CN114184793A - 锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法 - Google Patents
锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法 Download PDFInfo
- Publication number
- CN114184793A CN114184793A CN202111498554.5A CN202111498554A CN114184793A CN 114184793 A CN114184793 A CN 114184793A CN 202111498554 A CN202111498554 A CN 202111498554A CN 114184793 A CN114184793 A CN 114184793A
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- Prior art keywords
- transport protein
- antigen
- zinc transport
- zinc
- monomer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- 229910052725 zinc Inorganic materials 0.000 title claims abstract description 128
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Abstract
本发明涉及一种锌转运蛋白8抗体化学发光免疫检测试剂盒,包括:标记有化学发光基团的锌转运蛋白8单体抗原,锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。本发明采用标记有化学发光基团的锌转运蛋白8单体抗原,对锌转运蛋白8抗体进行检测,由于锌转运蛋白8单体抗原由锌转运蛋白8多聚体抗原还原得到,具有充分暴露的抗原位点,能够结合更多的化学发光基团,从而使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。
Description
技术领域
本发明涉及生物医学检测技术领域,具体而言,涉及一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法。
背景技术
锌转运蛋白8(Zinc transporters member 8,ZnT8),以二聚体形式定位于胰岛β细胞,能将胞浆锌离子转运至胰岛素储存/分泌性囊泡内。锌转运蛋白8转运功能降低会影响胰岛素合成、储存和分泌,能增加1型糖尿病(type 1 1diabetes mellitus,T1DM)的发病风险,ZnT8蛋白也可作为抗原引起β细胞自身免疫损伤,诱发1型糖尿病(T1DM)。1型糖尿病的特征是患者血清中存在循环性自身抗体,包括胰岛细胞抗体(ICA)、谷氨酸脱羧酶抗体(GADA)、酪氨酸磷酸酶抗体(IA-2A)、人胰岛素抗体(IAA)和锌转运蛋白8抗体(ZnT8A)。
ZnT8A是诊断1型糖尿病的特异性抗体,在1型糖尿病初发病例中阳性率仅次于抗GAD抗体。检测ZnT8A可有助于评估1型糖尿病的发病危险,对糖尿病患者的一级亲属具有很高的诊断及预后评估价值。
目前,临床上检测锌转运蛋白8抗体的常用方法是磁微粒化学发光法,利用双抗原夹心法原理进行检测,测定相关发光值RLU。化学发光免疫分析(CLIA),是用化学发光剂直接标记抗原或抗体的免疫分析方法。常用于标记的化学发光物质有吖啶酯类化合物——acridinium ester(AE),是有效的发光标记物,其通过启动发光试剂(NaOH、H2O2)作用而发光,其发光在一秒钟内完成,为快速的闪烁发光。
然而,锌转运蛋白8抗原分子结构复杂,常以多聚体形式存在,采用化学发光标记物对锌转运蛋白8抗原进行标记,锌转运蛋白8抗原活性不高,重复性差批间差大,标记率低,进而导致检测灵敏度较低。
发明内容
为了解决上述问题,本发明的第一目的在于提供一种锌转运蛋白8抗体化学发光免疫检测试剂盒,该试剂盒采用标记有化学发光基团的锌转运蛋白8单体抗原,对锌转运蛋白8抗体进行检测,由于锌转运蛋白8单体抗原具有充分暴露的抗原位点,能够结合更多的化学发光基团,从而使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。
本发明提供的一种锌转运蛋白8抗体化学发光免疫检测试剂盒,包括:标记有化学发光基团的锌转运蛋白8单体抗原,锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。
本发明的第二目的在于提供一种上述锌转运蛋白8抗体化学发光免疫检测试剂盒的制备方法,包括制备标记有化学发光基团的锌转运蛋白8单体抗原的步骤:
利用还原剂对锌转运蛋白8多聚体抗原进行还原处理,得到锌转运蛋白8单体抗原;
采用化学发光标记物对锌转运蛋白8单体抗原进行标记反应,得到化学发光标记物标记的锌转运蛋白8单体抗原。
本发明提供的锌转运蛋白8抗体化学发光免疫检测试剂盒,采用标记有化学发光基团的锌转运蛋白8单体抗原对锌转运蛋白8抗体进行检测,由于锌转运蛋白8单体抗原由锌转运蛋白8多聚体抗原还原得到,降低多聚体比例差异的影响,具有充分暴露的抗原位点,能够结合更多的化学发光基团,提高了化学发光标记物的标记率,从而使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。
具体实施方式
现将详细地提供本发明实施方式的参考,其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上,对本领域技术人员而言,显而易见的是,可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如,作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中,来产生更进一步的实施方式。
因此,旨在本发明覆盖落入所附权利要求的范围及其等同范围中的此类修改和变化。本发明的其它对象、特征和方面公开于以下详细描述中或从中是显而易见的。本领域普通技术人员应理解本讨论仅是示例性实施方式的描述,而非意在限制本发明更广阔的方面。
传统锌转运蛋白8(ZnT8)抗体的检测方法常采用磁微粒化学发光法(CLIA),其技术原理为:向锌转运蛋白8抗体待测样本依次加入包被于固相载体上的锌转运蛋白8抗原、化学发光标记物标记的锌转运蛋白8抗原,反应形成双抗原夹心抗体复合物,加入预激发液和激发液进行发光反应,根据发光强度和标准曲线计算锌转运蛋白8抗体的含量。
目前,锌转运蛋白8抗原的标记方法一般将吖啶酯直接标记在待结合物ZnT8抗原上,ZnT8抗原常以二聚体形式存在,这种标记方法存在标记率低,标记的ZnT8抗原稳定性差、活性不好、重复性差批间差大等问题。
为了至少部分解决上述技术问题的至少一个,本发明的第一方面提供了一种锌转运蛋白8抗体化学发光免疫检测试剂盒,包括:标记有化学发光基团的锌转运蛋白8单体抗原,锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。
本发明创造性地采用标记有化学发光基团的锌转运蛋白8单体抗原对锌转运蛋白8抗体进行检测,由于锌转运蛋白8单体抗原由锌转运蛋白8多聚体抗原还原得到,具有充分暴露的抗原位点,活性更高,能够结合更多的化学发光基团,从而提高了化学发光标记物的标记率,使得标记有化学发光基团的锌转运蛋白8单体抗原具有更高的检测灵敏度。
一些实施方案中,锌转运蛋白8单体抗原还修饰有封闭基团,封闭基团和锌转运蛋白8单体抗原相连接,用以起到封闭作用,防止锌转运蛋白8单体抗原再次聚合,影响锌转运蛋白8单体抗原的活性,降低锌转运蛋白8单体抗原和化学发光基团的结合效率。
一些实施方案中,封闭基团为巯基封闭基团,巯基封闭基团和锌转运蛋白8单体抗原的巯基相连接,用以起到封闭作用,防止锌转运蛋白8单体抗原通过巯基再次聚合,影响锌转运蛋白8单体抗原的活性,降低锌转运蛋白8单体抗原和化学发光基团的结合效率。
一些实施方案中,巯基封闭基团来自于N-乙基马来酰亚胺(NEM)、N-羟基马来酰亚胺和碘乙酰胺中的至少一种。可以理解的是,封闭基团的来源不受上述列举的封闭剂范围的限制,任何可以和巯基结合以起到封闭巯基的作用且不影响后续化学发光免疫检测的物质,均可以作为封闭剂以提供封闭基团。
一些实施方案中,化学发光基团来自于吖啶酯(AE-NHS、DMAE-NHS)、吖啶酸(9-吖啶甲酸)、吖啶酰胺或吖啶磺酰胺(NSP-SA-NHS)中的至少一种。其中,化学发光基团和锌转运蛋白8单体抗原上的氨基相连接,并且能够在激发液或者预激发液的激发下,快速发光,根据发光强度和标准曲线计算锌转运蛋白8抗体的含量。
本发明的第二方面提供了一种上述锌转运蛋白8抗体化学发光免疫检测试剂盒的制备方法,包括制备标记有化学发光基团的锌转运蛋白8单体抗原的步骤:
利用还原剂对锌转运蛋白8多聚体抗原进行还原处理,得到锌转运蛋白8单体抗原;
采用化学发光标记物对锌转运蛋白8单体抗原进行标记反应,得到标记有化学发光基团的锌转运蛋白8单体抗原。
具体地,将锌转运蛋白8多聚体抗原用PBS缓冲液稀释制成锌转运蛋白8多聚体抗原溶液,向ZnT8多聚体抗原溶液加入还原剂,达到还原剂的工作浓度后,进行一段时间的还原反应,反应结束后采用PBS平衡脱盐柱,对反应产物脱盐除杂,制得纯化后的ZnT8单体抗原溶液;
进一步,向ZnT8单体抗原溶液中加入溶解于DMSO溶剂的化学标记物进行一段时间的标记反应,将反应产物用截留分子量的脱盐柱,以TRIS(pH 7.4)缓冲液作为换液缓冲液,过柱,除去游离的化学标记物及反应副产物,得到标记有化学发光基团的ZnT8单体抗原溶液。
可以理解的是,通过本发明的标记方法,在对锌转运蛋白8多聚体抗原进行还原处理得到ZnT8单体抗原后,可以提高ZnT8抗原的活性,使得ZnT8抗原暴露更多的活性位点,对ZnT8单体抗原进行化学发光标记物的标记,能够增加化学发光标记物的标记率,进而采用化学发光标记物标记的ZnT8单体抗原检测抗体时,能够提高检测ZnT8单体抗体的灵敏度。
一些实施方案中,化学发光标记物的摩尔数是锌转运蛋白8单体抗原的摩尔数的1-5000倍,例如,可以是5-4000倍,10-3000倍,20-2000倍,进一步可以是30-1000倍,50-400倍,100-300倍,还可以是200倍,1500倍,2500倍,3500倍,4500倍。
一些实施方案中,还原剂包括二硫苏糖醇、巯基乙胺、巯基乙醇、三羧基乙基膦中的至少一种。
一些实施方案中,还原剂的摩尔数是锌转运蛋白8多聚体抗原摩尔数的1-2500倍,例如,可以是5-2000倍,10-1500倍,20-1000倍,进一步可以是30-600倍,40-300倍,还可以是50倍,100倍,200倍。
一些实施方案中,采用化学发光标记物对锌转运蛋白8单体抗原进行标记之前或之后还包括:采用封闭剂对锌转运蛋白8单体抗原进行封闭处理,得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原,以使得锌转运蛋白8单体抗原经化学反应连接于封闭基团。
具体地,向ZnT8单体抗原溶液加入封闭剂,达到封闭剂工作浓度后,在室温下反应一段时间,反应结束后采用PBS平衡脱盐柱,对反应产物脱盐除杂,制得纯化后的巯基封闭的ZnT8单体抗原溶液。可以理解的是,封闭巯基的步骤可以在标记ZnT8单体抗原之前进行,也可以在标记ZnT8单体抗原之后进行。
一些实施方案中,封闭剂的摩尔数是锌转运蛋白8多聚体抗原摩尔数的1-2000倍,优选为20-200倍。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1
本实施例提供了一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法,试剂盒包括巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原,试剂盒的制备方法包括:
1、吖啶酯标记的锌转运蛋白8单体抗原溶液的制备
(1)将锌转运蛋白8用0.05M PBS缓冲液稀释至0.5mg/mL,制得多聚体抗原溶液;
(2)在步骤(1)的多聚体抗原溶液中加入二硫苏糖醇(DTT),使其终浓度为1.5mg/mL,温和混匀后,在室温(25℃)条件下反应60min;采用PBS平衡脱盐柱,并对反应结束后的产物脱盐除杂,制得纯化后的锌转运蛋白8单体抗原溶液;
(3)在步骤(2)纯化后的锌转运蛋白8单体抗原溶液中加入N-乙基马来酰亚胺(NEM),使其终浓度为0.5mg/mL,温和混匀后,在室温(25℃)条件下反应60min。采用PBS平衡脱盐柱,并对反应结束后的产物脱盐除杂,制得纯化后的巯基封闭的锌转运蛋白8单体抗原溶液;
(4)向锌转运蛋白单体抗原溶液中加入10μL溶解于DMSO溶剂的6.0mg/mL吖啶酯,于25℃反应60min,用2mL 7KD截留分子量的脱盐柱(Thermo fisher公司)以50mM TRIS(pH7.4)缓冲液作为换液缓冲液,过柱3遍,除去游离的吖啶酯及反应副产物,得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原。
2、锌转运蛋白8单体抗原包被的纳米磁珠的制备
取50mg羧基化的磁微粒(粒径为0.05-1um)悬浮液,磁分离去上清,用0.02M,pH为5.5MES缓冲液重悬,加入0.5-2mL新配置的10mg/mL的EDC水溶液,活化磁珠表面羧基,加入3-5mg锌转运蛋白8,室温下混悬2-10h,磁分离,去除上清,用含2%BSA的0.1M pH为8.0的Tris缓冲液重悬到1mg/mL,得到锌转运蛋白8包被的磁微粒,每瓶5mL分装保存于4℃备用。
3、锌转运蛋白8抗体定标品的制备
用标准品缓冲液(40mM Tris-HCl,0.5%BSA,1%NaCl,pH 8.0)将锌转运蛋白8抗体配置成浓度为5AU/mL、20AU/mL、80AU/mL、200AU/mL、800AU/mL、2000AU/mL,每瓶0.5mL分装冻干,4℃保存备用。
4、化学发光预激发液的配制
量取1.0升纯化水,依次加入80μL质量分数为20%的双氧水(H2O2)、1.0克叠氮化钠、1.5克吐温20,摇匀后避光存放。
5、化学发光激发液的配制
量取1.0升纯化水,依次加入0.6克氢氧化钠、0.5克PC300、0.5g叠氮化钠、1.5克Triton 405,摇匀后避光存放。
实施例2
本实施例提供了一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法,试剂盒包括巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原,试剂盒的制备方法包括:
1、吖啶酯标记的锌转运蛋白8单体抗原溶液的制备
(1)将锌转运蛋白8抗原用0.05M PBS缓冲液稀释至0.5mg/mL,制得锌转运蛋白8多聚体抗原溶液;
(2)在步骤(1)的锌转运蛋白8抗原溶液中加入二硫苏糖醇(DTT),使其终浓度为1.5mg/mL,温和混匀后,在室温(25℃)条件下反应60min;采用PBS平衡脱盐柱,并对反应结束后的产物脱盐除杂,制得纯化后的锌转运蛋白8单体抗原溶液;
(3)向抗原溶液中加入10μL溶解于DMSO溶剂的6.0mg/mL吖啶酯,于25℃反应60min,用2mL 7KD截留分子量的脱盐柱(Thermo fisher公司)以50mM TRIS(pH 7.4)缓冲液作为换液缓冲液,过柱3遍,除去游离的吖啶酯及反应副产物,得到吖啶酯标记的锌转运蛋白8单体抗原溶液;
(4)在步骤(3)的结合产物吖啶酯-抗原溶液中加入N-乙基马来酰亚胺(NEM),使其终浓度为0.5mg/mL,温和混匀后,在室温(25℃)条件下反应60min,采用PBS平衡脱盐柱,并对反应结束后的产物脱盐除杂,制得纯化后的巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原溶液。
2、锌转运蛋白8单体抗原包被的纳米磁珠的制备
取50mg羧基化的磁微粒(粒径为0.05-1um)悬浮液,磁分离去上清,用0.02M,pH为5.5MES缓冲液重悬,加入0.5-2mL新配置的10mg/mL的EDC水溶液,活化磁珠表面羧基,加入3-5mg锌转运蛋白8单体抗原,室温下混悬2-10h,磁分离,去除上清,用含2%BSA的0.1M pH为8.0的Tris缓冲液重悬到1mg/mL,得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原包被的磁微粒,每瓶5mL分装保存于4℃备用。
3、锌转运蛋白8抗体定标品的制备
用标准品缓冲液(40mM Tris-HCl,0.5%BSA,1%NaCl,pH 8.0)将锌转运蛋白8抗体配置成浓度为5AU/mL、20AU/mL、80AU/mL、200AU/mL、800AU/mL、2000AU/mL,每瓶0.5mL分装冻干,4℃保存备用。
4、化学发光预激发液的配制
量取1.0升纯化水,依次加入80μL质量分数为20%的双氧水(H2O2)、1.0克叠氮化钠、1.5克吐温20,摇匀后避光存放。
5、化学发光激发液的配制
量取1.0升纯化水,依次加入0.6克氢氧化钠、0.5克PC300、0.5g叠氮化钠、1.5克Triton 405,摇匀后避光存放。
对比例1
本实施例提供了一种锌转运蛋白8抗体化学发光免疫检测试剂盒及其制备方法,试剂盒包括吖啶酯标记的锌转运蛋白8多聚体抗原,试剂盒的制备方法包括:
1、吖啶酯标记的锌转运蛋白8抗原溶液的制备
(1)将锌转运蛋白8抗原用0.05M PBS缓冲液稀释至0.5mg/mL,制得抗原溶液;
(2)向抗原溶液中加入10μL溶解于DMSO溶剂的6.0mg/mL吖啶酯,于25℃反应60min,用2mL 7KD截留分子量的脱盐柱(Thermo fisher公司)以50mM TRIS(pH 7.4)缓冲液作为换液缓冲液,过柱3遍,除去游离的吖啶酯及反应副产物,得到吖啶酯标记的锌转运蛋白8抗原溶液。
2、吖啶酯标记的锌转运蛋白8抗原包被的纳米磁珠的制备
取50mg羧基化的磁微粒(粒径为0.05-1um)悬浮液,磁分离去上清,用0.02M,pH为5.5MES缓冲液重悬,加入0.5-2mL新配置的10mg/mL的EDC水溶液,活化磁珠表面羧基,加入3-5mg锌转运蛋白8单体抗原,室温下混悬2-10h,磁分离,去除上清,用含2%BSA的0.1M pH为8.0的Tris缓冲液重悬到1mg/mL,得到锌转运蛋白8抗原包被的磁微粒,每瓶5mL分装保存于4℃备用。
3、锌转运蛋白8抗体定标品的制备
用标准品缓冲液(40mM Tris-HCl,0.5%BSA,1%NaCl,pH 8.0)将锌转运蛋白8抗体配置成浓度为5AU/mL、20AU/mL、80AU/mL、200AU/mL、800AU/mL、2000AU/mL,每瓶0.5mL分装冻干,4℃保存备用。
4、化学发光预激发液的配制
量取1.0升纯化水,依次加入80μL质量分数为20%的双氧水(H2O2)、1.0克叠氮化钠、1.5克吐温20,摇匀后避光存放。
5、化学发光激发液的配制
量取1.0升纯化水,依次加入0.6克氢氧化钠、0.5克PC300、0.5g叠氮化钠、1.5克Triton 405,摇匀后避光存放。
二、锌转运蛋白8抗体化学发光免疫检测
本实施例以全自动化学发光免疫分析仪为检测工具,本实施例的检测方法为双抗原夹心法,即仪器依次加入25μL的样品、50μL的锌转运蛋白8包被的磁微粒以及50μL的吖啶酯标记锌转运蛋白8或者巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原,反应10min后,进行磁分离,仪器将反应混合物送入暗室,依次加入50μL化学发光预激发液、50μL化学发光激发液进行发光反应,最后记录发光强度,从标准曲线计算出被测样品的锌转运蛋白8抗体含量。
三、锌转运蛋白8抗体化学发光免疫检测试剂盒的性能评价
1、灵敏度的检测
对相同浓度的样本分别采用实施例1、2及对比例1中的试剂盒进行锌转运蛋白8抗体化学发光免疫检测,灵敏度用检测信号值表征,在相同样本浓度下,信号值越高,说明灵敏度越高,具体检测结果如表1所示。根据表1可知,在6.83~285.93AU/mL的浓度范围内,实施例1和实施例2的试剂盒灵敏度明显大于对比例1的试剂盒。
表1
2、特异性检测
分别采用实施例1、2及对比例1中的试剂盒检测含ZnT8A的样本或不含ZnT8A的样本,根据检测结果判断样本是否含ZnT8A。特异性以将实际无病者正确地判断为阴性的百分率标示,即通过检测阴性样本的正确率来表征特异性,正确率越高,说明准确度越高,特异性越好,具体检测结果如表2所示。根据表2可知,实施例1和实施例2的试剂盒的特异性明显高于比对例1的试剂盒。
表2
实施例1 | 实施例2 | 实施例3 | |
阳性 | 115 | 116 | 116 |
阴性 | 3 | 2 | 10 |
总计 | 118 | 118 | 126 |
特异性 | 96.9% | 98.0% | 89.8% |
3、稳定性检测
将实施例1、2及对比例1制备的试剂盒中抗原溶液分别放至4℃和37℃恒温箱7天,检测不同浓度样本的化学发光信号值,以4℃抗原溶液为对照,计算抗原溶液37℃加速7天稳定性。稳定性偏差绝对数值越小,说明工作液稳定性越好,具体检测结果如表3、表4和表5所示。根据表3、表4和表5可知,实施例1和实施例2的试剂盒中的抗原溶液的稳定性明显高于对比例1的试剂盒中的抗原溶液。
表3
表4
表5
4、批间差
使用三批不同批次抗原分别进行吖啶标记制备实施例1和实施例2以及对比例1的试剂盒,测试不同浓度样本的化学发光信号值,检测不同批次的原料制备的试剂盒差异,以第一批试剂盒为对照,计算第二批、第三批相对偏差,相对偏差越小说明批间差越小,结果如下表6、表7和表8所示。根据表6、表7和表8的数据可知,实施例1和实施例2制备的试剂盒的批间差异远小于对比例1的试剂盒的批间差异。
表6
表7
表8
以上实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种锌转运蛋白8抗体化学发光免疫检测试剂盒,其特征在于,包括:标记有化学发光基团的锌转运蛋白8单体抗原,所述锌转运蛋白8单体抗原通过还原锌转运蛋白8多聚体抗原得到。
2.根据权利要求1所述的试剂盒,其特征在于,所述锌转运蛋白8单体抗原还修饰有封闭基团,所述封闭基团和所述锌转运蛋白8单体抗原经化学键相连接。
3.根据权利要求2所述的试剂盒,其特征在于,所述封闭基团为巯基封闭基团,所述封闭基团和所述锌转运蛋白8单体抗原的巯基经化学键相连接。
4.根据权利要求3所述的试剂盒,其特征在于,所述化学发光基团来自于吖啶酯、吖啶酸、吖啶酰胺和吖啶磺酰胺中的至少一种;
所述巯基封闭基团来自于N-乙基马来酰亚胺、N-羟基马来酰亚胺和碘乙酰胺中的至少一种。
5.一种如权利要求1~4任一项所述的试剂盒的制备方法,其特征在于,包括如下步骤:
利用还原剂对锌转运蛋白8多聚体抗原进行还原处理,得到锌转运蛋白8单体抗原;
采用化学发光标记物对锌转运蛋白8单体抗原进行标记反应,得到标记有化学发光基团的锌转运蛋白8单体抗原。
6.根据权利要求5所述的制备方法,其特征在于,所述化学发光标记物的摩尔数是锌转运蛋白8单体抗原的摩尔数的1-5000倍。
7.根据权利要求5所述的制备方法,其特征在于,所述还原剂包括二硫苏糖醇、巯基乙胺、巯基乙醇以及三羧基乙基膦中的至少一种。
8.根据权利要求7所述的制备方法,其特征在于,所述还原剂的摩尔数是所述锌转运蛋白8多聚体抗原摩尔数的1-2500倍。
9.根据权利要求6所述的制备方法,其特征在于,所述采用化学发光标记物对锌转运蛋白8单体抗原进行标记之前或之后还包括:
采用封闭剂对锌转运蛋白8单体抗原进行封闭处理,得到巯基封闭的吖啶酯标记的锌转运蛋白8单体抗原,以使得锌转运蛋白8单体抗原经化学反应连接于封闭基团。
10.根据权利要求9所述的制备方法,其特征在于,所述封闭剂的摩尔数是所述锌转运蛋白8多聚体抗原摩尔数的1-2000倍。
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