CN116621978A - 一种用于β-CTX检测的抗体、试剂盒及其应用 - Google Patents
一种用于β-CTX检测的抗体、试剂盒及其应用 Download PDFInfo
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- CN116621978A CN116621978A CN202211243489.6A CN202211243489A CN116621978A CN 116621978 A CN116621978 A CN 116621978A CN 202211243489 A CN202211243489 A CN 202211243489A CN 116621978 A CN116621978 A CN 116621978A
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- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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Abstract
本发明公开了一种用于β‑CTX检测的抗体、试剂盒及其应用,属于体外诊断试剂、生物工程技术和医疗器械交叉领域,试剂盒包括发光试剂、固相试剂、校准品及质控品,其中发光试剂为吖啶酯标记的β‑CTX抗体溶液,固相试剂为包被有β‑CTX抗体的磁微粒的混悬液,本发明所述试剂盒通过双抗体夹心法原理进行检测,可搭载全自动化学发光检测仪进行自动化检测,具有灵敏度高、特异性强、检测范围宽、检测效率高的特点,本发明提供的试剂盒主要用于人体β‑CTX的检测。
Description
技术领域
本发明涉及体外诊断试剂、生物工程技术和医疗器械交叉领域,具体而言,涉及一种用于β-CTX检测的抗体、试剂盒及其应用。
背景技术
骨转换,是破骨细胞不断吸收旧骨和成骨细胞不断形成新骨的自我更新过程,只有当骨组织不断进行骨塑建和骨重建,才可以维持骨骼生长和结构完整。骨转换对于修复骨骼疲劳损伤和维持机体矿物质平衡至关重要。骨转换生化标志物或骨转换标志物(boneturnover markers,BTMs)是骨转换过程中产生的代谢产物或酶类,分为骨形成指标和骨吸收指标,前者反映成骨细胞活性及骨形成状态,后者代表破骨细胞活性及骨吸收水平。BTMs浓度对于多种骨骼疾病的诊断与鉴别、骨折风险预测、药物疗效评价等具有重要参考价值。近年来,随着骨质疏松症和代谢性骨病的研究进展,特别是国内外BTMs的循证医学证据不断涌现,BTMs的临床应用日趋广泛。
目前广泛应用的BTMs包括β-CTX、P1NP、OC和b-ALP,国内主要临床应用的是β-CTX、P1NP、OC、ALP。其中,β-CTX(β-Cross laps)是目前国际上公认的代表骨吸收标志物。β-CTX是一种由8个氨基酸组成的I型胶原分解片段,由C端肽的α-天冬氨酸转变成β型而形成。血清β-CTX水平的增高表明患者的骨吸收程度增加,骨吸收抑制治疗后血清β-CTX水平会恢复正常。由于昼夜节律和食物的影响,β-CTX水平在不同时间段会显示出显著的变化,可以通过在早上禁食状态下的标准化样本收集最小化检测差异。
在研究和临床实践中,β-CTX测量的大多数应用都是在血液样本上进行的。血清和血浆β-CTX方法的特异性差异导致了标准化β-CTX商业测定的困难。另一方面,现有的检测手段存在诸多缺陷,如检测成本较高、灵敏度较低等。在国内,β-CTX检测临床上主要以罗氏公司试剂应用为主,但试剂价格昂贵,并不利于在基层普及。因此,有待开发一种灵敏度高、特异性强、检测范围宽、检测效率高的β-CTX检测试剂盒,用于监测骨质疏松症或其他骨疾病的抗吸收治疗疗效。
发明内容
本发明所要解决的问题是开发一种灵敏度高、特异性强、检测范围宽、检测效率高的β-CTX检测试剂盒,用于监测骨质疏松症或其他骨疾病的抗吸收治疗疗效。
为解决上述问题,本发明第一方面提供一种用于β-CTX检测的抗体。
本发明所述的用于β-CTX检测的抗体为采用杂交瘤细胞生产的单克隆抗体,其制备步骤为:
抗原合成:在如SEQ ID No.1所示的多肽序列的多肽基础上偶联大分子载体蛋白,所述大分子载体蛋白为BSA、OVA中的一种或多种,制备出具有抗原性和免疫原性的重组免疫原;
SEQ ID No.1:Cys Glu Lys Ala His Asp Gly Gly Arg;
单独的多肽其分子量通常过小不足以激起充分的免疫反应,而当多肽与带有很多抗原表位的载体蛋白诸如KLH、BSA、OVA进行偶联后,重组抗原相较于多肽,更加有利于刺激后续实验中小鼠的TH细胞,进而诱导B淋巴细胞产生免疫反应。
动物免疫:使用前述大分子载体蛋白制备的重组免疫原和佐剂,通过皮下注射对小鼠进行首次免疫,首次免疫后进行加强免疫,最后一次加强免疫后进行不加佐剂的腋下注射冲击免疫;
动物免疫利用的是生物体自身的体液免疫应答机制,通过抗原的持续刺激先后启动初级免疫反应和次级免疫反应,实际操作中以血清中抗体效价来衡量实际免疫效果。抗原刺激机体后启动初级反应一般会有一个滞后期,此时幼B细胞经过克隆筛选、增殖,然后分化成记忆细胞或浆细胞;经过滞后期,血清效价呈指数增长达到一个峰值然后下降;初级免疫反应中,会首先分泌IgM,然后经过抗体亚型转换,IgG比例会升高,次级免疫反应的启动依赖于记忆B细胞和记忆T细胞的数目,记忆细胞再次遇到相同抗原就能被激活,从而导致次级免疫反应的发生;当记忆细胞数目远大于幼B细胞时,次级反应启动更快、强度更大;次级反应相比初级反应滞后期更短,浆细胞和血清效价的峰值更高,持续时间更长;同时,此时分泌的抗体经过亲和力成熟和亚型转换,质量更高;进行动物免疫时需要用到佐剂,佐剂的作用主要是延缓抗原的释放,增强吞噬细胞的吞噬和对抗原的呈递。
细胞融合:提取前述小鼠的脾细胞与骨髓瘤细胞进行混合,加入PEG,诱导细胞进行细胞融合;
PEG是人工合成药物,在细胞融合中有如下作用:使细胞凝结;破坏互相接触处的细胞膜的磷脂双分子层,从而使相互接触的细胞膜之间发生融合,进而细胞质沟通,形成一个打和的双核或多核融合细胞。
单克隆抗体制备:SEQ ID No.1所示的多肽-OVA偶联物和SEQ ID No.2所示多肽的OVA偶联物以1μg/ml包被,间接法ELISA检测挑选双阳性克隆,经过亚克隆,确保形成单克隆时扩大培养,纯化得单克隆抗体;
SEQ ID No.2:Cys Glu Lys Ala His Asp Gly Gly Arg Glu Lys Ala His AspGly Gly Arg;
抗体标记:将单克隆抗体与生物素进行混匀、反应后获得生物素标记的单克隆抗体;
抗体标记是指将标记物共价连接到抗体上,与待检测物特异性反应形成多元复合物,并借助于荧光显微镜、射线测量仪、酶标检测仪、电子显微镜和发光免疫测定仪等精密仪器对试验结果直接镜检观察或进行自动化测定,生物素都可与蛋白质、荧光素等分子结合而不影响后者的生物活性,是理想的标记剂。一个抗体分子可偶联数个生物素分子,而生物素分子又可与酶或荧光素结合,从而组成一个生物放大***,显著提高检测的灵敏度。
抗体配对筛选:将未标记的单克隆抗体孵育包被、封闭,加入SEQ ID No.2所示多肽的多肽-OVA偶联物进行孵育,加入生物素标记的单克隆抗体,孵育,加入SA-HRP孵育显色,检测,挑选特异性强和灵敏度高的阳性配对。
通常情况下,一个抗原分子拥有多个抗原决定簇,将抗原注射入动物体内进行免疫,针对不同的抗原决定簇会产生不同的抗体。产生的抗体具有特异性与专一性,即一个抗体分子只会特异性地结合一个抗原决定簇,两个针对不同抗原决定簇的抗体,有可能同时结合到抗原分子上,如果两个抗体同时结合到同一个抗原分子上,那么这两个抗体就是配对抗体。抗体配对常应用于双抗体夹心ELISA实验,能够对目的抗原进行定性定量检测,具有高灵敏度,高特异性及高稳定性的优点。
本发明第二方面提供一种用于β-CTX检测的试剂盒。
进一步地,所述试剂盒包含前述β-CTX抗体制备方法制得的单克隆抗体。
进一步地,本发明提供的用于β-CTX检测的试剂盒为包括发光试剂、固相试剂、标准品以及质控品的化学发光免疫试剂盒。
进一步地,本发明所述试剂盒的固相试剂为包被有β-CTX单克隆抗体的磁微粒混悬液,磁微粒浓度为0.05-0.5μg/ml。
优选地,本发明所述试剂盒固相试剂中的磁微粒为具有活性化学基团的超顺磁性纳米磁性微球,其表面活性基团为羧基、甲苯磺酰基、环氧基、氨基中的任意一种。
进一步地,本发明所述试剂盒的发光试剂为标记有化学发光试剂的β-CTX单克隆抗体溶液,浓度为0.05-1.0μg/ml,所述发光试剂中的β-CTX抗体和所述固相试剂中的β-CTX抗体为两株针对不同抗原靶点的可进行配对的单克隆抗体。
优选地,本发明所述试剂盒发光试剂为吖啶酯、异鲁米诺、碱性磷酸酶、过氧化物酶、三联吡啶钌中任意一种。
进一步地,本发明所述试剂盒的稀释液配方由缓冲盐,无机盐,表面活性剂,蛋白保护剂,化学保护剂,防腐剂组成,Ph为6-8,缓冲盐为HEPES、磷酸盐缓冲液,TRIS缓冲中的一种或多种;无机盐为氯化钠,氯化钾,氯化镁,硫酸锌中的一种或多种;表面活性剂为吐温120曲拉通405,NP-40和十二烷基硫酸钠中的一种或多种;蛋白保护剂为HSA、BSA,明胶,聚多卡醇,α环糊精中的一种或多种,化学保护剂为聚乙烯吡咯烷酮,Biolipidure,乙二醇,硫酸葡聚糖钠盐中的一种或多种,防腐剂为proclin300,庆大霉素,左氧氟沙星,苯氧乙醇中的一种或多种。
进一步地,所述校准品为由SEQ ID No.2所示蛋白的二聚体多肽配制的浓度在0-2000pg/ml范围内的2个不同浓度点的液体校准品;所述质控品为由序列2蛋白表达得到的二聚体多肽配制的浓度在0-1000pg/ml范围内的2个不同浓度点的液体质控品。
本发明第三方面提供一种用于β-CTX检测的试剂盒的应用。
本发明所述试剂盒通过双抗体夹心法原理进行检测,主要反应流程为仪器样本针自动吸取30μl血清样本于反应管中,试剂针分别吸取40μl固相试剂和60μl发光试剂于反应管中后,充分混合后孵育15分钟,分析仪自动转入清洗站进行3次重复清洗,最终转入测量池中,通过管路泵入预激发液和激发液,测量池中的PMT收集反应管中的产生的光信号并上传至软件中,通过仪器中输入的校准曲线给出最终的测量浓度。
本发明具备的有益效果:(1)本发明提供的β-CTX检测试剂盒为化学发光免疫试剂盒,分析时不需要激发光,检测下限低且灵敏度高;(2)本发明提供的β-CTX检测试剂盒使用磁微球作为固相载体,方便清洗,去除非特异性结合,有利于提高检测准确度;(3)本发明提供的β-CTX检测试剂盒还具有特异性强、重复性好、稳定性高的特点。
附图说明
图1为本发明具体实施方式中相关性检测结果;
图2为本发明具体实施方式中线性检测结果;
图3为本发明具体实施方式中热稳定性检测结果;
图4为本发明具体实施方式中机载稳定性实验结果。
具体实施方式
为使本发明的上述目的、特征和优点能够更为明显易懂,下面对本发明的具体实施例做详细的说明。需要说明的是,以下各实施例仅用于说明本发明的实施方法和典型参数,而不用于限定本发明所述的参数范围,由此引申出的合理变化,仍处于本发明权利要求的保护范围内。
需要说明的是,在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
本发明实施例提供一种用于β-CTX检测的抗体、试剂盒及其应用,抗体是通过如SEQ ID No.1和SEQ ID No.2序列所示多肽经载体蛋白包被后制备筛选的β-CTX单克隆抗体。试剂盒为为包括发光试剂、固相试剂、标准品以及质控品的化学发光免疫试剂盒,其中固相试剂为包被有前述β-CTX单克隆抗体的磁微粒浓度为0.05-0.5μg/ml的混悬液;发光试剂为浓度为0.05-1.0μg/ml标记有化学发光试剂的β-CTX单克隆抗体溶液;校准品为由SEQID No.2所示蛋白的二聚体多肽配制的浓度在0-2000pg/ml范围内的2个不同浓度点的液体校准品;质控品为由序列2蛋白表达得到的二聚体多肽配制的浓度在0-1000pg/ml范围内的2个不同浓度点的液体质控品。
实施例1β-CTX单克隆抗体的制备
1.多肽合成
合成SEQ ID No.1和SEQ ID No.2所示的多肽,多肽合成委托相关CXO公司完成,最终纯度≥95%。
SEQ ID No.1:Cys Glu Lys Ala His Asp Gly Gly Arg
SEQ ID No.2:Cys Glu Lys Ala His Asp Gly Gly Arg Glu Lys Ala His AspGly Gly Arg
2.重组免疫原制备
用PBS(pH=7.4)配置5mg/ml载体蛋白(BSA和OVA)溶液,每5mg载体蛋白以摩尔比1:10加入sulfo-SMCC(sulfo-SMCC,Thermo)混合,在室温下反应30分钟,或在4℃下反应过夜,使用PBS(pH=7.4)平衡的脱盐柱除去过量的交联剂,将多肽干粉复溶到5mg/ml,以活化后载体:多肽的摩尔比1:10混合,在室温下反应30分钟,或在4℃下反应过夜。将反应物置于PBS(pH=7.4)透析3次,每4小时换液一次,得到偶联物。
3.动物免疫
免疫原为SEQ ID No.1-BSA偶联物,免疫小鼠为6-8周龄身体健康的雌性Balb/c小鼠。首次免疫时,免疫剂量为100ug/只小鼠,使用弗氏完全佐剂混合,皮下多点注射;3周后进行加强免疫,50ug/只小鼠,使用弗氏不完全佐剂混合,皮下多点注射,此后间隔2周进行再次加强免疫;首免后共进行3次加强免疫。首免、三免及四免的1周后进行眼眶采血,分别用SEQ ID No.1-OVA偶联物及SEQ ID No.2-OVA偶联物以1ug/ml包被酶标板,间接ELISA方法检测鼠抗血清的效价。最后一次加强免疫的10天后,使用不加佐剂抗原100ug/只小鼠进行冲击腋下注射;冲击免3天后进行融合实验。
4.细胞融合及单克隆抗体制备实验
将冲击的小鼠断颈处死,于75%乙醇中浸泡灭菌。超净工作台中,无菌条件下取出小鼠脾脏,用已灭菌处理的毛玻璃片挤压并轻轻研磨脾脏分离脾脏细胞,脾脏细胞计数。以SP2/0细胞和脾细胞1:5的比例混合,室温下500g离心5分钟,弃上清。加入1.5ml PEG,让PEG和细胞充分均匀混合,此过程3分钟内完成。加50ml新鲜DMEM培养基,室温500g离心5分钟,HAT培养基重悬,轻轻混匀。100ul/孔加入到96孔板中,放入培养箱中培养,4-5天后96孔板中细胞长至1/3底面积时更换HT培养基,SEQ ID No.1-OVA偶联物及SEQ ID No.2-OVA偶联物以1ug/ml包被,间接法ELISA检测挑选双阳性克隆,经过3-4次亚克隆,确保形成单克隆时扩大培养,并进行腹水细胞注射,纯化得到单克隆抗体。
5.抗体标记
将单克隆抗体用1xPBS稀释至浓度1mg/ml,每2mg抗体以摩尔比1:5(抗体:生物素)加入生物素(EZ-Link NHS-PEG4-Biotin,Thermo)并迅速混匀,混匀后,盛有混合液的离心管放在垂直搅拌器上反应30min,或4℃过夜,生物素化后的抗体用1xPBS透析3次,每4小时换液一次。得到生物素标记的单克隆抗体。
6.抗体配对筛选
将未标记的单抗每孔2μg/mlx100μl,37℃孵育1h包被;200μL 2%BSA封闭,37℃孵育1h,PBST清洗3次,每孔加入1ug/ml的SEQ ID No.2-OVA偶联物,每个配对设置不加抗原的阴性对照,37℃孵育1h,PBST清洗3次。加入生物素标记的单抗(1:200),37℃孵育1h,PBST清洗3次;加入SA-HRP 1:2000,100μl,37℃孵育30min,PBST清洗4次;显色,450nm波长读取OD值,OD大于阴性对照孔的2.1倍,判定为阳性配对,挑选灵敏度高和特异性强的抗体对用于制备化学发光试剂进行样本测试。
实施例2β-CTX化学发光试剂盒制备
制备稀释液:将缓冲剂、无机盐、金属螯合剂、蛋白保护剂、防腐剂、表面活性剂依次溶解到纯化水中,调节pH至6.0~8.0范围内,按照配制量进行定容,经0.22μm滤膜过滤后备用;
制备固相试剂:将β-CTX抗体包被在磁微粒(购自日本JSR公司)表面制备成中间品,再取适量的磁微粒中间品加入到稀释液中,配制成浓度为0.05~0.5mg/ml的工作液;
制备发光试剂稀释液:将缓冲剂、无机盐、金属螯合剂、蛋白保护剂、防腐剂、表面活性剂依次溶解到纯化水中,调节pH至5.0~7.0范围内,按照配制量进行定容,经0.22μm滤膜过滤后备用;
制备发光试剂:将吖啶酯(NSP-SA-NHS,Sigma)按一定比例标记在β-CTX抗体上,制备成中间品,再取适量的发光试剂中间品加入到发光试剂稀释液中,配制成浓度为0.05~1.0μg/ml的发光试剂工作液;
制备校准品稀释液:将缓冲剂、无机盐、金属螯合剂、蛋白保护剂、防腐剂、表面活性剂依次溶解到纯化水中,调节pH至6.0~8.0范围内,按照配制量进行定容,经0.22μm滤膜过滤后备用;
制备校准品:将β-CTX重组抗原按照一定的比例添加到校准品稀释液中,配制成浓度分别为200pg/ml、5000pg/ml的校准品溶液,分别记为CAL1、CAL2;
制备质控品:将β-CTX重组抗原按照一定的比例添加到校准品稀释液中,配制成浓度分别为200pg/ml、2000pg/ml的质控品溶液,分别记为QC1、QC2。
实施例3相关性检测
梯度稀释重组β-CTX抗原,用罗氏β-CTX检测试剂盒检测重新赋值,选择0-6000pg/mL范围内2点作为定标点,并用自配发光试剂及固相试剂检测定标MS-i3080化学发光免疫分析仪,测试条件:一步法,60μL发光试剂、40μL固相试剂、30μL样本,37℃孵育15min。
搜集40份样本,理论值覆盖线性范围0-6000pg/mL,用罗氏β-CTX检测试剂盒,参照说明书条件测试。测试条件同上,记录测试结果。计算本试剂盒与罗氏试剂盒测试相关性系数R2。
据图1结果显示,本试剂盒与罗氏试剂盒的检测结果相关性较好,相关性系数R2为0.9915。
实施例4灵敏度检测
以5%牛血清做空白样本,连续测定25次,测试条件同上。记录测试结果,分别计算平均值AV、标准差SD,计算最低检测线LOB=AV+2SD;以5%牛血清为基质,分别添加浓度约为10pg/ml的5支低浓度样本,每支样本连续测定5次,测试条件同上,记录测试结果。
表1灵敏度检测结果
灵敏度 | 批次1(pg/ml) | 批次2(pg/ml) | 批次3(pg/ml) |
LOB | 0 | 0 | 0 |
LOD | 9.89 | 9.58 | 9.93 |
据表1结果显示,本试剂盒的LOB为0,最低检出限LOD<10pg/ml。
实施例5特异性检测
对1μg/mL的骨钙素、甲状旁腺素和骨ALP样本进行检测,用自配发光试剂及固相试剂进行检测,测试条件同上,记录测试结果,结果如表2所示。
表2特异性检测结果
如表2所示,检测结果显示均不高于10pg/mL,即未检出交叉反应。
实施例6重复性检测
连续测试10次浓度为200pg/ml、2000pg/ml的样本,测试条件同上,记录测试结果。分别计算平均值AV、标准差SD,计算最低检测线CV=SD/AV,检测结果如表3所示。
表3重复性检测
结果显示用本试剂盒连续测试浓度为200pg/ml、2000pg/ml的样本,计算CV均满足≤5%的要求,甚至可以控制在2%以内。
实施例7线性检测
用接近线性范围上限的高浓度样品和接近线性范围下限的低浓度样品,混合成8-11个稀释浓度,每个稀释浓度测试3次,测试条件同上,记录测试结果,结果如图2所示。
结果显示线性回归方程中的线性回归系数符合r≥0.99的要求。
实施例8热稳定性检测
将在37℃条件下放置7天并在2~8℃平衡1天后的试剂与2~8℃放置的试剂同时检测校准品与40份样本,测试条件同上,记录测试结果,结果如表4和图3所示。
表4热稳定性检测
据表4和图3的结果显示,两组试剂测两点校准的值与靶值偏差均在5%以内,校准后测样本的结果也基本没有偏差。
实施例9机载稳定性检测
将β-CTX检测试剂盒开瓶后放入仪器,使仪器处于开机状态以保持试剂盘温度,每个工作日测定质控品3次,结果取均值,连续监测28天,所测浓度与质控品靶值进行比较,测试条件同上,记录测试结果,结果如图4所示。
结果显示,机载连续监测28天且不经过校准,质控品准确度仍能满足偏差≤±10%的要求。
实施例10检测
1.正常参考值范围建立
使用本发明提供的β-CTX检测试剂盒分别对1250份不同年龄段的正常人样本血清内β-CTX进行检验。将正常参考值范围设立如表5所示。
表5正常人血清β-CTX检测结果统计学分析
2.参考范围验证
使用本发明的β-CTX检测试剂盒检测60例体检健康人群样本,对样本结果进行分析,采用Dixon的方法剔除离群值(若有,则另选参考个体补足),计算超出参考区间的数据。其中符合58例,符合率为96.67%,参考范围适用。
虽然本公开披露如上,但本公开的保护范围并非仅限于此。本领域技术人员,在不脱离本公开的精神和范围的前提下,可进行各种变更与修改,这些变更与修改均将落入本发明的保护范围。
Claims (10)
1.一种β-CTX单克隆抗体的制备方法,其特征在于,包括如下步骤:
抗原合成:在如SEQ ID No.1所示的多肽序列的多肽基础上偶联大分子载体蛋白,所述大分子载体蛋白为BSA、OVA中的一种或多种,制备出具有抗原性和免疫原性的重组免疫原;
SEQ ID No.1:Cys Glu Lys Ala His Asp Gly Gly Arg;
动物免疫:使用前述大分子载体蛋白制备的重组免疫原和佐剂,通过皮下注射对小鼠进行首次免疫,首次免疫后进行加强免疫,最后一次加强免疫后进行不加佐剂的腋下注射冲击免疫;
细胞融合:提取前述小鼠的脾细胞与骨髓瘤细胞进行混合,加入PEG,诱导细胞进行细胞融合;
单克隆抗体制备:SEQ ID No.1所示的多肽-OVA偶联物和SEQ ID No.2所示多肽的OVA偶联物以1μg/ml包被,间接法ELISA检测挑选双阳性克隆,经过亚克隆,确保形成单克隆时扩大培养,纯化得单克隆抗体;
SEQ ID No.2:Cys Glu Lys Ala His Asp Gly Gly Arg Glu Lys Ala His Asp GlyGly Arg;
抗体标记:将单克隆抗体与生物素进行混匀、反应后获得生物素标记的单克隆抗体;
抗体配对筛选:将未标记的单克隆抗体孵育包被、封闭,加入SEQ ID No.2所示多肽的多肽-OVA偶联物进行免疫反应,清洗后加入生物素标记的单克隆抗体,清洗后加入SA-HRP孵育显色,450nm处读取OD值,挑选特异性强和灵敏度高的阳性配对。
2.一种用于β-CTX检测的试剂盒,其特征在于,包含如权利要求1所述β-CTX抗体制备方法制得的单克隆抗体。
3.如权利要求2所述的用于β-CTX检测的试剂盒,其特征在于,所述试剂盒为包括发光试剂、固相试剂、标准品以及质控品的化学发光免疫试剂盒。
4.如权利要求3所述的用于β-CTX检测的试剂盒,其特征在于,所述固相试剂为包被有β-CTX单克隆抗体的磁微粒混悬液,磁微粒浓度为0.05-0.5μg/ml。
5.如权利要求4所述的用于β-CTX检测的试剂盒,其特征在于,所述磁微粒为具有活性化学基团的超顺磁性纳米磁性微球,其表面活性基团为羧基、甲苯磺酰基、环氧基、氨基中的任意一种。
6.如权利要求3所述的用于β-CTX检测的试剂盒,其特征在于,所述发光试剂为标记有化学发光试剂的β-CTX单克隆抗体溶液,浓度为0.05-1.0μg/ml,所述发光试剂中的β-CTX抗体和所述固相试剂中的β-CTX抗体为两株针对不同抗原靶点的可进行配对的单克隆抗体。
7.如权利要求6所述的用于β-CTX检测的试剂盒,其特征在于,所述化学发光试剂包括吖啶酯、异鲁米诺、碱性磷酸酶、过氧化物酶、三联吡啶钌中任意一种。
8.如权利要求3所述的用于β-CTX检测的试剂盒,其特征在于,所述试剂盒的稀释液配方由缓冲盐,无机盐,表面活性剂,蛋白保护剂,化学保护剂,防腐剂组成,Ph为6-8,缓冲盐为HEPES、磷酸盐缓冲液,TRIS缓冲中的一种或多种;无机盐为氯化钠,氯化钾,氯化镁,硫酸锌中的一种或多种;表面活性剂为吐温120曲拉通405,NP-40和十二烷基硫酸钠中的一种或多种;蛋白保护剂为HSA、BSA,明胶,聚多卡醇,α环糊精中的一种或多种,化学保护剂为聚乙烯吡咯烷酮,Biolipidure,乙二醇,硫酸葡聚糖钠盐中的一种或多种,防腐剂为proclin300,庆大霉素,左氧氟沙星,苯氧乙醇中的一种或多种。
9.如权利要求3所述的用于β-CTX检测的试剂盒,其特征在于,所述校准品为由SEQ IDNo.2所示蛋白的二聚体多肽配制的浓度在0-2000pg/ml范围内的2个不同浓度点的液体校准品;所述质控品为由序列2蛋白表达得到的二聚体多肽配制的浓度在0-1000pg/ml范围内的2个不同浓度点的液体质控品。
10.一种如权利要求2-9任一所述的用于β-CTX检测的试剂盒的检测方法,其特征在于,通过双抗体夹心法原理进行检测,主要反应流程为仪器样本针自动吸取30μl血清样本于反应管中,试剂针分别吸取40μl固相试剂和60μl发光试剂于反应管中后,充分混合后孵育15分钟,分析仪自动转入清洗站进行3次重复清洗,最终转入测量池中,通过管路泵入预激发液和激发液,测量池中的PMT收集反应管中的产生的光信号并上传至软件中,通过仪器中输入的校准曲线给出最终的测量浓度。
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