CN114181908B - 抗人s100b蛋白鼠单克隆抗体及其应用 - Google Patents
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Abstract
本发明涉及生物技术领域,公开了鼠抗人S100B蛋白的单克隆抗体OTI6A6和OTI3G6,所述的抗体是由保藏号分别为:CGMCCNo.22344和CGMCCNo.22345的杂交瘤细胞株OTI6A6和OTI3G6分泌产生。所述鼠单克隆抗体OTI6A6和OTI3G6的免疫原为真核细胞293T表达的S100B全长蛋白,这2个鼠单克隆抗体可以识别人S100B蛋白表面的不同抗原决定簇,其OTI6A6抗体轻链(VL)的氨基酸序列如SEQIDNO.1所示;OTI6A6抗体的重链(VH)的氨基酸序列如SEQIDNo.2所示。其OTI3G6轻链(VL)的氨基酸序列如SEQIDNO.11所示;OTI3G6的重链(VH)的氨基酸序列如SEQIDNo.12所示。本发明的抗人S100B蛋白鼠单克隆抗体可应用于检测S100B蛋白的各种免疫检测试剂盒的制备,包括但不限于双抗体夹心ELISA或化学发光法试剂盒的制备,为临床上评估脑损伤的程度和预后提供辅助诊断。
Description
技术领域
本发明属于生物技术领域,尤其涉及抗人S100B蛋白鼠单克隆抗体及其在免疫检测方面应用。
背景技术
S100B蛋白又叫中枢神经特异蛋白,由2个β亚基通过半胱氨酸残基形成二硫键,以二聚体活性形式存在于神经系统中,是分子量为21KD的酸性钙结合蛋白,主要由星形胶质细胞产生。
S100B蛋白具有广泛的生物学活性,在细胞增生、分化、基因表达、细胞凋亡中具有重要作用。生理状态下脑中S100B蛋白在胚胎期第14d就有微弱表达,随后与神经系统生长发育呈平行增加的关系,成年后相对稳定。正常成人血清中含量小于0.2ng/mL,生理状态下是一种神经营养因子,影响神经胶质细胞的生长、增殖、分化,维持钙稳态,并对学习记忆等发挥一定作用,促进脑的发育;当人在脑梗塞、脑外伤或心脏外科手术时,S100B蛋白从胞液中渗出进入脑脊液,再经受损的血脑屏障进入血液,从而导致血液中S100B蛋白的浓度升高。
近年来,S100B被广泛应用于评价脑外伤、脑出血、缺血性脑血管病、麻醉、休克等引起的脑损伤,也是恶性黑色素瘤标志物。S100B蛋白作为脑损伤的生化标志物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又紧密相关,稳定性较好,其浓度值的检测有助于临床上判断神经组织的病灶大小、治疗效果和判断人的预后等。
中国专利关于S100B蛋白单克隆抗体制备的条目基本没有,申请公布号CN111487409 A涉及S100B蛋白化学发光检测试剂盒及其使应方法,未提到抗体的制备方法也没有公开抗体CDR序列。申请公布号CN 111487407 A也涉及一种S100B蛋白的检测试剂盒及其使用方法,未提到抗体的制备方法也没有公开抗体CDR序列。国际专利申请利用S100B或S100B+monoclonal antibody作为关键词,有关与S100B具有高亲和力和特异性结合的单克隆抗体的专利没有查到,有关检测利用抗S100B鼠单克隆抗体OTI6A6和OTI3G6制备免疫检测试剂盒也未查到。
发明内容
本发明的目的是提供特异性好、亲和力高的抗人S100B蛋白鼠单克隆抗体OTI6A6和OTI3G6及其在双抗体夹心ELISA或化学发光法制备的免疫检测试剂盒中的应用,为临床上评估脑损伤的程度和预后提供辅助诊断。
所述鼠单克隆抗体为鼠单克隆抗体OTI6A6或鼠单克隆抗体OTI3G6。
所述鼠单克隆抗体OTI6A6和鼠单克隆抗体OTI3G6以真核细胞293T表达的S100B全长蛋白为免疫原,通过免疫注射小鼠,取免疫小鼠脾脏制备悬液,并使之与骨髓瘤细胞进行细胞融合获得。
所述鼠单克隆抗体OTI6A6,其轻链可变区(VL)含112aa,其氨基酸序列如SEQ IDNO.1所示;所述抗体的重链(VH)含116aa,其氨基酸序列如SEQ IDNo.2所示。
所述鼠单克隆抗体OTI6A6,其中VL区域包括3个抗原决定簇:CDR1、CDR2和CDR3,抗原决定簇的区域相应地分别为27aa-38aa,56aa-58aa和95aa-101aa。其氨基酸序列分别如SEQ ID No.3-5所示。
所述鼠单克隆抗体OTI6A6,其中VH区域包括3个抗原决定簇:CDR1、CDR2和CDR3,抗原决定簇的区域相应地分别为26aa-35aa,53aa-62aa和101aa-105aa。其氨基酸残基序列分别如SEQ ID No.6-8所示。
所述鼠单克隆抗体OTI3G6,其轻链可变区(VL)含110aa,其氨基酸序列如SEQ IDNO.11所示;所述抗体的重链(VH)含113aa,其氨基酸序列如SEQ IDNo.12所示。
所述鼠单克隆抗体OTI3G6,其中VL区域包括3个抗原决定簇:CDR1、CDR2和CDR3,抗原决定簇的区域相应地分别为27aa-37aa,55aa-57aa和94aa-99aa。其氨基酸序列分别如SEQ ID No.13-15所示。
所述鼠单克隆抗体OTI3G6,其中VH区域包括3个抗原决定簇:CDR1、CDR2和CDR3,抗原决定簇的区域相应地分别为26aa-34aa,52aa-58aa和97aa-102aa。其氨基酸残基序列分别如SEQ ID No.16-18所示。
所述的鼠单克隆抗体包括鼠单克隆抗体OTI6A6或鼠单克隆抗体OTI3G6可以与S100B蛋白高特异性结合,可通过本领域技术人员所公知的的方法,制备成检测S100B蛋白的各种免疫检测试剂盒。尤其是,应用于双抗体夹心ELISA或化学发光法制备的免疫检测试剂盒中。双抗体夹心ELISA检测S100B蛋白标准品,检测灵敏度能达到62.5pg/ml,标准曲线的R2=0.9934,推出样品浓度计算公式:y=0.0045x+0.1622。板式化学发光法检测S100B蛋白标准品,检测灵敏度能达到30pg/ml。
保藏信息
用于保藏的杂交瘤细胞株OTI6A6和OTI3G6的分类命名为:抗S100B鼠单克隆抗体的杂交瘤细胞株;
保藏单位全称:中国微生物菌种保藏管理委员会普通微生物中心;
保藏单位简称:CGMCC;
保藏单位地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所;
保藏日期:2021年5月21日;
保藏编号:CGMCC No.22344和CGMCC No.22345
附图说明
图1为鼠单克隆抗体OTI6A6和OTI3G6重链和轻链可变区扩增产物的电泳图,M为DNA分子量Marker。
图2为单克隆抗体OTI6A6特异识别完整的S100B(Full length S100B,S100B-FL)蛋白的Western blot检测结果图。泳道1到泳道13分别为S100B、S100A1、S100A2、S100A3、S100A4、S100A5、S100A6、S100A7、S100A9、S100A10、S100A12、S100A14、S100P蛋白;
图3为单克隆抗体OTI3G6特异识别完整的S100B(Full length S100B,S100B-FL)蛋白的Western blot检测结果图。泳道1到泳道13分别为S100B、S100A1、S100A2、S100A3、S100A4、S100A5、S100A6、S100A7、S100A9、S100A10、S100A12、S100A14、S100P蛋白;
图4为双抗体夹心ELISA法检测S100B标准曲线,横坐标为S100B蛋白浓度,纵坐标为检测的OD值。标准曲线的R2=0.9934,推出样品浓度计算公式:y=0.0045x+0.1622。
图5为板式化学发光检测S100B蛋白,横坐标为S100B蛋白浓度,纵坐标为检测的化学发光读数。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件、实验室手册中所述的条件或按照制造厂商所建议的条件。
实施例1:抗S100B蛋白鼠单克隆抗体的制备
一、S100B重组蛋白的生产
从Genebank中选调S100B基因NM_006272(NP_006263),该基因编码S100B全长92个氨基酸(aa)。以从美国傲锐东源生物科技有限公司获得的质粒RC200277(含S100B ORF276bp)为模板,设计两条引物并分别引入酶切位点SgfI和MluI,克隆入表达载体pCMV6-Entry,建立S100B重组表达质粒。采用本领域技术人员所知的技术方法将该基因转染HEK293T细胞,转染48小时后,进行细胞裂解,收集所有培养皿中的裂解液,4℃离心,DDK亲和层析柱纯化,SDS-PAGE电泳鉴定纯度。电泳后从凝胶上观察到分子量为10.5kDa左右的目的蛋白条带,纯度在85%以上,达到了制备单抗的纯度要求。
二、动物免疫
上述纯化的S100B重组蛋白以完全弗氏佐剂乳化,采用皮下或腹腔注射方法免疫6-8周龄BALB/c小鼠,免疫剂量为60μg/只,间隔两周后进行第二次免疫,以不完全弗氏佐剂乳化,免疫剂量为30μg/只。免疫两次后取尾血以ELISA法梯度稀释测定血清效价;依据ELISA效价12800时的OD450大于2.0为标准,根据结果判定是否加强免疫或是继续免疫,选取抗体效价最高的小鼠进行细胞融合。
三、细胞融合、克隆筛选取免疫小鼠脾细胞与小鼠骨髓瘤细胞SP2/0融合,脾细胞与SP2/0细胞以10:1混合,以50%PEG4,000介导融合,离心后重悬于HAT选择性培养基中,接种于含有饲养细胞的96孔微孔板中,置37℃、5%CO2培养箱培养,融合后第4d、第7d用HAT培养液半量换液,10d后换用HT培养液。观察杂交瘤细胞的生长情况,等克隆长至孔底面积的1/4~1/3,取培养上清液,用ELISA法进行抗体检测,筛选阳性克隆。以有限稀释法对阳性孔的杂交瘤细胞进行克隆化培养,直到克隆化细胞抗体阳性率为100%,选岀高分泌特异性细胞株OTI6A6和OTI3G6(即ELISA效价在1:104以上的阳性克隆),此时可将阳性克隆细胞进一步扩大培养,直至细胞系能稳定分泌单克隆抗体。
四、单克隆抗体的制备和纯化以无血清培养基悬浮培养杂交瘤细胞,1周左右当细胞上清体积扩培到任务量及细胞死亡率达到60%-70%时,收取细胞悬液,离心取上清,亲和层析法进行抗体纯化,根据抗体亚型选择相应柱料进行纯化。以BCA法测定纯化后的单抗浓度,再分装、冻存。
实施例2:抗S100B蛋白鼠单克隆抗体OTI6A6或OTI3G6的鉴定
一、鼠单克隆抗体的特异性鉴定
采用Western blot(WB)检测。S100B、S100A1、S100A2、S100A3、S100A4、S100A5、S100A6、S100A7、S100A9、S100A10、S100A12、S100A14、S100P等蛋白上样进行SDS-PAGE,蛋白上样量为20ng,转膜后进行WB检测。
结果显示,鼠单克隆抗体OTI6A6或OTI3G6仅特异识别S100B蛋白,而不识别其它12种S100家族蛋白,说明本发明的抗S100B蛋白鼠单克隆抗体OTI6A6或OTI3G6对S100B蛋白具有高度特异性。结果见图2和图3。
二、鼠单克隆抗体的效价
采用倍比稀释法稀释鼠单克隆抗体OTI6A6或OTI3G6,用间接ELISA测抗体效价,结果显示本发明涉及的鼠单克隆抗体OTI6A6或OTI3G6效价均为2×106。
三、抗体配对
为了挑选包被抗体和检测抗体的最佳组合,采用棋盘组合,将获得的多株S100B鼠单克隆抗体相互配对。基本步骤:多株单克隆抗体分别包被酶标板,4℃过夜。第二天取出酶标板,PBST洗涤一次,1%BSA溶液37℃封闭2小时,PBST洗涤3次;每孔加入S100B全长蛋白100μl,浓度分别为20、5、1和0ng/ml,37℃孵育1小时;孵育完成后取出酶标板,PBST洗涤3次,分别加入HRP标记的上述多株抗体作为检测抗体,37℃孵育1小时。PBST洗涤5次,加入TMB底物,37℃显色10min。取出后加终止液,在酶标仪上测定OD450读数。根据样品的OD值与阴性对照的背景值,选出最为理想的鼠单克隆抗体对,配对筛选结果见表1。本发明涉及的抗体OTI6A6作为包被抗体,抗体OTI3G6作为检测抗体最佳。抗人S100B蛋白鼠单克隆抗体OTI6A6和鼠单克隆抗体OTI3G6,可以识别人S100B蛋白表面的不同抗原决定簇。
表1抗体配对实验筛选结果
实施例3:鼠单克隆抗体OTI6A6或OTI3G6可变区基因与氨基酸序列分析
从Takara Bio USA公司采购 RACE 5’/3’试剂盒,采用5’RACE(RapidAmplification of cDNA Ends,快速扩增cDNA末端)技术扩增杂交瘤细胞功能性抗体的可变区轻链与重链基因序列。具体实验过程参见Takara Bio USA公司/> RACE5’/3’Kit使用者手册。
根据所述的抗体OTI6A6或OTI3G6为IgG1亚型,设计针对其Ig和Kapa恒定区3’端的特异性基因引物pRace-H-GSP和pRace-K-GSP,引物序列如下:
pRace-H-GSP:CATCDGTCTATCCACTGGCCCCTG
pRace-K-GSP:CTTCCCACCATCCAGTGAGCAGTT
从杂交瘤细胞OTI6A6或OTI3G6中提取mRNA,逆转录为cDNA,RACE扩增抗体重链和轻链的DNA片段,扩增产物如图1所示,基因大小符合鼠抗体特征。分别将扩增的轻链与重链通过酶切连于克隆载体PUC119,通过蓝白斑挑取阳性克隆,纯化阳性质粒进行测序,采用测序仪ABI 3730,测序引物为通用引物M13f和M13r。
利用互联网,在http://www.imgt.org上使用IMGT/V-QUEST分析软件,分别将轻链与重链的核苷酸序列进行测序结果数据分析,得到鼠单克隆抗体OTI6A6的轻链氨基酸序列如SEQ ID NO.1所示,重链氨基酸序列如SEQ ID NO.2所示。VL全长为112个氨基酸,其FR的4个结构域氨基酸数分别为26、17、36和11,CDR的3个结构域氨基酸数分别为12、3和7,CDR1、CDR2和CDR3的区域相应地分别为27aa-38aa,56aa-58aa和95aa-101aa,其氨基酸序列分别:QSLLNSNNQKNY、LAS、QQYYSIP。
通过分析,所述鼠单克隆抗体OTI6A6VH全长为116个氨基酸,其FR的4个结构域氨基酸数分别为25、17、38和11,CDR的3个结构域氨基酸数分别为10、10和5,CDR1、CDR2和CDR3分别为26aa-35aa,53aa-62aa和101aa-105aa,其氨基酸序列分别为GFTFTFSDYY、INTKANGYTT、AREGG。
通过分析,所述鼠单克隆抗体OTI3G6的轻链氨基酸序列如SEQ ID NO.11所示,重链氨基酸序列如SEQ ID NO.12所示。VL全长为110个氨基酸,其FR的4个结构域氨基酸数分别为26、17、36和11,CDR的3个结构域氨基酸数分别为11、3和6,CDR1、CDR2和CDR3的区域相应地分别为27aa-37aa,55aa-57aa和94aa-99aa,其氨基酸序列分别:QTIVYSNGNTY、WAS、FQGSHV。
通过分析,所述鼠单克隆抗体OTI3G6 VH全长为113个氨基酸,其FR的4个结构域氨基酸数分别为25、17、38和11,CDR的3个结构域氨基酸数分别为9、7和6,CDR1、CDR2和CDR3分别为25aa-34aa,52aa-58aa和97aa-102aa,其氨基酸序列分别为GYSITSGYA、INYSGET、ARANDA。
实施例4:抗S100B蛋白鼠单克隆抗体OTI6A6或OTI3G6用于制备双抗体夹心ELISA检测试剂盒
采用本领域技术人员所公知的基于ELISA双抗体夹心法原理的检测技术,制作S100B蛋白检测试剂盒,用于临床上与S100B蛋白相关疾病辅助诊断。
一、试剂盒组成
1.包被OTI6A6抗体的板条:用PBS缓冲液稀释抗体至5μg/ml,包被到微孔板上,每孔100μl,4℃孵育过夜,以PBST洗涤3次,甩干;加含有1%BSA、5%蔗糖、0.05%Proclin300的PBS进行封闭,37℃反应2h,弃去孔内封闭液,甩干;将包被板置于37℃烘箱2h,即完成包被,以铝箔袋密封,存放于4℃保存备用。
2.试剂配制
1.酶结合物配制采用简易过碘酸钠法标记抗S100B蛋白鼠单克隆抗体OTI3G6,滴定工作浓度,配制成1:20的浓缩液,稀释液为含1%BSA、5%甘油、0.05%Proclin 300的PBS,过滤除菌。
2.洗涤缓冲液为常规的pH7.4的PBST,含0.05%Proclin300,配制成20倍浓缩液。
3.酶的底物液(显色液)单组分TMB(从市场采购)。
4.样本稀释液含1%BSA、0.05%Proclin 300的PBS,过滤除菌。
5.终止液用10ml HCl(36-38%)加入110ml蒸馏水中,混合过程需缓慢进行,配置成1N的HCl。
6.标准品全长S100B蛋白,以含1%BSA、5%蔗糖、10%甘油、0.05%Proclin300的PBS为稀释液,将样品稀释为20μg/ml,过滤除菌,无菌分装。
二、检测操作要点
1、为保证检测结果的准确性,建议标准品及样本均设双孔测定。每次检测均需做标准曲线。
2、如标本中待测物质含量过高,请先用样本稀释液进行稀释,以使样本符合试剂盒的检测范围,最后计算时再乘以相应的稀释倍数。
3、加样:加样时,请使用一次性的洁净吸头,避免交叉污染。加样时应尽量轻缓,避免起泡,将样本加于酶标板孔底部,切勿沿孔壁加样。
4、温育:为防止样本蒸发或污染,温育过程中酶标板必须覆上板贴,实验过程中酶标板应避免处于干燥的状态。温育过程中应随时观察温箱温度是否恒定于37℃,及时调整。温育过程中,温箱不易开启太多次,以免影响温度平衡。
5、洗涤:洗涤过程非常重要,不充分的洗涤易造成假阳性。
6、显色:为保证实验结果的准确性,底物反应时间到后应尽快加入终止液。可在加入底物溶液后每隔一段时间观察一下显色情况以控制反应时间(比如每隔10分钟)。当肉眼可见标准品前3-4孔有明显梯度蓝色,后3-4孔显色不明显时,即可加入终止液终止反应,此时蓝色立刻变为黄色。终止液的加入顺序应尽量与底物溶液的加入顺序相同。
7、底物溶液应为浅蓝色或无色,如果颜色严重变深则必须弃用。底物溶液易受污染,请避光妥善保存。
三、标准S100B蛋白不同稀释度标准曲线的确定
上述制备的检测S100B蛋白双抗体夹心ELISA检测试剂盒从4度冰箱取出,平衡到室温。将标准品用PBS从20μg/ml稀释到500pg/ml,制备成0、31.25、62.5、125、250、500pg/ml共6个系列浓度标准品,按照上述的检测方法在每孔中加入标准品100μl,进行温育,然后弃去液体,加入HRP标记的检测抗体工作液进行温育,弃去孔内液体,甩干后每孔加入底物溶液,避光显色后每孔加入终止溶液,在反应终止后5分钟内用酶标仪在450nm波长依序测量各孔的光密度OD值做标准曲线,结果见表2。依据表2制作S100B检测标准曲线,见附图4。标准曲线的R2=0.9934,推出样品浓度计算公式:y=0.0045x+0.1622。
表2.双抗体夹心ELISA法检测S100B标准蛋白
实施例5:抗S100B蛋白鼠单克隆抗体OTI6A6或OTI3G6用于板式化学发光免疫检测
采用本领域技术人员所公知的基于双抗体夹心法原理的板式化学发光免疫检测技术,制作S100B蛋白检测试剂盒,用于临床上与S100B蛋白相关疾病辅助诊断。
一、检测原理与方法
采用基于双抗体夹心法原理的板式化学发光免疫检测技术。在不透光的白色微孔板内包被抗S100B蛋白的鼠单克隆抗体OTI6A6,将封闭好的包被板放置到科斯迈全自动发化学发光仪上,设置仪器程序,将标准品或待测标本与辣根过氧化物酶(HRP)标记的鼠单克隆抗体OTI3G6同时进行温育,则三者形成抗体-抗原-抗体复合物。洗涤除去未结合的物质,加入北京科跃中楷化学发光底物,反应10min后科斯迈全自动发化学发光仪显示化学发光值。依据信噪比大于2.0判读结果为阳性。发光值的大小反映了结合的酶标抗体的量,它与标本中的S100B蛋白的浓度成正比。根据所测定的标准品的发光值绘制标准曲线,待测标本中的S100B蛋白浓度值即可从标准曲线上获得。
二、检测S100B蛋白的板式化学发光检测试剂盒组成
1.包被OTI6A6抗体的板条:同实施例4。
2.试剂配制
1.酶结合物配制同实施例4。
2.洗涤缓冲液同实施例4。
3.化学发光显色液A液和B液,购于北京科跃中楷生物技术有限公司。
4.样本稀释液含1%BSA、0.05%Proclin 300的PBST,过滤除菌。
5.标准品S100B全长蛋白,以含1%BSA、5%蔗糖、10%甘油、0.05%Proclin300的PBS为稀释液,将样品稀释为5μg/ml,过滤除菌,无菌分装。
三、对S100B全长蛋白的检测
把S100B全长蛋白进行梯度稀释,分别为0pg/ml、10pg/ml、30pg/ml、100pg/ml、300pg/ml、1000pg/ml,以OTI6A6抗体为包被抗体、OTI3G6抗体为检测抗体,用上述检测方法检测6个梯度稀释的抗原,检测结果见表3。根据信噪比大于2.0判定,本发明所述的鼠单克隆抗体用于板式化学发光免疫检测试剂,最低灵敏度小于30pg/ml,线性范围为10-1000pg/ml。依据表3制作S100B蛋白检测标准曲线,见图5
表3.板式化学发光法检测S100B全长蛋白
OTI6A6序列表
<110> 无锡傲锐东源生物科技有限公司
<120> 抗人S100B蛋白鼠单克隆抗体及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 112
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 1
DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSNNQKNYLAWYQQKPGQSPKLLVYLASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQYYSIPFGAGTKLELKR
<210> 2
<211> 116
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 2
EVKLVESGGGLVQPGGSLRLSCATSGFTFTFSDYYMSWVRQPPGKALEWLGLINTKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCAREGGWGQGTLVTVSV
<210> 3
<211> 12
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 3
QSLLNSNNQKNY
<210> 4
<211> 3
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 4
LAS
1
<210> 5
<211> 7
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 5
QQYYSIP
<210> 6
<211> 10
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 6
GFTFTFSDYY
<210> 7
<211> 10
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 7
INTKANGYTT
<210> 8
<211> 5
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 8
AREGG
OTI3G6序列表
<110> 无锡傲锐东源生物科技有限公司
<120> 抗人S100B蛋白鼠单克隆抗体及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 11
<211> 110
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 11
DVLMTQTPLSLPVILGDQASISCRSSQTIVYSNGNTYLEWYLQKPGQSPKPLIYWASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVFGGGTKLEIKR
<210> 12
<211> 113
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 12
DVQLQESGPGLVKPSQSLSLTCTVTGYSITSGYAWNWIRQFPGNKLEWMGYINYSGETTYNPSLKSRFSITRDTSRNQFFLQLNSVTTEDTATYYCARANDAWGQGTSVTVSS
<210> 13
<211> 11
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 13
QTIVYSNGNTY
<210> 14
<211> 3
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 14
WAS
<210> 15
<211> 6
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 15
FQGSHV
<210> 16
<211> 9
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 16
GYSITSGYA
<210> 17
<211> 7
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 17
INYSGET
<210> 18
<211> 6
<212> PRT
<213> Mus musculus x Rattus norvegicus
<400> 18
ARANDA
Claims (8)
1.一株分泌抗人S100B单克隆抗体的杂交瘤细胞株OTI6A6,于2021年5月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.22344。
2.一株分泌抗人S100B的单克隆抗体的杂交瘤细胞株OTI3G6,于2021年5月21日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.22345。
3.根据权利要求1所述的杂交瘤细胞株OTI6A6分泌的抗人S100B单克隆抗体,其特征在于:其轻链可变区包括3个抗原决定簇:CDR1、CDR2和CDR3,其氨基酸序列分别如SEQ IDNo.3-5所示;其重链可变区包括3个抗原决定簇:CDR1、CDR2和CDR3,其氨基酸序列分别如SEQ ID No.6-8所示。
4.根据权利要求1所述的杂交瘤细胞株OTI6A6分泌的抗人S100B单克隆抗体,其特征在于:其轻链可变区含112aa,其氨基酸序列如SEQ ID NO.1所示;其重链可变区含116aa,其氨基酸序列如 SEQ IDNo.2所示。
5.根据权利要求2所述的杂交瘤细胞株OTI3G6分泌的抗人S100B单克隆抗体,其特征在于:其轻链可变区包括3个抗原决定簇:CDR1、CDR2和CDR3,其氨基酸序列分别如SEQ IDNo.13-15所示;其重链可变区包括3个抗原决定簇:CDR1、CDR2和CDR3,其氨基酸序列分别如SEQ ID No.16-18所示。
6.根据权利要求2所述的杂交瘤细胞株OTI3G6分泌的抗人S100B单克隆抗体,其特征在于:其轻链可变区含110aa,其氨基酸序列如SEQ ID NO.11所示;其重链可变区含113aa,其氨基酸序列如SEQ IDNo.12所示。
7.权利要求3-6任一项所述的抗人S100B单克隆抗体的应用,其特征在于:用于人S100B免疫检测试剂盒的制备。
8.根据权利要求7所述的抗人S100B单克隆抗体的应用,其特征在于:所述试剂盒为双抗体夹心ELISA试剂盒或化学发光免疫检测试剂盒。
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