CN117434257A - 一种PM/Scl抗体联合检测试剂盒及方法 - Google Patents
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Abstract
本发明提供了一种PM/Scl抗体联合检测试剂盒及方法,制备方法包括:磁微粒试剂的制备和酶标记物试剂的制备,磁微粒试剂包括包被有PM/Scl‑75抗原与PM/Scl‑100抗原两种抗原的磁微粒。本发明可对使用单一抗原作为检测原料而漏检的重叠综合征样本起到显著的改善作用,能将多数因漏检而产生的假阴性样本和信号值偏低的样本检出,且阴性样本不会受到干扰。本发明涉及的检测平台为化学发光平台,与传统的免疫印迹法相比,具有自动化程度高,线性范围宽,灵敏度高,结果重复性好,定量结果更精确等特点。
Description
技术领域
本发明属于抗体检测技术领域,涉及一种PM/Scl抗体联合检测试剂盒及方法。
背景技术
特发性炎性肌病(IIMs)是一组以肌肉受损为主的慢性自身免疫病,包括多发性肌炎(PM)、皮肌炎(DM)、无肌病性皮肌炎(CADM)、包涵体肌炎(IBM)和坏死性自身免疫性肌病(NAM)等亚型,其中PM和DM是临床常见类型。科学家在皮肌炎(PM)与系统性硬化症相重叠的患者血清中首先报道了抗PM-1抗体,此自身抗体因多见于PM患者而得名,但后来研究发现该自身抗体更多见于PM和系统性硬化症(Scl)相重叠的患者中,故又称为抗PM/Scl抗体。
抗PM-Scl抗体是一种自身抗体,其主要的检测方法包括免疫双扩散法、酶联免疫吸附测定法、蛋白印迹法和免疫沉淀法。PM-Scl抗原主要位于核仁的颗粒部分,是由11~16种蛋白多肽组成的复合物,分子量20~100kD。PM/Scl-100是PM/Scl的主要靶抗原,故实验室大部分检测都是基于重组PM/Scl-10蛋白进行,容易导致漏检。而膜条的检测方法具有操作复杂,耗时时间较长,自动化程度低,结果判读不精确,假阳性率较高等缺点。
发明内容
针对现有技术中存在的问题,本发明提供了一种PM/Scl抗体联合检测试剂盒及方法,通过以两种抗原作为原材料开发的化学发光检测方法,可以达到省时省力且准确度更高的目的。
本发明为实现上述目的采用的技术方案为:
本发明提供了一种PM/Scl抗体联合检测试剂盒的制备方法,包括:
(1)磁微粒标记物的制备:
在离心管中加入磁微粒包被缓冲液,将PM/Scl-75抗原与PM/Scl-100抗原分别与磁微粒按照1~10μg/mg的比例进行混合,25~45℃下包被6~12h,然后用封闭液封闭6~12h,用磁微粒清洗液清洗包被后的两种磁微粒;
(2)酶标记物的制备:
2-1)取鼠抗人单克隆抗体加入2-亚氨基硫烷盐酸盐溶液,得到浓度为0.05~0.2g/mL的混合溶液,室温静置30~120min,加入1M甘氨酸溶液封闭10~30min,除盐,收集活化后的鼠抗人单克隆抗体;将4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液加入至碱性磷酸酶中,得到浓度为0.05~0.2g/mL的混合溶液,室温静置30~120min,加入1M甘氨酸溶液封闭10~30min,除盐,收集活化后的碱性磷酸酶;
2-2)将活化的鼠抗人单克隆抗体与活化的碱性磷酸酶按照设定比例进行混合,常温下静置2~4h,再纯化偶联物,获得鼠抗人单克隆抗体-碱性磷酸酶标记物溶液。
优选地,步骤(1)还包括:将两种包被后的磁微粒按照设定配比投料,用磁微粒稀释液稀释形成终浓度为0.05~0.2mg/mL的磁微粒试剂;步骤(2)还包括:将步骤2-2)所得产物按照1:1000~1:8000的体积比添加入酶标记物稀释液中,过滤后即为酶标记物试剂。
更优选地,所述磁微粒稀释液中包含十二水磷酸氢二钠29g/L,二水磷酸二氢钠2.96g/L,氯化钠9g/L,牛血清白蛋白10g/L,Proclin-3000.1%(体积百分数,v/v),吐温-200.1%(体积百分数,v/v),pH 7.4±0.1;和/或所述酶标记物稀释液中包含取2-吗啉乙磺酸19.52g/L,氯化钠9g/L,牛血清白蛋白10g/L,Proclin-3000.1%(体积百分数,v/v),吐温-20 0.1%(体积百分数,v/v),pH 6.0±0.1。
更优选地,将两种包被后的磁微粒按照质量比1:1的比例进行投料。
优选地,步骤2-1)中,所述2-亚氨基硫烷盐酸盐溶液是将2-亚氨基硫烷盐酸盐用纯化水稀释至5mg/mL;和/或所述4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液是将4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯用N,N-二甲基甲酰胺稀释至5mg/mL。
优选地,步骤2-1)中,每mg鼠抗人单克隆抗体用2~5μL 1M甘氨酸溶液封闭;每mg碱性磷酸酶用2~5μL 1M甘氨酸溶液封闭。
优选地,步骤2-1)中,两次除盐均采用G-25凝胶柱;和/或步骤2-2)中,用Supperdex200凝胶纯化柱纯化偶联物。
优选地,步骤2-2)中,将活化的鼠抗人单克隆抗体与活化的碱性磷酸酶按照质量比1:1进行混合。
本发明还提供了一种PM/Scl抗体联合检测试剂盒,通过任一上述的制备方法制得。
本发明还提供了采用上述一种PM/Scl抗体联合检测试剂盒进行检测的方法,包括:将试剂与样本一起放入全自动化学发光仪中,仪器先加入样本与磁微粒试剂,孵育设定时间后,仪器自动进行清洗;然后加入酶标记物试剂,孵育设定时间后,仪器自动开始清洗,
等待清洗完成,再加入发光底物液,孵育设定时间后,进行测试,待测物信号与待测物浓度成正比,待测物浓度越高,则检测结果的信号值越大。
本发明可对使用单一抗原作为检测原料而漏检的重叠综合征样本起到显著的改善作用,能将多数因漏检而产生的假阴性样本和信号值偏低的样本检出,且阴性样本不会受到干扰。本发明涉及的检测平台为化学发光平台,与传统的免疫印迹法相比,具有自动化程度高,线性范围宽,灵敏度高,结果重复性好,定量结果更精确等特点。
具体实施方式
为了更清楚地说明本发明,下面结合实施例对本发明作进一步详细说明。本领域技术人员应当理解,下面所具体描述的内容是说明性的而非限制性的,不应以此限制本发明的保护范围。
本发明中,“常温”或“室温”通常约为25℃,可以是20℃±5℃。
本发明涉及试剂组分共两种:磁微粒试剂M,酶标记物试剂R1。
本发明涉及检测方法为间接法。测试时,将试剂与样本一起放入全自动化学发光仪中,仪器先加入样本与磁微粒试剂M,孵育一段时间后,仪器自动进行清洗;然后加入酶标记物试剂R1,孵育一段时间后,仪器自动开始清洗,等待清洗完成,再加入发光底物液,孵育一段时间后,进行测试,待测物信号与待测物浓度成正比,待测物浓度越高,则检测结果的信号值越大。
如未特殊说明,以下实施例中所用的试剂均为市售常规试剂,具体操作可采用常规技术手段来实现。
实施例
一、磁微粒标记物的制备
磁微粒偶联过程所需试剂配制
1)包被液A:称取29g十二水磷酸氢二钠,2.96g二水磷酸二氢钠,9g氯化钠,加入800mL纯化水,充分搅拌至完全溶解,再加入纯化水至体积为1L,并将pH调整为7.4±0.1,将溶液用0.22μm的滤膜进行过滤,即为包被液A。
2)包被液B:称取29g十二水磷酸氢二钠,2.96g二水磷酸二氢钠,9g氯化钠,硫酸铵264g,加入800mL纯化水,充分搅拌至完全溶解,再加入纯化水至体积为1L,并将pH调整为7.4±0.1,将溶液用0.22μm的滤膜进行过滤,即为包被液B。
3)封闭液:称取29g十二水磷酸氢二钠,2.96g二水磷酸二氢钠,9g氯化钠,加入800mL纯化水,充分搅拌至完全溶解;
加入5g酪蛋白,充分搅拌至完全溶解;
加入1mL的Proclin-300,1mL的吐温-20充分搅拌至完全溶解;再加入纯化水至体积为1L,并将pH调整为7.4±0.1,将溶液用0.22μm的滤膜进行过滤,即为封闭液。
4)磁微粒清洗液:称取29g十二水磷酸氢二钠,2.96g二水磷酸二氢钠,9g氯化钠,加入800mL纯化水,充分搅拌至完全溶解;
加入1mL的Proclin-300,1mL的吐温-20充分搅拌至完全溶解;再加入纯化水至体积为1L,并将pH调整为7.4±0.1,将溶液用0.22μm的滤膜进行过滤,即为磁微粒清洗液。
5)磁微粒保存液:称取29g十二水磷酸氢二钠,2.96g二水磷酸二氢钠,9g氯化钠,加入800mL纯化水,充分搅拌至完全溶解;
加入10g牛血清白蛋白,充分搅拌至完全溶解;
加入1mL的Proclin-300,1mL的吐温-20充分搅拌至完全溶解;再加入纯化水至体积为1L,并将pH调整为7.4±0.1,将溶液用0.22μm的滤膜进行过滤,即为磁微粒保存液。
磁微粒偶联流程如下:
取选定的磁微粒(杭州博岳生物技术有限公司,货号:M1000T,粒径1.2μm)两管,每管5mg,加入1.5mL的离心管中,将PM/Scl-75抗原(货号:PRO-126,购自prospecbio)与PM/Scl-100抗原(货号:PRO-444,购自prospecbio)与磁微粒按照5μg/mg的比例进行包被,流程如下:
表1.磁微粒偶联所用试剂及用量
试剂 | 体积 |
包被液A | 100μL |
包被液B | 100μL |
封闭液 | 200μL |
磁微粒清洗液 | 200μL |
磁微粒保存液 | 500μL |
1-1)取表1中相应体积的包被液A与B,将所需磁微粒与抗原进行混合后,恒温金属浴37℃下进行包被,时长10h。
1-2)加入封闭液A,恒温金属浴37℃下进行封闭,时长6h。
1-3)封闭完成后取出,取表1中相应体积的磁微粒清洗液清洗三次,最后加入表1中相应体积的磁微粒保存液在2~8℃进行保存。
二、酶标记物的制备
酶标记物制备所需溶液的配制
2-亚氨基硫烷盐酸盐溶液:称取2-亚氨基硫烷盐酸盐11.5mg,并用纯化水2.3mL溶解为5mg/mL的溶液备用,此溶液需现配现用。
4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液:称取13.7mg的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯,并用N,N-二甲基甲酰胺2.74mL溶解为5mg/mL的溶液备用。
1M甘氨酸:称取15g甘氨酸,溶解于200mL纯化水中可得。
酶标记物的标记流程如下:
取碱性磷酸酶(采购自Sigma-Aldrich,货号:P7640)与鼠抗人单克隆抗体(武汉奥克博泰生物科技有限公司,货号:A0032)各1mg,分别加入两个1.5mL离心管内进行标记,流程如下:
表2.酶标记物的制备所用试剂及用量
试剂 | 体积 |
2-亚氨基硫烷盐酸盐溶液 | 10μL |
4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液 | 20μL |
1M甘氨酸 | 5μL |
2-1)分别取表2中相应体积的2-亚氨基硫烷盐酸盐溶液和4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液加入鼠抗人单克隆抗体和碱性磷酸酶中。混匀后,25℃静置反应1.5h。
2-2)活化后的产物分别加入表2中相应体积的1M甘氨酸在25℃下封闭10min,然后分别用G-25凝胶柱对活化后的鼠抗人单克隆抗体和碱性磷酸酶进行脱盐,收集活化后的产物。
2-3)将纯化后的鼠抗人单克隆抗体和碱性磷酸酶按质量比1:1进行混合,混匀后25℃静置反应3h,Supperdex200凝胶纯化柱纯化偶联物,获得鼠抗人单克隆抗体-碱性磷酸酶标记物,加入与产物等体积的甘油,-20℃下保存。
测试例
测试前需配制磁微粒标记物与酶标记物稀释液
1)磁微粒标记物稀释液配制过程:
取33.9g十二水磷酸氢二钠,0.827g二水磷酸二氢钠,9g氯化钠,加入800mL纯化水,充分搅拌至完全溶解;
加入10g牛血清白蛋白,充分搅拌至完全溶解;
加入1mLProclin-300,1mL吐温-20充分搅拌至完全溶解,并补加纯化水至体积为1L,并将pH调整为8.0±0.1。
将溶液用0.22μm的滤膜进行过滤,即为磁微粒标记物稀释液。
2)酶标记物稀释液配制过程:
取19.52g 2-吗啉乙磺酸,9g氯化钠,加入800mL纯化水,充分搅拌至完全溶解;用6M氢氧化钠溶液调节pH至6.0;
加入10g牛血清白蛋白,Proclin-3001mL,吐温-20 1mL充分搅拌至完全溶解;并补加纯化水至体积为1L,并将pH调整为6.0±0.1。
将溶液用0.22μm的滤膜进行过滤,即为酶标记物稀释液。
3)将实施例步骤一中的两种磁微粒标记物按照质量比1:1进行配比,用磁微粒标记物稀释液稀释为终浓度0.1mg/mL的工作液,此即为磁微粒试剂M。将实施例步骤二中的酶标记物与酶标记物稀释液按照体积比1:4000的比例进行稀释,用0.22μm的滤膜进行过滤,滤后所得溶液即为酶标记物试剂R1。测试前,需将磁微粒试剂M与酶标记物试剂R1装入试剂盒中组装为测试所需试剂,配套使用。
4)测试时,将试剂与30μL样本一起放入全自动化学发光仪中,仪器先加入样本与磁微粒试剂M,孵育设定时间后,仪器自动进行清洗;然后加入酶标记物试剂R1,孵育设定时间后,仪器自动开始清洗,等待清洗完成,再加入发光底物液,孵育设定时间后,进行测试,待测物信号与待测物浓度成正比,待测物浓度越高,则检测结果的信号值越大。欧蒙膜条和市售化学发光产品则按照其提供的说明书进行操作。
表3.本发明产品与市售产品对比结果
表3为检测结果的对比,以欧蒙膜条测试结果为标准,与市售发光产品相比,本发明所测试的弱阳性结果的检测信号有明显的提升,部分阳性与强阳性的检测结果也有明显改善,且阴性结果没有明显的升高,整体检测结果与欧蒙膜条测试结果匹配程度明显提高。
显然,本发明的上述实施例仅仅是为更清楚地说明本发明所作的举例,而并非是对本发明的实施方式的限定,对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化或变动,这里无法对所有的实施方法予以穷举,凡是属于本发明的技术方案所引申出的显而易见的变化或变动仍处于本发明的保护范围之列。
Claims (10)
1.一种PM/Scl抗体联合检测试剂盒的制备方法,包括:
(1)磁微粒标记物的制备:
在离心管中加入磁微粒包被缓冲液,将PM/Scl-75抗原与PM/Scl-100抗原分别与磁微粒按照1~10μg/mg的比例进行混合,25~45℃下包被6~12h,然后用封闭液封闭6~12h,用磁微粒清洗液清洗包被后的两种磁微粒;
(2)酶标记物的制备:
2-1)取鼠抗人单克隆抗体加入2-亚氨基硫烷盐酸盐溶液,得到浓度为0.05~0.2g/mL的混合溶液,室温静置30~120min,加入1M甘氨酸溶液封闭10~30min,除盐,收集活化后的鼠抗人单克隆抗体;将4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液加入至碱性磷酸酶中,得到浓度为0.05~0.2g/mL的混合溶液,室温静置30~120min,加入1M甘氨酸溶液封闭10~30min,除盐,收集活化后的碱性磷酸酶;
2-2)将活化的鼠抗人单克隆抗体与活化的碱性磷酸酶按照设定比例进行混合,常温下静置2~4h,再纯化偶联物,获得鼠抗人单克隆抗体-碱性磷酸酶标记物溶液。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)还包括:将两种包被后的磁微粒按照设定配比投料,用磁微粒稀释液稀释形成终浓度为0.05~0.2mg/mL的磁微粒试剂;步骤(2)还包括:将步骤2-2)所得产物按照1:1000~1:8000的体积比添加入酶标记物稀释液中,过滤后即为酶标记物试剂。
3.根据权利要求2所述的制备方法,其特征在于,所述磁微粒稀释液中包含十二水磷酸氢二钠29g/L,二水磷酸二氢钠2.96g/L,氯化钠9g/L,牛血清白蛋白10g/L,Proclin-3000.1%,吐温-20 0.1%,pH 7.4±0.1;和/或所述酶标记物稀释液中包含取2-吗啉乙磺酸19.52g/L,氯化钠9g/L,牛血清白蛋白10g/L,Proclin-3000.1%,吐温-20 0.1%,pH 6.0±0.1。
4.根据权利要求2所述的制备方法,其特征在于,将两种包被后的磁微粒按照质量比1:1的比例进行投料。
5.根据权利要求1所述的制备方法,其特征在于,步骤2-1)中,所述2-亚氨基硫烷盐酸盐溶液是将2-亚氨基硫烷盐酸盐用纯化水稀释至5mg/mL;和/或所述4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液是将4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯用N,N-二甲基甲酰胺稀释至5mg/mL。
6.根据权利要求1所述的制备方法,其特征在于,步骤2-1)中,每mg鼠抗人单克隆抗体用2~5μL 1M甘氨酸溶液封闭;每mg碱性磷酸酶用2~5μL 1M甘氨酸溶液封闭。
7.根据权利要求1所述的制备方法,其特征在于,步骤2-1)中,两次除盐均采用G-25凝胶柱;和/或步骤2-2)中,用Supperdex200凝胶纯化柱纯化偶联物。
8.根据权利要求1所述的制备方法,其特征在于,步骤2-2)中,将活化的鼠抗人单克隆抗体与活化的碱性磷酸酶按照质量比1:1进行混合。
9.一种PM/Scl抗体联合检测试剂盒,通过权利要求1~8任意一项所述的制备方法制得。
10.采用权利要求9所述的一种PM/Scl抗体联合检测试剂盒进行检测的方法,包括:将试剂与样本一起放入全自动化学发光仪中,仪器先加入样本与磁微粒试剂,孵育设定时间后,仪器自动进行清洗;然后加入酶标记物试剂,孵育设定时间后,仪器自动开始清洗,等待清洗完成,再加入发光底物液,孵育设定时间后,进行测试,待测物信号与待测物浓度成正比,待测物浓度越高,则检测结果的信号值越大。
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