CN117003869A - 抗il-10蛋白的单克隆抗体组合物及应用 - Google Patents
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Abstract
本发明公开了抗IL‑10蛋白的单克隆抗体组合物及应用，单克隆抗体组合物包括IL‑10‑Clone1和IL‑10‑Clone2，IL‑10‑Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示，IL‑10‑Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示；IL‑10‑Clone2的重链可变区氨基酸序列如SEQ ID NO.3所示，IL‑10‑Clone2的轻链可变区氨基酸序列如SEQ ID NO.4所示。制备方法：将亲和纯化后的IL‑10与弗氏佐剂进行等体积混合乳化，采集小鼠血液分离血清，筛选免疫较好的小鼠进行杂交瘤融合实验，以IL‑10作为酶标板包被抗原，取上清进行酶联免疫吸附测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆。同时也提供了上述抗体的化学发光诊断检测试剂盒，从而提高诊断试剂的特异性和灵敏度。
Description
技术领域
本发明属于抗体的制备及序列测定领域，具体涉及抗IL-10蛋白的单克隆抗体组合物及应用。
背景技术
白细胞介素-10（IL-10）是一种具有重要免疫调节功能的多效性细胞因子。IL-10是一种由两个非共价键结合的单体组成的同型二聚体分子，分子量约36Kd，每个单体由160个氨基酸组成，在单体之间存在两个二硫键连接。IL-10参与免疫系统中多种类型细胞的活动。IL-10主要由活化的T细胞、单核细胞、巨噬细胞、树突状细胞、自然杀伤细胞和B细胞分泌，T细胞是IL-10的主要来源。
IL-10 具有双向免疫调节作用，可以通过抗原提呈细胞（APC）发挥免疫抑制，通过T 细胞发挥负调节作用，在肿瘤环境中对免疫应答具有负向调节作用；另外，IL-10对T、B淋巴细胞具有刺激作用，并且在肿瘤环境中IL-10 也能发挥刺激作用；IL-10的双向调节作用自从被发现起就一直被关注，它不仅影响免疫系统，而且可以通过调节生长因子、细胞因子影响许多病理生理过程，包括血管生成、肿瘤形成和感染，还能通过诱导调节性T 细胞在外周耐受中建立作用；对克罗恩病、类风湿性关节炎、银屑病、HCV 感染、HIV 感染等，都发挥着重要作用。
IL-10参与免疫反应和炎症反应的调节。炎症因子免疫诊断用于帮助医生制定抗生素的使用范围，有利于抗菌药物的合理使用和感染疾病的快速筛查。炎症因子检测在医院层面的临床价值和使用量将不断提升，同时对诊断的时效性和准确度要求也随之增加。
开发性能优异的IL-10免疫诊断试剂的核心原料是抗IL-10单克隆抗体，一般厂商的特异性和灵敏度不高，所以开发开发性能优异的IL-10诊断试剂非常有必要，同时也期望能够进一步推动诊断试剂的国产化。
发明内容
为解决灵敏性、特异性的问题，本发明提供了抗IL-10蛋白的单克隆抗体组合物。该抗体性能优异、准确性高。同时提供了基于上述IL-10抗体的化学发光试剂盒。
本发明提供如下技术方案：
抗IL-10蛋白的单克隆抗体组合物，包括IL-10-Clone1和IL-10-Clone2，
IL-10-Clone1的重链可变区氨基酸序列：
DVQLVESGGDLVQPGGSLKFSCAASGFSFSKYGMSWVRQTPDKRLELVANINSNKASTYYPDRVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGSSDYQWYFDVWGAGTTVIVSS
IL-10-Clone1的轻链可变区氨基酸序列：
DIGGTQTPLSLPVSLGDQASISCRSSQSPVHQNGNTYLHWYLQKPDQSPKLLIYKKQNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSPHVPFTFGSGTKLEIKR
IL-10-Clone2的重链可变区氨基酸序列：
EVPLQQSGAELVRPGSSVKISCKASASTFSRYWMNWVKQRPGQGLEWIGQIFTDDGDTNYNGKFKGKATLTSDKSSSTAYMQLSSLTSEDSAVFFCGQRVNYYGSLYAMDYWGQGTSVIVSS
IL-10-Clone2的轻链可变区氨基酸序列：
DVQMNQSPKMMSTSIKDIVSITCKASQDVSTAVAWKQSKPGQSPKLLIYWAATRHTGVPDRFTGSGSGTDFTLTISSVQAEDLALYYCQQHYTEPWTFGGGTKLEIKR
抗IL-10蛋白的单克隆抗体组合物的制备方法：
IL-10蛋白的表达生产：将pTT5-IL10表达质粒与转染试剂PEI混匀后，转染HEK293细胞，同时在OPM-293 CD03 Medium细胞培养基中加入增强剂和辅料进行培养，当细胞活率<70%后，收集细胞培养物，离心并收集细胞上清。
IL-10蛋白的亲和纯化：细胞上清用盐和咪唑充分重悬过滤后，用预装重力柱进行亲和纯化，通过梯度咪唑浓度进行洗杂和洗脱，SDS-PAGE跑胶检测蛋白纯度，将蛋白透析至磷酸盐缓冲液中，0.22μm滤器无菌过滤后，BCA测定蛋白浓度。
将亲和纯化后IL-10蛋白与弗氏佐剂进行等体积混合乳化，免疫计量为100ug/只小鼠，3周后进行加强免疫，免疫计量为50ug/只小鼠，后期间隔2周每次，采集小鼠血液分离血清，酶联免疫吸附测定抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过HAT筛选培养基筛选培养后，以IL-10蛋白作为酶标板包被抗原，取上清进行酶联免疫吸附测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株，用无血清培养和发酵细胞株，收集培养上清。
抗IL-10蛋白的单克隆抗体组合物在制备炎症因子免疫诊断试剂中的应用。
单克隆抗体组合物用于IL-10化学发光试剂盒。试剂盒使用IL-10-Clone1和IL-10-Clone2分别作为包被和检测抗体。
与现有技术相比，本发明的有益效果是：
本发明提供了抗IL-10蛋白，用于制备诊断用单克隆抗体，本发明的抗IL-10蛋白的单克隆抗体组合物包括IL-10-Clone1和IL-10-Clone2，能够识别免疫原，此抗体组合具有特异性和高亲和力的特点，同时也提供了基于上述抗体的化学发光检测试剂盒，从而提高诊断试剂的特异性和灵敏度，炎症因子免疫诊断能够快速帮助医生制定抗生素的使用范围，在临床治疗手术后患者或门诊感染患者时，精准的检测结果具有指导用药的医学价值。
附图说明
图1为本发明检测试剂盒与同类产品相关性对比图。
实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 IL-10蛋白表达载体的构建
在NCBI官网上查找人IL-10氨基酸序列，在蛋白氨基酸的C端加上组氨酸标签，利用密码子优化软件在哺乳动物细胞表达系统中优化密码子，基因合成后利用EcoRI/BamHI限制性内切酶酶切位点，将基因克隆至pTT5表达载体中，构建pTT5-IL-10表达质粒，并测序验证基因序列。
实施例2 IL-10蛋白的表达生产
转染前一天，细胞以0.8×106/ml接种于细胞培养瓶，以OPM-293 CD03 Medium培养液培养，置于37℃，5%CO2培养箱中，至细胞融合度达到80%-90%，活力大于95%时进行转染，pTT5-IL-10表达载体与聚醚酰亚胺混匀后，室温孵育15-30min，缓慢加入到HEK293细胞中，加入增强剂和辅料，于 37℃，5%CO2培养箱中继续培养。当细胞活率<70%后，收集细胞培养物，8000 g离心10 min，收集上清液，再通过12000 g，4℃离心30 min，收集细胞上清。
实施例3 IL-10蛋白的亲和纯化
细胞上清中加入NaCl和咪唑，并将工作浓度分别调整至300 mM和5 mM后再用0.22μm滤膜过滤，Ni TED-6FF beads预装重力柱进行亲和纯化，20mM PB，300mM NaCl，10%甘油，50mM咪唑，pH8.0溶液进行洗杂，20mM PB，300mM NaCl，10%甘油，250mM咪唑，pH8.0溶液进行洗脱，SDS-PAGE跑胶检测蛋白纯度，将蛋白透析至20mM PB，300mM NaCl，10%甘油，pH8.0缓冲液中，0.22μm滤器无菌过滤后，BCA测定蛋白浓度。
实施例4 单克隆抗体的制备
将IL-10蛋白与弗氏佐剂进行等体积混合乳化，免疫计量为100ug/只小鼠，皮下多点注射，3周后进行加强免疫，免疫计量为50ug/只小鼠，后期间隔2周每次，采集小鼠血液分离血清，酶联免疫吸附法测定抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过10天左右的HAT筛选培养基筛选培养后，以IL-10蛋白作为酶标板包被抗原，取上清进行酶联免疫吸附法测定，筛选出优质阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株，用无血清培养和发酵细胞株，收集培养上清，进行抗体纯化，将纯化后的抗体进行诊断性能分析。
实施例5 单克隆抗体性能分析
基于双抗体夹心法和吖啶酯发光法原理制备试剂盒。
1、羧基磁珠包被抗体
将购买的羧基磁珠充分混匀，取出相应量的磁珠置于离心管，使用磁力架去除上清液，清洗后加入包被活化液，混匀后加入包被活化液溶解的EDC，充分混匀后室温颠倒活化0.5-1h；
超声分散磁珠，使用包被活化液清洗三次，加入本发明的包被抗体，充分混匀后室温颠倒反应1-2h；
超声分散磁珠，加入封闭剂，充分混匀后室温颠倒封闭1-2h；
超声分散磁珠，使用磁力架去除上清液，清洗三次，加入磁珠保存液将磁珠稀释至浓度为0.25mg/mL待用。
上述磁珠保存液配方为50mM Tris、0.2% Tween-20、0.85% NaCl、2% BSA、0.2%Proclin-300。
2、吖啶酯标记抗体
将NHS酯化的吖啶酯（NSP-DMAE-NHS）与本发明的包被抗体按照分子摩尔比10：1的比例在1×PBS中颠倒混匀标记反应1-2h；
反应结束后将标记液转移至14kD透析袋中，1×PBS中旋转透析，换液6次，每次1-2h；
透析过夜后取出标记液，使用分光光度计测试标记液中蛋白含量，计算并添加保护液，充分混匀后可置于18-20℃保存，上机测试时使用标记保存液将蛋白浓度稀释至0.5ug/mL待用。
上述标记保存液的配方为0.1M MES缓冲液、0.2% Tween-20、0.85% NaCl、2% BSA、0.2% Proclin-300。
3、配制校准品和质控品
使用抗原稀释液将IL-10抗原分别稀释至0、1、2、10、20、100、200、1000 pg/mL，分装至校准品管中。
使用抗原稀释液将IL-10抗原稀分别释液至6pg/mL、100 pg/mL，分装至质控品管中。
上述抗原稀释液配方为1×PBS、1% 牛血清白蛋白。
检测试剂盒与同类产品对比：
实施例中的IL-10化学发光检测试剂盒与罗氏公司的产品在120例临床标本上进行了相关性对比，数据如图1所示。相关系数R2为0.9864。说明本发明的试剂盒能准确检测IL-10含量，为临床诊断提供依据，能够充分满足临床体外诊断检测需求。
检测试剂盒灵敏度和精密度考察：
按照临床和实验室标准协会灵敏度验证分析指南文件CLSI:EP17-A2对本发明中的试剂盒的空白限和检测限进行了研究，结果表明空白限和检测限分别为0.5 pg/mL和1pg/mL。
按照临床和实验室标准协会精密度验证分析指南文件CLSI:EP5-A3对本发明的试剂盒进行精密度研究，使用3个批号的试剂和校准品，对2个水平的质控品进行了检测，每天上下午各检测两次，连续考察5天，结果表明各批次室内总精密度小于5%。
检测试剂盒抗干扰能力分析：
在IL-10浓度约为6 pg/mL和100 pg/mL浓度水平的两个血清样本中分别添加不同浓度的干扰物，以未添加组为对照，添加组和对照组两者相对偏差小于5%为干扰可接受标准，研究不同干扰物的最大浓度水平（不超过临床可见最高浓度）。
表1 检测试剂盒抗干扰能力分析数据
结果表明表1中的浓度时对检测结果干扰不明显。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.抗IL-10蛋白的单克隆抗体组合物，其特征在于：包括IL-10-Clone1和IL-10-Clone2，
所述IL-10-Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示，IL-10-Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示；
所述IL-10-Clone2的重链可变区氨基酸序列如SEQ ID NO.3所示，IL-10-Clone2的轻链可变区氨基酸序列如SEQ ID NO.4所示。
2.权利要求1所述的抗IL-10蛋白的单克隆抗体组合物在制备炎症因子免疫诊断试剂中的应用。
3.根据权利要求2所述的抗IL-10蛋白的单克隆抗体组合物在制备炎症因子免疫诊断试剂中的应用，其特征在于：单克隆抗体组合物用于IL-10化学发光试剂盒。
4. IL-10化学发光试剂盒，其特征在于：含有权利要求1中的IL-10-Clone1和IL-10-Clone2。
5.根据权利要求4所述的IL-10化学发光试剂盒，其特征在于：试剂盒使用IL-10-Clone2和IL-10-Clone1分别作为包被抗体和检测抗体。
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