WO2024055498A1 - 一种能定量检定人载脂蛋白apoc1的检测试剂盒及其检测方法 - Google Patents
一种能定量检定人载脂蛋白apoc1的检测试剂盒及其检测方法 Download PDFInfo
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Abstract
一种定量检定人载脂蛋白APOC1的检测试剂盒及检测方法,试剂盒包括已包被捕获抗体的酶标板、校准品、阴性质控品试剂、阳性质控品试剂、酶标二抗试剂、显色液试剂、终止液试剂、浓缩洗涤液试剂。试剂盒及其检测方法的检测限为0.005μg/mL;检测准确度参考品,定量结果的相对偏差在±15%范围内;检测浓度为6.25ng/mL-180ng/mL的样本,剂量-反应曲线相关系数绝对值(r)不低于0.99;检测高、低重复性参考品各1份,变异系数(CV)均不高于15%(n=10)。
Description
本发明涉及人载脂蛋白APOC1的检测领域,特别涉及一种能定量检定人载脂蛋白APOC1的检测试剂盒及其及其检测方法。
孤独症又叫自闭症,儿童孤独症或者是孤独谱系障碍。孤独症的症状在医学上面主要是表现在两大类。第一类,就是社会交往、沟通的障碍。第二大类,就叫行为的刻板,兴趣的狭窄。目前我国在临床上针对小儿孤独症的筛查方式是医生根据《ASD行为量表》、《社交沟通问卷》、《幼儿ASD筛查量表》、《儿童ASD测试量表》、《社交反应量表》、《ASD评定量表》以及《克氏ASD行为量表》等筛查工具,对幼年儿童进行小儿孤独症的筛查。而诊断方式也仅仅是医生根据《儿童ASD评定量表》、《ASD诊断观察量表》以及《ASD诊断访谈量表修订版》这三个主要的诊断评估工具,对幼年儿童进行临床诊断。这些筛查、诊断方式往往由于耗时长、影响判断因素多(环境、幼年儿童心情、医生主观因素等),导致出现误诊的几率较高,错失治疗的最佳时间。
而后经过学术界不断的研究,也有采用不同的标志物进行检测,以应用于孤独症的辅助诊断,如专利文献ZL201610096458.0公开了一种孤独症血清多肽标志物APOC1-A及其应用,其公开了APOC1-A多肽可作为血清标志物用于孤独症的诊断,但由于ApoC1全长蛋白的分解产物较多,在检测结合ApoC1-A短肽时,经常会结合其他的多肽,导致检测结果不准确。
因此,亟需一种能够提供更明确的理化指标的技术手段,用于辅助诊断,可对患者的病情严重程度提供理化数据,为临床医生提供更有指向性的判断依据,为患者争取到更好地治疗时机。
发明内容
本发明要解决的技术问题是提供一种能定量检定人载脂蛋白APOC1的检测试剂盒,可对患者的病情严重程度提供理化数据。
为了解决上述技术问题,本发明的技术方案为一种能定量检定人载脂蛋白APOC1的检测试剂盒,包括已包被捕获抗体的酶标板、校准品、阴性质控品试剂、阳性质控品试剂、
酶标二抗试剂、显色液试剂、终止液试剂、浓缩洗涤液试剂。
优选地,酶标板的包被捕获抗体是以人ApoC1-A短肽为抗原,采用杂交瘤技术制备得到,人ApoC1-A短肽的氨基酸序列如下所示:
优选地,已包被捕获抗体的酶标板的制备方法为:首先采用CBS缓冲液(碳酸盐缓冲液)稀释捕获抗体,后加入酶标板中,低温过夜至少16小时,然后用PBS缓冲液(磷酸盐缓冲液)清洗数次后拍干,最后采用封闭液进行封闭。
进一步优选地,所述CBS缓冲液0.05mol/L的Na2CO3-NaHCO3溶液,pH为9.6;PBS缓冲液为1xPBS缓冲液。
优选地,酶标二抗试剂的制备方法为:用ELISA稀释液按1:2000比例稀释已标好辣根过氧化物酶(HRP)的检测抗体。
优选地,ELISA稀释液每100份中包含如下重量份的组分:
优选地,浓缩洗涤液试剂由20xPBS缓冲液与吐温-20混合而成,吐温-20的体积百分比为1%。
优选地,所述阴性质控品试剂为ELISA稀释液,阳性质控品试剂为标准品采用ELISA稀释液稀释至25~50ng/mL的稀释液。
优选地,所述酶标板采用封闭液进行封闭,封闭液组分包括蔗糖、BSA、酪蛋白、PEG2w、硫柳汞钠。
进一步优选地,封闭液每100份的组成按重量份如下:
进一步优选地,封闭液的制备方法为:称取相应比例的封闭液组分置于烧杯中,搅拌至组分完全溶解后,调节PH为6.50~7.2,用5μm滤膜进行抽滤,得到封闭液。
优选地,每100份1xPBS缓冲液中包含如下重量份的组分:
氯化钠 0.8份
十二水合磷酸氢二钠 0.323份
二水磷酸二氢钠 0.045份
氯化钠 0.8份
十二水合磷酸氢二钠 0.323份
二水磷酸二氢钠 0.045份
其余为超纯水。
优选地,终止液试剂为2mol/L的硫酸试剂。
一种人载脂蛋白APOC1的检测方法,其操作步骤如下:
加样:将校准品、阴性质控品试剂、阳性质控品试剂和已稀释的待测样本(疑似患者的血清/血浆)加入对应的实验孔中,每孔加入100μL,盖上封板膜,轻轻振荡1min混匀,置恒温培养箱(37℃±1℃)孵育45min;
洗涤:倾倒孔内液体,浓缩洗涤液试剂300μL/孔洗涤5次,每次45秒,每次洗完在吸水纸上拍干;
加入酶标二抗试剂::100μL/孔加入酶标二抗试剂,盖上封板膜,轻轻振荡混匀,置恒温培养箱(37℃±1℃)孵育30min;
二次洗涤:倾倒孔内液体,浓缩洗涤液试剂300μL/孔洗涤5次,每次45秒,每次洗完在吸水纸上拍干;
显色:加入显色液试剂100μL/孔,盖上封板膜,轻轻振荡混匀,置恒温培养箱(37℃±1℃)孵育,避光显色15min;
终止显色:取出微孔板,直接加入终止液试剂50μL/孔,终止反应(此时蓝色立即转为黄色);
检测:终止结束后,用酶标仪于主波长450nm及参考波长620~650nm进行检测,OD450减OD630的读取结果为OD值;
结果判定:首先,检验阴性质控品试剂的OD值≦0.02、阳性质控品试剂的OD值>0.25,即可判定为次检测的所有数据有效;再者,以不同梯度稀释的校准品的浓度为横坐标,相应的OD值为纵坐标,做标准曲线,计算出标准曲线的线性回归方程式(计算时忽略零浓度标准品),将样本的OD值代入方程式计算出其浓度。
采用上述技术方案,由于采用酶标板、校准品、阴性质控品试剂、阳性质控品试剂、酶标二抗试剂、显色液试剂、终止液试剂、浓缩洗涤液试剂的组合,并配合检测方法可以达到如下的性能指标:
1、通过阴性质控品试剂、阳性质控品试剂可预判数据的有效性与否;
2、检测限:0.005μg/mL;
3、准确度:用准确度参考品作为样本进行检测,其定量结果的相对偏差在±15%范围内;
4、线性范围:在6.25ng/mL-180ng/mL范围内,用4 Parameter Logisitcs或其他适当的曲线方法拟合,剂量-反应曲线关系数聚堆值(r)不低于0.99;
5、精密度:用高、低重复性参考品各1份分别进行检测,变异系数(CV)均不高于15%(n=10)。
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明的基本原理是:将含有已知抗体的抗血清吸附在微量滴定板上的小孔里,洗涤一次;加待测抗原,如两者是特异的,则发生结合,再加入与待测抗原呈特异反应的酶联抗体,使一单位的抗原同时结合两单位的抗体形成“夹心”,最后加入该酶的底物,根据产生的有色酶解产物颜色的深浅来判断待测抗原的含量。
而在如下的具体实施方式中用到的试剂及其制备方法如下:
碳酸盐缓冲液(CBS缓冲液):0.05mol/L的Na2CO3-NaHCO3溶液,pH为9.6。在称量室按配方用电子天平或分析天平准确称取各固体化学试剂,装入烧杯后转移到配液室中;往烧杯中加入需配液溶液体积80%的超纯水,置于搅拌器上搅拌至完全溶解,用超纯水补至需配制的体积,测量溶液的pH值并记录(正常范围为9.60±0.30)。
1xPBS缓冲液(0.01mol/L):
每100份中包含如下重量份的组分:
氯化钠 0.8份
十二水合磷酸氢二钠 0.323份
二水磷酸二氢钠 0.045份
氯化钠 0.8份
十二水合磷酸氢二钠 0.323份
二水磷酸二氢钠 0.045份
其余为超纯水
配制方法:按比例称取各固体化学试剂,装入烧杯后转移到配液室中;往烧杯中加入需配液溶液体积80%的超纯水,置于搅拌器上搅拌至完全溶解,用超纯水补至需配制的体积。
浓缩洗涤液试剂:20xPBS缓冲液(0.2mol/L),然后加入吐温-20,吐温-20占浓缩洗涤液试剂体积的1%。
ELISA稀释液
每100份中包含如下重量份的组分:
配制方法:称取各固体化学试剂,装入烧杯后转移到配液室中,往烧杯中加入需配体积量约80%的纯水,置于搅拌器中搅拌至完全溶解,后继续补加纯水至配制体积。
而本发明的试剂盒中的阴性质控品试剂即为ELISA稀释液。
酶标二抗试剂:用ELISA稀释液按1:20000比例稀释已标好辣根过氧化物酶(HRP)的检测抗体,检测抗体由西安海娜生物科技有限公司购得,稀释后的检测抗体即为酶标二抗试剂。
封闭液
每100份中包含如下重量份的组分:
配制方法:称取各固体化学试剂装入烧杯后转移到配液室中,往烧杯中加入需配体积量约80%的1xPBS缓冲液,盖上锡纸,置于搅拌器中搅拌至完全溶解,后继续补加1xPBS至配制体积,测量溶液的pH值并记录(正常范围为6.50-7.20),用5μm滤膜进行抽滤,标
识,储存于2~8℃后备用。使用前室温自然平衡至室温,约0.5-1h。
终止液:按常规方法配制2mol/L的硫酸试剂,即为终止液。
显色液试剂:采用湖州英创生物科技有限公司购买的TMB显色液(3,3',5,5'-四甲基联苯胺)。
校准品:以人载脂蛋白C1作为校准品,校准品定量后即为标准品。阳性质控品试剂为ELISA稀释后浓度为25~50ng/mL的标准品。以下的具体实施例中采用的校准品为abcam生产的ISA试剂盒(APOCI)(ab108808),但此处只是作为试验的例子,并不是限制只能使用该校准品,只要人载脂蛋白C1经定量后均可。
包被酶标板:首先采用CBS缓冲液(碳酸盐缓冲液)稀释捕获抗体至4μg/mL,后加入酶标板中,低温过夜至少16小时,然后用PBS缓冲液(磷酸盐缓冲液)清洗数次后拍干,最后采用封闭液进行封闭,封闭数小时,甩干液体并拍干,晾干后用铝箔袋加入干燥剂后抽真空封口,低温保存。
实施例:
1)、收集孤独症患病儿童血清50例(男性31例,女性19例;平均年龄4.5岁)、正常对照儿童血清40例(男性24例;女性16例;平均年龄4.5岁)进行本试剂盒的血清验证分析。
2)、准备样品
用ELISA稀释液,将标准品稀释至180ng/mL、100ng/mL、50ng/mL、25ng/mL、12.5ng/mL,每管分装至合适的体积;
阴性质控品试剂:ELISA稀释液;
阳性质控品试剂:稀释至25~50ng/mL的标准品;
待测样本:将步骤1)中收集的血清按1:2000比例进行稀释,每份血清三个平行样;
3)、检测
将步骤2)中所有样品加入酶标板的实验孔中,每孔加入100μL,盖上封板膜,轻轻振荡1min混匀,置恒温培养箱(37℃±1℃)孵育45min;
倾倒孔内液体,洗涤液300μL/孔洗涤5次,每次45秒,每次洗完在吸水纸上拍干,如果用洗板机洗涤,洗涤次数增加一次;
加入酶标二抗试剂100μL/孔,盖上封板膜,轻轻振荡混匀,置恒温培养箱(37℃±1℃)孵育30min;
倾倒孔内液体,浓缩洗涤液试剂300μL/孔洗涤5次,每次45秒,每次洗完在吸水纸
上拍干;
加入显色液试剂100μL/孔,盖上封板膜,轻轻振荡混匀,置恒温培养箱(37℃±1℃)孵育,避光显色15min;
取出微孔板,直接加入终止液试剂50μL/孔,终止反应(此时蓝色立即转为黄色);
终止结束后,用酶标仪于主波长450nm及参考波长620~650nm进行检测,OD450减OD630的读取结果为OD值;
结果判定:
首先,检验阴性质控品试剂的OD值≦0.02、阳性质控品试剂的OD值>0.25,即可判定为次检测的所有数据有效;
再者,根据标准品的OD值及其浓度绘制标准曲线,浓度为横坐标,OD值为纵坐标
以不同梯度稀释的校准品的浓度为横坐标,相应的OD值为纵坐标,做标准曲线,计算出标准曲线的线性回归方程式(计算时忽略零浓度标准品),将样本的OD值代入方程式计算出其浓度。
上述结果均为样品多个平行测试后的平均结果,而其中患病儿童和正常对照儿童中每例均测试3个平行样。
上述结果均为样品多个平行测试后的平均结果,而其中患病儿童和正常对照儿童中每例均测试3个平行样。
结果表明ApoC1在孤独症患病儿童及正常儿童对照检测组中的表达水平分别为:患病儿童110.96ng/mL,正常对照儿童58.14ng/mL,具有显著性差异。而由于测试时对样品进行稀释,稀释倍数为1:2000,则ApoC1在孤独症患病儿童及正常儿童对照检测组中正常的表达水平分别为患病儿童221.92ug/mL,正常对照儿童116.28ug/mL。
而通过对标准品加大稀释倍数,可以检验出本发明的检测限,即标准品稀释后,检测
至OD值稳定大于0.02的浓度即可判定为检测限。
另外,经多次试验,本发明的试剂盒可以达到以下性能指标:
1、检测限:0.005μg/mL;
2、准确度:用准确度参考品作为样本进行检测,其定量结果的相对偏差在±15%范围内;
3、线性范围:在6.25ng/mL-180ng/mL范围内,用4Parameter Logisitcs或其他适当的曲线方法拟合,剂量-反应曲线关系数聚堆值(r)不低于0.99;
4、精密度:用高、低重复性参考品各1份分别进行检测,变异系数(CV)均不高于15%(n=10)。
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (10)
- 一种能定量检定人载脂蛋白APOC1的检测试剂盒,其特征在于:包括已包被捕获抗体的酶标板、校准品、阴性质控品试剂、阳性质控品试剂、酶标二抗试剂、显色液试剂、终止液试剂、浓缩洗涤液试剂。
- 根据权利要求1所述的检测试剂盒,其特征在于,所述标板的制备方法为:首先采用CBS缓冲液稀释捕获抗体,后加入酶标板中,低温过夜至少16小时,然后用PBS缓冲液清洗数次后拍干,最后采用封闭液进行封闭。
- 根据权利要求2所述的检测试剂盒,其特征在于:所述CBS缓冲液为0.05mol/L的Na2CO3-NaHCO3溶液,pH为9.6;PBS缓冲液为1xPBS缓冲液。
- 根据权利要求2所述的检测试剂盒,其特征在于:所述封闭液每100份的组成按重量份如下:
- 根据权利要求4所述的检测试剂盒,其特征在于:所述封闭液的制备方法为:称取相应比例的封闭液组分置于烧杯中,搅拌至组分完全溶解后,调节PH为6.50~7.2,用5μm滤膜进行抽滤,得到封闭液。
- 根据权利要求1所述的检测试剂盒,其特征在于:所述的酶标二抗试剂为ELISA稀释液按1:2000比例稀释已标好辣根过氧化物酶的检测抗体。
- 根据权利要求6所述的检测试剂盒,其特征在于:所述ELISA稀释液每100份中包含如下重量份的组分:
- 根据权利要求1所述的检测试剂盒,其特征在于,每100份1xPBS缓冲液中包含如下重量份的组分:
氯化钠 0.8份
十二水合磷酸氢二钠 0.323份
二水磷酸二氢钠 0.045份
其余为超纯水。 - 根据权利要求1所述的检测试剂盒,其特征在于:所述浓缩洗涤液试剂由20xPBS缓冲液与吐温-20混合而成,吐温-20的体积百分比为1%。
- 一种人载脂蛋白APOC1的检测方法,其特征在于,操作步骤如下:加样:将校准品、阴性质控品试剂、阳性质控品试剂和已稀释的待测样本加入对应的实验孔中,每孔加入100μL,盖上封板膜,轻轻振荡1min混匀,置恒温培养箱(37℃±1℃)孵育45min;洗涤:倾倒孔内液体,浓缩洗涤液试剂300μL/孔洗涤5次,每次45秒,每次洗完在吸水纸上拍干;加入酶标二抗试剂::100μL/孔加入酶标二抗试剂,盖上封板膜,轻轻振荡混匀,置恒温培养箱(37℃±1℃)孵育30min;二次洗涤:倾倒孔内液体,浓缩洗涤液试剂300μL/孔洗涤5次,每次45秒,每次洗完在吸水纸上拍干;显色:加入显色液试剂100μL/孔,盖上封板膜,轻轻振荡混匀,置恒温培养箱(37℃±1℃)孵育,避光显色15min;终止显色:取出微孔板,直接加入终止液试剂50μL/孔,终止反应(此时蓝色立即转为黄色);检测:终止结束后,用酶标仪于主波长450nm及参考波长620~650nm进行检测,OD450减OD630的读取结果为OD值;结果判定:首先,检验阴性质控品试剂的OD值≦0.02、阳性质控品试剂的OD值>0.25,即可判定为次检测的所有数据有效;再者,以不同梯度稀释的校准品的浓度为横坐标,相应的OD值为纵坐标,做标准曲线,计算出标准曲线的线性回归方程式,将样本的OD值代入方程式计算出其浓度。
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