CN113150157A - 一种用于检测血清中vegf含量的抗体对及其用途 - Google Patents
一种用于检测血清中vegf含量的抗体对及其用途 Download PDFInfo
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Abstract
本申请提供一种用于检测血清中VEGF含量的抗体对及其用途,包括与VEGF的N端表位即SEQ I D NO:2所示氨基酸序列相结合的单克隆抗体Ant iVEGF_N和与VEGF的C端表位即SEQ I D NO:3所示氨基酸序列相结合的单克隆抗体Ant iVEGF_C,所述单克隆抗体Ant iVEGF_N包括SEQ I D NO:7所示氨基酸序列的轻链可变区和SEQ I D NO:11所示氨基酸序列的重链可变区,所述Ant iVEGF_C包括SEQ I D NO:17所示氨基酸序列的轻链可变区和SEQ I DNO:21所示氨基酸序列的重链可变区。本申请将VEGF抗原分成N端(SEQ I DNO:2)和C端(SEQ I D NO:3)两段序列,并用这两段序列分别免疫小鼠,筛选到高特异性和高灵敏性的抗体对Ant iVEGF_N和Ant iVEGF_C,通过该抗体对可以有效的测定血清中VEGF的含量,进而便于肿瘤广谱及早期筛查,大大提高了肿瘤早期筛查的准确性。
Description
技术领域
本申请涉及医药领域,尤其涉及一种用于检测血清中VEGF含量的抗体对及其用途。
背景技术
血管内皮生长因子(Vascular endothelial growth Factor,VEGF)是一种具有高度生物活性的功能性糖蛋白。它是特异性的、生理作用强大的内皮细胞有丝分裂原,能作用于血管内皮细胞,使其增殖、迁移、管腔形成,参与血管生成并使毛细血管通透性增加;它可导致肿瘤血管异常生长,阻碍抗肿瘤药物有效输送至肿瘤组织内,并可刺激新生血管生长因子增加。
VEGF最早在1983年由Senger等[Senger DR,PerruzziCA,FederJ.A highlyconserved vascular permeability factor secreted by a variety of human androdent tumor celllines[J].CancerRes,1986,46(11):5629-5632.]发现,由两条8个外显子和7个内含子组成的23kD的单链蛋白构成。1994年,Kondo[Kondo S,A sano M,M atsuoK,et a1.Vascular endothelia l growth fac—tor/vascu l ar perm eability factoris d etectable in the sera of tum or—bearing m ice and cancer patients[J].Biochim Biophys Acta,1994,1221(2):211—214.]等人首次报道了癌症病人血清VEGF水平高于正常对照,从而开始了VEGF血液(包括血清和血浆)含量与肿瘤的发生、发展、以及治疗过程中VEGF的变化的实验性临床研究并获得了大量的研究资料。VEGF近些年被作为一种能反应肿瘤发生发展变化的物质,研究较为广泛。在检索英文280余篇,中文230余篇的VEGF相关文献中,95%以上的文献证实了肿瘤病人血清VEGF水平增高的事实。肿瘤类型几乎涵盖了目前临床大部分实体肿瘤和部分非实体肿瘤。VEGF血液水平不仅在肿瘤各期均增加,而且在其早期0-1期即增加,常规肿瘤标志物只有4期癌症和正常对照才有显著性差异。VEGF被认为是目前肿瘤广谱及早期筛查最有意义的标记物。由于VEGF的抗原较大,抗原制备的难度较大,同时,截止目前,还没有针对VEGF临床检测的配对抗体报道。
发明内容
本申请为解决上述技术问题而提供一种用于检测血清中VEGF含量的抗体对及其用途。
本申请所采取的技术方案是:本申请第一方面提供一种用于检测血清中VEGF含量的抗体对,其特征在于:包括与VEGF的N端表位即SEQ ID NO:2所示氨基酸序列相结合的单克隆抗体AntiVEGF_N和与VEGF的C端表位即SEQ ID NO:3所示氨基酸序列相结合的单克隆抗体AntiVEGF_C,所述单克隆抗体AntiVEGF_N包括SEQ ID NO:7所示氨基酸序列的轻链可变区和SEQ ID NO:11所示氨基酸序列的重链可变区,所述AntiVEGF_C包括SEQ ID NO:17所示氨基酸序列的轻链可变区和SEQ ID NO:21所示氨基酸序列的重链可变区。
进一步的,所述单克隆抗体AntiVEGF_N轻链氨基酸序列包括SEQ ID NO:4所示的LCDR1或与其至少90%同源的氨基酸序列,SEQ ID NO:5所示的LCDR2或与其至少90%同源的氨基酸序列,以及SEQ ID NO:6所示的LCDR3或与其至少90%同源的氨基酸序列;所述单克隆抗体AntiVEGF_N重链氨基酸序列包括SEQ ID NO:8所示的HCDR1或与其至少90%同源的氨基酸序列,SEQ ID NO:9所示的HCDR2或与其至少90%同源的氨基酸序列,以及SEQ IDNO:10所示的HCDR3或与其至少90%同源的氨基酸序列。
进一步的,所述单克隆抗体AntiVEGF_C轻链氨基酸序列包括SEQ ID NO:14所示的LCDR1或与其至少90%同源的氨基酸序列,SEQ ID NO:15所示的LCDR2或与其至少90%同源的氨基酸序列,以及SEQ ID NO:16所示的LCDR3或与其至少90%同源的氨基酸序列;所述单克隆抗体AntiVEGF_C重链氨基酸序列包括SEQ ID NO:18所示的HCDR1或与其至少90%同源的氨基酸序列,SEQ ID NO:19所示的HCDR2或与其至少90%同源的氨基酸序列,以及SEQ IDNO:20所示的HCDR3或与其至少90%同源的氨基酸序列。
进一步的,所述单克隆抗体AntiVEGF_N和AntiVEGF_C均为IgG型单克隆抗体,且一个为捕获抗体,一个为检测抗体。
进一步的,所述AntiVEGF_N为捕获抗体,所示AntiVEGF_C为检测抗体。
进一步的,所述检测抗体的检测标记物为检测标记蛋白、荧光基团、放射性同位素中的一种。
进一步的,所述检测抗体的检测标记蛋白包括辣根过氧化物酶、碱性磷酸酶、荧光素酶、β-半乳糖苷酶、葡萄糖氧化酶、溶菌酶和苹果酸脱氢酶,且检测抗体与检测标记蛋白重组表达。
进一步的,所述检测抗体的检测方法包括酶联免疫(ELISA)检测法、化学发光检测检测法、蛋白质印迹检测检测法和免疫组化(IHC)检测法和免疫层析检测检测法。
进一步的,所述抗体对选自与VEGF结合的Fab、F(ab’)2、Fab’、scFv和di-scFv的抗体片段。
本申请的第二方面提供一种DNA分子,其特征在于,它编码权利要求1或2或3所述的抗体。
本申请的第三方面提供一种上述任一一种抗体对在制备检测血清中VEGF含量的试剂盒中的应用。
本申请具有的优点和积极效果是:本申请将VEGF抗原分成N端(SEQ ID NO:2)和C端(SEQ ID NO:3)两段序列,并用这两段序列分别免疫小鼠,筛选到高特异性和高灵敏性的抗体对AntiVEGF_N和AntiVEGF_C,通过该抗体对可以有效的测定血清中VEGF的含量,进而便于肿瘤广谱及早期筛查,大大提高了肿瘤早期筛查的准确性。
除了上面所描述的本申请解决的技术问题、构成技术方案的技术特征以及由这些技术方案的技术特征所带来的优点之外,本申请所能解决的其他技术问题、技术方案中包含的其他技术特征以及这些技术特征所带来的优点,将在下文中作进一步详细的说明。
附图说明
图1是本申请实施例提供的AntiVEGF_N和AntiVEGF_C抗体对检测示意图;
图2是本申请实施例提供的构建的pcDNA3.1-NL-LFc载体电泳图;
图3是本申请实施例提供的构建的pcDNA3.1-NH-HFc载体电泳图;
图4是本申请实施例提供的重组AntiVEGF_N蛋白纯化电泳图;
图5是本申请实施例提供的构建的pcDNA3.1-CL-LFc载体电泳图;
图6是本申请实施例提供的构建的pcDNA3.1-CL-HFc载体电泳图;
图7是本申请实施例提供的重组AntiVEGF_C蛋白纯化电泳图;
图8是本申请实施例提供的AntiVEGF_N和AntiVEGF_C抗体对制备的试剂盒验证流程示意图;
图9是本申请实施例四提供的剂量-反应曲线的线性测试中的第一次测定曲线;
图10是本申请实施例四提供的剂量-反应曲线的线性测试中的第二次测定曲线;
图11是本申请实施例四提供的剂量-反应曲线的线性测试中的第三次测定曲线;
图12是本申请实施例提供的临床诊断中本申请抗体制成试剂盒与对照试剂盒检测VEGF的剂量-反应曲线图。
具体实施方式
下面结合附图和实施例对本申请作进一步的详细说明。可以理解的是,此处所描述的具体实施例仅仅用于解释相关发明,而非对该发明的限定。另外还需要说明的是,为了便于描述,附图中仅示出了与发明相关的部分。
需要说明的是,在不冲突的情况下,本发明中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
实施例一:单克隆抗体AntiVEGF_N和AntiVEGF_C的制备
1、对VEGF(SEQ ID NO:1)蛋白抗原性分析,将VEGF抗原分成N端(SEQ ID NO:2)和C端(SEQ ID NO:3)两段序列,分别合成VEGF的N端表位(SEQ ID NO:2)和VEGF的C端表位(SEQID NO:3),分别偶联KLH,获得N端免疫原和C端免疫原。
2、动物免疫:采用N端免疫原和C端免疫原分别免疫8周龄的雌性Balb/c小鼠,100ug的抗原免疫小鼠4次,每次间隔4周。
3、细胞融合:断颈处死小鼠,置于75%乙醇溶液中5-10min,在无菌条件下取出脾脏,用已灭菌处理的毛玻璃片挤压并轻轻研磨脾脏细胞,脾脏细胞计数,将免疫后的小鼠脾细胞和小鼠的骨髓瘤细胞按1:1的比例混合,在50ml离心管中用无血清不完全培养液洗1次,1000rpm/min离心10分钟,弃去上清,用吸管吸净残留液体(为了避免影响PEG的浓度),轻轻弹击离心管底,使细胞沉淀略松动。
4、用吸管在60s内加预热至40℃的50%PEG(PH 8.0)1ml,边加边轻轻搅拌。
5、用10ml吸管在90s内加20-30ml预热的不完全培养基(终止PEG作用);20-27℃静置10分钟。
6、1000r/min离心5分钟,弃上清
7、加入5ml HAT培养基,轻轻吹吸沉淀细胞,使其悬浮并混匀,然后补加含腹腔巨噬细胞的HAT培养基至80-100ml。
8、分装96孔细胞培养板(孔板内有饲养细胞层),每孔0.1-0.15ml(或分装24孔板,每孔1.0-1.5ml),然后将培养板置37℃,6%CO2培养箱内培养。
9、5天后用HAT培养基换出1/2培养基
10、7-10天后用HT培养基换出HAT培养基;
11、经常观察杂交瘤细胞的生长情况,待其长至孔底面积1/10以上时吸出上清通过ELISA检测其活性(阳性克隆),筛选抗体效价高的杂交瘤细胞,经过3-4次亚克隆,确保形成单克隆时扩大培养,并进行腹水细胞注射,抗体纯化。
实施例二:对实施例1获得的单克隆抗体进行验证
(1)将VEGF全抗原(SEQ ID NO:1)、VEGF的N端抗原(SEQ ID NO:2)和VEGF的C端抗原(SEQ ID NO:3)分别进行包被,包被浓度2ug/ml,4℃2小时,用0.9%NaCl洗涤液洗板3次,拍干,加封闭液(0.5mol PBS+1%BSA+2.5%蔗糖)200μl,37℃封闭2小时,之后倒尽板孔中液体,拍干;
(2)加入不同体积的实施例1获得的AntiVEGF_N和AntiVEGF_C(如表1所示),37℃反应1小时;
(3)0.9%NaCl洗涤液洗板3次,拍干;
(4)加入HRP标记的羊抗小鼠IgG的二抗,37℃反应1小时;
(5)0.9%NaCl洗涤液洗板5次,拍干;
(6)最后加入底物液鲁米诺,测定其发光值,测定结果如表1所示:
表1
由表1可知,AntiVEGF_N能特异性的结合VEGF的N端抗原(SEQ ID NO:2),而不结合VEGF的C端抗原(SEQ ID NO:3);AntiVEGF_C能特异性的结合VEGF的C端抗原(SEQ ID NO:3),而不结合VEGF的N端抗原(SEQ ID NO:2)。
实施例三:单克隆抗体AntiVEGF_N和AntiVEGF_C的测序及通过杂交瘤细胞制备相应抗体
1、提取实施例2中AntiVEGF_N杂交瘤细胞的总RNA,采用cDNA合成逆转录试剂盒,以RNA为模板合成第一条链cDNA,再以cDNA为模板,扩增单克隆抗体AntiVEGF_N可变区基因。对可变区PCR产物序列进行T/A克隆,后挑选阳性菌落进行测序,并对测序结果进行氨基酸翻译分析。
结果表明,AntiVEGF_N单抗轻链可变区的LCDR1,LCDR2和LCDR3的氨基酸序列分别列于SEQ ID NO:4,5和6。此外,包含上述可变区的轻链可变区基因及编码的氨基酸序列如SEQ ID NO:12和7所示。
AntiVEGF_N单抗重链可变区的HCDR1,HCDR2和HCDR3的氨基酸序列分别于SEQ IDNO:8,9和10。此外,包含上述可变区的重链可变区基因及编码的氨基酸序列如SEQ ID N0:13和11所示。
优化AntiVEGF_N单抗轻链可变区基因和重链可变区基因,将轻链可变区基因克隆到pcDNA3.1-LFc载体中,构建pcDNA3.1-NL-LFc载体,如图2所示;将重链可变区基因克隆到pcDNA3.1-HFc载体中,构建pcDNA3.1-NH-HFc载体,如图3所示,本实施例中,pcDNA3.1-LFc和pcDNA3.1-HFc中已经含有人源的轻链恒定区和重链恒定区,将pcDNA3.1-NL-LFc和pcDNA3.1-NH-HFc按照1:1共转染到CHO-S细胞中,重组表达抗VEGF的N端抗体AntiVEGF_N,如图4所示。
2、提取实施例2中AntiVEGF_C杂交瘤细胞的总RNA,采用cDNA合成逆转录试剂盒,以RNA为模板合成第一条链cDNA,再以cDNA为模板,扩增单克隆抗体AntiVEGF_C可变区基因。对可变区PCR产物序列进行T/A克隆,后挑选阳性菌落进行测序,并对测序结果进行氨基酸翻译分析。
结果表明,AntiVEGF_C单抗轻链可变区的LCDR1,LCDR2和LCDR3的氨基酸序列分别列于SEQ ID NO:14,15和16。此外,包含上述可变区的轻链可变区基因及编码的氨基酸序列如SEQ ID NO:22和17所示。
AntiVEGF_C单抗重链可变区的HCDR1,HCDR2和HCDR3的氨基酸序列分别于SEQ IDNO:18,19和20。此外,包含上述可变区的重链可变区基因及编码的氨基酸序列如SEQ IDN0:23和21所示。
优化AntiVEGF_C单抗轻链可变区基因和重链可变区基因,将轻链可变区基因克隆到pcDNA3.1-LFc载体中,构建pcDNA3.1-CL-LFc载体,如图5所示;将重链可变区基因克隆到pcDNA3.1-HFc载体中,构建pcDNA3.1-CH-HFc载体,如图6所示,本实施例中,pcDNA3.1-LFc和pcDNA3.1-HFc中已经含有人源的轻链恒定区和重链恒定区,将pcDNA3.1-CL-LFc和pcDNA3.1-NH-CFc按照1:1共转染到CHO-S细胞中,重组表达抗VEGF的C端抗体AntiVEGF_C,如图7所示。
实施例四:AntiVEGF_N和AntiVEGF_C抗体对在化学发光法诊断试剂盒中的应用
按照现有化学发光法诊断试剂生产工艺,将本发明中AntiVEGF_C作为捕获抗体进行包被配置成VEGF包被板,将本发明中AntiVEGF_N标记作为结合抗体进行配置成VEGF酶结合物,将VEGF抗原配置成系列校准品(0pg/ml、50pg/ml、200pg/ml、800pg/ml、1600pg/ml、3200pg/ml)形成一套完整试剂盒并对其进行一系列验证。
1、性能验证指标
参照YY/T1175-2010肿瘤标志物定量测定试剂(盒)化学发光法行业标准,制定对VEGF免洗检测试剂的性能验证指标
(1)最低检测限
应不高于40pg/ml。
(2)剂量-反应曲线的线性
在线性区间0pg/ml~3200pg/ml内,剂量-反应曲线线性相关系数(r)应不小于0.9900。
(3)准确度
以VEGF校准品检测试剂检测国际标准品,准确度应在90%~110%范围内。
(4)精密度
精密度(CV)应不大于10%。
(5)特异性
20μg/ml的人成纤维细胞生长因子2(FGF2),30ng/ml的表皮生长因(EGF),检测表观值应不大于50pg/ml。
2、实验配置
(1)VEGF包被板配置
将AntiVEGF_C作为捕获抗体,加入到包被缓冲液(0.05mol PBS),按照1ug/ml比例加入。每个空白发光板板孔加入100ul,4℃过夜,取出,用洗板洗涤液(0.9%NaCl)洗涤2次,加入每孔200ul封闭液(0.5mol PBS+1%BSA+2.5%蔗糖),4℃过夜,取出,弃掉板中液体,干燥后制成VEGF包被板。
(2)VEGF酶结合物配置
A、与检测抗体结合的酶为辣根过氧化物酶、碱性磷酸酶、荧光素酶、β-半乳糖苷酶、葡萄糖氧化酶、溶菌酶和苹果酸脱氢酶中的一种,根据与检测抗体结合的酶选择相应的检测方法。
B、本实施例中,AntiVEGF_N抗体标记HRP,取1mg/mlAntiVEGF_N抗体,并用醋酸溶液活化1mgHRP备用,抗体和HRP按1:1混合后颠倒混匀,2h后加入硼氢化钠终止反应,得到AntiVEGF_N-HRP结合抗体。
将AntiVEGF_N-HRP作为检测抗体,加入到酶稀释液(50Mmol Tris+1%BSA),按照1/1000比例加入。
(3)校准品配置
将VEGF抗原(久丰润达,20190306,0.15mg/ml)用抗原稀释液(0.5mol PBS+1%BSA)稀释6个梯度(0pg/ml、50pg/ml、200pg/ml、800pg/ml、1600pg/ml、3200pg/ml)。
(4)国际标准品(NIBSC 02/286)配置
将国际标准品用抗原稀释液(0.5mol PBS+1%BSA)稀释5个梯度(50pg/ml、200pg/ml、800pg/ml、1600pg/ml、3200pg/ml)。
(5)质控品配置
将VEGF抗原(购自久丰润达,20190306,0.15mg/ml)用抗原稀释液(0.5mol PBS+1%BSA)稀释成QC1(200pg/ml±10%),QC2(1750pg/ml±10%)。
(6)特异性样本配置
将FGF2(NIBSC 91/550)和EGF(NIBSC 90/712)用稀释液(0.5mol PBS+1%BSA)分别稀释成20μg/ml和30ng/ml。
3、加样操作,如图8所示
(1)分别取上述配置的(3)-(6)VEGF校准品、国际标准品,质控品及特异性样本各50μl加入上述配置的(1)VEGF包被板相应包被板板孔内,再取上述配置的(2)VEGF酶结合物50μL加入各孔振荡器振荡30秒钟,使其充分混合均匀。
(2)盖上封板膜,置37℃温育60分钟。
(3)取出,用应用洗涤液(0.5mol PBS+0.025%T-20)洗板5次。
(4)每孔加入底物液100ul(购买自ThermoFisher T2142)。
(5)用中生百克BHP9504化学发光仪检测化学发光强度(RLU)。
4、性能指标考核
(1)最低检测限
表2
由表2可知,最低检测限为4.73pg/ml,不高于40pg/ml,符合指标。
(2)剂量-反应曲线的线性
复孔测定试剂盒的参考品(S0~S5,浓度依次为0pg/ml、50pg/ml、200pg/ml、800pg/ml、1600pg/ml、3200pg/ml),平行测定三次,用四参数进行拟合,三次测定的剂量-反应曲线如图9、图10和图11所示(需要咱们补下图)。测定的剂量-反应曲线线性相关系数(R2)如表3所示。
表3
第一次测 | 第二次测 | 第三次测 | |
线性相关系数R<sup>2</sup> | 0.9999 | 0.9997 | 0.9997 |
由图7、图8和图9,以及表3可知,连续三次测定0-3200pg/ml浓度范围VEGF抗原曲线,线性相关系数R2均大于0.9990,符合指标。
(3)准确度
将国际标准品(13ug/支)稀释成理论浓度为50pg/ml、200pg/ml、800pg/ml、1600pg/ml、3200pg/m标本,检测3次,计算测定浓度与理论浓度的比值,结果如表4所示。
表4
由表4可知,连续三次检测由VEGF国际标准品配制的准确性标本,结果准确度均在95~105%范围内,在90%~110%要求内,符合指标。
(4)精密度
平行测定质控品Q1和质控品Q2各10孔,分别按照公式计算CV,结果如表5所示。
SD—质控品测定浓度值的标准差
表5
由表5可知,连续三次平行测定QC1和QC2,结果均不大于10%要求,符合指标。
(4)特异性
在稀释液中分别加入人成纤维细胞生长因子2(FGF2)和表皮生长因子(EGF),配制成含20g/ml的FGF2样本和30ng/ml的EGF样本,进行检测,测定结果如表6所示:
表6
项目 | 发光值 | 浓度值(pg/mL) |
人成纤维细胞生长因子2(20μg/ml) | 14858 | 11.265 |
表皮生长因子(30ng/ml) | 8774 | 6.571 |
由表6可知,人成纤维细胞生长因子2(20μg/ml)和表皮生长因子(30ng/ml),检测表观值分别为11.265pg/ml和6.571pg/ml,不大于50pg/ml,符合要求。
本实施例中,VEGF检测试剂盒的最低检测限、剂量-反应曲线、准确性、精密度和特异性性能指标均符合现有技术要求并优于市场现有方法学试剂。
实施例五:临床检测
选取40份健康人血清标本及10份经确诊的恶性肿瘤病例血清,用本试剂盒及电化学发光法VEGF试剂盒对40份健康人血清标本及10份经确诊的恶性肿瘤病例血清进行检测,在临床上健康人血清发光值均小于200;性肿瘤病例血清发光值均高于400。
1)40份健康人血清标本检测发光强度如表7所示:
表7
(2)10份恶性肿瘤血清标本检测发光强度如表8所示:
表8
根据表7和表8绘制健康人血清与性肿瘤病例血清剂量-反应曲线,如图12所示。
结果由表7和表8所示,基于本申请试剂盒与现有电化学发光法VEGF试剂盒临床诊断结果符合率100%。
如图10所示,基于本申请试剂盒检测浓度结果与已有电化学发光法VEGF试剂盒检测浓度结果相关性R2为0.9930,符合YY/T1175-2010肿瘤标志物定量测定试剂(盒)化学发光法行业标准。
本申请中,检测抗体可以与多种检测标记蛋白结合,检测抗体的检测方法包括酶联免疫(ELISA)检测法、化学发光检测检测法、蛋白质印迹检测检测法、免疫组化(IHC)检测法和免疫层析检测检测法,便于根据需要选择合适的检测标记蛋白,大大提高了本申请抗体对制成的试剂盒的便捷性。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明的申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
序列表
<110> 廊坊天光生物技术有限公司
<120> 一种用于检测血清中VEGF含量的抗体对及其用途
<150> 2020103896789
<151> 2020-05-11
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ggtaatgccc caaggcttct gattagcggt gctacaaact tggagactgg agtgcccagc 180
agattttccg gatctggata cggtaccaac tattctttga ctattagcaa tcttgagcag 240
gaggacatcg ccacatactt ctgccaacag agaaacacat tgccatacac ctttggcggc 300
gggaccaagc tcgaaatcaa a 321
<210> 13
<211> 363
<212> DNA
<213> 小鼠(Mus musculus)
<400> 13
gaagttcagc tggttgaaag cggtggcgat cttgtgaaac ctggaggcag tttgaagttg 60
agctgcgccg catctgggtt tactttttct gcctattata tgtattgggt caggcagaca 120
ccagaaaaga ggctggaatg ggtggcatat attcgcctgc ggagtaataa ttatactaac 180
aactataacc ccagccttaa gaaccgactg tcaatcacac gggataacgc taagaataat 240
ctgtacttgc acatgtcctc cctcaggagc gaagataccg ccatgtacta ttgtgcccgc 300
tactccaatt acgcttacgc tctcgattac tgggggcagg gaaccaccgt gacagtgtcc 360
agc 363
<210> 14
<211> 5
<212> PRT
<213> 小鼠(Mus musculus)
<400> 14
Ser Ser Val Ser Tyr
1 5
<210> 15
<211> 3
<212> PRT
<213> 小鼠(Mus musculus)
<400> 15
Asp Thr Ser
1
<210> 16
<211> 10
<212> PRT
<213> 小鼠(Mus musculus)
<400> 16
Gln Gln Trp Ser Arg His Pro Pro Ile Thr
1 5 10
<210> 17
<211> 107
<212> PRT
<213> 小鼠(Mus musculus)
<400> 17
Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Val Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg His Pro Pro Ile
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 18
<211> 8
<212> PRT
<213> 小鼠(Mus musculus)
<400> 18
Gly Phe Thr Phe Ser Ser Phe Gly
1 5
<210> 19
<211> 8
<212> PRT
<213> 小鼠(Mus musculus)
<400> 19
Ile Asn Ser Asp Ser Ser Ala Phe
1 5
<210> 20
<211> 12
<212> PRT
<213> 小鼠(Mus musculus)
<400> 20
Ala Arg Ala Gly Asn His Tyr Tyr Gly Gly Ala Tyr
1 5 10
<210> 21
<211> 119
<212> PRT
<213> 小鼠(Mus musculus)
<400> 21
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val
35 40 45
Ala Phe Ile Asn Ser Asp Ser Ser Ala Phe Tyr Tyr Ala Asp Thr Val
50 55 60
Gln Asp Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Ala Gly Asn His Tyr Tyr Gly Gly Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 22
<211> 321
<212> DNA
<213> 小鼠(Mus musculus)
<400> 22
cagatagtcc tttcacagag ccctgcaatc cttagcgcct ctcctggtga gaaggtcacc 60
atgacctgtc gggcatcatc atccgtctcc tacatgcatt ggtaccagca gagacctggc 120
agctccccta agccctggat atacgataca agcaacctgg caagcggtgt gcctgtgaga 180
tttagcggat caggcagcgg cacatcatat agcttgacta tttccagggt agaagtggaa 240
gatgccgcca catattactg ccagcagtgg tccaggcacc caccaatcac attcggatcc 300
ggcacaaagc tggagatcaa a 321
<210> 23
<211> 357
<212> DNA
<213> 小鼠(Mus musculus)
<400> 23
gaagttcagc ttgttgaatc cggaggtgga ttggttcagc ctggaggatc tcgtaaactg 60
tcttgtgccg ccagcggctt caccttcagt agtttcggaa tgcactgggt gcgtcaggca 120
cccgagaaag gcctcgaatg ggtggctttc ataaatagcg actcatccgc tttctactat 180
gccgatactg tccaggacag atttaccatc agtcgagaca accccaagaa tacactgttc 240
ctgcagatga cctccctgcg aagtgaggac actgccatgt actactgtgc acgagctggg 300
aatcattact atggaggcgc atactggggg cagggcacac ttgtgactgt cagcgct 357
Claims (11)
1.一种用于检测血清中VEGF含量的抗体对,其特征在于:包括与VEGF的N端表位即SEQID NO:2所示氨基酸序列相结合的单克隆抗体AntiVEGF_N和与VEGF的C端表位即SEQ IDNO:3所示氨基酸序列相结合的单克隆抗体AntiVEGF_C,所述单克隆抗体AntiVEGF_N包括SEQ ID NO:7所示氨基酸序列的轻链可变区和SEQ ID NO:11所示氨基酸序列的重链可变区,所述AntiVEGF_C包括SEQ ID NO:17所示氨基酸序列的轻链可变区和SEQ ID NO:21所示氨基酸序列的重链可变区。
2.如权利要求1所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述单克隆抗体AntiVEGF_N轻链氨基酸序列包括SEQ ID NO:4所示的LCDR1,SEQ ID NO:5所示的LCDR2,以及SEQ ID NO:6所示的LCDR3;所述单克隆抗体AntiVEGF_N重链氨基酸序列包括SEQ ID NO:8所示的HCDR1,SEQ ID NO:9所示的HCDR2,以及SEQ ID NO:10所示的HCDR3。
3.如权利要求2所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述单克隆抗体AntiVEGF_C轻链氨基酸序列包括SEQ ID NO:14所示的LCDR1,SEQ ID NO:15所示的LCDR2,以及SEQ ID NO:16所示的LCDR3;所述单克隆抗体AntiVEGF_C重链氨基酸序列包括SEQ ID NO:18所示的HCDR1,SEQ ID NO:19所示的HCDR2,以及SEQ ID NO:20所示的HCDR3。
4.如权利要求3所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述单克隆抗体AntiVEGF_N和AntiVEGF_C均为IgG型单克隆抗体,且一个为捕获抗体,一个为检测抗体。
5.如权利要求4所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述AntiVEGF_N为捕获抗体,所示AntiVEGF_C为检测抗体。
6.如权利要求4所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述检测抗体的检测标记物为检测标记蛋白、荧光基团、放射性同位素中的一种。
7.如权利要求6所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述检测抗体的检测标记蛋白包括辣根过氧化物酶、碱性磷酸酶、荧光素酶、β-半乳糖苷酶、葡萄糖氧化酶、溶菌酶和苹果酸脱氢酶,且检测抗体与检测标记蛋白重组表达。
8.如权利要求7所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述检测抗体的检测方法包括酶联免疫(ELISA)检测法、化学发光检测检测法、蛋白质印迹检测检测法和免疫组化(IHC)检测法和免疫层析检测检测法。
9.如权利要求1所述的一种用于检测血清中VEGF含量的抗体对,其特征在于,所述抗体对选自与VEGF结合的Fab、F(ab’)2、Fab’、scFv和di-scFv的抗体片段。
10.一种DNA分子,其特征在于,它编码权利要求1或2或3所述的抗体。
11.如权利要求1-10所述的抗体对在制备检测血清中VEGF含量的试剂盒中的应用。
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