CN113533733A - 用于检测人血中aep蛋白水平的化学发光免疫检测试剂盒 - Google Patents
用于检测人血中aep蛋白水平的化学发光免疫检测试剂盒 Download PDFInfo
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Abstract
一种用于检测人血中AEP蛋白水平的化学发光免疫检测试剂盒,包括:酶标液、AEP标准品、包被抗AEP单克隆抗体的免疫磁珠、稀释液、化学发光底物和浓缩洗涤液。本发明使用化学发光免疫分析技术,解决了对临床快速检测病人血液内AEP蛋白水平的需求,能够实现高灵敏度,重复性好,自动化程度高,操作便捷地检测AEP的水平,更适合临床POCT检测时,短时间内高灵敏度地检测一定数量的病人样品的需求。为临床快速检测AEP水平提供有力帮助。
Description
技术领域
本发明涉及的是一种化学检测领域的技术,具体是一种用于检测人血中天冬酰胺内肽酶(Asparaginyl Endopeptidase,AEP)水平的化学发光免疫检测试剂盒。
背景技术
现有ELISA方法,抗原,抗体的结合反应是在固相(ELISA板反应孔)表面进行的,该方法操作繁琐,灵敏度低于化学发光,重复性差,且检测用时相比化学发光长。
发明内容
本发明针对现有AEP的ELISA试剂盒,操作繁琐,且不适合大规模临床快速检测的不足,提出一种用于检测人血中AEP蛋白水平的化学发光免疫检测试剂盒,使用化学发光免疫分析技术,解决了对临床快速检测病人血液内AEP蛋白水平的需求,能够实现高灵敏度,重复性好,自动化程度高,操作便捷地检测AEP的水平,更适合临床POCT检测时,短时间内高灵敏度地检测一定数量的病人样品的需求。为临床快速检测AEP水平提供有力帮助。
本发明是通过以下技术方案实现的:
本发明涉及一种AEP酶促化学发光免疫检测试剂盒,包括:酶标液、AEP标准品、包被抗AEP单克隆抗体的免疫磁珠、稀释液、化学发光底物和浓缩洗涤液。
所述的酶标液中含有辣根过氧化物酶标记的AEP单克隆抗体,该AEP单克隆抗体为兔抗或鼠抗,靶点为D6S4H。
所述的AEP标准品,其溶液浓度采用但不限于:0.01μg/mL、0.1μg/mL、0.5μg/mL、1.0μg/mL。
所述的化学发光底物液包括:辣根过氧化物酶底物溶液A液与B液,即发光增强剂和发光底物为HRP酶的底物。使用时将A液与B液混合,再与磁珠孵育1分钟即可。
所述的稀释液为含0.3-0.5M NaCl的50mM的磷酸盐缓冲液。
所述的浓缩洗涤液为20倍浓缩液,是pH值为7-8,含有终浓度为0.01-0.1%Proclin300,0.5-1.5%吐温-20和0.01-0.05mol/L的磷酸盐缓冲液,使用时用去离子水稀释至1倍洗涤液。
所述的包被抗AEP单克隆抗体的免疫磁珠,即含有标记有抗AEP单克隆抗体的磁珠,其具体通过将磁珠经活化剂活化后,与超滤后的抗AEP单克隆抗体在MEM溶液中偶联,后通过终洗液清洗后得到。
上述AEP酶促化学发光免疫检测试剂盒具体组分含量为:每升包含酶标液20mL、包被抗AEP单克隆抗体的免疫磁珠5mL、AEP标准品1.5mL、稀释液100mL、化学发光底物A液50mL、化学发光底物B液50mL、浓缩洗涤液20mL,余量为水。
本发明涉及上述AEP酶促化学发光免疫检测试剂盒的应用,将其用于检测人血中AEP蛋白水平,即通过将抗AEP单克隆抗体包被在磁珠表面,与样品中的AEP结合后,再与酶标AEP单克隆抗体试剂结合,形成固相-抗体-AEP抗原-酶标抗体夹心免疫复合物,通过加入发光底物后检测发光强度,对应标准曲线得到AEP含量。
所述的应用具体为:在样品中加入定量的包被抗AEP单克隆抗体的免疫磁珠和HRP标记抗AEP单克隆抗体。37℃孵育后,包被抗AEP单克隆抗体的免疫磁珠与HRP标记抗AEP单克隆抗体分别与标本中AEP分子的不同表位结合,形成磁珠-抗体-抗原-抗体复合物;在外加磁场中直接沉淀,即可分离;弃去上清液,清洗沉淀的复合物,然后加入促化学发光底物HRP。底物在酶的作用下催化裂解,形成不稳定的激发态中间体,当激发态中间体回到基态时便发出了光子,形成发光反应,即可使用发光仪检测反应的发光强度,根据标准曲线即可算出样品中AEP含量。
优选地,检测时血浆样品应添加在肝素抗凝管中。
技术效果
本发明整体解决了现有AEP检测试剂盒均使用ELISA方法,该方法易受到自身抗体,嗜异性抗体等干扰继而出现假阳性结果且操作繁琐,干扰因素较多,易出现假阳性,重复性差,且检测用时较长,不满足临床POCT快速检测的要求。本方法通过免疫磁珠化学发光法实现,具有操作便捷,灵敏性高,检测速度快,结果可靠等特点,能够满足临床快速检测的要求。与现有AEP检测试剂盒相比,本发明通过磁珠能够偶联抗体的能力,实现AEP单克隆抗体和磁珠偶联,进而在形成磁珠-抗体-AEP抗原-抗体复合物的过程中,使用外加磁场精准地分离复合物且单克隆抗体生产批间差异相对小,更容易保证产品的批间稳定。另外,使用的捕获抗体和酶标抗体均为单克隆抗体,使反应的亲和力更高,非特异性结合更少。
附图说明
图1为包被抗体后的1mg磁珠对于不同浓度AEP抗原的发光趋势线。
具体实施方式
本实施例涉及一种用于检测人血中AEP蛋白水平的化学发光免疫检测试剂盒,包括酶标液、AEP标准品、包被抗AEP单克隆抗体的免疫磁珠、稀释液、化学发光底物A液、B液和浓缩洗涤液,其中:
所述的包被抗AEP单克隆抗体的免疫磁珠,通过以下方法制备得到:
一、制备磁珠活化缓冲液:
1.初洗缓冲液的配制:
1.1称取0.1952g的MEShygrate于100mL的纯水中,调整pH至5.0。
1.2加入50μL吐温-20,配成0.01mol/LMES缓冲液,调整pH至5.0。
2.偶联缓冲液的配制:
2.1称取0.38138gNa2B4O7·10H2O溶于40mL纯水中,得到0.05mol/L的硼砂溶液。
2.2称取0.1237gH3BO3溶于10mL纯水中,得到0.2mol/L的硼酸溶液。
2.3取18mL0.05mol/L的Na2B4O7溶液与2mL0.2mol/L的H3BO3溶液混合,定容至80mL得0.05moi/L硼酸缓冲液。
3.终洗液的配制:
3.1称取2.78gNa2HPO4溶于100mL纯水得0.2mol/LNa2HPO4溶液。
3.2称取1.2gNaH2PO4溶于50mL纯水得0.2mol/LNaH2PO4溶液。
3.3取81mL0.2moi/LNa2HPO4溶液与19mL0.2mol/LNaH2PO4溶液混合,得0.2mol/LPB缓冲溶液得0.2mol/LPB缓冲溶液。
3.3取50mL0.2mol/LPB缓冲液,2倍稀释到100mL,加入0.9gNaCl,0.5g0.5%-1%BSA、0.02-0.03%生物防腐剂、0.05%吐温-20,充分溶解混匀。
4.封闭液的配制:取100mLTris缓冲液,将溶液的pH调节成8.5-9.0。向其加入2%BSA。
5.EDC溶液和NHS溶液的配制:
5.1称取25mg/mL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC),溶解于1mL的初洗缓冲液中,配制成25mg/mL的EDC溶液。
5.2称取25mg/mL的2羟基琥珀酰亚胺溶解于初洗缓冲液中,配置成25mg/mLNHS溶液。
二、磁珠活化缓冲液的操作步骤:
1.初洗:
1.1取100μL磁珠,将其稀释成10mg/mL的磁珠浓度,从中吸取100μL充分混匀。
1.2磁分离架上分离,去上清。
1.3用初洗液250μL洗涤三次,磁分离,去上清。
2.活化:
2.1取初洗完成的磁珠加入150μLMES,再加入50μLEDC及50μL的NHS溶液,充分混匀。室温下25℃活化30min。
2.2活化完成磁分离去上清,以250μL偶联缓冲液重悬洗涤三次,分离去上清。
3.稀释抗AEP单克隆抗体:将抗AEP单克隆抗体从-20℃冰箱中取出,待其融化后取出20μL,用硼酸缓冲液1:20稀释到400μL。
4.偶联:
4.1加入400μL用偶联液稀释的不同浓度的抗体重悬活化完的磁珠,充分混匀。
4.237℃偶联2h,37℃摇床摇晃恒温混匀。
5.封闭
5.1将偶联结束的磁珠于磁分离架上分离,去上清。
5.2加入400μL封闭液,37℃摇床封闭1h。
5.3封闭结束后去除上清。
6.终洗保存
6.1以400μL终洗液洗涤封闭完成的磁珠,重复三次。
6.2最终定容到250μL,用时将其稀释到500μL。
所述的稀释液,通过以下方法制备得到:在纯化水中加入Nacl、Na2HPO4、NaH2PO4配置成含0.3-0.5M NaCl的50mM的磷酸盐缓冲液。
所述的AEP标准品,通过以下方法制备得到:配制4瓶AEP标准品,含有0.1%ProCline300为防腐剂。每批试剂盒中AEP标准液的浓度为0.01μg/mL、0.1μg/mL、0.5μg/mL、1.0μg/mL。
所述的酶标液,通过以下方法制备得到:配制15mLHRP标记抗AEP单克隆抗体,保存在0.02%methylosothiazolone,0.02%bromonitrodioxne稳定剂及0.002%activeisothiazolone防腐剂中。
所述的洗涤液,通过以下方法制备得到:配制20mL(20倍稀释后使用)的浓缩磷酸盐缓冲液,含有吐温-20和0.1%ProCline-300为防腐剂。
所述的底物液,通过以下方法制备得到:
1.配制底物缓冲液:
称取4.542gTris,称取0.458g硼酸钠,定容到300mL,调节pH至8.7-9.0。
2.配制底物A液
2.1称取0.023g过氧化氢脲,称取0.024g4-Morpholinopyridine(稳定剂)。
2.2用Tris-HCl加硼酸钠缓冲液定容到100mL,室温保存。
3.配制底物B液
3.1称取0.088g鲁米诺,称取0.051g3-(10-phenothiazinyl)propane-l-sulfonate。
3.2用Tris-HCl加硼酸钠缓冲液定容到100mL。
3.3B液置于棕色瓶中,避光室温保存。
本实施例涉及上述方法制备得到的化学发光免疫检测试剂盒的应用,即检测人血浆,具体为检测受蛋白质刺激的餐后血清或血浆中AEP的含量。
所述的受蛋白质刺激是指:通过隔夜的空腹(至少10小时)刺激AEP的分泌。在服用蛋白质物刺激之前及20分钟之后采集静脉血样,或只采集餐后血样。
所述的检测具体为:将血样置于肝素抗凝管内,及时上下颠倒试管5-6次以混匀样品。血清测试管的凝结在室温20-25℃不应超过30分钟。由于AEP在较高温下会变质,建议采集到的血清管应置于碎冰(或冰水)浴内凝结,冰浴时间不超过60分钟。血清凝集后,立即用离心方法(如用塑料试管,加速至2000G,离心10-15分钟)分离血清和血浆。将样品分成几份至不同管中,如需更长时间的储存应冷冻,适宜在-70℃-20℃储存。样品在解冻后应混匀。避免反复冻融。避免使用含有溶血素、油脂或污浊不纯的样品。
标本采自500例正常体检、供血者。血清样品体检结果均无肝、脑、肾、消化道疾病,半年内无输液和大手术史,妇女不在妊娠期和哺乳期。将测值进行统计学分析。正常人群AEP含量为0.01-0.2μg/mL。
灵敏度:最低检出值0.002μg/mL;精密性:批内变异CV%<10.0%、批间变异CV%<15.0%;线性系数:r>0.9900;线性范围:0.005-5μg/mL
实施效果验证
本实施例采用宁波紫园化学发光免疫分析仪,根据上述上海仁济医院采集的肿瘤病人血浆样品与正常人血浆样品(-80℃保存),得到以下实验结果:
发光值 | 阴性样品 | 阳性样品 |
400729 | 1583991 | |
575065 | 1215558 | |
408776 | 1862972 | |
236821 | 1717730 | |
217649 | 1192359 | |
575065 | 3898802 | |
408776 | 1288094 |
将试剂配制成小样,37℃加速3天内,检测试剂盒检测时空白限、准确度、线性范围及重复性。要求空白限≤0.2ng/mL,准确度:回收实验的回收率85%~115%。线性范围:[0.2~200]ng/mL范围内,线性相关系数(r)应不低于0.9900。重复性:CV<10%。稳定性:37℃加速3天内,空白限、准确度、线性范围及重复性满足要求。
加速天数 | 空白限 | 准确度 | 线性范围 | 重复性 |
0 | 0.018 | 91.1% | 0.9999 | 2.80% |
1 | 0.018 | 94.8% | 0.9999 | 2.00% |
3 | 0.016 | 91.6% | 0.9999 | 1.80% |
由实验结果来看,本试剂盒空白限、准确度、线性、重复性、稳定性均满足要求。
(2)本实施例抗干扰实验的验证
分别往样本中添加一定浓度干扰物为实验组,同时往样本中添加等体积基质为对照组。
比较相对偏差,应不超过±10%。
干扰物 | 相对偏差 | 干扰物 | 相对偏差 |
30mg/dL胆红素 | 1.5% | 500mg/dL血红蛋白 | 0.3% |
由实验结果来看,本试剂盒抗干扰能力满足要求。
上述具体实施可由本领域技术人员在不背离本发明原理和宗旨的前提下以不同的方式对其进行局部调整,本发明的保护范围以权利要求书为准且不由上述具体实施所限,在其范围内的各个实现方案均受本发明之约束。
Claims (10)
1.一种AEP酶促化学发光免疫检测试剂盒,其特征在于,包括:酶标液、AEP标准品、包被抗AEP单克隆抗体的免疫磁珠、稀释液、化学发光底物和浓缩洗涤液;
所述的包被抗AEP单克隆抗体的免疫磁珠,即含有标记有抗AEP单克隆抗体的磁珠,其具体通过将磁珠经活化剂活化后,与超滤后的抗AEP单克隆抗体在MEM溶液中偶联,后通过终洗液清洗后得到。
2.根据权利要求1所述的AEP酶促化学发光免疫检测试剂盒,其特征是,所述的酶标液中含有辣根过氧化物酶标记的AEP单克隆抗体,该AEP单克隆抗体为兔抗或鼠抗,靶点为D6S4H。
3.根据权利要求1所述的AEP酶促化学发光免疫检测试剂盒,其特征是,所述的AEP标准品,其溶液浓度采用:0.01μg/mL、0.1μg/mL、0.5μg/mL、1.0μg/mL。
4.根据权利要求1所述的AEP酶促化学发光免疫检测试剂盒,其特征是,所述的化学发光底物液包括:辣根过氧化物酶底物溶液A液与B液,即发光增强剂和发光底物为HRP酶的底物。
5.根据权利要求1所述的AEP酶促化学发光免疫检测试剂盒,其特征是,所述的稀释液为含0.3-0.5M NaCl的50mM的磷酸盐缓冲液。
6.根据权利要求1所述的AEP酶促化学发光免疫检测试剂盒,其特征是,所述的浓缩洗涤液为20倍浓缩液,是pH值为7-8,含有终浓度为0.01-0.1%Proclin300,0.5-1.5%吐温-20和0.01-0.05mol/L的磷酸盐缓冲液,使用时用去离子水稀释至1倍洗涤液。
7.根据权利要求1~6中任一所述的AEP酶促化学发光免疫检测试剂盒,其特征是,每升包含酶标液20mL、包被抗AEP单克隆抗体的免疫磁珠5mL、AEP标准品1.5mL、稀释液100mL、化学发光底物A液50mL、化学发光底物B液50mL、浓缩洗涤液20mL,余量为水。
8.一种基于上述权利要求1~7中任一所述的AEP酶促化学发光免疫检测试剂盒的应用,其特征在于,用于检测人血中AEP蛋白水平,即通过将抗AEP单克隆抗体包被在磁珠表面,与样品中的AEP结合后,再与酶标AEP单克隆抗体试剂结合,形成固相-抗体-AEP抗原-酶标抗体夹心免疫复合物,通过加入发光底物后检测发光强度,对应标准曲线得到AEP含量。
9.根据权利要求8所述的应用,其特征是,具体为:在样品中加入定量的包被抗AEP单克隆抗体的免疫磁珠和HRP标记抗AEP单克隆抗体;37℃孵育后,包被抗AEP单克隆抗体的免疫磁珠与HRP标记抗AEP单克隆抗体分别与标本中AEP分子的不同表位结合,形成磁珠-抗体-抗原-抗体复合物;在外加磁场中直接沉淀,即可分离;弃去上清液,清洗沉淀的复合物,然后加入促化学发光底物HRP;底物在酶的作用下催化裂解,形成不稳定的激发态中间体,当激发态中间体回到基态时便发出了光子,形成发光反应,即可使用发光仪检测反应的发光强度,根据标准曲线即可算出样品中AEP含量。
10.根据权利要求8或9所述的应用,其特征是,检测时血浆样品应添加在肝素抗凝管中。
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