CN117430701A - 抗人钙卫蛋白的单克隆抗体组合物及应用 - Google Patents
抗人钙卫蛋白的单克隆抗体组合物及应用 Download PDFInfo
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Abstract
本发明公开了抗人钙卫蛋白的单克隆抗体组合物及应用,单克隆抗体组合物包括1号克隆和2号克隆,1号克隆的重链可变区氨基酸序列如SEQ ID NO.1所示,轻链可变区氨基酸序列如SEQ ID NO.2所示;2号克隆的重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区氨基酸序列如SEQ ID NO.4所示。制备方法:将亲和纯化后的人钙卫蛋白S100A8/A9复合物与弗氏佐剂进行等体积混合乳化,筛选免疫较好的小鼠进行杂交瘤融合实验,利用亲和纯化后的人钙卫蛋白S100A8亚基、S100A9亚基和S100A8/A9复合物筛选特异性针对人钙卫蛋白S100A8亚基、S100A9亚基和S100A8/A9复合物的抗体,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。同时提供了钙卫蛋白化学发光测定试剂盒,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,具体涉及抗人钙卫蛋白的单克隆抗体组合物及应用。
背景技术
钙卫蛋白,又称MRP8-MRP14、卡尔钙调蛋白A和B、囊性纤维化抗原等,是哺乳动物蛋白S100A8/A9的复合物,属于S100家族,广泛分布于人体细胞,组织和体液中。钙卫蛋白以S100A8/A9异二聚体或异四聚体的形式存在,具有抗微生物、促炎症和促血栓形成的特性,人钙卫蛋白分子量大小约为24 kDa,由蛋白单体 S100A8(10,835 Da)和 S100A9(13,242Da)组成。在钙离子存在下,钙卫蛋白能够通过螯合作用结合过渡金属铁、锰和锌,这种金属螯合赋予钙卫蛋白抗微生物特性。
钙卫蛋白检测的临床意义:
1. 血清学钙卫蛋白检测:
血清钙卫蛋白的检测在临床上相对较少,通常用于评估全身性炎症状态,尤其是与风湿性疾病、全身性感染和白血病等相关的情况。血清钙卫蛋白水平的升高可能暗示全身性炎症的存在,但对于评估肠道炎症活动程度不如粪便检测的钙卫蛋白直接、准确。
2. 评估肠道炎症和炎症性肠病(IBD)的活动程度:
钙卫蛋白主要用于评估肠道炎症的程度,如溃疡性结肠炎和克罗恩病。炎症活动时,肠道黏膜受损,导致钙卫蛋白释放增加。检测粪便中的钙卫蛋白能够客观反映肠道黏膜的炎症程度,为评估炎症性肠病的活动程度提供依据。
3 鉴别炎症性肠病和非炎症性肠疾病:
钙卫蛋白检测有助于鉴别肠道炎症性疾病(如炎症性肠病)和非炎症性肠疾病,如功能性胃肠疾病(如肠易激综合征),以确保正确的诊断和治疗方案。
钙卫蛋白的粪便检测具有非侵入性、敏感性高、特异性强和操作简便等优点,已成为评估肠道炎症的重要工具,对临床医生制定个性化治疗方案、监测疾病进展和预后评估具有重要帮助。
综合来说,血清钙卫蛋白和粪便钙卫蛋白检测在临床上具有广泛的应用价值,能够提供重要的辅助诊断、治疗监测和预后评估信息,有助于医生制定更合理的治疗方案,改善患者的生存率和生活质量。
开发性能优异的钙卫蛋白免疫诊断试剂的核心原料是一株特异性识别鼠抗人钙卫蛋白S100A8/A9 复合物构象表位的单克隆抗体和一株特异性识别人钙卫蛋白S100A8亚基或者S100A9亚基的抗体,单克隆抗体以人钙卫蛋白S100A8/A9 复合物为免疫原,人钙卫蛋白S100A8亚基和S100A9亚基作为筛选原,筛选高亲和力的单克隆抗体同时,还需要确保特异性识别鼠抗人钙卫蛋白S100A8/A9 复合物的单克隆抗体不识别单独的人钙卫蛋白S100A8亚基和S100A9亚基,从而提高诊断试剂的特异性和灵敏度,对于钙卫蛋白临床诊断具有很大的临床意义。市面上鼠抗钙卫蛋白单克隆抗体质量良莠不齐,无法兼顾高亲和力和特异性。
发明内容
为解决亲和力和特异性的问题,本发明提供了抗人钙卫蛋白的单克隆抗体组合物。具有高亲和力的单克隆抗体序列,且抗体性能优异、准确性高。同时提供了基于上述钙卫蛋白化学发光测定试剂盒。
本发明提供如下技术方案:
抗人钙卫蛋白的单克隆抗体组合物,包括1号克隆和2号克隆,
1号克隆的重链可变区氨基酸序列:
EVQLQQSGPELVKPGASVEMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNYDTKYNEKFKGKASLTSDKSSSTAYMELSSLTSEDSAVYYCTRGGDFDYWGQGTTLTVSS
1号克隆的轻链可变区氨基酸序列:
QAVVIQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGSNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCGLLYSNNWVFGGGTKLTVL
2号克隆的重链可变区氨基酸序列:
EVQLQQSGPELVKPGASVKMSCKASGYTFTLYVIHWVKQKPGQGLEWIGYINPYIDGTKYNEKFKGKATLTSDKSSSTAFMELSSLTSEDSAVYYCARSGYGNYGLAWLAYWGQGTLVTVSA
2号克隆的轻链可变区氨基酸序列:
QIVLTQSPAIMSASLGERVTMTCTASSSVYPSYLYWYQQKPGSSPKLWIYTTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSRTFGGGTKLEIK
抗人钙卫蛋白的单克隆抗体组合物的制备方法,具体步骤包括:
S1、人钙卫蛋白S100A8亚基、S100A9亚基和S100A8/A9 复合物的表达生产:
S11、人钙卫蛋白S100A8亚基表达生产
将密码子已经优化成大肠杆菌偏好的密码子的S100A8基因导入pET30a表达质粒,转化到大肠杆菌BL21(DE3)感受态细胞,挑取单菌落分别进行诱导表达,在含有卡那霉素的LB培养基中培养,培养至OD600nm至0.6-0.8时,加入IPTG进行诱导表达后分别收集菌体;
S12、人钙卫蛋白S100A9亚基表达生产
将密码子已经优化成大肠杆菌偏好的密码子的S100A9基因导入pET30a表达质粒,转化到大肠杆菌BL21(DE3)感受态细胞,挑取单菌落分别进行诱导表达,在含有卡那霉素的LB培养基中培养,培养至OD600nm至0.6-0.8时,加入IPTG进行诱导表达后分别收集菌体;
S13、人钙卫蛋白S100A8/A9 复合物
将密码子已经优化成大肠杆菌偏好的密码子的S100A9亚基基因和S100A8亚基基因分别导入pRSFDuet-1载体多克隆位点1和多克隆位点2,构建双表达载体质粒,转化到大肠杆菌BL21(DE3)感受态细胞,挑取单菌落进行诱导表达,在含有卡那霉素的LB培养基中培养,培养至OD600nm至0.6-0.8时,加入IPTG进行诱导表达后收集菌体。
S2、人钙卫蛋白S100A8亚基、S100A9亚基和S100A8/A9 复合物的纯化:
S21、人钙卫蛋白S100A8亚基的纯化
菌体用磷酸盐缓冲液重悬,高压均质机进行高压均质后,离心取上清,用预装柱NiSmart-6FF beads进行亲和纯化,用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,0.22um滤器无菌过滤后,BCA测定蛋白浓度;
S22、人钙卫蛋白S100A9亚基的纯化
菌体用磷酸盐缓冲液重悬,高压均质机进行高压均质后,离心取上清,用预装柱NiSmart-6FF beads进行亲和纯化,用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,0.22um滤器无菌过滤后,BCA测定蛋白浓度;
S23、人钙卫蛋白S100A8/A9 复合物的纯化
第一步:纯化S100A9亚基
菌体用磷酸盐缓冲液重悬,高压均质机进行高压均质后,离心取上清,用预装柱NiSmart-6FF beads进行亲和纯化,用磷酸盐缓冲液洗杂,用500mM咪唑浓度直接一步洗脱,收集洗脱液;
第二步:纯化S100A8亚基
将收集好的洗脱液,离心取上清,用预装柱Streptactin Beads 4FF进行亲和纯化,用磷酸盐缓冲液洗杂,用2.5mM脱硫生物素浓度洗脱,收集洗脱液,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。
S3、单克隆抗体的制备:
将亲和纯化后的人钙卫蛋白S100A8/A9 复合物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,取上清进行酶联免疫吸附测定,利用亲和纯化后的人钙卫蛋白S100A8亚基、S100A9亚基和S100A8/A9 复合物筛选特异性针对人钙卫蛋白S100A8亚基、S100A9亚基和S100A8/A9 复合物的抗体,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。
抗人钙卫蛋白的单克隆抗体组合物在制备检测钙卫蛋白的试剂中的应用。单克隆抗体组合物用于制备检测钙卫蛋白的化学发光试剂盒。
钙卫蛋白化学发光测定试剂盒,使用1号克隆和2号克隆分别作为包被抗体和检测抗体。
与现有技术相比,本发明的有益效果是:
本发明提供了抗人钙卫蛋白,用于制备诊断用单克隆抗体,本发明的抗人钙卫蛋白的单克隆抗体组合物包括1号克隆和2号克隆,能特异性识别鼠抗人钙卫蛋白S100A8/A9复合物的单克隆抗体不识别单独的人钙卫蛋白S100A8亚基和S100A9亚基,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钙卫蛋白化学发光测定试剂盒,从而提高诊断试剂的特异性和灵敏度,对于肿瘤的早期发现具有很大的临床意义。
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 人钙卫蛋白S100A8亚基表达载体的构建、表达生产和亲和纯化
在NCBI官网上查找人钙卫蛋白S100A8亚基氨基酸序列,在蛋白氨基酸的N端或者C端加上组氨酸标签,利用密码子优化软件在大肠杆菌表达***中优化密码子,基因合成后利用NdeI&XhoI限制性内切酶酶切位点,将基因克隆至pET30a表达载体中,构建pET30a-S100A8,并测序验证基因序列。
将pET30a-S100A8 表达质粒,转化到大肠杆菌BL21(DE3)感受态细胞,挑取单菌落进行诱导表达,在含有50ug/ml的卡那霉素的LB培养基中培养,培养温度区间为20-22℃,200-220rpm条件下培养至OD600nm至0.6-0.8时,加入0.2 mM IPTG进行诱导表达,诱导15-18小时,8000g,离心10min,收集菌体。
菌体用20mM PB,300mM NaCl,pH8.0的缓冲液重悬,用800bar压力进行高压均质后,12000g,离心30min,取上清,用预装柱Ni Smart-6FF beads进行亲和纯化,20mM PB,300mM NaCl,10mM咪唑,pH8.0溶液进行洗杂,20mM PB,300mM NaCl,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,pH8.0缓冲液中,0.22um滤器无菌过滤后,BCA测定蛋白浓度。
实施例2 人钙卫蛋白S100A9亚基表达载体的构建、表达生产和亲和纯化
在NCBI官网上查找人钙卫蛋白S100A9亚基氨基酸序列,在蛋白氨基酸的N端或者C端加上组氨酸标签,利用密码子优化软件在大肠杆菌表达***中优化密码子,基因合成后利用NdeI&XhoI限制性内切酶酶切位点,将基因克隆至pET30a表达载体中,构建pET30a-S100A9,并测序验证基因序列。
将pET30a-S100A9表达质粒,转化到大肠杆菌BL21(DE3)感受态细胞,挑取单菌落进行诱导表达,在含有50ug/ml的卡那霉素的LB培养基中培养,培养温度区间为20-22℃,200-220rpm条件下培养至OD600nm至0.6-0.8时,加入0.2 mM IPTG进行诱导表达,诱导15-18小时,8000g,离心10min,收集菌体。
菌体用20mM PB,300mM NaCl,pH8.0的缓冲液重悬,用800bar压力进行高压均质后,12000g,离心30min,取上清,用预装柱Ni Smart-6FF beads进行亲和纯化,20mM PB,300mM NaCl,10mM咪唑,pH8.0溶液进行洗杂,20mM PB,300mM NaCl,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,pH8.0缓冲液中,0.22um滤器无菌过滤后,BCA测定蛋白浓度。
实施例3 人钙卫蛋白S100A8/A9 复合物双表达载体的构建、表达生产和亲和纯化
在NCBI官网上查找人钙卫蛋白S100A8亚基氨基酸序列,在蛋白氨基酸序列的N端或者C端加上Strep-tag II标签;在NCBI官网上查找人钙卫蛋白S100A8亚基氨基酸序列,在蛋白氨基酸的N端或者C端加上组氨酸标签,利用密码子优化软件在大肠杆菌表达***中优化密码子。基因合成后将S100A8亚基导入pRSFDuet1质粒多克隆位点1,利用BamHⅠ&HindⅢ作为限制性内切酶酶切位点;将S100A9亚基导入pRSFDuet1质粒多克隆位点2,利用NdeI&XhoI限制性内切酶酶切位点,构建双蛋白表达质粒pRSFDuet1-S100A8&S100A9,并测序验证基因序列。
将pRSFDuet1-S100A8&S100A9表达质粒,转化到大肠杆菌BL21(DE3)感受态细胞,挑取单菌落进行诱导表达,在含有50ug/ml的卡那霉素的LB培养基中培养,培养温度区间为20℃,220rpm条件下培养至OD600nm至0.6-0.8时,加入0.2 mM IPTG进行诱导表达,诱导15-18小时,8000g,离心10min,收集菌体。
菌体用20mM PB,300mM NaCl,pH8.0的缓冲液重悬,用800bar压力进行高压均质后,12000g,离心30min,取上清,用预装柱Ni Smart-6FF beads进行亲和纯化,用20mM PB,300mM NaCl,10mM咪唑,pH8.0溶液进行洗杂,20mM PB,300mM NaCl,500mM咪唑,pH8.0溶液进行洗脱,洗脱完成后;将收集好的洗脱液离心取上清,用预装柱Streptactin Beads 4FF进行亲和纯化,用20mM PB,300mM NaCl,pH8.0洗杂,用20mM PB,300mM NaCl,2.5mM脱硫生物素,pH8.0浓度洗脱,收集洗脱液。SDS-PAGE跑胶检测蛋白纯度,将蛋白透析至20mM PB,300mM NaCl,pH8.0缓冲液中,0.22um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
将人钙卫蛋白S100A8和S100A9复合物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,皮下多点注射,后期间隔2周每次,采集小鼠血液分离血清,ELISA检测抗体效价,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,取上清进行ELISA检测,包被抗原选择人钙卫蛋白S100A8&S100A9复合物、人钙卫蛋白S100A8亚基和人钙卫蛋白S100A9亚基,筛选出性能较优的阳性细胞株,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
钙卫蛋白化学发光测定试剂盒的制备
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1.5-2h;
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)与本发明的检测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1-1.5h;
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-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PRL抗原分别稀释至0、0.6、2、6、20、60、200、600、1000、2000ng/mL,分装至校准品管中。
使用抗原稀释液将PRL抗原稀分别释液至200、1000 ng/mL,分装至质控品管中。
上述抗原稀释液配方为1×PBS、1% BSA。
检测试剂盒与同类产品对比:
实施例中的钙卫蛋白化学发光测定试剂盒与对照公司的人钙卫蛋白 ELISA试剂盒在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0.992。说明本发明的试剂盒能准确检测钙卫蛋白含量,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-A2:2012《临床实验室测量程序检测能力的评估》,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0.3 ng/mL和0.6 ng/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-A3:2014《定量测量程序精度的评估指南》,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:
在钙卫蛋白浓度约为200 ng/mL和1000 ng/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:如表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.抗人钙卫蛋白的单克隆抗体组合物,其特征在于:包括1号克隆和2号克隆,
所述1号克隆的重链可变区氨基酸序列如SEQ ID NO.1所示,1号克隆的轻链可变区氨基酸序列如SEQ ID NO.2所示;
所述2号克隆的重链可变区氨基酸序列如SEQ ID NO.3所示,2号克隆的轻链可变区氨基酸序列如SEQ ID NO.4所示。
2.权利要求1所述的抗人钙卫蛋白的单克隆抗体组合物在制备检测钙卫蛋白的试剂中的应用。
3.根据权利要求2所述的抗人钙卫蛋白的单克隆抗体组合物在制备检测钙卫蛋白的试剂中的应用,其特征在于:单克隆抗体组合物用于制备检测钙卫蛋白的化学发光试剂盒。
4.钙卫蛋白化学发光测定试剂盒,其特征在于:含有权利要求1中的1号克隆和2号克隆。
5.根据权利要求4所述的钙卫蛋白化学发光测定试剂盒,其特征在于:试剂盒使用1号克隆和2号克隆分别作为包被抗体和检测抗体。
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