CN112557660A - 一种磁微粒化学发光法her2检测试剂盒及制备方法 - Google Patents
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Abstract
本发明公开了一种磁微粒化学发光法HER2检测试剂盒及制备方法,属于生物免疫技术领域。本发明公开的一种磁微粒化学发光法HER2检测试剂盒,包括如下组分:磁分离试剂、异硫氰酸荧光素标记抗HER2抗体、碱性磷酸酶标记抗HER2抗体、校准品。本发明采用血清学检测,具有方便、可重复测量、定量、客观以及可以实时检测等特点,不失为组织学检测的一种补充;整个反应体系反应时间短,避免了放射性污染,简化了实验操作流程,采样方式降低了病人的痛苦,操作便捷。
Description
技术领域
本发明涉及生物免疫技术领域,更具体的说是涉及一种磁微粒化学发光法HER2检测试剂盒及制备方法。
背景技术
人表皮生长因子受体-2(HER2)在20世纪80年代被三个研究小组独立发现,是迄今为止被研究的比较透彻的乳腺癌基因之一。HER2基因是临床治疗监测的预后指标,也是肿瘤靶向治疗药物选择的一个重要靶点。血清HER2与乳腺癌患者的肿瘤负荷、组织学HER2、淋巴结状况均有一定关联,并可能是一个独立的预后因素,可能对化疗或内分泌治疗疗效产生一定影响。
HER2蛋白通常只在胎儿时期表达,成年以后只在极少数组织内低水平表达。然而有研究表明30%以上的人类肿瘤中存在HER2基因的扩增/过度表达(如乳腺癌、卵巢癌、子宫内膜癌、输卵管癌、胃癌和前列腺癌等);其中20%-30%的原发性浸润性乳腺癌有HER2基因的扩增/过度表达。
乳腺癌CISH与FISH检测结果的符合率达90%以上;FISH和IHC检测结果的符合率较高。由于这三种方法都属于形态学检测,受主观的影响很大,并且该项目检测是为了筛选对身体有一定毒害作用并且昂贵的药物适宜人群,因此2007年美国临床肿瘤学会(ASCO)和美国病理学会(CAP)推荐了HER2检测的标准规程,以期能够降低误差的发生。HER2检测所用的组织主要来自固定在福尔马林缓冲液或石蜡中的肿瘤组织。IHC与FISH的检测结果有较好的一致性,但IHC结果为(++)样本与FISH结果差异较大。因此IHC是初步筛查的首选方法,而由于FISH法检测乳腺癌HER2/neu基因敏感性及稳定性较高,可作为最终确诊HER2基因状态的方法。
由于组织学检测需取得病理标本,对未取得癌组织病理的患者就无法判断其HER2状态,并且也难以多次检测。血清学检测具有方便、可重复测量、定量、客观以及可以实时检测等特点,不失为组织学检测的一种补充。
细胞表面HER2蛋白胞外段会受蛋白酶裂解脱落进入血液循环,循环中有HER-2蛋白的胞外段的存在为血清学检测HER2提供了可能。检测血清HER2的方法有:酶联免疫分析(EIA)、酶联免疫吸附试验(ELISA)、化学发光免疫分析法等。近期发现的NIDS快速反应法则有待进一步研究。
近来研究表明,血清学与组织学HER2的检测结果一致性较好,约有20%-30%乳腺癌患者血清中可溶性HER2蛋白水平升高。血清HER2可作为反映肿瘤生长、复发或者转移的检测指标。高血清HER2水平提示肿瘤的高侵袭性,与临床分期、病情进展、无瘤生存期和总生存期有关,并能监测化疗药的治疗效果,是重要的预后因素。
通过检测血清中HER2的水平,有助于诊断、预测患者组织HER2的状态,在接受化疗的晚期乳腺癌患者中监测血清HER2含量有助于预测疗效、无病生存期和总生存期。在指导靶向药物应用方面,血清HER2的检测也可以作为组织学检测的一个补充,并可能使靶向治疗的适应人群得以放宽。血清HER2的检测也可以作为重要的预后因素,可在乳腺癌随访监测、疗效观察尤其是靶向治疗后的疗效观察中发挥其独到的作用。
因此,提供一种磁微粒化学发光法HER2检测试剂盒及制备方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种磁微粒化学发光法HER2检测试剂盒及制备方法。
为了实现上述目的,本发明采用如下技术方案:
一种磁微粒化学发光法HER2检测试剂盒,包括如下组分:磁分离试剂(异硫氰酸荧光素免疫磁微粒)、异硫氰酸荧光素标记抗HER2抗体、碱性磷酸酶标记抗HER2抗体、校准品。
进一步,所述异硫氰酸荧光素标记抗HER2抗体的制备步骤如下:
(1)量取1mg抗HER2抗体;
(2)通过抗体分子量(150000)和异硫氰酸荧光素分子量(389.4)进行计算,按照抗HER2抗体与异硫氰酸荧光素的摩尔比为1:30称量异硫氰酸荧光素;
(3)称取5mg异硫氰酸荧光素,用0.05M pH 9.0碳酸钠缓冲液溶解至终浓度为5mg/ml;
(4)在步骤(1)量取的抗HER2抗体中加入15.6μl步骤(3)溶解的异硫氰酸荧光素,充分混匀后,室温反应15-18h;
(5)用截留分子量为1000道尔顿的透析袋透析步骤(4)获得的反应产物,获得混合物;所用透析缓冲液为pH 7.5的0.15M PBS缓冲液;
(6)用抗试剂缓冲液将步骤(5)获得的混合物稀释至1μg/ml作为工作液使用;所述抗试剂缓冲液包括0.15M pH7.5磷酸盐缓冲液,1%牛血清白蛋白,0.1%ProCin300。
进一步,所述碱性磷酸酶标记抗HER2抗体的制备步骤如下:
①将2mg抗HER2抗体用pH7.5的0.1M PBS进行透析,再用0.7mg 2-亚氨基硫烷盐酸盐进行活化;
②将2mg碱性磷酸酶用1mg(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)进行活化;
③将步骤①活化后的2mg抗HER2抗体和步骤②活化后的2mg碱性磷酸酶混合,2-8℃反应15-20h,获得反应物;
④将步骤③获得的反应物用superdex 200层析柱进行纯化,收集I峰II峰进行混合,获得碱性磷酸酶标记抗HER2抗体;
⑤用抗试剂缓冲液将步骤④获得的碱性磷酸酶标记抗HER2抗体稀释至0.5μg/ml作为工作液使用;所述抗试剂缓冲液包括0.15M pH7.5磷酸盐缓冲液,1%牛血清白蛋白,0.1%ProClin300。
进一步,所述校准品的制备步骤如下:
A、配制HER2校准品缓冲液:0.05M pH7.4的TRIS缓冲液中加入1%的牛血清白蛋白,0.2%防腐剂;
B、将HER2纯品用校准品缓冲液进行稀释,校准品浓度分别为0ng/mL、10ng/mL、20ng/mL、80ng/mL、200ng/mL和400ng/mL的人表皮生长因子受体-2溶液,校准品形态为液态且具有良好的稳定性。
试剂盒的使用方法如下:
1)将10μl样本与50μl异硫氰酸荧光素标记抗HER2抗体、50μl碱性磷酸酶标记抗HER2抗体,共同孵育15min;
2)加入50μl磁分离试剂,孵育5min;
3)磁分离2min,去上清;
4)洗涤3次,每次300μl洗液;
5)底物200μl,测值。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种磁微粒化学发光法HER2检测试剂盒及制备方法,采用血清学检测,具有方便、可重复测量、定量、客观以及可以实时检测等特点,不失为组织学检测的一种补充。整个反应体系反应时间短,避免了放射性污染,简化了实验操作流程,采样方式降低了病人的痛苦,操作便捷。异硫氰酸荧光素与抗异硫氰酸素抗体间的结合具有极高的亲和力,其反应呈高度专一性。因此,在提高灵敏度的同时,并不增加非特异性干扰。而且结合特性不会因反应试剂的高度稀释而受影响,人体内不存在异硫氰酸荧光素,使其在实际应用中可最大限度地降低反应试剂的非特异作用。人表皮生长因子受体2抗原与异硫氰酸荧光素标记抗体或者酶标记抗体之间连接,解决了开发此项目的关键问题即偶联问题,可以方便地建立人表皮生长因子受体2的磁微粒酶促化学发光体系。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术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,还可以根据提供的附图获得其他的附图。
图1附图为本发明FITC体系曲线拟合;
图2附图为本发明FITC体系测试血清与西门子HER2试剂盒测定值相关性。
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。
实施例1
一种磁微粒化学发光法HER2检测试剂盒,包括如下组分:磁分离试剂(异硫氰酸荧光素免疫磁微粒)(北京泽诚)、异硫氰酸荧光素标记抗HER2抗体、碱性磷酸酶标记抗HER2抗体、校准品(添博生物)。
其中,异硫氰酸荧光素标记抗HER2抗体的制备步骤如下:
(1)量取1mg抗HER2抗体(单克隆配对抗体,添博生物);
(2)通过抗HER2抗体分子量(150000)和异硫氰酸荧光素分子量(389.4)进行计算,按照抗HER2抗体与异硫氰酸荧光素的摩尔比为1:30称量异硫氰酸荧光素(添博生物);
(3)称取5mg异硫氰酸荧光素,用0.05M pH 9.0碳酸钠缓冲液溶解至终浓度为5mg/ml;
(4)在步骤(1)量取的抗HER2抗体中加入15.6μl步骤(3)溶解的异硫氰酸荧光素,充分混匀后,室温反应15-18h;
(5)用截留分子量为1000道尔顿的透析袋透析步骤(4)获得的反应产物,获得混合物;所用透析缓冲液为pH 7.5的0.15M PBS缓冲液;
(6)用抗试剂缓冲液将步骤(5)获得的混合物稀释至1μg/ml作为工作液使用;所述抗试剂缓冲液包括0.15M pH7.5磷酸盐缓冲液,1%牛血清白蛋白(罗氏),0.1%ProCin300(西格玛)。
碱性磷酸酶标记抗HER2抗体的制备步骤如下:
①将2mg抗HER2抗体用pH7.5的0.1M PBS进行透析,再用0.7mg 2-亚氨基硫烷盐酸盐(西格玛)进行活化;
②将2mg碱性磷酸酶(罗氏)用1mg(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC,西格玛)进行活化;
③将步骤①活化后的2mg抗HER2抗体和步骤②活化后的2mg碱性磷酸酶混合,2-8℃反应15-20h,获得反应物;
④将步骤③获得的反应物用superdex 200层析柱(美国通用)进行纯化,收集I峰II峰进行混合,获得碱性磷酸酶标记抗HER2抗体;
⑤用抗试剂缓冲液将步骤④获得的碱性磷酸酶标记抗HER2抗体稀释至0.5μg/ml作为工作液使用;所述抗试剂缓冲液包括0.15M pH7.5磷酸盐缓冲液,1%牛血清白蛋白(罗氏),0.1%ProClin300(西格玛)。
校准品的制备步骤如下:
A、配制HER2校准品缓冲液:0.05M pH7.4的TRIS缓冲液中加入1%的牛血清白蛋白,0.2%防腐剂;
B、将HER2纯品用校准品缓冲液进行稀释,校准品浓度分别为0ng/mL、10ng/mL、20ng/mL、80ng/mL、200ng/mL和400ng/mL的人表皮生长因子受体-2溶液,校准品形态为液态且具有良好的稳定性。
本发明对0ng/mL、10ng/mL、20ng/mL、80ng/mL、200ng/mL和400ng/mL的人表皮生长因子受体-2样品做检测,进行样本测试。
1)将10μl样本与50μl异硫氰酸荧光素标记抗HER2抗体、50μl碱性磷酸酶标记抗HER2抗体,共同孵育15min;
2)加入50μl磁分离试剂,孵育5min;
3)磁分离2min,去上清;
4)洗涤3次,每次300μl洗液(添博生物);
5)底物200μl,测值。
FITC体系曲线拟合见图1。
图1结果显示,各浓度点对应的发光值拟合的曲线相关系数R2为0.9979。
进一步与西门子HER2试剂盒测定值进行比对,结果见表1。
表1 FITC体系下测值与给值之间对比
注:样本为血清,来自添博生物。
FITC体系测试血清与西门子HER2试剂盒测定值相关性结果见图2。
由图2可以看出,对检测结果进行对比研究,所获得的回归方程y=0.9871x,其相关系数R为0.995。
因此,本发明的技术方案与西门子试剂盒具有良好的一致性。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (4)
1.一种磁微粒化学发光法HER2检测试剂盒,其特征在于,包括如下组分:磁分离试剂、异硫氰酸荧光素标记抗HER2抗体、碱性磷酸酶标记抗HER2抗体、校准品。
2.根据权利要求1所述的一种磁微粒化学发光法HER2检测试剂盒,其特征在于,所述异硫氰酸荧光素标记抗HER2抗体的制备步骤如下:
(1)量取1mg抗HER2抗体;
(2)按照抗HER2抗体与异硫氰酸荧光素的摩尔比为1:30称量异硫氰酸荧光素;
(3)称取5mg异硫氰酸荧光素,用0.05MpH 9.0碳酸钠缓冲液溶解至终浓度为5mg/ml;
(4)在步骤(1)量取的抗HER2抗体中加入15.6μl步骤(3)溶解的异硫氰酸荧光素,充分混匀后,室温反应15-18h;
(5)用截留分子量为1000道尔顿的透析袋透析步骤(4)获得的反应产物,获得混合物;所用透析缓冲液为pH 7.5的0.15MPBS缓冲液;
(6)用抗试剂缓冲液将步骤(5)获得的混合物稀释至1μg/ml作为工作液使用;所述抗试剂缓冲液包括0.15MpH7.5磷酸盐缓冲液,1%牛血清白蛋白,0.1%ProCin300。
3.根据权利要求1所述的一种磁微粒化学发光法HER2检测试剂盒,其特征在于,所述碱性磷酸酶标记抗HER2抗体的制备步骤如下:
①将2mg抗HER2抗体用pH7.5的0.1M PBS进行透析,再用0.7mg 2-亚氨基硫烷盐酸盐进行活化;
②将2mg碱性磷酸酶用1mg(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯进行活化;
③将步骤①活化后的2mg抗HER2抗体和步骤②活化后的2mg碱性磷酸酶混合,2-8℃反应15-20h,获得反应物;
④将步骤③获得的反应物用superdex 200层析柱进行纯化,收集I峰II峰进行混合,获得碱性磷酸酶标记抗HER2抗体;
⑤用抗试剂缓冲液将步骤④获得的碱性磷酸酶标记抗HER2抗体稀释至0.5μg/ml作为工作液使用;所述抗试剂缓冲液包括0.15MpH7.5磷酸盐缓冲液,1%牛血清白蛋白,0.1%ProClin300。
4.根据权利要求1所述的一种磁微粒化学发光法HER2检测试剂盒,其特征在于,所述校准品的制备步骤如下:
A、配制HER2校准品缓冲液:0.05MpH7.4的TRIS缓冲液中加入1%的牛血清白蛋白,0.2%防腐剂;
B、将HER2纯品用校准品缓冲液进行稀释,校准品浓度分别为0ng/mL、10ng/mL、20ng/mL、80ng/mL、200ng/mL和400ng/mL的人表皮生长因子受体-2溶液。
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