CN116874596B - 抗S100β蛋白的单克隆抗体及其制备方法和应用 - Google Patents
抗S100β蛋白的单克隆抗体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了抗S100β蛋白的单克隆抗体及其制备方法和应用,包括S100β‑Clone1和S100β‑Clone2,S100β‑Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示,S100β‑Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示;S100β‑Clone2的重链可变区氨基酸序列如SEQ ID NO.3所示,S100β‑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S100ββ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S100ββ和S100αβ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脑损伤的早期诊断检测试剂盒,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S100β蛋白的单克隆抗体。
本发明还涉及抗S100β蛋白的单克隆抗体的制备方法和应用。
背景技术
S100蛋白最初由Moore等于1965年首先在牛脑组织中发现,因该蛋白在中性饱和硫酸铵中100%溶解而得名。S100蛋白相对分子量较小(9-14 kDa),生理上主要以不同的同源二聚体形式存在。
S100家族有25个成员组成,属于钙离子结合蛋白。当钙离子与S100结合后暴露于疏水基团表面,从而参与信息传递。S100家族包含α和β亚基的各种组合,最常见于S100A(α-α) 或S100B (α-β和β-β) 亚型。
S100B为该家族最具活性的成员,其96%的含量存在于脑内,因此被认为是脑组织特异性蛋白,也是该家族中被研究最多的成员。
S100B蛋白主要在神经***星形胶质细胞和施万细胞中表达,在细胞内外发挥作用,其作用取决于浓度。在生理水平下,由于脑细胞及血脑屏障的完整性,血液、脑脊液中S100B的水平极低且相对稳定,S100B蛋白促使神经突延伸,保护神经元存活;当血脑屏障结构或功能受损时,S100B从胞内进入脑脊液,然后经受损的血脑屏障进入血液,使血液中S100B含量增加,进而刺激促炎细胞因子的表达,引起细胞凋亡,发挥神经毒性作用。
临床意义:由于S100B蛋白分子量小,易通过血脑屏障,无论在血清,血浆或脑脊液中,均可灵敏的反应中枢神经***损害程度,因此检测S100B蛋白含量对患者脑损伤的早期诊断,病情评估具有重要意义。
作为脑损伤的生化标志物,S100B蛋白在脑损伤后有一定的时间变化规律,而且与脑损伤程度及预后紧密相关。在临床上可用于新生儿缺血缺氧性脑病 (HIE)、急性脑卒中(脑梗死和脑出血)、创伤性颅脑损伤、中枢神经***感染等疾病的早期诊断、病情评估和治疗指导。
开发性能优异的S100β化学发光试剂盒的核心原料是鼠抗S100的单克隆抗体,需要抗体在保持高亲和力的同时能识别S100B (α-β)和S100B(β-β) 两种亚型的β亚基,从而提高诊断试剂的特异性和灵敏度,对于患者脑损伤的早期诊断、病情评估具有重要意义。
发明内容
为解决灵敏性、特异性的问题,本发明提供了抗S100β蛋白的单克隆抗体。该抗体性能优异、准确性高。
同时提供S100β化学发光试剂盒,能准确检测患者血液中S100β蛋白的含量,为患者脑损伤诊断提供依据。
本发明提供如下技术方案:
抗S100β蛋白的单克隆抗体,包括S100β-Clone1和S100β-Clone2,
S100β-Clone1的重链可变区氨基酸序列:
EVQLQQSGAEPVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIDPADGDTKYDPKFQGKATITADTSSNTAYLQLSRLTSEDTAVYYCARPSLYDGYYPFAYWGQGTLVTVSA
S100β-Clone1的轻链可变区氨基酸序列:
DVVMTQTPLSLSVSLGDQASISCRSSQSLIYSNRHTYLHWYLQKPGQSPKLLIFQVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHLPFTFGSGTKLEIK
S100β-Clone2的重链可变区氨基酸序列:
EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWMKQRTEQGLDWIGRIDPEDGETKYAPEFQGKATITADTSSNTAYLQLSTLTSEDTAVYYCARYYSSYVPFVYWGQGTLVTASA
S100β-Clone2的轻链可变区氨基酸序列:
ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKAGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMFSEDVADYYCLRSDKLPYTFGGGTKLEIK
抗S100β蛋白的单克隆抗体的制备方法,
将亲和纯化后的S100ββ与弗氏佐剂进行等体积混合乳化,免疫计量为100μg/只小鼠,3周后进行加强免疫,免疫计量为50μg/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S100ββ和S100αβ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。
S100ββ的亲和纯化:菌体用磷酸盐缓冲液充分重悬,高压均质机进行高压均质后,离心取上清,用预装柱进行亲和纯化,通过梯度咪唑浓度进行洗杂和洗脱,SDS-PAGE跑胶检测蛋白纯度和Native PAGE检测蛋白是否自发形成二聚体,即S100ββ,后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。
S100αβ的亲和纯化:菌体用磷酸盐缓冲液充分重悬,高压均质机进行高压均质后,离心取上清,将用预装柱进行亲和纯化,通过磷酸盐缓冲液进行洗杂,然后用2.5 mM脱硫生物素洗脱,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测定蛋白浓度后,将纯化后的S100α亚基和β亚基等量混合,置于2-5℃摇床孵育过夜,得到S100αα、S100ββ、S100αβ的混合物,将三者的混合物用Ni Smart-6FF beads进行亲和纯化,通过梯度咪唑浓度进行洗杂和洗脱,得到S100ββ、S100αβ的混合物,再将两者的混合物用预装柱进行亲和纯化,通过磷酸缓冲液溶液进行洗杂,然后用2.5 mM脱硫生物素洗脱,SDS-PAGE跑胶检测蛋白纯度和Native PAGE检测蛋白是否形成二聚体S100αβ。
S100β亚基的表达生产:将pET30a-S100β表达质粒,转化大肠杆菌感受态细胞,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。
S100α亚基的表达生产:将pET30a-S100α表达质粒,转化大肠杆菌感受态细胞,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。
抗S100β蛋白的单克隆抗体在制备患者脑损伤早期诊断试剂中的应用。
单克隆抗体用于S100β化学发光试剂盒。试剂盒使用S100β-Clone1和S100β-Clone2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S100β蛋白,用于制备诊断用单克隆抗体,本发明的抗S100β蛋白的单克隆抗体,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,对于临床患者脑损伤的早期诊断、病情评估具有重要意义。
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 S100α和S100β亚基表达载体的构建
在NCBI官网上查找人S100β亚基的氨基酸序列,在β亚基蛋白氨基酸加上组氨酸标签,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-S100β表达质粒,并测序验证基因序列。
在NCBI官网上查找人S100α亚基的氨基酸序列,在α亚基蛋白氨基酸加上Strep-tag II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-S100α表达质粒,并测序验证基因序列。
实施例2 S100β亚基的表达生产
将pET30a-S100β表达质粒,转化大肠杆菌BL21(DE3)感受态细胞,挑取单菌落进行诱导表达,在含有50μg/ml的卡那霉素的LB培养基中培养,37℃,220 rpm条件下培养至OD600nm至0.6-0.8时,加入0.2mM IPTG进行诱导表达,37℃诱导6小时,8000 g,离心10 min收集菌体。
实施例3 S100α亚基的表达生产
将pET30a-S100α表达质粒,转化大肠杆菌BL21(DE3)感受态细胞,挑取单菌落进行诱导表达,在含有50μg/ml的卡那霉素的LB培养基中培养,37℃,220 rpm条件下培养至OD600nm至0.6-0.8时,加入0.2mM IPTG进行诱导表达,37℃诱导6小时,8000 g,离心10 min收集菌体。
实施例4 S100ββ的亲和纯化
菌体用20mM PB,300mM NaCl,10%甘油,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,20mMPB,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,Native PAGE检测蛋白是否形成二聚体S100ββ,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5 S100αβ的亲和纯化
菌体用20mM PB,300mM NaCl,10%甘油,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Streptactin Beads 4FF进行亲和纯化,20mM PB,300mM NaCl,pH8.0溶液进行洗杂,然后用2.5 mM脱硫生物素洗脱,0.22μm滤器无菌过滤后,BCA测定蛋白浓度。将纯化后的S100α亚基和β亚基等量混合,置于2-5℃摇床孵育过夜,得到S100αα、S100ββ、S100αβ的混合物,将三者的混合物用Ni Smart-6FF beads进行亲和纯化,通过梯度咪唑浓度进行洗杂和洗脱,得到S100ββ、S100αβ的混合物,再将两者的混合物用预装柱Streptactin Beads 4FF进行亲和纯化,通过PB溶液进行洗杂,然后用2.5mM脱硫生物素洗脱,SDS-PAGE跑胶检测蛋白纯度和Native PAGE检测蛋白是否形成二聚体S100αβ。
实施例6 单克隆抗体的制备
将亲和纯化后的S100ββ与弗氏佐剂进行等体积混合乳化,免疫计量为100μg/只小鼠,3周后进行加强免疫,免疫计量为50μg/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S100ββ和S100αβ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,进行抗体纯化,将纯化后的抗体进行诊断性能分析。
实施例7 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、制备链霉亲和素磁微粒
使用磁微粒缓冲液(50mM PB、150mM Nacl、3% Trehalose、0.1% Proclin300)将购买的赛默飞公司链霉亲和素磁微粒稀释至固含量0.02%,作为R1试剂。
2、吖啶酯标记抗体
将吖啶酯和S100β抗体按照分子摩尔比3:1的比例在PBS中标记反应2h;反应结束后用30kDa超滤离心管进行超滤离心以去除游离吖啶酯;离心结束后,收集超滤管中的标记物并使用BCA法检测吖啶酯-抗体浓度;将吖啶酯-抗体使用R2配制液稀释至1.5μg/mL,稀释后的溶液即为R2试剂,上述R2配制液配方为20mM Tris、200mM KCl、2%BSA、0.1%Tween20、0.1%Proclin300。
3、生物素标记抗体
将NHS酯化的生物(赛默飞,EZ-link NHS-PEG4-biotin)与本发明的抗S100β抗体按照摩尔比5:1比例在PBS中反应标记1h;反应结束后用30kDa超滤离心管进行超滤离心,离心结束后在超滤管上部加入PBS再次离心,重复两次;收集超滤管中的生物素-抗体标记产物,并使用BCA法检测抗体浓度;使用R3配制稀释液稀释上述产物至抗体浓度2μg/mL作为R3试剂,R3配制稀释液的配方为20mM Tris、50mM KCl、2%Casein、0.1%Tween20、0.1%Proclin300。
4、配制校准品和质控品
使用抗原稀释液将S100β抗原分别稀释至0、0.005、0.05、0.5、5、50 ng/mL,分装至校准品管中。
使用抗原稀释液将S100β抗原稀分别释液至0.08、1 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S100β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0.9948。说明本发明的试剂盒能准确检测S100β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0.003 ng/mL和0.005 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S100β浓度约为0.08 ng/mL和1 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:表1中的干扰物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S100β蛋白的单克隆抗体组合物,其特征在于:包括S100β-Clone1和S100β-Clone2,
所述S100β-Clone1的重链可变区氨基酸序列如SEQ ID NO.1所示,S100β-Clone1的轻链可变区氨基酸序列如SEQ ID NO.2所示;
所述S100β-Clone2的重链可变区氨基酸序列如SEQ ID NO.3所示,S100β-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S100β蛋白的单克隆抗体组合物在制备患者脑损伤早期诊断试剂中的应用。
3.根据权利要求2所述的抗S100β蛋白的单克隆抗体组合物在制备患者脑损伤早期诊断试剂中的应用,其特征在于:单克隆抗体用于S100β化学发光试剂盒。
4. S100β化学发光试剂盒,其特征在于:含有权利要求1中的S100β-Clone1和S100β-Clone2。
5.根据权利要求4所述的S100β化学发光试剂盒,其特征在于:试剂盒使用S100β-Clone1和S100β-Clone2分别作为包被抗体和检测抗体。
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ELISA法测定血清S100B蛋白;孙志;荣墨克;金淑杰;单桂芬;;中国实验诊断学(第07期);第73-74页 * |
Functional expression, characterization, and application of human S100B;LEI Wu等;ONCOLOGY REPORTS;第2309-2316页 * |
血清S100B蛋白水平的研究进展;周艳;张亚美;庞小峰;;生命科学仪器(第09期);第11-14页 * |
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