
本发明公开了一种IgA抗体检测试剂盒及检测方法,IgA抗体检测试剂盒包括试剂和盒体,所述试剂设置于所述盒体内;所述试剂包括特异性荧光编码微球、标准物质和荧光素标记二抗,所述特异性荧光编码微球是将抗IgA多克隆抗体偶联于具有荧光强度的微球表面。IgA抗体检测方法是将特异性荧光编码微球分别与待检样本、标准物质孵育反应,加入荧光素标记二抗继续孵育反应,反应结束后分析,得到检测结果。本发明可实现一次反应快速完成IgA抗原/抗体特异性定量检测,检测过程简便、准确。
Description
技术领域
本发明涉及免疫分析检测领域,特别是涉及一种IgA抗体检测试剂盒及检测方法。
背景技术
免疫球蛋白A(IgA)不仅在粘膜免疫中是重要的抗体,而且在血液中是继免疫球蛋白G(IgG)的第2个主要的抗体家族,发挥对于细菌、病毒感染情况的防御功能。IgA分两种类型:即血清型IgA和分泌型IgA。血清型IgA存在于血清中,血清型IgA具有某些IgG和IgM的免疫功能,特异性IgA能中和血液中的抗原,同时也能出现替代性补体免疫***。分泌型IgA存在于分泌液中,例如:唾液、泪液以及乳汁当中,均含有IgA抗体。分泌型IgA是机体局部黏膜抗感染免疫的主要抗体,故又称黏膜免疫抗体。IgA不能通过胎盘产生,新生畜血清中无IgA抗体,但可从母乳中获得分泌型IgA。
IgA正常含量值根据不同医院采用不同的检测技术而有所不同,并且在成人和儿童中亦有差异。一般来说,成人中免疫球蛋白体液免疫检测正常范围在80-350mg/dL,在实验室的研究中多将IgA缺乏标准定为血清含量少于5mg/dL,当含量低于0.05mg/dL时则称之为IgA完全缺乏。我国不同地区六个民族选择性IgA缺乏症的流行病学调查显示患病率为1:4100,且不同民族患病率也有差别,占总人口数较多的汉族为1:2600。
IgA引起的输血免疫反应根据抗体的不同分为两种:一种是对于完全缺乏IgA的患者输入含IgA的血浆、全血或IVIG(含有微量的IgA)时刺激机体产生很强的抗IgA抗体,再次输注含有IgA的血液或血浆制品时,则导致严重的过敏反应,甚至死亡。另一种是IgA的不同亚类、同种异型之间的血型不相容性输血,也会产生免疫性输血反应。
抗IgA在选择性IgA缺乏症(sIgAD)受者中可引起输血反应,如果受者输入了含IgA的血液成分会导致过敏性反应,严重者甚至休克、死亡。IgA输血过敏反应发生率大约为1:20000-47000,占输血相关性死亡的3.1%。我国近年来,已有多篇关于IgA输血反应的报道,但IgA过敏性输血反应还没有可靠的评估。因为诊断未标准化,大多数严重过敏性输血反应患者缺乏实验室检查与随访。我国缺乏对IgA缺乏者患者的检测,更缺乏提供IgA(-)的血液。所以目前我国IgA缺乏症患者的输血存在很大风险。
为了避免输血过敏反应发生的风险,需要快速有效的检测出IgA含量和IgA抗体。很多IgA诊断和IgA抗体测定方法已有报道,如放射免疫扩散法、被动凝集实验、被动凝集抑制实验、酶联免疫吸附反应、固相红细胞粘附试验、颗粒胶免疫分析法等。但是以上方法中,有些对实验室要求高,有放射污染,有些操作复杂,实验时长较久,不适合针对献血员的筛查工作,不利于推广应用。
发明内容
本发明主要解决的技术问题是提供一种IgA抗体检测试剂盒及检测方法,应用简便、高效,结果准确、可靠。
为解决上述技术问题,本发明采用的一个技术方案是:提供一种IgA抗体检测试剂盒,包括试剂和盒体,所述试剂设置于所述盒体内;所述试剂包括特异性荧光编码微球、标准物质和荧光素标记二抗,所述特异性荧光编码微球是将抗IgA多克隆抗体偶联于具有荧光强度的微球表面。
在本发明一个较佳实施例中,所述抗IgA多克隆抗体为鼠抗IgA单克隆抗体、羊抗IgA多克隆抗体或兔抗IgA多克隆抗体。
在本发明一个较佳实施例中,所述标准物质为从母乳中提取的分泌型IgA。
在本发明一个较佳实施例中,所述标准物质为五组。
在本发明一个较佳实施例中,五组所述标准物质的反应蛋白浓度分别为2.0μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0μg/mL。
在本发明一个较佳实施例中,所述荧光素标记二抗中的荧光素为FITC系列荧光物质、PE系列荧光物质、APC系列荧光物质或CY系列荧光物质。
提供一种IgA抗体检测方法,采用所述IgA抗体检测试剂盒检测,包括步骤为:(1)将特异性荧光编码微球分别与待检样本、标准物质孵育反应;(2)加入荧光素标记二抗继续孵育反应;(3)反应结束后分析,得到检测结果。
在本发明一个较佳实施例中,步骤(1)中所述待检样本为血清或血浆。
在本发明一个较佳实施例中,步骤(3)中所述分析为采用流式细胞仪分析。
本发明的有益效果是:本发明的IgA抗体检测试剂盒及检测方法,可实现一次反应快速完成IgA抗原/抗体特异性定量检测,检测过程简便、准确。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图,其中:
图1是本发明IgA抗体检测方法实施例一的流程示意图;
图2是本发明IgA抗体检测方法实施例一的检测结果对比图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
实施例一:
提供一种IgA抗体检测试剂盒,包括试剂和盒体,所述试剂设置于所述盒体内;所述试剂包括特异性荧光编码微球、标准物质和荧光素标记二抗,所述特异性荧光编码微球是将抗IgA多克隆抗体偶联于具有荧光强度的微球表面。
所述抗IgA多克隆抗体为鼠抗IgA单克隆抗体、羊抗IgA多克隆抗体或兔抗IgA多克隆抗体,在本实施例中,所述抗IgA多克隆抗体选取鼠抗IgA单克隆抗体,后续方法也同样是采用此抗体进行检测。所述标准物质为人IgA抗原标准品。在本实施例中,所述标准物质为五组,可通过这五种标准物质的反应蛋白浓度对待检样本的IgA反应蛋白浓度进行定量,其中五组所述标准物质的反应蛋白浓度分别为2.0μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0μg/mL。所述荧光素标记二抗中的荧光素为FITC系列荧光物质、PE系列荧光物质、APC系列荧光物质或CY系列荧光物质,在本实施例中,所述荧光素标记二抗中的荧光素为FITC系列荧光物质。
请参阅图1和图2,提供一种IgA抗体检测方法,采用所述IgA抗体检测试剂盒检测,包括步骤为:
(1)对于试剂盒中的特异性荧光编码微球,是将抗IgA多克隆抗体偶联于对应具有荧光强度的微球表面,以制备成特异性荧光编码微球;具体过程:首先将荧光微球进行活化处理,得到活化荧光微球;向活化荧光微球中添加含有甘油的MES缓冲液、超声混匀,随后再添加IgA抗体溶液、超声反应、避光旋转混匀;待反应结束后,向沉淀物中添加封闭液、超声混匀、避光旋转混匀、离心并弃去上清液,随后继续添加封闭液、超声混匀、离心;收集离心后的沉淀物,并置于甘氨酸溶液中、超声分散,即得荧光微球与抗体的复合物
(2)将特异性荧光编码微球分别与待检样本、标准物质孵育反应,其中所述待检样本为血清或血浆,五组所述标准物质与特异性荧光编码微球孵育反应作为对照组;孵育过程为37℃下20分钟,或常温45分钟。
(3)加入荧光素标记二抗继续孵育反应;孵育过程为37℃下20分钟,或常温45分钟。
(4)反应结束后采用流式细胞仪分析,得到检测结果。计算IgA标准物质浓度和对应荧光信号值,拟合标准曲线,得出曲线方程,其中零值标准品的荧光值作为本地扣除。将样本的荧光信号值代入曲线方程,乘以相应的稀释倍数,即可算出样本中IgA的浓度。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效结构或等效流程变换,或直接或间接运用在其它相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (9)
1.一种IgA抗体检测试剂盒,其特征在于,包括试剂和盒体,所述试剂设置于所述盒体内;所述试剂包括特异性荧光编码微球、标准物质和荧光素标记二抗,所述特异性荧光编码微球是将抗IgA多克隆抗体偶联于具有荧光强度的微球表面。
2.根据权利要求1所述的IgA抗体检测试剂盒,其特征在于,所述抗IgA多克隆抗体为鼠抗IgA单克隆抗体、羊抗IgA多克隆抗体或兔抗IgA多克隆抗体。
3.根据权利要求1所述的IgA抗体检测试剂盒,其特征在于,所述标准物质为从母乳中提取的分泌型IgA。
4.根据权利要求1所述的IgA抗体检测试剂盒,其特征在于,所述标准物质为五组。
5.根据权利要求4所述的IgA抗体检测试剂盒,其特征在于,五组所述标准物质的反应蛋白浓度分别为2.0μg/mL、1μg/mL、0.5μg/mL、0.25μg/mL、0μg/mL。
6.根据权利要求1所述的IgA抗体检测试剂盒,其特征在于,所述荧光素标记二抗中的荧光素为FITC系列荧光物质、PE系列荧光物质、APC系列荧光物质或CY系列荧光物质。
7.一种IgA抗体检测方法,其特征在于,采用权利要求1-6任一所述的IgA抗体检测试剂盒检测,包括步骤为:(1)将特异性荧光编码微球分别与待检样本、标准物质孵育反应;(2)加入荧光素标记二抗继续孵育反应;(3)反应结束后分析,得到检测结果。
8.根据权利要求7所述的IgA抗体检测方法,其特征在于,步骤(1)中所述待检样本为血清或血浆。
9.根据权利要求7所述的IgA抗体检测方法,其特征在于,步骤(3)中所述分析为采用流式细胞仪分析。
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