CN113447659A - 一种检测抗蛋白酶体亚基α1-IgG抗体的试剂盒 - Google Patents
一种检测抗蛋白酶体亚基α1-IgG抗体的试剂盒 Download PDFInfo
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Abstract
本发明提供一种检测抗蛋白酶体亚基α1‑IgG抗体的试剂盒，由抗原蛋白Proteasomesubunit alpha type 1(蛋白酶体亚基α1)、固相载体、标记抗体、抗原和抗体稀释液、样品稀释缓冲液、底物显色剂、洗涤液、标准品、阳性和阴性质控品组成。本发明试剂盒利用间接法反应原理结合磁微粒化学发光免疫分析检测待检血清中的抗蛋白酶体亚基α1‑IgG抗体。本发明首次鉴定出自身免疫性肾病综合征患者血清中针对靶抗原蛋白酶体亚基α1的自身抗体。本发明为国内外针对与蛋白酶体亚基α1和蛋白酶体亚基α1‑IgG自身抗体有关的自身免疫性肾病综合征的分子机制研究及临床诊疗提供依据。
Description
技术领域
本发明属于生物医学技术领域，涉及一种检测抗蛋白酶体亚基α1-IgG抗体的试剂盒。
背景技术
近年来儿童肾脏疾病种类越来越多，其中以自身免疫性肾病综合症发病率最高，严重危害了儿童身心健康。自身免疫性肾病综合征即一类因为肾小球滤过膜通透性增加，使得血浆蛋白滤出增多，引起大量蛋白尿，并由此引发病人主要表现为大量蛋白尿、低蛋白血症、高度水肿等特征的临床综合症。Ali等学者观察到，移植了来自难治性微小病变性肾病综合征病人的肾脏后，受者肾脏功能正常未出现任何蛋白尿，由此可见微小病变性肾病综合征病因并非都在肾脏自身，而可能主要是病人的内环境出现了问题。此外，除了部分由基因缺陷而引起的患儿，大部分自身免疫性肾病综合征患儿在经过激素和免疫抑制剂治疗后病情都能好转，这间接证明了该疾病与患者的自身免疫有着密切的联系。
近年来发现B细胞功能异常在自身免疫性肾病综合征中也具有重要的作用。近些年，全球多个多中心的临床研究成果表明，利妥昔单抗(Rituximab，RTX)能成功用于微小病变性肾病综合征的治疗，特别是在难治性肾病综合征的治疗方面取得了很好的治疗效果。然而研究也发现使用利妥昔单抗治疗激素依赖型肾病综合征的过程中发现，其清除B细胞的作用大约能维持5个月左右的时间，在6到7个月随着B细胞数量的回升患者的病情也会复发。由此提示自身免疫性肾病综合征患者体内存在病理性的B细胞克隆，对这些病理性的B细胞克隆进行鉴别和精准的清除，不仅有利于自身免疫性肾病综合征病情恢复，也减少因为采用利妥昔单抗等手段进行无差别的B细胞清除而给病人带来体液免疫缺陷的风险。然而，迄今为止，自身免疫性肾病综合征患儿体内病理性B细胞针对的靶抗原仍然不是很清楚。从病理特征上来说，微小病变型肾病或局灶节段性肾小球硬化被认为是由于足细胞功能丧失或改变，导致大量蛋白尿的足细胞病。足细胞为肾脏肾小球上皮细胞，附着在肾小球的基底膜的外侧，是阻止蛋白质丢失的最后屏障，足细胞损伤通常会引起大量的蛋白尿。
蛋白酶体(Proteasome subunit)是细胞内降解蛋白质的大分子复合体，其主要作用是降解细胞不需要的或受到损伤的蛋白质。蛋白酶体亚基α-1(Proteasome subunitalpha type 1，Proteasome subunit alpha type 1)是蛋白酶体家族一种，目前关于Proteasome subunit alpha type1蛋白要在癌症方面研究比较多。如有研究显示在蛋白酶体通路中的Proteasome subunit alpha type 1蛋白与控制细胞周期进程和凋亡密切相关，Proteasome subunit alpha type 1在肝细胞癌肿瘤组织中表达明显下调，Proteasomesubunit alpha type 1可能是一种肝细胞癌潜在生物标志物(Jian Qin1*，etal.Identification of proteasome subunit alpha type-1as a novel biomarker inHBV-associated hepatocellμlar carcinoma tissue interstitial fluid byproteomic analysis.Int J Clin Exp Pathol 2017；10(7)：7812-7820.)。Yang Qian等报道Proteasome subunit alpha type 1蛋白可能是一种结肠癌潜在生物标志物(YangQian，Roehrl Michael H，Wang J，et al.Proteomic profiling of antibody-inducingimmunogens in tumor tissue identifies Proteasome subunit alpha type 1，LAP3，ANXA3，and maspin as colon cancer markers.Journal[J]Oncotarget Volume 9，Issue3.2018.PP 3996-4019.)。
但目前关于Proteasome subunit alpha type 1的表达情况和Proteasomesubunit alpha type1的自身抗体的存在情况在肾病综合征中未见报道。另外现有技术中，没有涉及基于靶点Proteasome subunit alpha type 1或其自身抗体作为一种血清学标志物在自身免疫性肾病综合征中的应用。通过检测血清抗Proteasome subunit alpha type1-IgG抗体鉴别出自身免疫性肾病综合征的研究是空白的。
发明内容
本发明的目的是提供一种检测抗蛋白酶体亚基α1-IgG抗体的试剂盒，是一种基于靶点Proteasome subunit alpha type 1及其相应自身抗体的检测试剂盒。所述试剂盒可通过与抗原蛋白Proteasome subunit alpha type 1(特别根据序列识别号SEQ ID NO.1所示)的免疫反应对来自组织(肾活检组织)或体液(特别是血液、血浆、血清)中的自身抗体进行检测。
本发明所述试剂盒由抗原蛋白Proteasome subunit alpha type 1、固相载体、标记抗体如酶标记或化学发光剂标记或生物素标记的二抗、抗原稀释液、样品稀释缓冲液、抗体稀释液、底物显色剂、洗涤液、标准品、阳性质控品、阴性质控品组成。
所述抗原蛋白Proteasome subunit alpha type 1的序列如SEQ ID NO.1所示：MFRNQYDNDVTVWSPQGRIHQIEYAMEAVKQGSATVGLKSKTHAVLVALKRAQSELAAHQKKILHVDNHIGISIAGLTADARLLCNFMRQECLDSRFVFDRPLPVSRLVSLIGSKTQIPTQRYGRRPYGVGLLIAGYDDMGPHIFQTCPSANYFDCRAMSIGARSQSARTYLERHMSEFMECNLNELVKHGLRALRETLPAEQDLTTKNVSIGIVGKDLEFTIYDDDDVSPFLEGLEERPQRKAQPAQPADEPAEKADEPMEH。
本发明的Proteasome subunit alpha type 1抗原蛋白可以是融合蛋白，使用具有某些生物学或物理功能的标签，特别是N-末端或C-末端。这些标签的存在有利于抗原蛋白纯化，固定，沉淀。在一个优选的实施方案中，标签是能够特异性结合配体的序列或结构域，例如，所述标签肽选自：GST标签、c-Myc标签、His标签、Flag标签或生物素标签。
根据本发明，所述抗原蛋白Proteasome subunit alpha type 1固定于固相载体上，利用物理吸附或共价键结合或化学结合将Proteasome subunit alpha type 1抗原蛋白通过直接的方式固定于固相载体上。优选的固相载体包括：硝酸纤维素膜、磁珠、酶标微孔板。
本发明一个实施方案中，所述标准品和阳性质控品，标准品和阳性质控品为重组人抗标签肽免疫球蛋白G或其片段、或从病人血清中提取抗Proteasome subunit alphatype 1-IgG抗体作为阳性质控品和标准品，健康体检者血清为阴性质控品。
根据本发明，所述抗原蛋白Proteasome subunit alpha type 1可表达于细菌如大肠杆菌、酵母、哺乳动物细胞中。
根据本发明，所述抗原蛋白Proteasome subunit alpha type 1经Ni柱亲和层析、分子筛层析、离子交换层析、疏水柱纯化。
根据本发明，所述生物样本为包含自身抗体的样品，选自全血、血清、血浆、尿液、淋巴液、胸腹水。优选为哺乳动物(人类)血清。
所述检测试剂盒还包括底物显色剂、抗原稀释液、样品稀释缓冲液、抗体稀释液、洗涤液。所述底物显色剂为TMB、AMPPD、4-MUP、BCIP；所述抗原稀释液为含有163mM NaCL、1％TritonX-100的1x PBS pH7.4；所述样品稀释缓冲液为含有10％BSA的0.01M PBSpH7.4；所述抗体稀释液为含有1M D-glucose、2％甘油、0.35％Tween20的0.01M PBSpH7.4；所述洗涤液为：含有163mM NaCL、10％甘油、1％TritonX-100的1x PBS pH7.4。
在一个优选实施方案中，如本文中所述“固定”是指与Proteasome subunit alphatype 1抗原蛋白不溶于水的固相载体结合，该固相载体或支持物不溶于水，更优选地通过共价键合，静电相互作用，疏水相互作用、或通过二硫键相互作用，最优选通过一个或多个共价键。固定可以是直接固定方式，例如通过过滤，离心或层析，将固定化的分子与不溶性载体一起从水溶液中分离出来。还包括可逆或不可逆的方式固定Proteasome subunitalpha type 1抗原蛋白。例如，抗原蛋白通过可裂解的共价键(如可添加含硫醇的试剂来裂解的二硫键)固定于载体，这种固定是可逆的。另外，如抗原蛋白通过在水性溶液中不会裂解的共价键(通过环氧化物基团与将赖氨酸侧链偶联至亲和柱的胺基团的反应形成的键)固定于载体，则固定是不可逆的。固定还可以是间接方式：如固定对所述抗原蛋白具有特异性亲和力的抗体，然后形成抗原蛋白-抗体复合物以达到固定的效目的。
本发明所述的抗原蛋白Proteasome subunit alpha type 1固定方法为直接包被法：(1)抗原蛋白Proteasome subunit alpha type 1通过物理吸附方式或非共价键结合到硝酸纤维素膜或聚苯乙烯微孔板上；(2)带有羧基功能团的磁微粒与抗原蛋白Proteasomesubunit alpha type1的氨基结合，抗原蛋白Proteasome subunit alpha type 1通过化学偶联方式结合在磁微粒上。
本发明所选的标记抗体可以是辣根过氧化物酶(Horseradish Peroxidase，HRP)标记抗人IgG抗体、吖啶酯标记抗人IgG抗体、生物素标记抗人IgG抗体。
本发明采用基因重组原核表达方法成功表达并纯化出重组蛋白Proteasomesubunit alpha type 1，以此作为试剂盒中抗原蛋白，研发出一套适合于检测自身免疫性肾病综合征患者血清抗Proteasome subunit alpha type 1-IgG抗体的试剂盒。包括是一种定性或定量分析检测人血清中抗Proteasome subunit alpha type 1-IgG抗体的检测试剂盒。
一种检测血清中抗Proteasome subunit alpha type 1-IgG抗体试剂盒的原理利用间接法反应原理，首先将Proteasome subunit alpha type 1抗原吸附于固相载体作为包被抗原，然后加入阳性质控品或标准品或待检血清样本进行孵育，再加入标记二抗反应后，若待检血清中含有抗Proteasome subunit alpha type 1-IgG抗体，则形成包被抗原Proteasome subunit alpha type1-待检血清抗Proteasome subunit alpha type 1-IgG抗体-标记抗人IgG抗体三元复合物，最后利用光显色法、化学发光法、荧光发光法来检测光信号，以达到定性或定量分析人血清中抗Proteasome subunit alpha type 1-IgG抗体的目的。
应用本发明试剂盒首次在部分自身免疫性肾病综合征患者的体内检测到了一种抗Proteasome subunit alpha type 1-IgG自身抗体，且确定了该自身抗体针对的靶抗原为足细胞上蛋白酶体亚基α-1(Proteasome subunit alpha type 1)。因此，本发明的试剂盒可用于抗Proteasome subunit alpha type 1-IgG自身抗体的检测，为研究自身免疫性肾病综合征提供依据。
与现有技术相比本发明试剂盒的有益之处如下：
(1)目前国内外关于肾脏疾病患者有关Proteasome subunit alpha type 1、抗Proteasome subunit alpha type 1-IgG抗体仅限于分子机制研究，并没有定量检测其在患者血清中水平。本发明首次鉴定了针对Proteasome subunit alpha type 1的自身抗体，并针对该抗Proteasome subunit alpha type 1-IgG自身抗体发明了检测试剂盒，填补了国内外空白。
(2)本发明试剂盒涉及到定性分析人血清中抗Proteasome subunit alpha type1-IgG抗体，其中固相膜免疫定性检测操作简单，试剂用量较少，比传统ELISA节约近10倍；另外NC膜吸附能力极强接近100％，微量抗原能够完全吸附固定在NC膜上；吸附抗原或抗体或已有结果的NC膜可长期保存(-20℃可保存半年)，且不影响其活性；另外本发明固相膜免疫定性检测人血清中抗Proteasome subunit alpha type 1-IgG抗体的试剂盒引入生物素-亲和素放大系统，大大提高了检测灵敏度。
(3)本发明涉及的磁微粒化学发光免疫分析定量检测人血清中抗Proteasomesubunit alpha type 1-IgG抗体试剂盒，利用磁微粒为固相载体，其直径仅为1.0μm，这就大大增加了包被表面积，增加了抗原的吸附量，提高了反应速度，也使清洗分离更简便，从而减少污染，降低交叉感染概率。另一方面，采用吖啶酯发光剂直接标记抗人IgG，其化学反应简单、快速、无须催化剂；吖啶酯化学发光为闪光型，其通过起动发光试剂(H2O2、NaOH)0.4s后发射强度即可达到最大，半衰期为0.9s，2s内基本结束，便于快速检测。
附图说明
图1：足细胞上的Proteasome subunit alpha type 1蛋白是自身免疫性肾病综合征病人体内自身抗体针对的靶抗原。图1A：一抗为健康人血清的二维电泳蛋白点；图1B：一抗为自身免疫性肾病综合征患者血清的二维电泳蛋白点；图1C：靶抗原Proteasomesubunit alpha type1蛋白的质谱鉴定。
图2：表达的重组蛋白Proteasome subunit alpha type 1的SDS-PAGE鉴定图。
图3：固相膜免疫试剂盒检测自身免疫性肾病综合征患者血清中抗Proteasomesubunit alpha type 1-IgG抗体。
图:4：磁微粒化学发光免疫分析试剂盒检测抗Proteasome subunit alpha type1-IgG抗体的原理示意图。
图5：抗原蛋白Proteasome subunit alpha type 1包被羧基磁微粒示意图。
图6：各类肾病病人中抗Proteasome subunit alpha type 1-IgG抗体的检测情况，其中NS：自身免疫性肾病综合征，HSP：过敏性紫癜，HSPN：紫癜性肾炎，IgAN：IgA肾病，NC：健康儿童。
图7：ROC曲线评估抗Proteasome subunit alpha type 1抗体作为自身免疫性肾病综合征患者诊断的血清学标志物的应用价值。
具体实施方式
以下结合附图和具体实施例，对本发明作进一步说明。以下实施例仅用于阐述本发明而非用于限制本发明的范围。
实施例1足细胞上的Proteasome subunit alpha type 1蛋白是自身免疫性肾病综合征病人体内自身抗体针对的靶抗原
本发明通过前期大量临床和分子机制研究，首次发现肾病综合征患者血清IgG水平较高，并证实足细胞上的Proteasome subunit alpha type 1是自身免疫性肾病综合征病人体内自身抗体针对的靶抗原。因此检测血清中抗Proteasome subunit alpha type 1-IgG抗体的存在及其定量水平有助于自身免疫性肾病综合征的早期鉴别、尤其对有相关症状的患者进行筛查是有利的。具体实施如下(1)肾小球足细胞总蛋白的提取：培养足细胞株(MPC5)，用PBS洗涤2-3次，然后用聚焦超声仪(Covaris S220,Gene)在含有30mm Tris-HCl、8m尿素、4％CHAPS和蛋白酶抑制剂(#ab65621；Abcam，1：200稀释)的裂解缓冲液中冰上进行充分裂解，然后将样本置于离心机，12000g，4℃，离心30min。收集上清，即为收集到的肾小球足细胞总蛋白。利用BCA蛋白浓度测定试剂盒测定收集到的肾小球足细胞总蛋白浓度。(2)二维电泳：提取肾小球足细胞总蛋白进行二维电泳后转到硝酸纤维素膜上，分别用健康人和自身免疫性肾病综合征病人的血清作为一抗进行孵育，之后加上二抗进行显影，见图1A，1B。(3)基质辅助激光解吸/电离飞行时间质谱分析：将步骤(2)显影后进行阳性点的差异分析，选取二维电泳胶上肾病综合征病人强阳性，而健康人阴性或者弱阳性的蛋白点，从凝胶上剪下选中的蛋白点，将干燥后的凝胶用胰蛋白酶(0.1μg/μl)进行消化，然后向反应混合物中加入10μl的25mM碳酸氢铵，37℃孵育过夜，然后用三氟乙酸(0.1％)从凝胶中提取肽。用基质辅助激光解吸/电离飞行时间质谱分析(MALDI-TOF-MS)质谱仪对提取的肽进行分析，得到肽质量图谱，鉴定为Proteasome subunit alpha type 1蛋白，见图1C。
实施例2重组抗原蛋白Proteasome subunit alpha type 1表达及纯化
利用基因工程的方法以编码Proteasome subunit alpha type 1蛋白的基因为模板，进行PCR扩增，然后构建表达载体进行蛋白表达。本发明表达的抗原蛋白上含有GST标签的标签肽。表达的重组蛋白经镍柱亲和层析、离子亲和层析法、疏水柱、分子筛等进行纯化，最后利用SDS-PAGE鉴定重组蛋白Proteasome subunit alpha type 1的分子量为56KDa，见图2。
实施例3本发明采用正交试验设计对试剂盒反应条件进行优化
根据抗原Proteasome subunit alpha type 1包被浓度(50μg/ml、90μg/ml、120μg/ml、150μg/ml四个包被浓度)、各反应时间(15min、30min、45min)和温度(25℃、37℃)、酶标二抗最佳稀释度(1:100、1:500、1:1000、1:1500四个稀释度)等4个因素选择正交表，每个因素按2水平重复测定标准阳性血清和标准阴性血清，选择阳性血清的最高光信号值(P)和阴性血清的最低光信号值(N)的比值(P/N)。通过正交设计我们得到了本试剂盒最佳抗原包Proteasome subunit alpha type 1被浓度为90μg/ml、固相膜免疫检测抗Proteasomesubunit alpha type 1-IgG抗体试剂盒最佳抗原抗体反应温度为25℃、最佳抗原抗体反应时间30min及最佳标记抗人IgG抗体最佳工作稀释度为1：1000；磁微粒化学发光免疫分析检测抗Proteasome subunit alpha type1-IgG抗体试剂盒最佳抗原抗体反应温度为37℃、最佳抗原抗体反应时间15min及最佳标记抗人IgG抗体最佳工作稀释度为1：1000。
实施例4用于检测抗Proteasome subunit alpha type 1-IgG抗体的固相膜免疫试剂盒的制备
4.1用于检测抗Proteasome subunit alpha type 1-IgG抗体的固相膜免疫试剂盒的组成：
1.抗原：重组蛋白Proteasome subunit alpha type 1
2.固相载体：Satourius CN140硝酸纤维素膜
3.阳性质控品(标准品)：人抗GST标签免疫球蛋白G(购自湖州英创)
4.阴性质控品：健康体检者血清
5.标记抗体：生物素标记抗人IgG抗体
6.抗原稀释液
7.样品稀释缓冲液
8.抗体稀释液
9.洗涤液
10.酶工作液：碱性磷酸酶-链霉亲和素
11.底物显色液：BCIP显色液。
4.2用于检测抗Proteasome subunit alpha type 1-IgG抗体的固相膜免疫试剂盒的检测步骤如下：
4.2.1包被、封闭：将8μl浓度为90μg/ml的Proteasome subunit alpha type 1抗原直接点于硝酸纤维素膜上置37℃孵育箱中干燥30min，将硝酸纤维素膜置于检测板中，加入200μl5％BSA于37℃温盒中封闭30min，弃去封闭液后用洗涤液洗2次；
4.2.2抗原孵育：向检测板内加入10μl用稀释液稀释的抗体标准品或待检血清，同时做阴性对照、阳性对照，25℃孵育30min，每个样品设置3个平行孔；
4.2.3二抗孵育：弃去检测板内液体，洗涤液洗5次×1min，加入20μl 1:1000生物素标记抗人IgG抗体，25℃孵育30min；
4.2.4显色：弃去检测板内液体，洗涤液洗5次×1min，加500μl碱性磷酸酶-链霉亲和素，室温孵育20min，弃去检测板内液体，洗涤液洗5次×1min，然后加入BCIP显色液，室温反应20min，用流水冲洗检测板，终止酶反应。取出测试硝酸纤维素膜条用吹风机吹干膜条，用比色卡肉眼定性判定，出现明显棕色斑点者为阳性见图3，或将膜条置于显影仪上扫描，显影仪自带的分析软件以参考标准品浓度作为纵坐标、仪器读取的灰度值作为横坐标，绘制标准曲线对血清中抗Proteasome subunit alpha type 1-IgG抗体水平进行半定量分析。
实施例5、用于检测抗Proteasome subunit alpha type 1-IgG抗体的磁微粒化学发光免疫分析试剂盒的制备
5.1用于检测抗Proteasome subunit alpha type 1-IgG抗体的磁微粒化学发光免疫分析试剂盒组成：
1.抗原：重组蛋白Proteasome subunit alpha type 1
2.固相载体：羧基功能团的磁微粒
3.阳性质控品(标准品)：人抗GST标签免疫球蛋白G(购自湖州英创)
4.阴性质控品：健康体检者血清
5.标记抗体：吖啶酯标记抗人IgG抗体
6.抗原稀释液
7.样品稀释缓冲液
8.抗体稀释液
9.洗涤液
10.预激发液：H2O2
11.激发液：NaOH
5.2用于检测抗Proteasome subunit alpha type 1-IgG抗体的磁微粒化学发光免疫分析试剂盒的检测原理
本发明化学发光免疫分析试剂盒是将磁性分离技术、免疫分析技术、化学发光技术三者结合起来的一种分析方法。本发明试剂盒采用间接法定量分析检测人血清中抗Proteasome subunit alpha type 1-IgG抗体：首先将磁微粒液与稀释样本混合，特异性抗Proteasome subunit alpha type 1-IgG抗体结合到Proteasome subunit alpha type 1抗原包被的磁微粒上，洗涤后，加入吖啶酯标记抗人IgG抗体，形成Proteasome subunitalpha type 1抗原包被磁微粒-抗Proteasome subunit alpha type 1-IgG抗体-吖啶酯标记抗人IgG抗体复合物，在外加磁场作用下，将未结合的物质与免疫反应形成的复合物进行分离，弃上清后清洗沉淀的复合物，加入预激发液(H2O2)和激发液(NaOH)进行发光反应，在碱性条件下，吖啶酯分子受到过氧化氢攻击生成二氧乙烷，二氧乙烷不稳定而分解为CO2和电子激发态的N-甲基吖啶酮，当其回到基态时发出波长为430nm的光，使用化学发光仪收集发光强度。抗Proteasome subunit alpha type 1-IgG抗体浓度与发光值成正比，通过校准曲线计算待检血清中抗Proteasome subunit alpha type 1-IgG抗体浓度，见图4。
5.3Proteasome subunit alpha type 1抗原包被磁微粒的制备
5.3.1Proteasome subunit alpha type 1抗原包被磁微粒的原理：基于磁微粒表面所含的羧基功能团与EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺)溶液发生反应而生成不稳定的氨基活性O-酰基脲中间体，该中间体与NHS(N-羟基琥珀酰亚胺)发生反应生成半稳定的氨基反应活性NHS酯，半稳定的氨基反应活性NHS酯再与抗原蛋白Proteasomesubunit alpha type 1上的氨基发生反应，形成抗原Proteasome subunit alpha type 1包被磁微粒，见图5。
5.3.2EDC/NHS活化羧基磁微粒，具体步骤如下：
a)称取10mg磁微粒，用20mM MES清洗磁微粒3次后，磁铁分离，弃去上清；
b)将清洗过的磁微粒重悬于100μl的20mM MES中，使磁微粒终浓度为100mg/ml；
c)向清洗后的磁微粒依次加入磷酸盐缓冲液配制的50μl 20mg/ml的EDC和50μl24mg/ml的Sμlfo-NHS，充分混匀，室温静置活化30min；
d)在外加磁场作用后，弃去上清，取400μl 0.05M磷酸盐缓冲液洗涤磁微粒，加入400μl保存液定容保存备用。
5.3.3活化磁微粒与抗原蛋白Proteasome subunit alpha type 1交联向上述活化好的磁微粒溶液中加入预冷的1ml 20mM MES继续清洗磁微粒2次；取200μl 2mg/ml的抗原蛋白Proteasome subunit alpha type 1加入到活化好的磁微粒中，充分混匀，室温静置反应16小时；反应结束后，加入含有0.2％Tween20的pH 7.4PBS缓冲液，重复清洗磁微粒2次；然后再加入含有0.2％Tween20、0.2％BSA的的pH 7.4PBS缓冲液，至磁微粒终浓度为10mg/ml，充分混匀，室温静置反应30min；反应结束后，弃上清，并用含有0.2％Tween20、0.2％BSA的的pH 7.4PBS缓冲液重悬磁微粒，这样活化磁微粒与抗原蛋白Proteasomesubunit alpha type1交联完成。
5.4吖啶酯标记抗人IgG抗体制备，具体步骤如下：
a)利用二甲基甲酰胺配制2mg/mL吖啶酯溶液；
b)利用0.2M(pH8.0)碳酸盐缓冲液配制1mg/mL抗人IgG抗体；
c)取摩尔比为4:1的吖啶酯与抗人IgG抗体充分混匀，反应40min；
d)加入20μl含有5％赖氨酸的碳酸盐缓冲液终止反应；
e)通过脱盐除杂，得到纯度较高的吖啶酯标记抗人IgG抗体溶液。
5.5磁微粒化学发光免疫分析试剂盒检测血清中抗Proteasome subunit alphatype1-IgG抗体的步骤
5.5.1待检血清加入取100μl稀释后的待检血清或抗GST标签的IgG标准品，加入100μl包被抗原蛋白Proteasome subunit alpha type 1的磁微粒溶液中，37℃反应15min，同时做阴阳性对照；
5.5.2标记抗体的加入400μl洗涤液洗涤3次x1 min，加入100μl 1:1000稀释的吖啶酯标记抗人IgG抗体，37℃反应15min；
5.5.3信号检测弃上清后清洗沉淀的复合物，400μl洗涤液洗涤3次x1 min，加入100μl预激发液(H2O2)和100μl激发液(NaOH)进行反应。通过化学发光仪检测发光信号，记录发光值。待检血清中抗Proteasome subunit alpha type 1-IgG抗体浓度与发光值成正比，通过标准曲线计算待检血清中抗Proteasome subunit alpha type 1-IgG抗体浓度。
实施例6检测血清抗Proteasome subunit alpha type 1-IgG抗体试剂盒的临床应用
6.1受试者纳入从2018年6月到2020年6月诊断出各类肾病的患者，包括466例自身免疫性肾病综合征(NS)、168例过敏性紫癜(HSP)、137例紫癜性肾炎(HSPN)、133例IgA肾病(IgAN)及同时期195例健康儿童(NC)。血清样本取自各类肾病患者和健康对照组。所有受试者在没有进行免疫抑制治疗之前进行第一次血清样本采集。
6.2各类肾病病人中抗Proteasome subunit alpha type 1-IgG抗体的检测情况利用本发明试剂盒检测从2018年6月到2020年6月诊断出各类肾病的患者血清中抗Proteasome subunit alpha type 1-IgG抗体水平，包括466例自身免疫性肾病综合征、168例过敏性紫癜、137例紫癜性肾炎、133例IgA肾病及同时期195例健康儿童，结果显示自身免疫性肾病综合征病人中抗Proteasome subunit alpha type 1-IgG抗体阳性，而紫癜性肾炎、过敏性紫癜、IgA肾病病人以及健康儿童中抗Proteasome subunit alpha type 1-IgG抗体为阴性，见图6。
6.3ROC曲线评估抗Proteasome subunit alpha type 1-IgG抗体作为一种血清学标志物用于自身免疫性肾病综合征患者诊断的价值对6.2中的自身免疫性肾病综合征患者中抗Proteasome subunit alpha type 1-IgG抗体的检测结果采用ROC曲线进行分析，以评估抗Proteasome subunit alpha type 1-IgG抗体在诊断自身免疫性肾病综合征中的应用价值。结果显示抗Proteasome subunit alpha type 1-IgG抗体是用于自身免疫性肾病综合征患者的诊断的一种很好的血清学标志物，抗Proteasome subunit alpha type 1-IgG抗体(用界值大于32.2作为标准)作为一种血清学标志物用于自身免疫性肾病综合征患者诊断的敏感性为74.4％，特异性为80.8％，曲线下面积为0.832，见图7。
序列表
<110> 浙江大学医学院附属儿童医院
<120> 一种检测抗蛋白酶体亚基α1-IgG抗体的试剂盒
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<213> Proteasome subunit alpha type 1蛋白(人工序列Unknow)
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Met Phe Arg Asn Gln Tyr Asp Asn Asp Val Thr Val Trp Ser Pro Gln
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Gly Arg Ile His Gln Ile Glu Tyr Ala Met Glu Ala Val Lys Gln Gly
20 25 30
Ser Ala Thr Val Gly Leu Lys Ser Lys Thr His Ala Val Leu Val Ala
35 40 45
Leu Lys Arg Ala Gln Ser Glu Leu Ala Ala His Gln Lys Lys Ile Leu
50 55 60
His Val Asp Asn His Ile Gly Ile Ser Ile Ala Gly Leu Thr Ala Asp
65 70 75 80
Ala Arg Leu Leu Cys Asn Phe Met Arg Gln Glu Cys Leu Asp Ser Arg
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Phe Val Phe Asp Arg Pro Leu Pro Val Ser Arg Leu Val Ser Leu Ile
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Gly Ser Lys Thr Gln Ile Pro Thr Gln Arg Tyr Gly Arg Arg Pro Tyr
115 120 125
Gly Val Gly Leu Leu Ile Ala Gly Tyr Asp Asp Met Gly Pro His Ile
130 135 140
Phe Gln Thr Cys Pro Ser Ala Asn Tyr Phe Asp Cys Arg Ala Met Ser
145 150 155 160
Ile Gly Ala Arg Ser Gln Ser Ala Arg Thr Tyr Leu Glu Arg His Met
165 170 175
Ser Glu Phe Met Glu Cys Asn Leu Asn Glu Leu Val Lys His Gly Leu
180 185 190
Arg Ala Leu Arg Glu Thr Leu Pro Ala Glu Gln Asp Leu Thr Thr Lys
195 200 205
Asn Val Ser Ile Gly Ile Val Gly Lys Asp Leu Glu Phe Thr Ile Tyr
210 215 220
Asp Asp Asp Asp Val Ser Pro Phe Leu Glu Gly Leu Glu Glu Arg Pro
225 230 235 240
Gln Arg Lys Ala Gln Pro Ala Gln Pro Ala Asp Glu Pro Ala Glu Lys
245 250 255
Ala Asp Glu Pro Met Glu His
260
Claims (9)
1.一种检测抗蛋白酶体亚基α1-IgG抗体的试剂盒，其特征在于，所述试剂盒由抗原蛋白Proteasome subunit alpha type 1、固相载体、标记抗体、抗原稀释液、样品稀释缓冲液、抗体稀释液、底物显色剂、洗涤液、标准品、阳性质控品、阴性质控品组成。
2.根据权利要求1所述试剂盒，其特征在于，所述抗原蛋白Proteasome subunit alphatype1的序列如SEQ ID:1所示。
3.根据权利要求1所述检测试剂盒，其特征在于，所述标记抗体为酶标记、化学发光剂标记或生物素标记的二抗。
4.根据权利要求1所述试剂盒，其特征在于，所述的抗原蛋白Proteasome subunitalpha type1上带有标签肽，所述的标签肽包括：GST标签、c-Myc标签、His标签、Flag标签或生物素标签。
5.根据权利要求1所述的试剂盒，其特征在于，所述Proteasome subunit alpha type1抗原蛋白经镍柱亲和层析、分子筛层析、凝胶过滤层析、离子交换柱层析、疏水柱纯化。
6.根据权利要求1所述的试剂盒，其特征在于，所述标准品和阳性质控品为重组人抗标签肽免疫球蛋白G或其片段，或从血清中提取抗Proteasome subunit alpha type 1-IgG抗体作为阳性质控品和标准品，阴性质控品为健康体检者血清。
7.根据权利要求1所述的试剂盒，其特征在于，所述抗原蛋白Proteasome subunitalpha type1固定于固相载体上，所述固相载体选用硝酸纤维素膜、尼龙膜、离子交换树脂、聚苯乙烯微孔板、磁珠。
8.根据权利要求7所述的试剂盒，其特征在于，利用物理吸附或共价键结合或化学结合将Proteasome subunit alpha type 1抗原蛋白通过直接的方式固定于固相载体上。
9.根据权利要求1所述的检测试剂盒，其特征在于，所述底物显色剂为TMB、AMPPD、4-MUP、BCIP；所述抗原稀释液为含有163mM NaCL、1％TritonX-100的1x PBS pH7.4；所述样品稀释缓冲液为含有10％BSA的0.01M PBS pH7.4；所述抗体稀释液为含有1MD-glucose、2％甘油、0.35％Tween20的0.01M PBS pH7.4；所述洗涤液为：含有163mM NaCL、10％甘油、1％TritonX-100的1x PBS pH7.4。
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| CN202110743551.7A CN113447659B (zh) | 2021-07-01 | 2021-07-01 | 一种检测抗蛋白酶体亚基α1-IgG抗体的试剂盒 |
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| US17/856,033 US20230333115A1 (en) | 2021-07-01 | 2022-07-01 | KIT FOR DETECTING ANTI-PROTEASOME SUBUNIT ALPHA TYPE 1-IMMUNOGLOBULIN G (IgG) ANTIBODY |
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| CN114910650A (zh) * | 2022-05-07 | 2022-08-16 | 浙江大学 | 检测抗膜突蛋白-IgG抗体的试剂在制备检测血管内皮损伤的试剂盒中的应用 |
| CN114924081A (zh) * | 2022-05-07 | 2022-08-19 | 浙江大学 | 神经母细胞分化相关蛋白-IgG在制备血管内皮损伤试剂盒中的应用 |
| CN114994330A (zh) * | 2022-05-07 | 2022-09-02 | 浙江大学 | 检测抗HSP 90-beta-IgG自身抗体的试剂盒及其应用 |
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| CN113447659B (zh) | 2022-05-17 |
| CN114895041A (zh) | 2022-08-12 |
| US20230333115A1 (en) | 2023-10-19 |
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