CN117405470A - 一种免疫磁珠试剂盒及其使用方法 - Google Patents
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Abstract
本发明涉及生物材料技术领域,且公开了一种免疫磁珠试剂盒及其使用方法,所述免疫磁珠试剂盒包括:溴化氰活化的免疫磁珠、黄曲霉毒素单克隆抗体、偶联缓冲溶液和封闭试剂。该免疫磁珠试剂盒及其使用方法,通过该免疫试剂盒能够富集、净化与提取样品中的黄曲霉毒素B1、B2、G1、G2、M1、M2,具有提取效率高、操作方法简单等优点,能够实现样品中黄曲霉毒素含量的准确检测,偶联缓冲溶液的pH控制为6.8‑8.2,所得每100uL免疫磁珠对黄曲霉毒素的最大载量为460‑560ng,免疫磁珠的偶联率>80%;且进一步将偶联剂缓冲溶液的pH控制在6.8‑7.5之间时,免疫磁珠对黄曲霉毒素的最大载量更大,免疫磁珠与黄曲霉毒素抗体的偶联率更高。
Description
技术领域
本发明涉及生物材料技术领域,具体为一种免疫磁珠试剂盒及其使用方法。
背景技术
黄曲霉毒素是由黄曲霉和寄生曲霉等产生的一组结构类似的次生代谢产物,是一组以二吠喃香豆素为基本结构的化合物,目前已分离鉴定出12种,主要存在于谷物、坚果、中药材、饲料相关的产品中。在农作物被收获后,极容易被B1、B2、G1、G2这几种黄曲霉毒素污染,当人类或动物食用被黄曲霉毒素污染的上述产品后,黄曲霉毒素会在体内富集,进而对人体和动物的肝脏、肾脏等组织和器官造成危害。1993年黄曲霉毒素被世界卫生组织(WHO)的癌症研究机构划定为I类致癌物,是一种毒性极强的剧毒物质。
目前,黄曲霉毒素的检测方法主要有薄层层析法、酶联免疫吸附法、金标试纸法及高效液相色谱法。其中,薄层层析法是将样品在薄层板上分离,然后在一定紫外波长下,根据薄层板上显示的荧光最低检出量来测定黄曲霉毒素的含量,但是其样品前处理过程复杂、检测操作步骤多、且重现性较差。酶联免疫吸附法是基于抗原与抗体的特异性结合以及后续的酶对底物显色反应的高效催化作用,利用仪器检测被酶催化生成产物的量,从而获得黄曲霉毒素的含量,目前该方法主要用于黄曲霉毒素B1、M1的检测,对其他黄曲霉毒素以及有机溶液中的黄曲霉毒素校测效果不佳。金标试纸法是利用胶体金标记单克隆抗体而设计的定性或定量检测方法,该方法对于低含量的黄曲霉毒素的检测准确度不高。而高效液相色谱法不受限于有机溶剂,并且样品适用性广、结果准确度与重现性高,无论利用上述何种检测方法,均需要对待测样品进行前处理,使样品中的黄曲霉毒素被富集并提取出来。目前,黄曲霉毒素的提取主要是采用固相萃取柱净化提取,然而利用上述方法提取过程中,一方面会消耗大量的有机溶剂,对人员与环境造成损伤;另一方面其提取效率并不理想,导致后续分析检测的灵敏度和特异性较差,因此本发明提供了一种免疫磁珠试剂盒及其使用方法。
发明内容
(一)解决的技术问题
针对现有技术的不足,本发明提供了一种免疫磁珠试剂盒及其使用方法,解决了上述背景技术中提出的问题。
(二)技术方案
为实现以上目的,本发明提供如下技术方案:一种免疫磁珠试剂盒及其使用方法,所述免疫磁珠试剂盒包括:溴化氰活化的免疫磁珠、黄曲霉毒素单克隆抗体、偶联缓冲溶液和封闭试剂。
优选的,所述溴化氰活化的免疫磁珠是溴化氰活化的磁性微球,所述磁性微球表面包覆有琼脂糖高分子层,所述磁性微球的粒径为20-60μm。
优选的,所述偶联缓冲溶液选自柠檬酸缓冲液、磷酸盐缓冲液、Tris-HCL缓冲液、碳酸盐缓冲液和硼砂缓冲液;所述封闭试剂选自为Tris-HCl、牛血清蛋白、酪蛋白、乙胺和乙醇胺。
优选的,所述黄曲霉毒素单克隆抗体由杂交瘤细胞分泌获得。
优选的,包括以下步骤:偶联、封闭;偶联:将盐酸溶液加入到所述溴化氰活化的免疫磁珠中,离心去上清,并加入pH为6.8-8.2的偶联缓冲溶液进行复溶,获得溶胀的磁珠微球;然后将所述黄曲霉毒素单克隆抗体加入到所述溶胀的磁珠微球中,充分混匀,离心,弃上清液,获得磁珠微球基质。
优选的,所述封闭步骤为:将磁珠微球基质转移至封闭试剂中,静置然后离心,弃上清液,最后用pH为6.5-7.5的PBS溶液复溶,获得免疫磁珠。
优选的,包括以下步骤:中药材前处理、提取磁吸;中药材前处理:向中药材试样中加入提取试剂,并采用高速涡旋的方法进行提取,过滤,获得样品滤液;所述提取试剂为甲醇水溶液或乙腈水溶液。
优选的,所述提取磁吸步骤为:首先配制免疫磁珠溶液;然后向其中加入样品滤液,磁吸、弃上清液;再加入含有吐温-20的PBS溶液,磁吸、弃上清液;最后加入甲醇,涡旋混匀、磁吸、收集洗脱液,即获得黄曲霉毒素。
(三)有益效果
与现有技术相比,本发明提供了一种免疫磁珠试剂盒及其使用方法,具备以下有益效果:
该免疫磁珠试剂盒及其使用方法,通过该免疫试剂盒能够富集、净化与提取样品中的黄曲霉毒素B1、B2、G1、G2、M1、M2,具有提取效率高、操作方法简单等优点,能够实现样品中黄曲霉毒素含量的准确检测,偶联缓冲溶液的pH控制为6.8-8.2,所得每100uL免疫磁珠对黄曲霉毒素的最大载量为460-560ng,免疫磁珠的偶联率>80%;且进一步将偶联剂缓冲溶液的pH控制在6.8-7.5之间时,免疫磁珠对黄曲霉毒素的最大载量更大,免疫磁珠与黄曲霉毒素抗体的偶联率更高,进一步选用50-90%乙腈水溶液作为提取试剂对中药材进行前处理,获得的黄曲霉毒素B1回收率高达80.2-91.2%、黄曲霉毒素B2的回收率高达82.4-93.5%、黄曲霉毒素G1的回收率高达81.5-90.4%、黄曲霉毒素G2的回收率高达79.6-89.6%。
附图说明
图1为本发明提出的一种免疫磁珠试剂盒及其使用方法流程图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,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他实施例,都属于本发明保护的范围。
请参阅图1,本发明提供一种技术方案:所述免疫磁珠试剂盒包括:溴化氰活化的免疫磁珠、黄曲霉毒素单克隆抗体、偶联缓冲溶液和封闭试剂,所述溴化氰活化的免疫磁珠是溴化氰活化的磁性微球,所述磁性微球表面包覆有琼脂糖高分子层,所述磁性微球的粒径为20-60μm,所述偶联缓冲溶液选自柠檬酸缓冲液、磷酸盐缓冲液、Tris-HCL缓冲液、碳酸盐缓冲液和硼砂缓冲液;所述封闭试剂选自为Tris-HCl、牛血清蛋白、酪蛋白、乙胺和乙醇胺,所述黄曲霉毒素单克隆抗体由杂交瘤细胞分泌获得,包括以下步骤:偶联、封闭;偶联:将盐酸溶液加入到所述溴化氰活化的免疫磁珠中,离心去上清,并加入pH为6.8-8.2的偶联缓冲溶液进行复溶,获得溶胀的磁珠微球;然后将所述黄曲霉毒素单克隆抗体加入到所述溶胀的磁珠微球中,充分混匀,离心,弃上清液,获得磁珠微球基质,所述封闭步骤为:将磁珠微球基质转移至封闭试剂中,静置然后离心,弃上清液,最后用pH为6.5-7.5的PBS溶液复溶,获得免疫磁珠,包括以下步骤:中药材前处理、提取磁吸;中药材前处理:向中药材试样中加入提取试剂,并采用高速涡旋的方法进行提取,过滤,获得样品滤液;所述提取试剂为甲醇水溶液或乙腈水溶液,所述提取磁吸步骤为:首先配制免疫磁珠溶液;然后向其中加入样品滤液,磁吸、弃上清液;再加入含有吐温-20的PBS溶液,磁吸、弃上清液;最后加入甲醇,涡旋混匀、磁吸、收集洗脱液,即获得黄曲霉毒素。
制备例1:上述黄曲霉毒素单克隆抗体的制备方法为:将冻存的5506杂交瘤细胞于T75培养瓶中扩大培养,当细胞生长密度达到5~10×106/mL时,将细胞转入到1L的培养瓶中培养,连续培养10天左右,待细胞活率<50%时,收取培养液,8000rpm离心10min收集上清,使用蛋白A柱子纯化抗体,最后得到黄曲霉毒素单克隆抗体。
制备例2:配制得到的免疫磁珠具体包括以下步骤:(1)偶联:将0.1M盐酸溶液加入到1.0g溴化氰活化的免疫磁珠中,2000rpm离心去上清,并加入pH为7的磷酸盐缓冲液进行复溶,获得溶胀的磁珠微球;然后将5.0g黄曲霉毒素单克隆抗体加入到溶胀的磁珠微球中,在25°C下采用end-over-end的方式充分混匀,时间为12h,2000rpm离心去上清,获得磁珠微球基质。(2)封闭:将磁珠微球基质转移至0.1M封闭试剂中,在25°C下静置2-4h,然后2000rpm下离心5min,弃上清液,最后用pH为7.0的PBS溶液复溶,获得免疫磁珠。
对比制备例1:对比制备例2提供一种免疫磁珠,其制备方法的具体步骤为:取表面具有氨基的磁性微球,用0.1M、pH为8.4的硼砂缓冲液洗涤5次,按照每毫克磁性微球加入10%抗体的比例,加入黄曲霉毒素抗体,在25°C的条件下充分混匀,反应1小时,反应结束后,采用硼砂缓冲液清洗磁性微球,以洗去未反应的黄曲霉毒素抗体;加入10%牛血清蛋白,在25°C的条件下充分混匀,反应1-5小时,反应结束后,获得免疫磁株。
综上所述,该免疫磁珠试剂盒及其使用方法,通过该免疫试剂盒能够富集、净化与提取样品中的黄曲霉毒素B1、B2、G1、G2、M1、M2,具有提取效率高、操作方法简单等优点,能够实现样品中黄曲霉毒素含量的准确检测,偶联缓冲溶液的pH控制为6.8-8.2,所得每100uL免疫磁珠对黄曲霉毒素的最大载量为460-560ng,免疫磁珠的偶联率>80%;且进一步将偶联剂缓冲溶液的pH控制在6.8-7.5之间时,免疫磁珠对黄曲霉毒素的最大载量更大,免疫磁珠与黄曲霉毒素抗体的偶联率更高,进一步选用50-90%乙腈水溶液作为提取试剂对中药材进行前处理,获得的黄曲霉毒素B1回收率高达80.2-91.2%、黄曲霉毒素B2的回收率高达82.4-93.5%、黄曲霉毒素G1的回收率高达81.5-90.4%、黄曲霉毒素G2的回收率高达79.6-89.6%。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (8)
1.一种免疫磁珠试剂盒及其使用方法,其特征在于:所述免疫磁珠试剂盒包括:溴化氰活化的免疫磁珠、黄曲霉毒素单克隆抗体、偶联缓冲溶液和封闭试剂。
2.根据权利要求1所述的一种免疫磁珠试剂盒及其使用方法,其特征在于:所述溴化氰活化的免疫磁珠是溴化氰活化的磁性微球,所述磁性微球表面包覆有琼脂糖高分子层,所述磁性微球的粒径为20-60μm。
3.根据权利要求1所述的一种免疫磁珠试剂盒及其使用方法,其特征在于:所述偶联缓冲溶液选自柠檬酸缓冲液、磷酸盐缓冲液、Tris-HCL缓冲液、碳酸盐缓冲液和硼砂缓冲液;所述封闭试剂选自为Tris-HCl、牛血清蛋白、酪蛋白、乙胺和乙醇胺。
4.根据权利要求1所述的一种免疫磁珠试剂盒及其使用方法,其特征在于:所述黄曲霉毒素单克隆抗体由杂交瘤细胞分泌获得。
5.根据权利要求1所述的一种免疫磁珠试剂盒及其使用方法,其特征在于:包括以下步骤:偶联、封闭;偶联:将盐酸溶液加入到所述溴化氰活化的免疫磁珠中,离心去上清,并加入pH为6.8-8.2的偶联缓冲溶液进行复溶,获得溶胀的磁珠微球;然后将所述黄曲霉毒素单克隆抗体加入到所述溶胀的磁珠微球中,充分混匀,离心,弃上清液,获得磁珠微球基质。
6.根据权利要求1所述的一种免疫磁珠试剂盒及其使用方法,其特征在于:所述封闭步骤为:将磁珠微球基质转移至封闭试剂中,静置然后离心,弃上清液,最后用pH为6.5-7.5的PBS溶液复溶,获得免疫磁珠。
7.根据权利要求1所述的一种免疫磁珠试剂盒及其使用方法,其特征在于:包括以下步骤:中药材前处理、提取磁吸;中药材前处理:向中药材试样中加入提取试剂,并采用高速涡旋的方法进行提取,过滤,获得样品滤液;所述提取试剂为甲醇水溶液或乙腈水溶液。
8.根据权利要求1所述的一种免疫磁珠试剂盒及其使用方法,其特征在于:所述提取磁吸步骤为:首先配制免疫磁珠溶液;然后向其中加入样品滤液,磁吸、弃上清液;再加入含有吐温-20的PBS溶液,磁吸、弃上清液;最后加入甲醇,涡旋混匀、磁吸、收集洗脱液,即获得黄曲霉毒素。
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