CN114152753B - 一种用于检测人Dsg3 IgG抗体的ELISA试剂盒及其应用 - Google Patents
一种用于检测人Dsg3 IgG抗体的ELISA试剂盒及其应用 Download PDFInfo
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- CN114152753B CN114152753B CN202111341905.1A CN202111341905A CN114152753B CN 114152753 B CN114152753 B CN 114152753B CN 202111341905 A CN202111341905 A CN 202111341905A CN 114152753 B CN114152753 B CN 114152753B
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Abstract
本发明公开了一种用于检测人Dsg3 IgG抗体的ELISA试剂盒及其应用。所述的试剂盒中含有Dsg3微孔条带，所述的Dsg3微孔条带中分别包被有SEQ ID NO.1‑20所示的多肽或所述多肽的混合物。优选的，所述的试剂盒中还含有标准血清1和2、酶标抗体、稀释液、反应缓冲液、清洗缓冲液、酶基质液以及终止液。本发明利用多个抗原多肽来替代长片段的完整蛋白作为ELISA的包被蛋白，在充分保障了抗体识别的抗原表位的同时，节约了大量的成本，且相对于重组蛋白而言，敏感性会更高。本发明利用ELISA方法检测人Dsg3 IgG抗体，相对于现有的免疫印迹法，更容易应用于临床对寻常型天疱疮的诊断。
Description
技术领域
本发明涉及一种ELISA试剂盒及其应用，特别涉及一种用于检测人Dsg3 IgG抗体的ELISA试剂盒及其应用。本发明属于医药技术领域。
背景技术
人皮肤自身免疫性水疱病主要分为两类，分别是天疱疮和类天疱疮。天疱疮的诊断主要依赖于实验室对Desmoglein 1(Dsg1)和Desmoglein 3(Dsg3)自身抗体的检测。其中可疑天疱疮患者如果只检测到Dsg1的自身抗体就会被诊断为落叶型天疱疮，如果同时检测到Dsg1和Dsg3的自身抗体就会被诊断为寻常型天疱疮(Pemphigus vulgaris，PV)，如果只检测到Dsg3的自身抗体就会被诊断为黏膜优位的寻常型天疱疮。
现有的检测人Dsg3的IgG自身抗体的方法主要是免疫印迹法和ELISA法两种。免疫印迹法主要使用的是表皮抽提物作为抗原。表皮抽提物的制备过程非常复杂，目前全世界也只有非常专业的实验室才能制备，重组蛋白获得较容易，但是费用较高，最关键的问题是免疫印迹的方法比较复杂，不适宜在临床进行广泛应用。目前的商品化的检测Dsg3 IgG自身抗体的ELISA试剂盒因其只针对Dsg3的胞外区进行检测，这个区域被认为是寻常型天疱疮患者血清中的自身抗体主要识别的区域。但是，近期发现，一些天疱疮患者体内还存在着识别Dsg3胞内区的抗体，而目前商品化识别Dsg3 IgG抗体检测试剂盒对识别Dsg3其它区域的抗体无法检出，会出现假阴性的情况，从而影响患者的诊断及后续的治疗，不能满足未来天疱疮的诊断需求。
有鉴于此，本发明提出了一种新型的Dsg3 IgG抗体检测ELISA试剂盒，该试剂盒能够对识别Dsg3全长的全部抗原表位的抗体进行检测，提高了自身抗体的检出率，进而提高了临床上对天疱疮的诊断准确性。
发明内容
本发明的目的在于提供一种用于检测人Dsg3 IgG抗体的ELISA试剂盒及其应用。
为了达到上述目的，本发明采用了以下技术手段：
本发明的一种用于检测人Dsg3 IgG抗体的ELISA试剂盒，所述的试剂盒中含有Dsg3微孔条带，所述的Dsg3微孔条带中分别包被有SEQ ID NO.1-20所示的多肽或所述多肽的混合物。
其中，优选的，所述的试剂盒中还含有标准血清1和2、酶标抗体、稀释液、反应缓冲液、清洗缓冲液、酶基质液以及终止液。
其中，优选的，所述的标准血清1为含0.05％叠氮钠w/v的反应缓冲液；所述的标准血清2为含有100U/ml的Dsg3 IgG抗体以及0.05％w/v叠氮钠的反应缓冲液；所述的酶标抗体为辣根过氧化物酶标记的兔抗人IgG抗体；所述的反应缓冲液为含0.05％v/v Tween20和0.05％w/vNaN3的1xPBS，所述的清洗缓冲液为含0.5％v/vTween20的10xPBS；所述的酶基质液为TMB显色液；所述的终止液为0.5N盐酸溶液。
其中，优选的，所述的含有Dsg3微孔条带通过以下方法制备得到：
1)将SEQ ID NO.1-20所示的多肽分别用PBS溶解，得到储存浓度为4mg/ml的多肽溶液，所有溶解后的多肽等体积混合，获得4mg/ml的多肽混合物，包被前使用PBS进行1000倍稀释，使得多肽混合物的终浓度达到4μg/ml；取96孔板进行抗原多肽的包被，每孔加入多肽混合物100μl，4℃进行包被12-16h；或
2)将SEQ ID NO.1-20所示的多肽分别用PBS溶解，得到储存浓度为4mg/ml的多肽溶液，包被前分别使用PBS进行1000倍稀释，使得多肽的终浓度达到4μg/ml；取96孔板进行抗原多肽的包被，每孔分别加入一种多肽溶液100μl，4℃进行包被12-16h。
本发明的一种人Dsg3的IgG抗体检测试剂盒(ELISA)，利用ELISA方法定性测定血清中的Dsg3的IgG抗体。检测时，患者血清和标准血清加入到包被有Dsg3抗原多肽的微孔条带，Dsg3的IgG抗体与抗原结合。洗涤后，去除未结合的血清蛋白，然后微孔中加入辣根过氧化物酶标记的抗人IgG抗体与人IgG结合，洗涤后，加入辣根过氧化物酶底物与辣根过氧化物酶反应，最后通过加入酸溶液终止酶反应。使用酶标仪测定吸光度，量化检测结果。
其中，优选的，所述的用于检测人Dsg3 IgG抗体时，按照以下步骤进行：
(1)试剂准备
实验前，将所有检测材料置于室温下(20-30℃)，根据需要用蒸馏水稀释适量的清洗缓冲液；
(2)样本准备
1:50稀释每个患者血清：10μl血清加500μl反应缓冲液，混匀；
(3)检测步骤
1)微孔条带加入100μl稀释后样本，室温下孵育60分钟；
2)洗涤微孔条带：使用200μl/孔稀释后的清洗缓冲液，洗涤4次；
3)加入100μl/孔酶标记抗体液，室温下孵育45分钟；
4)洗涤微孔条带：使用200μl/孔稀释后的清洗缓冲液，洗涤4次；
5)加入100μl/孔酶基质液，室温下孵育30分钟；
6)加入100μl/孔终止液；
7)读取吸光度(450nm)；
8)结果计算和判定
单位值(U/ml)计算公式：
(A450<样品>-A450<标准血清1>)*100/(A450<标准血清2>-A450<标准血清1>)
判定标准：单位值<21为阴性；单位值≥21为阳性。
质量控制：每个检测结果必须符合下列条件，否则结果无效：
标准血清1的OD450小于等于0.100
标准血清2的OD450大于等于0.500。
进一步的，本发明还提出了所述的ELISA试剂盒在制备检测Dsg3 IgG抗体的试剂中的用途。
相较于现有技术，本发明的有益效果是：
1)本发明利用多个Dsg3抗原多肽来替代全长片段的完整蛋白作为ELISA的包被蛋白，在充分保障了抗体识别的抗原表位的同时，节约了大量的成本；若合成人Dsg3的全长蛋白，或者分几段进行合成，然后混合在一起使用，作为包被蛋白，进行ELISA检测。但是这样做的话，会导致2个问题：(1)成本大量提高；(2)需要包被更大量的蛋白，且可能会导致检测敏感性降低(因为包被的抗原表位相当于被稀释了)。
2)采用ELISA方法检测Dsg3 IgG抗体，相对于现有的免疫印迹法，ELISA方法更容易应用于临床对天疱疮的诊断；
3)本试剂盒的微孔条带中包被的是Dsg3的全长的抗原多肽，相对于Dsg3重组蛋白等而言，成本低，且含有全部需要的抗原位点，因为包被的抗原表位相对更多，所以敏感性会更高。
具体实施方式
下面通过实验并结合实施例对本发明做进一步说明，应该理解的是，这些实施例仅用于例证的目的，决不限制本发明的保护范围。
实施例1用于检测Dsg3 IgG抗体的ELISA试剂盒的制备
1、组成：
ELISA试剂盒包括：
(1)Dsg3微孔条带(48孔)
(2)0U/ml的标准血清1(含0.05％叠氮钠w/v的反应缓冲液，1.5ml)
(3)100U/ml的标准血清2(Dsg3 IgG自身抗体阳性的患者血清稀释于含0.05％w/v叠氮钠的反应缓冲液，1.5ml)
(4)酶标抗体(辣根过氧化物酶标记的兔抗人IgG抗体，8ml)
(5)1x反应缓冲液(含0.05％v/vTween20和0.05％w/vNaN3的0.01M PBS，50ml)
(6)10x清洗缓冲液(含0.5％v/vTween20的0.1M PBS，100ml)
(7)酶基质液(即用型TMB显色液，8ml)
(8)终止液(0.5N盐酸溶液，8ml)
2、Dsg3微孔条带的包被
(1)包被的材料包括如下表1所示的20个Dsg3抗原多肽：
表1 20个Dsg3抗原多肽
(2)多肽溶解和包被
表1中的每个多肽用PBS溶解，储存浓度为4mg/ml。所有溶解后的多肽等体积混合，获得4mg/ml的多肽混合物。包被前使用PBS进行1000倍稀释，使得多肽混合物的终浓度达到4μg/ml。取96孔板进行抗原多肽的包被，每孔加入多肽混合物(4μg/ml)100μl，4℃过夜(12-16小时)进行包被。
3、检测方法
(1)试剂准备
实验前，将所有检测材料置于室温下(20-30℃)；根据需要用蒸馏水稀释适量的清洗缓冲液(1:10)。
(2)样本准备
1:50稀释每个患者血清：10μl血清加500μl反应缓冲液，混匀。
(3)检测步骤
1)微孔条带加入100μl稀释后样本(1:50)，室温下(20-30℃)孵育60分钟；
2)洗涤微孔条带：200μl/孔稀释后的清洗缓冲液，洗涤4次；
3)加入100μl/孔酶标记抗体液，室温下(20-30℃)45分钟；
4)洗涤微孔条带：200μl/孔稀释后的清洗缓冲液，洗涤4次；
5)加入100μl/孔酶基质液，室温下(20-30℃)孵育30分钟；
6)加入100μl/孔终止液；
7)读取吸光度(450nm)；
8)结果计算和判定
单位值(U/ml)计算公式：
(A450<样品>-A450<标准血清1>)*100/(A450<标准血清2>-A450<标准血清1>)
判定标准：单位值<21为阴性；单位值≥21为阳性。
质量控制：每个检测结果必须符合下列条件，否则结果无效：
(1)标准血清1的OD450小于等于0.100
(2)标准血清2的OD450大于等于0.500。
实施例2试剂盒的性能指标检测
(1)样本来源
落叶型天疱疮、寻常型天疱疮、大疱性类天疱疮患者血清各50份，一部分来自于日本久留米大学皮肤科，由Takashi Hashimoto教授赠与(收集的来自于日本和世界各地的患者)，另一部分来自于中国的三甲医院皮肤科或者皮肤病专科医院提供的进行皮肤自身免疫性水疱病的血清学诊断后的剩余患者血清。正常人血清300份来自于医院的正常体检的剩余血清。
血清均采用1：50稀释。
(2)特异性和敏感性
将50份寻常型天疱疮患者血清分别使用商品化ELISA试剂盒(MBL公司)以及本发明建立的ELISA试剂盒进行检测，结果发现，使用商品化ELISA试剂盒50份患者血清中有30份是检测到Dsg3抗体阳性的，即为寻常型天疱疮，另外20份检测阴性，但免疫印迹法确认Dsg3抗体阳性的。即商品化试剂盒的检测Dsg3抗体阳性率为60％。
当使用本发明的ELISA试剂盒检测Dsg3抗体的时候，得到的结果显示50份患者血清中有41份(82％)Dsg3抗体检测阳性，而对落叶型天疱疮、大疱性类天疱疮患者血清检测结果均为阴性。结果如下表2所示：
表2
由此可见，相较于商品化ELISA试剂盒，本发明的试剂盒敏感性更高，并且特异性良好。
(3)重复性
对5个样本，分别进行了6次的重复，每个样本重复的CV％值均小于15％。
(4)检测范围
本试剂盒的检测范围为5-150U/ml。
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Claims (5)
1.用于检测人Dsg3 IgG抗体的ELISA试剂盒，其特征在于，所述的试剂盒中含有Dsg3微孔条带，所述的Dsg3微孔条带中分别包被有SEQ ID NO.1-20所示的多肽或是所述多肽的混合物。
2.如权利要求1所示的ELISA试剂盒，其特征在于，所述的试剂盒中还含有标准血清1、标准血清2、酶标抗体、稀释液、反应缓冲液、清洗缓冲液、酶基质液以及终止液。
3.如权利要求2所示的ELISA试剂盒，其特征在于，所述的标准血清1为含0.05%w/v叠氮钠的反应缓冲液；所述的标准血清2为含有100 U/ml的Dsg3 IgG抗体以及0.05%w/v叠氮钠的反应缓冲液；所述的酶标抗体为辣根过氧化物酶标记的兔抗人IgG抗体；所述的反应缓冲液为含0.05%v/v Tween20和0.05%w/vNaN3的1xPBS，所述的清洗缓冲液为含0.5%v/vTween20的10xPBS；所述的酶基质液为TMB显色液；所述的终止液为0.5N盐酸溶液。
4.如权利要求1所示的ELISA试剂盒，其特征在于，所述的含有Dsg3微孔条带通过以下方法制备得到：
1）将SEQ ID NO.1-20所示的多肽分别用PBS溶解，得到储存浓度为4mg/ml的多肽溶液，所有溶解后的多肽等体积混合，获得4mg/ml的多肽混合物，包被前使用PBS进行1000倍稀释，使得多肽混合物的终浓度达到4µg/ml；取96孔板进行抗原多肽的包被，每孔加入多肽混合物100µl，4℃进行包被12-16h；或
2）将SEQ ID NO.1-20所示的多肽分别用PBS溶解，得到储存浓度为4mg/ml的多肽溶液，包被前分别使用PBS进行1000倍稀释，使得多肽的终浓度达到4µg/ml；取96孔板进行抗原多肽的包被，每孔分别加入一种多肽溶液100µl，4℃进行包被12-16h。
5.权利要求1-4任一项所述的ELISA试剂盒在制备检测人Dsg3 IgG抗体的装置中的用途。
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