CN114874329A - 一种补体活化C1s酶荧光检测试剂盒及检测方法与应用 - Google Patents
一种补体活化C1s酶荧光检测试剂盒及检测方法与应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种补体活化C1s酶荧光检测试剂盒及检测方法与应用。本发明以检测样本中补体活化C1s的酶活性水平为目的，建立一种补体活化C1s酶荧光检测试剂盒及检测方法与应用。本发明基于重组C1s抗体预包被磁珠，对C1s标准品或待测样品中的C1s进行特异性捕获，磁吸分离，去除非特异性结合物后，加入FRET荧光物标记的活化C1s酶切底物肽，然后用酶标仪检测反应体系中的荧光信号；通过检测荧光强度变化，对活化C1s酶活性进行量化检测。具有灵敏度高、特异性强、误差小、操作简单方便等特点。可对人或动物血液及体液中C1s活化后的酶活性进行检测，以准确评估机体的补体经典活化状态。
Description
技术领域
本发明属于生物技术领域，具体涉及一种补体活化C1s酶荧光检测试剂盒及检测方法与应用。
背景技术
补体是一类天然的免疫分子，由多种补体分子及其调节分子组成，在免疫应答中起重要作用。正常生理状态下，补体分子多处于非活性状态；当免疫应答激活后，补体分子经活化后才表现出各种生物学活性。从补体分子C1（简称C1）开始的补体激活途径称为补体活化经典途径。C1是由C1q、C1r和C1s三种亚单位在Ca2+作用下按1:2:2组成的五聚体大分子复合物（C1qC1r2C1s2）。其中，C1q有6个相同的免疫球蛋白（Ig）结合点。当其中两个及以上结合点与免疫复合物中的IgM或IgG的Fc段补体结合位结合后，C1q构型发生改变，并继而引起亚单位C1r的活化。活化的C1r进一步使得C1s活化。而活化的C1s具有酶活性，可裂解补体C4和C2，最终使后续活化的补体组份在靶细胞膜上形成一个跨膜的孔道（即攻膜复合物）。C1s的活化与感染、肿瘤、冷凝集素病、血小板减少性紫癜、系统性红斑狼疮等疾病的发生、发展和预后相关。
近年来，针对C1s为靶点开发的治疗性药物已开始进入临床试验，所涉疾病包括冷凝集素病（CAD）、温抗体型自身免疫性溶血性贫血（WAIHA）、大疱性类天疱疮（BP）、以及抗体介导的移植排斥反应（AMR）。正常生理条件下的C1s呈酶原状态存在，只有当该C1s酶原经活化，成为活化C1s后才能发挥其生物活性效应。由此可见，只有准确检测样本中的活化C1s才能正确反映补体经典途径的活化及活化C1s的功能状态。因此，检测体内C1s活性的变化对了解疾病的发生机制、疾病状态及临床疾病的个体化治疗有着重要的意义。目前，C1s的检测方法包括双向扩散试验、ELISA、明胶酶谱技术等，但这些方法仅能检测临床血液样本中C1s的总蛋白含量，不能有效区分C1s的活化或非活化状态，即不能客观反映C1s的酶活性。因此，有必要建立灵敏、特异的活化C1s酶活性检测方法，以满足临床上对样本中活化C1s的检测需求。
发明内容
有鉴于此，本发明提供了一种补体活化C1s酶荧光检测试剂盒及其对补体活化C1s的检测方法。所提供的试剂盒可以应用于人和动物血液、体液（胸水、腹水、脑脊液等）中补体活化C1s的检测，以评估体内补体活化状态，满足临床疾病诊断、个体化治疗和预后评估、疾病机制研究等应用中对补体活性检测的需求。
为实现上述目的，本发明采用如下技术方案：
本发明提供了一种重组C1s特异性抗体，所述特异性抗体重链可变区的氨基酸序列如SEQ ID NO：1所示，所述特异性抗体的轻链可变区的氨基酸序列如SEQ ID NO：2所示。
本发明还提供了一种免疫磁珠，所述磁珠与上述重组C1s特异性抗体偶联。
本发明还提供了一种补体活化C1s的酶荧光检测试剂盒，所述试剂盒包括上述免疫磁珠；所述试剂盒还包括有C1s标准品、底物肽、磷酸缓冲液和Tris缓冲液。
进一步地，所述底物肽的FRET（荧光能量共转移）荧光物标记的完整序列为2Abz-GYLGRSYKVG-Lys(Dnp)D-OH；所述底物肽的氨基酸序列来自于噬菌体肽库。
所述底物肽的N端连接有Abz（荧光基团R）、C端连接有Dnp（荧光淬灭基团Q ）。
所述的C1s标准品为人源或重组的C1s标准品。
所述的磷酸缓冲液（PBS，pH7.4），其包括如下组份：0.137M NaCl，0.0027M KCl，0.01 M Na2HPO4，0.002 M KH2PO4。
所述的Tris缓冲液，其包括如下组份：0.05M Tris，0.15M NaCl，0.2%(w/v)聚乙二醇8000。
进一步地，本发明还提供了一种用于补体活化C1s的酶荧光检测方法，所述的检测方法包括步骤有：
（1）重组C1s抗体偶联免疫磁珠的制备；
（2）将待检测样本或标准品与重组C1s抗体偶联免疫磁珠混合得到重悬免疫磁珠；
（3） 重悬免疫磁珠加入底物肽进行酶切反应；
（4）用酶标仪检测上清中荧光信号；
（5）以浓度为横坐标，以反应速度为纵坐标，用双对数Log-Log数学模型最小二乘法进行直线拟合制作标准曲线。根据标准品的浓度，计算得到待检测样本中活化C1s浓度。
进一步地， 步骤（3）中，所述底物肽的N端连接有Abz、C端连接有Dnp；所述底物肽的序列为2Abz-YVGRSYRG-Lys(Dnp)-NH2。
本发明还提供了上述底物肽在制备用于检测C1s活化状态的试剂盒中的用途。
本发明还提供了一种重组C1s特异性抗体在制备用于检测C1s活性状态的试剂盒中的用途。
本发明还提供了一种免疫磁珠在制备用于检测C1s活化状态的试剂盒中的用途。
与现有技术相比，本发明的有益效果是：
本发明以检测样本中活化C1s的酶活性水平为目的，基于重组C1s抗体预包被免疫磁珠，对C1s标准品或待测样品中的C1s进行特异捕获，磁吸分离，去除非特异性结合物后，加入荧光物标记的活化C1s酶切底物肽，然后用酶标仪检测反应体系中的荧光信号；通过检测荧光强度变化，对待测样本中活化C1s酶活性进行量化检测。具有灵敏度高、特异性强、误差小、操作简单方便等特点。可对人或动物血液及体液中C1s的酶活性进行检测，准确评估机体的补体经典活化状态。
附图说明
图1是活化C1s酶荧光检测试剂盒的方法原理图；
图2是重组C1s抗体的SDS-PAGE电泳图；
图3是重组C1s抗体的SEC-HPLC图；
图4是重组C1s抗体特异性检测原理图；
图5是重组C1s抗体与检测抗原的结合特异性对比图；
图6是不同浓度的C1s标准品对底物肽作用图；图中，A是活化C1s标准品对底物肽作用的酶反应动力学；B是活化C1s浓度与反应速度相关性关系；
图7是不同C1s标准品浓度与荧光强度值的标准曲线图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。下列实施例中未注明具体条件的实验方法，按照本领域常规方法和条件，或按照商品说明书选择。下述实施例中未注明具体成分的试剂和原料均市售可得。
本发明所述血清样本来自于泰州市人民医院；所述干扰检查A试剂盒来自于希森美康医用电子（上海）有限公司，所述SDS-PAGE还原胶、非还原胶均来自于BBI生命科学有限公司，所述耦合剂为1-乙基-3-[3-二甲氨基丙基]碳化二亚胺盐酸盐（EDC）来自于Diamond，所述磁珠来自于默克制药（江苏）有限公司，所述C1r蛋白、双链C1r、未活化C1s蛋白、双链活化C1s蛋白均来自于Complement Technology，所述MASP1、MASP2均来自于Abnova。
本发明提供一种重组C1s特异性抗体以及荧光共振能量转移（FRET)（Abz/Dnp染料组合）标记的底物肽，并基于此，提供一种补体活化C1s酶荧光检测试剂盒及检测方法与应用。将重组抗C1s抗体包被于磁珠，制备成免疫磁珠；洗涤、磁吸去上清后，加入C1s标准品或待测样本；免疫磁珠结合的抗C1s抗体可捕获液相中的C1s。经洗涤、磁吸分离，去除没有结合的其它组份后，再加入底物肽。活化C1s是补体经典活化途径启动后的重要酶活性分子，补体系统中的活化C1s具有丝氨酸蛋白酶活性，能特异性地切割补体C4和C2蛋白。选择C4和C2蛋白的切割位点作为酶切底物肽的氨基酸序列中心点，在该中心氨基酸序列的左右各选择增加若干个氨基酸，制备成基本的酶切底物肽。继而，在此肽的左、右两边分别连接Abz（荧光基团R）和Dnp（荧光淬灭基团Q），制成酶切荧光底物肽。当待测体系中没有活性C1s时，底物肽完整，底物肽标记的R基团与Q基团间距离在10–100埃（Å），Q基团可淬灭R基团发出的荧光，待测体系里检测不到荧光信号。当待测体系中有活性C1s时，该底物肽被活化C1s酶切，底物肽上Q基团和R基团分别位于不同酶切片段上，Q基团不再被R基团淬灭，Q基团可发出可检测荧光信号，其荧光强度与活化C1s的量相关。
因此，可通过检测荧光强度变化，对待测样本中活化C1s酶活性进行量化检测。同时，考虑到血清、血浆中其它蛋白（如MASP2）可能对酶切底物肽的干扰，本发明采用重组C1s抗体偶联磁珠（免疫磁珠）对C1s进行特异性捕获，然后磁吸洗脱非接合的其它干扰物，再加入特异性活化C1s酶切底物肽，通过检测活化C1s酶切底物肽荧光强度的变化计算活化C1s的活性。
图1是活化C1s酶荧光检测的方法原理图；由图1可见，用免疫磁珠对加入的C1s标准品或待测样品中的C1s进行特异捕获，磁吸分离，去除非特异性结合物后，加入FRET荧光物标记的活化C1s酶切底物肽，进行酶切反应，然后用酶标仪检测反应体系中的荧光信号。
实施例1：C1s抗体的重组表达和鉴定
根据如SEQ ID NO：1所示的重组C1s抗体重链氨基酸序列，即：EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATISSGGSHTYYLDSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCARLFTGYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；如SEQ ID NO：2所示的所述重组C1s抗体轻链氨基酸序列，即：QIVLTQSPATLSLSPGERATMSCTASSSVSSSYLHWYQQKPGKAPKLWIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCHQYYRLPPITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。委托泰州百英生物技术有限公司采用HEK293T系统重组表达C1s抗体。并分别采用聚丙烯酰胺凝胶电泳（SDS-PAGE）和分子排阻色谱-高效液相色谱法（SEC-HPLC）分析、鉴定制备的重组C1s抗体。图2是重组C1s抗体的SDS-PAGE电泳图；如图2所示，重组C1s抗体在SDS-PAGE还原胶上可检测出两条带，其分子量分别在49KD和23KD处；而在非还原胶上仅为一条144KD条带。图3是重组C1s抗体的SEC-HPLC图；如图3所示， SEC-HPLC结果显示得到的重组C1s抗体的纯度为95.5%。
实施例2：重组C1s抗体的抗原结合特异性分析
图4是重组C1s抗体特异性检测原理图。在本实施例中，用ELISA间接法根据图4所示的重组C1s抗体特异性检测原理图进行重组C1s抗体的抗原结合特异性分析。具体包括如下步骤：
向酶标板各孔中分别加入200μL（1μg/ml）检测抗原（单链未活化C1s蛋白、双链活化C1s蛋白或C1r蛋白），轻轻振荡酶标板，使抗体覆盖酶标板孔底。4℃过夜孵育；PBS温缓冲液洗板3次后，加入200μL 封闭液（10mM PBS，1% BSA，0.1%酪蛋白）进行封闭；37℃孵育1h；PBS缓冲液冲洗板5次；加100μL 重组C1s抗体（第一个孔的初始浓度是10μg/mL。然后按1/4梯度的比例往下稀释7个孔，设置复孔）；37℃作用1h；将酶标板内的液体甩干，200μL PBS温缓冲液冲洗板5次；向酶标板各孔中加入100μL 稀释后的二抗（Goat anti Human IgG-HRP)；37℃下作用30min；将酶标板内的液体甩干，200μL PBS温缓冲液冲洗板5次；每孔加入100μL 显色液（TMB），37℃孵育10min之后，加入50μL 终止液（0.25 M HCl）终止显色反应；酶标仪450nm处读取光密度值。
分别对重组C1s抗体与单链未活化C1s蛋白、双链活化C1s蛋白、C1r蛋白的结合状况进行分析。图5是重组C1s抗体与检测抗原的结合特异性对比图；由图5所示，重组C1s抗体与双链活化的C1s蛋白有较强的结合能力，与单链未活化的C1s蛋白结合能力较弱，而与C1r蛋白无结合。可见，重组C1s抗体对活化的C1s蛋白有较好的抗原结合特异性，可以特异性识别活化C1s，实现对C1s进行酶活性状态进行检测的目的。
实施例3：底物肽的筛选
因补体系统中的活化C1s具有丝氨酸蛋白酶活性，能特异性地切割补体C4和C2蛋白。本发明选择C4和C2蛋白的切割位点作为酶切底物肽的氨基酸序列中心点，在中心氨基酸序列的左右各选择增加若干个氨基酸，制备成基本的酶切底物肽。继而，在此肽的左、右两边分别连接Abz（荧光基团R）和Dnp（荧光淬灭基团Q），制成酶切荧光底物肽。预设计三种底物肽，均委托上海科肽生物科技有限公司合成。三种底物肽的序列如下：
底物肽1：2Abz-GLQRALEI-Lys(Dnp)-NH2；
底物肽2：2Abz-SLGRKIQ-Lys(Dnp)-NH2；
底物肽3：2Abz-GYLGRSYKVG-Lys(Dnp)D-OH； 分别对上述三种底物肽的被活化C1s酶切的Km值和Kcat值进行测定，三次重复取平均值，测定步骤包括：
（1）第一孔中加入40μL底物肽标准品；其他孔中分别加入同体积的用Tris缓冲液进行倍比稀释成不同终浓度（245.7μM，122.9μM，61.4μM，30.7μM，7.7μM，0μM）的底物肽溶液；
（2）每孔中加入双链活化C1s 2.5μL（1mg/mL），补充Tris缓冲液至100μL。
（3）酶标仪于360/40激发光波，460/40发射光波检测，每5min检测一次，共检测2h，记录荧光信号变化。
（4）采用Graphpad prism 5.0进行数据处理，得出Vmax、Km值。根据公式Kcat=Vmax/分子量。计算Km值和Kcat值，以Kcat/Km值最高的作为候选底物肽。表1是活化C1s对不同底物肽作用的Kcat/Km值。
表1.活化C1s对不同底物肽作用的Kcat/Km比较
| 底物 | Kcat/Km |
| 底物肽1 | 1.67±0.89 |
| 底物肽2 | 1.45±0.61 |
| 底物肽3 | 15.38±2.69 |
结果如表1所示，底物肽1 vs 3：t=12.461，p<0.001；底物肽2 vs 3：t=12.664，p<0.001。底物肽3效果最佳，Kcat/Km值为15.38，故选择底物肽3作为活化C1s的底物肽。
当待测体系中没有活性C1s时，底物肽完整，底物肽标记的R基团与Q基团间距离在10–100埃（Å），Q基团可淬灭R基团发出的荧光，待测体系里检测不到荧光信号。当待测体系中有活性C1s时，该底物肽被活化C1s酶切，底物肽上Q基团和R基团分别位于不同酶切片段上，Q基团不再被R基团淬灭，Q基团可发出可检测荧光信号，其荧光强度与活化C1s的量相关。图6是不同浓度的C1s标准品对底物肽作用图；图中，A是活化C1s标准品对底物肽作用的酶反应动力学；B是活化C1s浓度与反应速度相关性关系；由图6可见，随着时间的变化，活化C1s对底物肽作用产生的荧光强度不断变化，而且不同浓度的活化C1s具有较好的分辨率。
实施例4：活化C1s酶荧光法检测的方法学建立及最佳反应时间
1、配制缓冲液：① Binging Buffer：0.1M吗啉乙磺酸(MES，PH5.0)；
② Washing Buffer：含0.05% Tween20的 TBS；
③ Blocking Buffer：含0.05M Tris ，1.0%的BSA，0.05% Tween20；
④ Desired Buffer：含0.05% Tween20和BSA的Tris缓冲液；
⑤ Tris缓冲液：0.05M Tris，0.15M NaCl，0.2% (w/v) 聚乙二醇8000。
2、免疫磁珠（重组C1s抗体偶联磁珠）的制备：
（1）取10mg 磁珠（Merck羧基磁珠，粒径1μm）用1mL Binging Buffer洗3次，涡旋振荡混匀。
（2）每1mg 磁珠添加20μg 实施例1得到的重组C1s抗体，混匀，室温（25℃）涡旋15min。
（3）每1mg 磁珠添加50μg 耦合剂，混匀，室温涡旋1h。
（4）加入1mL Binging Buffer，室温，涡旋3h。
（5）使用0.5mL Washing Buffer洗4次。
（6）用1mL Desired Buffer重悬磁珠，得到浓度为5mg/mL的重组C1s抗体偶联磁珠；4℃保存待用。
3、活化C1s的检测：
（1）取60μL重组C1s抗体偶联磁珠搅匀，磁吸去除上清，加入200μL PBS 缓冲液混匀后，再次磁吸去除上清，重复洗涤2次。
（2）用 PBS 缓冲液将10μL 的待测样品或不同浓度（200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、0μg/mL）的活化C1s标准品（稀释至100μL；与重组C1s抗体偶联磁珠混匀，室温垂直混匀10 min。磁吸，舍弃上清。
（3） 用100μL PBS 缓冲液重复洗涤3次；
（4）用90μL Tris缓冲液重悬磁珠；
（5）加入10μL 浓度为1μg/mL的底物肽3；
（6）用酶标仪于360/40激发光波，460/40发射光波进行检测，每5min检测一次，检测至2h，测定并记录荧光信号变化；以浓度为横坐标，反应速度为纵坐标，用双对数Log-Log数学模型最小二乘法进行直线拟合制作标准曲线。
以不同反应时间作终点，选择标准曲线中线性最好、误差最小的反应时间。图7是不同C1s标准品浓度与荧光强度值的标准曲线图。如图7所示，反应40min后具有比较好的荧光强度，60 min时荧光强度趋于最佳，确定最佳反应时间为60min，标准曲线的R2=0.9985。每个定标品的CV值均小于10%，且在剂量－反应曲线范围内未见hook效应。
实施例5：灵敏度实验
在本实施例中对活性C1s酶荧光检测方法进行灵敏度实验。参考实施例4步骤3中的实验步骤测定活化C1s的浓度，选择底物肽3作为反应底物，重复测定零值定标品（A）20次，计算其VA的平均值（M）和标准差（SD）。将VA的均值加上二倍标准差即M+2SD减去本底值后代入剂量-反应曲线，得到的浓度值为本方法测定活化C1s的灵敏度。经计算，最低检出限为0.81μg/mL。
实施例6：回收率实验
在本实施例中进行活性C1s酶荧光检测方法的回收率实验。
选择3份泰州市人民医院的血清样本，以底物肽3作为反应底物，参考实施例4步骤3中的实验步骤测定血清样本中活化C1s的浓度；经测定血清样本中活化C1s的浓度分别为36.8μg/mL、37.2μg/mL、4.6μg/mL。在血浆样本中添加50μg/mL、50μg/mL、100μg/mL浓度的活化C1s（按照1：1体积比），再次进行检测。每个样品平行测量10次，记录并计算其实测值，根据公式回收率=（加入标品后含量－样品含量）/加入标品量×100%，计算回收率。
结果显示，该检测方法的回收率分别为91.21%、102.75%、90.35%。可见，回收率结果在90%～110%范围内，不同浓度下的活化C1s回收率结果准确。
实施例7：精密度实验
参照美国临床实验室标准化委员会（NCCLS）的EP-5文件，从泰州市人民医院收集的血清样本中，选取高、中、低3个样品进行检测，以评价检测方法的精密度，实验数据见表2。
表2.活化C1s酶荧光检测法的批内和批间精密度试验
注：M—平均数；SD—标准差；CV—变异系数
由表2可见，高、中、低3个样品的批内CV值分别为6.68%、6.08%、8.86%。批间CV值分别为8.47%、9.36%、9.53%。其批内和批间变异系数均小于10.0%。可见，本发明所提供的检测方法具有良好的精密度，符合NCCLS的EP-5文件规定要求。
实施例8：交叉反应实验
在本实施例中对活化C1s酶的特异性进行实验。对血清中可能存在的干扰性的抗原物质（补体类似物）如双链C1r、MASP1、MASP2进行检测。使用PBS缓冲液分别对双链C1r、MASP1、MASP2进行稀释，稀释后的浓度依次为0.66 mg/mL、5 mg/mL、5 mg/mL；参考实施例4步骤3中的实验步骤检测各补体类似物。计算各种物质的交叉反应率。交叉反应率=实测值／理论值×100%。表3是活化C1s酶荧光检测法的交叉反应试验数据。如表3所示，双链C1r、MASP1、MASP2交叉反应率分别为0.3%、0.3%、0.04%。交叉反应率均小于0.5%。可见，与双链C1r、MASP1、MASP2的交叉反应较小，能特异性识别血清样本中的活化C1s，具有较高的检测特异性。
表3.活化C1s酶荧光检测法的交叉反应试验
| 干扰物 | 测得浓度 | 实际浓度 | 交叉反应率(%) |
| 双链C1r | 0.001 mg/mL | 0.66 mg/mL | 0.15 |
| MASP1 | 0.002 mg /mL | 5 mg/mL | 0.04 |
| MASP2 | 0.018 mg /mL | 5 mg/mL | 0. 36 |
实施例9：抗干扰实验
考虑临床样本中的胆红素、乳糜、血红蛋白对实验可能存在有潜在的干扰。参照干扰检查A试剂盒，在高、中、低浓度的标准品中分别加入不同浓度的胆红素（终浓度分别为0、0.06、0.14、0. 20 mg/mL）、乳糜（终浓度分别为0、600、1400、2000 FTU）、血红蛋白溶液（终浓度分别为0、1.5、3.5、5 mg/mL）。将所测含不同干扰物的高、中、低值平均浓度与不含干扰物的相应的平均浓度相减，求出差值及差值占不含干扰物的空白平均浓度的百分比。小于5%为该干扰物相应的浓度对检测无干扰，大于5%为对检测有干扰。
表4.活化C1s酶荧光检测法的胆红素干扰实验（n=4）
| 0 mg/mL | 0.06 mg/mL | 0.14 mg/mL | 0.20 mg/mL | |
| 低值（mg/mL） | 6.04±0.35 | 6.33±0.35 | 6.26±0.27 | 6.17±0.46 |
| 中值（mg/mL） | 32.73±3.10 | 34.11±2.55 | 33.85±1.44 | 31.59±2.30 |
| 高值（mg/mL） | 62.19±2.91 | 62.65±5.04 | 60.99±4.95 | 61.38±3.46 |
表5. 活化C1s酶荧光检测法的乳糜干扰实验（n=4）
| 0 FTU | 600 FTU | 1400 FTU | 2000 FTU | |
| 低值（mg/mL） | 6.35±0.63 | 6.45±0.33 | 6.24±0.75 | 6.08±1.04 |
| 中值（mg/mL） | 35.38±2.44 | 36.36±3.58 | 34.56±1.74 | 37.10±3.19 |
| 高值（mg/mL） | 62.16±2.31 | 61.4.9±2.53 | 59.56±5.93 | 60.83±6.05 |
注：FTU，福尔马肼浊度
表6.活化C1s酶荧光检测法的血红蛋白干扰实验（n=4）
| 0 mg/mL | 1.5 mg/mL | 3.5 mg/mL | 5 mg/mL | |
| 低值（mg/mL） | 6.86±0.47 | 6.59±0.65 | 6.73±0.62 | 7.00±0.56 |
| 中值（mg/mL） | 33.69±3.19 | 32.39±1.26 | 32.94±2.95 | 31.99±1.59 |
| 高值（mg/mL） | 63.76±4.50 | 66.56±4.02 | 61.79±5.03 | 65.38±5.64 |
如表4、5、6所示，在高浓度胆红素（0.20 mg/mL）、高乳糜（2000 FTU）、高浓度血红蛋白（4 mg/mL）存在的情况下，检测结果均在测量偏差允许范围内（结果以CV低于10％为标准）。结果表明，本发明所提供的检测方法不受胆红素（0.20 mg/mL）、乳糜混合物（2000FTU）、血红蛋白（5 mg/mL）的干扰。以上抗干扰实验所测得的数据均符合要求。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
序列表
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Claims (10)
1.一种重组C1s特异性抗体，其特征在于，所述特异性抗体的重链可变区的氨基酸序列如SEQ ID NO：1所示，所述特异性抗体的轻链可变区的氨基酸序列如SEQ ID NO：2所示。
2.一种免疫磁珠，其特征在于，所述磁珠与权利要求1所述重组C1s特异性抗体偶联。
3.一种补体活化C1s的酶荧光检测试剂盒，其特征在于，所述试剂盒包括权利要求2所述免疫磁珠，所述试剂盒还包括有C1s标准品、底物肽、磷酸缓冲液和Tris缓冲液。
4.根据权利要求3所述的试剂盒，其特征在于，所述底物肽的FRET荧光物标记完整序列为2Abz-GYLGRSYKVG-Lys(Dnp)D-OH；所述底物肽的氨基酸序列来自于噬菌体肽库。
5.根据权利要求3所述的试剂盒，其特征在于，所述底物肽的N端连接有荧光基团R、C端连接有荧光淬灭基团Q；所述的C1s标准品为人源或重组的C1s标准品。
6.根据权利要求3所述的试剂盒，其特征在于，所述的磷酸缓冲液包括如下组份：0.137M NaCl，0.0027M KCl，0.01 M Na2HPO4，0.002 M KH2PO4；所述的Tris缓冲液包括如下组份：0.05M Tris，0.15M NaCl，0.2%聚乙二醇8000。
7.一种补体活化C1s的酶荧光检测方法，其特征在于，所述的检测方法包括步骤有：
（1）重组C1s抗体偶联免疫磁珠的制备；
（2）将待检测样本或标准品与重组C1s抗体偶联免疫磁珠混合得到重悬免疫磁珠；
（3） 重悬免疫磁珠加入底物肽进行酶切反应，底物肽的序列为2Abz-GYLGRSYKVG-Lys(Dnp)D-OH；
（4）用酶标仪检测上清中荧光信号；
（5）以浓度为横坐标，反应速度为纵坐标，用双对数Log-Log数学模型最小二乘法进行直线拟合制作标准曲线，根据标准品浓度，计算得到待检测样本中活化C1s浓度。
8.权利要求1所述的重组C1s特异性抗体在制备用于检测C1s活化状态的试剂盒中的用途。
9.权利要求2所述的免疫磁珠在制备用于检测C1s活化状态的试剂盒中的用途。
10.权利要求7所述的底物肽在制备用于检测C1s活化状态的试剂盒中的用途。
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