CN116284415B - 一种纤溶酶-α2抗纤溶酶复合物单克隆抗体及其制备和应用 - Google Patents
一种纤溶酶-α2抗纤溶酶复合物单克隆抗体及其制备和应用 Download PDFInfo
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Abstract
本发明属于医学检验体外诊断领域,尤其涉及特异性纤溶酶‑α2抗纤溶酶复合物单克隆抗体及其制备和应用。所述抗体是特异性单克隆抗体PIC mAb 1,其轻链氨基酸序列如SEQ ID NO:7所示,重链氨基酸序列如SEQ ID NO:9所示。能够稳定表达PIC抗体的CHO细胞株,其命名为中国仓鼠卵巢细胞CHO PIC,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C202203。本发明PIC检测试剂盒的准确性与临床常用试剂盒一致,并且具有准确度高、精密度高、重复性好等特点。
Description
技术领域
本发明属于医学检验体外诊断领域,尤其涉及一种纤溶酶-α2抗纤溶酶复合物(PIC)抗体及其制备和应用。
背景技术
临床上将血栓形成和血栓栓塞两种病理过程引起的疾病称为血栓性疾病,血栓形成过程主要受血管及血流状态影响,而血流状态的稳定主要由凝血系统和抗凝血系统维持,其中纤维蛋白溶解系统在抗凝血系统中发挥重要作用。纤溶酶是纤维蛋白溶解系统的核心,游离的纤溶酶很快即被其特异性抑制剂(α2纤溶酶抑制剂)按1:1比例结合形成PIC复合物,是临床上检测血栓性疾病的重要生物标志物。
PIC是反映纤溶系统激活的早期标志物,提示纤溶酶原激活为纤溶酶。一方面,通过降解纤维蛋白,除去体内多余的血栓;另一方面,通过负反馈降低体内纤维蛋白原水平,从而减少血栓形成。由于纤溶酶半衰期短,体内不容易测定,而形成的PIC复合物血浆半衰期约6h,可直接测定。
在临床样本检测中,采用针对PIC的抗体可以检测PIC复合物含量。然而,常用的PIC检测方法采用酶标板为固相载体包被抗体,其与样品反应不充分、灵敏度低、检测效率低,且现有市售PIC复合物测定试剂盒多依赖进口。因此,亟需开发国产化、低成本的高灵敏度、高特异度的PIC抗体及检测试剂盒。
发明内容
本发明为了解决检测技术中存在的问题,制备特异性检测PIC复合物的抗体,开发新型PIC抗体免疫比浊试剂盒。
本发明是通过如下技术方案实现的:
第一方面,本发明提供了一种纤溶酶-α2抗纤溶酶复合物单克隆抗体,所述纤溶酶-α2抗纤溶酶复合物单克隆抗体的轻链氨基酸序列如SEQ ID NO:7所示,重链氨基酸序列如SEQ ID NO:9所示。
本发明提供的PIC单克隆抗体轻链氨基酸序列为:QAVVIQESALTTSPGDNASILHEVPTGAVTTSNYANWVQEKPDHLFTGLIGGSNNRAPGVPDKFIILEPKVKAALTITGAQTEDEAIYFCGLLYSNNWVFGGGTKLTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
重链氨基酸序列为:EVQLQQSGPELVKPGASVYEDNWAILLTFTLYVIHWVKQKPGQGLEWIGYINPYIDGTKYNEKFKGKDFSVWQNPLHTAFMELSSLTSEDSAVYYCARSGYGNYGLAWLAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
第二方面,本发明提供了一种纤溶酶-α2抗纤溶酶复合物单克隆抗体的制备方法,包括以下步骤:
S1.单克隆抗体的初步制备:
用完全弗氏佐剂和天然抗原免疫小鼠,分离脾脏细胞、在PEG融合剂作用下与SP2/0细胞融合,并在HAT选择培养基上筛选能产生单克隆抗体的杂交瘤细胞株;
S2.单克隆抗体序列的获得
提取杂交瘤细胞株mRNA、RT-PCR扩增抗体轻链和重链可变区DNA的序列、对扩增后的DNA序列测序;
S3.单克隆抗体序列的构建
轻链:将测序获得的轻链可变区DNA序列加上小鼠抗体IgK保守区的DNA序列;
重链:将测序获得的重链可变区DNA序列加上小鼠重链IgG1保守区的DNA序列;
S4.单克隆抗体的表达
利用基因工程技术构建构建抗体轻链和重链表达载体,将载体转染进入CHO细胞系,进行抗体稳定表达。
进一步的,所述步骤步骤S2中,5’端引物序列为:ATTGT CCTTA ATGGG GGG
轻链混合引物:
5’-ACAGT TGGTG CAGCA TCTGC-3’(IgK保守区5’端反向引物)
5’-GGCGA AGACT TGGGC TGGCC-3’(IgL1保守区5’端反向引物)
5’-GGAGT GGACT TGGGC TGACC-3’(IgL2保守区5’端反向引物)
重链混合引物:
5’-CTAGG GGGTG TCGTT TTAGC-3’(IgG1保守区5’端反向引物)
5’-GATGG GGCTG TTGTT TTAGC-3’(IgG2a保守区5’端反向引物)
5’-GATGG GGGTG TTGTT TTAGC-3’(IgG2b保守区5’端反向引物)
5’-GATGG GGCTG TTGTT GTAGC-3’(IgG3保守区5’端反向引物)。
进一步的,所述步骤S1中,与骨髓瘤细胞的融合的脾脏细胞为末次免疫后第三天取脾制成的细胞悬液,其骨髓瘤细胞的混合比例为1:5。
第三方面,本发明提供了纤溶酶-α2抗纤溶酶复合物单克隆抗体在免疫比浊体外诊断试剂盒中的应用。
第四方面,本发明提供了用于稳定表达纤溶酶-α2抗纤溶酶复合物单克隆抗体的CHO细胞株,所述CHO细胞株的保藏编号为CCTCC NO:C202203,其命名为中国仓鼠卵巢细胞CHO PIC,保藏于中国典型培养物保藏中心,保藏日期为2022年01月12日,保藏地址为:湖北省武汉市武昌区八一路299号武汉大学。
与现有技术相比,本发明的有益效果在于:
本发明制备的抗体是特异性单克隆抗体PIC mAb 1,其轻链氨基酸序列如SEQ IDNO:7所示,重链氨基酸序列如SEQ ID NO:9所示。能够稳定表达PIC抗体的CHO细胞株,其命名为中国仓鼠卵巢细胞CHO PIC,保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:C202203。本发明提供的抗体具有良好特异性,PIC检测试剂盒的准确性与临床常用试剂盒一致,并且具有准确度高、精密度高、重复性好等特点,适合免疫比浊法测定PIC推广使用。
附图说明
下面结合附图对本发明作进一步的说明。
图1为本发明ELISA检测PIC mAb1与纤溶酶交叉反应图。
具体实施方式
为了能够更清楚地理解本发明的目的、特征和优点,下面结合具体实施例对本发明做进一步说明。需要说明的是,在不冲突的情况下,本申请的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了具体细节以便于充分理解本发明。此外,本发明还可以采用不同于在此描述的其他方式来实施,因此,本发明并不限于下面公开说明书的具体实施例的限制。
实施例1
本实施例提供单克隆PIC抗体的制备过程。本实施例未特殊说明之处,采用生物领域抗体制备和细胞培养等过程的常规技术手段。
1. 免疫动物
1.1 免疫动物的选择
本实施例选用6-8周龄雌性BALB/c小鼠。
1.2免疫动物
采用天然PIC抗原(Kmaels)进行动物免疫。动物的免疫通常共4次,初次免疫将100μg抗原与等量的弗氏完全佐剂充分乳化后,腹腔或皮下多点注射。以后每隔2周,将50μg抗原与等量的不完全佐剂混合后注射,在最后一次加强免疫后第3天取脾进行融合。
2. 制备杂交瘤细胞
2.1 脾淋巴细胞制备
已经免疫的BALB/c小鼠,在无菌条件下去除脾脏,并完成脾淋巴细胞的制备。
2.2 骨髓瘤细胞制备
选取骨髓瘤细胞,用含10%-20%小牛血清的DMEM培养基进行培养,细胞浓度以104~105/mL为宜。当细胞处于对数生长的中期时,可按1:3~1:10的比例传代。
3.细胞融合
3.1 将骨髓瘤细胞与脾细胞按1:5的比例混合在一起,在50mL离心管中用无血清不完全培养液洗1次,1200r/min离心8 min,弃去上清,吸净残留液体,轻轻弹击离心管底,使细胞沉淀略松动。
3.2 用吸管在60s 内加入预热至40℃的50% PEG(PH= 8)1mL,边加边轻轻搅拌。
3.3 用10mL吸管在90s内加入20-30mL预热的不完全培养基(终止PEG作用);20-27℃静置10 min。
3.4 1000r/min 离心6 min,弃上清。
3.5 HAT培养基重悬,并进行单克隆筛选。
4.经3次融合,得到21株抗PIC特异的单克隆抗体株,分别按序号命名为命名为PICmAb 1至PIC mAb 21。
5.抗体的质量控制
骨髓瘤细胞:SP2/0本身不合成或不分泌免疫球蛋白,来源历史明确,保存条件符合要求。
细胞融合:末次免疫后第3天取脾制成细胞悬液,与骨髓瘤细胞按1:5的比例混合,在PEG作用下进行细胞融合。
克隆化:用ELISA法筛选出分泌目的抗体的杂交瘤,经有限稀释法对其进行克隆,直至获得产生单克隆抗体的杂交瘤细胞系。
6.杂交瘤细胞的鉴定
抗体分泌稳定性:经克隆化及连续传代检查,连续克隆化至检测单克隆抗体的阳性率达100%。
7.单克隆抗体的鉴定
特异性分析:将所得到的特异性单克隆抗体与系列无关抗原进行ELISA反应,检测所得抗体是否与无关免疫原产生交叉反应。检测结果如图1所示,本实施例制得的PIC mAb1与其他抗原基本没有交叉反应。
8.抗体DNA序列的获得
8.1 按Invitrogen™ PureLink™ RNA提取试剂盒说明书的方法,从单克隆细胞中提取总RNA。
8.2然后按照Thermo Scientific™ RevertAid RT 逆转录试剂盒说明书,按如下体系将抗体轻链可变区和重链可变区的RNA逆转录为cDNA,具体反应体系如下表1:
表1 反应体系 (42℃,30min)
8.3按照Invitrogen™末端脱氧核苷酸转移酶 (TdT) 说明书在逆转录产物后端加入polyC尾,具体反应体系如下表2:
表2 反应体系(37℃,15min)
8.4按Thermo Scientific™PCR Master Mix (2X)说明书用PCR技术分别扩增抗体轻链、重链DNA,PCR反应条件为:预热95℃ 5min,循环(变性95℃ 30s,退火55℃ 30s,延伸72℃ 30s),长时延伸(72℃ 10min)。
具体反应体系如下表3:
表3 反应体系
5’端引物序列为:ATTGT CCTTA ATGGG GGG
轻链混合引物:
5’-ACAGT TGGTG CAGCA TCTGC-3’(IgK保守区5’端反向引物)
5’-GGCGA AGACT TGGGC TGGCC-3’(IgL1保守区5’端反向引物)
5’-GGAGT GGACT TGGGC TGACC-3’(IgL2保守区5’端反向引物)
重链混合引物:
5’-CTAGG GGGTG TCGTT TTAGC-3’(IgG1保守区5’端反向引物)
5’-GATGG GGCTG TTGTT TTAGC-3’(IgG2a保守区5’端反向引物)
5’-GATGG GGGTG TTGTT TTAGC-3’(IgG2b保守区5’端反向引物)
5’-GATGG GGCTG TTGTT GTAGC-3’(IgG3保守区5’端反向引物)
8.5按ChargeSwitch™ PCR 纯化试剂盒说明书、纯化PCR产物。
9.可变区DNA测序
利用Sanger测序法对上述纯化后的PCR产物进行DNA测序,测序结果见序列表。
PIC mAb 1轻链可变区氨基酸序列如SEQ ID NO:1所示,其DNA序列如SEQ ID NO:2所示。
PIC mAb 1的重链可变区氨基酸序列如SEQ IDNO:3所示,其DNA序列如SEQ ID NO:4所示。
10.抗体基因的改造
在抗体轻链可变区DNA序列后加入小鼠抗体IgK轻链保守区,DNA序列如SEQ IDNO:5所示。
在抗体重链可变区Fab端DNA序列后加入小鼠重链IgG1保守区,DNA序列如SEQ IDNO:6所示。
PIC mAb 1抗体轻链氨基酸序列如SEQ ID NO:7所示,其DNA序列如SEQ ID NO:8所示。
PIC mAb 1抗体重链氨基酸序列如SEQ ID NO:9所示,其DNA序列如SEQ ID NO:10所示。
11.PIC抗体的制备
11.1人工合成DNA序列SEQ ID NO:8进行连接到pcDNA3.1质粒中;
11.2人工合成DNA序列SEQ ID NO:10进行连接到pcDNA3.1质粒中;
11.3将以上两个质粒进行1:1混合,按Gibco™ ExpiFectamine™ CHO 转染试剂盒说明书将质粒转染到CHO细胞系中表达。
11.4转染72h后收集细胞进行裂解,上清收集,蛋白表达鉴定。
11.5经非还原型SDS-PAGE电泳,采用半干式电转法将蛋白从凝胶转移到PVDF膜上,用HRP-羊抗人IgG-Fab酶标抗体检测蛋白表达情况。
11.6 收集的抗体裂解液经ProteinG亲和纯化,依次加入洗涤缓冲液和洗脱缓冲液,收集洗脱物。
11.7 抗体浓缩:汇集纯化洗脱物,超滤浓缩,最后分装冻存。
上述PIC mAb 1抗体的纯化过程,除了已特别说明的部分外,都在0-4℃进行。
实施例2
本实施例提供免疫比浊反应试剂的配制过程。
1.R1试剂配制
3-双(2-羟乙基)氨基-2-羟基丙磺酸(DIPSO)缓冲液浓度为0.05mol/L,用0.1mol/L HCl溶液调节pH至7.5。再加入0.15mol/L氯化钠,0.1%曲拉通X-100,0.02%ProClin300。其中,DIPSO缓冲液起到稳定反应体系pH值的作用,而氯化钠为反应体系提供离子环境,曲拉通X-100为表面活性剂,ProClin 300为防腐剂。
2. R2试剂配制
2.1 0.05mol/L磷酸盐缓冲液(pH=7.2)的配制
首先单独配制0.05mol/L的NaH2PO4溶液和0.05mol/L Na2HPO4的溶液。其中0.05mol/L的NaH2PO4配制如下:称取7.8015g的NaH2PO4-2H2O,加入到1L水中。同理,将17.911g Na2HPO4-12H2O溶于1L水中配制0.05mol/L Na2HPO4的溶液。之后取28mL NaH2PO4溶液与72mL Na2HPO4溶液,搅拌混合。
2.2 0.5mol/L MES缓冲液(pH=6)的配制
称取2-吗啉乙磺酸97.6g,加入1L纯化水中,室温下充分溶解,用氢氧化钠调节pH至6(0.5mol/L)。
2.3其他物料的称量
2.3.1 NHS:称取NHS 2g,加入5mL纯化水搅拌溶解,搅拌5min使其充分溶解。
2.3.2 EDC:称取EDC 1.4g,加入3mL纯化水搅拌溶解,搅拌5min使其充分溶解。
2.3.3聚苯乙烯乳胶珠:量取聚苯乙烯乳胶珠2.5mL(10%聚苯乙烯乳胶珠)。
2.3.4牛血清白蛋白:称取牛血清白蛋白10g(1%牛血清白蛋白)。
2.3.5 ProClin 300:量取ProClin300 0.2mL(0.02%ProClin 300)。
2.4聚苯乙烯乳胶微球的活化
2.4.1将溶解的EDC溶液加入NHS溶液中混合均匀。
2.4.2将上述聚苯乙烯乳胶珠加入溶解好的EDC和NHS的混合溶液中,再加入2.5mL0.5mol/L MES缓冲液(pH=6),然后加入5倍体积(聚苯乙烯乳胶珠)纯化水进行充分溶解,室温反应30min,不断搅拌。
2.4.3将配制好的聚苯乙烯乳胶溶液转移至离心瓶中,配平后离心,15000rpm,8℃,50min。
2.4.4弃上清,收集离心沉淀;加入40mL磷酸盐缓冲液,充分重悬聚苯乙烯乳胶珠,配平后离心,15000rpm,8℃,40min;连续两次。
2.4.5弃上清,收集离心沉淀;加入40mL磷酸盐缓冲液重新悬浮聚苯乙烯乳胶珠,超声20min(150W,3s,间隔10s,30次)使其分散。
2.5 PIC单克隆抗体的偶联
2.5.1按每2.5 mg聚苯乙烯乳胶珠加入0.2mg的PIC单克隆抗体,加入上述活化后乳胶微球,室温搅拌30min,然后将烧杯移至2-8℃过夜搅拌。
2.5.2 15000rmp离心50min,弃上清。用磷酸盐缓冲液洗涤两次,离心沉淀后,弃上清,用40mL磷酸盐缓冲液重悬抗PIC抗体乳胶,超声20min(150W,3s,间隔10s,30次)。
2.5.3加入牛血清白蛋白和ProClin 300,充分溶解,加入460mL磷酸盐缓冲液,即得R2试剂。
实施例3
实验室检测结果:
A.检测试剂的准确性
取50例临床标本,将本发明试剂盒与希森美康试剂盒做临床阴阳性比对,结果如下表4:
表4 准确性检验结果
检测结果表明,本发明试剂盒与完全符合标准诊断方法希森美康试剂盒的检测结果,具有100%的检测准确度。
B. 检测试剂的线性范围
将希森美康试剂盒检测值为38.74μg/mL的血样,用去除PIC的人基质血清溶液作为稀释液,配置浓度为30μg/mL,15μg/mL,5μg/mL,1μg/mL,0.5μg/mL,0.1μg/mL,0μg/mL的PIC标准品,使用本发明试剂盒进行检测,检测结果如下表5:
表5 线性范围检测结果
由此可见本发明检测试剂盒能够检测0-30μg/mL的抗原浓度,且与抗原理论浓度值相符,具有较高的可信度。
C.检测试剂的精密度
将希森美康试剂盒检测值为0.74μg/mL和25.17μg/mL的血样用本发明试剂盒测试10次,检测本发明试剂盒的精密度,结果如下表6:
表6 精密度检测结果
针对希森美康试剂盒检测值为0.74μg/mL和25.17μg/mL的血样,用本发明试剂盒进行10次重复检测,均值分别为0.73μg/mL和25.69μg/mL,CV分别为3.22%和1.90%,与标准化检测方法的结果高度一致。
序列表:
SEQ ID NO:1:
QAVVIQESALTTSPGDNASILHEVPTGAVTTSNYANWVQEKPDHLFTGLIGGSNNRAPGVPDKFIILEPKVKAALTITGAQTEDEAIYFCGLLYSNNWVFGGGTKLTVL
SEQ ID NO:2:
CAAGCTGTTGTTATTCAAGAGTCTGCTTTAACTACTTCTCCTGGAGATAATGCTTCTATTTTACATGAGGTTCCTACTGGAGCTGTTACTACTTCTAATTATGCTAATTGGGTTCAAGAGAAACCTGATCATTTATTTACTGGATTAATTGGAGGATCTAATAATCGTGCTCCTGGAGTTCCTGATAAATTTATTATTTTAGAGCCTAAAGTTAAAGCTGCTTTAACTATTACTGGAGCTCAAACTGAGGATGAGGCTATTTATTTTTGTGGATTATTATATTCTAATAATTGGGTTTTTGGAGGAGGAACTAAATTAACTGTTTTA
SEQ ID NO:3:
EVQLQQSGPELVKPGASVYEDNWAILLTFTLYVIHWVKQKPGQGLEWIGYINPYIDGTKYNEKFKGKDFSVWQNPLHTAFMELSSLTSEDSAVYYCARSGYGNYGLAWLAYWGQGTLVTVSA
SEQ ID NO:4:
GAGGTTCAATTACAACAATCTGGACCTGAGTTAGTTAAACCTGGAGCTTCTGTTTATGAGGATAATTGGGCTATTTTATTAACTTTTACTTTATATGTTATTCATTGGGTTAAACAAAAACCTGGACAAGGATTAGAGTGGATTGGATATATTAATCCTTATATTGATGGAACTAAATATAATGAGAAATTTAAAGGAAAAGATTTTTCTGTTTGGCAAAATCCTTTACATACTGCTTTTATGGAGTTATCTTCTTTAACTTCTGAGGATTCTGCTGTTTATTATTGTGCTCGTTCTGGATATGGAAATTATGGATTAGCTTGGTTAGCTTATTGGGGACAAGGAACTTTAGTTACTGTTTCTGCT
SEQ ID NO:5:
CGTGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG
SEQ ID NO:6:
GCTAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCGCAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA
SEQ ID NO:7:
QAVVIQESALTTSPGDNASILHEVPTGAVTTSNYANWVQEKPDHLFTGLIGGSNNRAPGVPDKFIILEPKVKAALTITGAQTEDEAIYFCGLLYSNNWVFGGGTKLTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO:8:
CAAGCTGTTGTTATTCAAGAGTCTGCTTTAACTACTTCTCCTGGAGATAATGCTTCTATTTTACATGAGGTTCCTACTGGAGCTGTTACTACTTCTAATTATGCTAATTGGGTTCAAGAGAAACCTGATCATTTATTTACTGGATTAATTGGAGGATCTAATAATCGTGCTCCTGGAGTTCCTGATAAATTTATTATTTTAGAGCCTAAAGTTAAAGCTGCTTTAACTATTACTGGAGCTCAAACTGAGGATGAGGCTATTTATTTTTGTGGATTATTATATTCTAATAATTGGGTTTTTGGAGGAGGAACTAAATTAACTGTTTTACGTGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG
SEQ ID NO:9:
EVQLQQSGPELVKPGASVYEDNWAILLTFTLYVIHWVKQKPGQGLEWIGYINPYIDGTKYNEKFKGKDFSVWQNPLHTAFMELSSLTSEDSAVYYCARSGYGNYGLAWLAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
SEQ ID NO:10:
GAGGTTCAATTACAACAATCTGGACCTGAGTTAGTTAAACCTGGAGCTTCTGTTTATGAGGATAATTGGGCTATTTTATTAACTTTTACTTTATATGTTATTCATTGGGTTAAACAAAAACCTGGACAAGGATTAGAGTGGATTGGATATATTAATCCTTATATTGATGGAACTAAATATAATGAGAAATTTAAAGGAAAAGATTTTTCTGTTTGGCAAAATCCTTTACATACTGCTTTTATGGAGTTATCTTCTTTAACTTCTGAGGATTCTGCTGTTTATTATTGTGCTCGTTCTGGATATGGAAATTATGGATTAGCTTGGTTAGCTTATTGGGGACAAGGAACTTTAGTTACTGTTTCTGCTGCTAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCGCAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA
以上所述,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例应用于其它领域,但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
Claims (3)
1.一种纤溶酶-α2抗纤溶酶复合物单克隆抗体,其特征在于,所述纤溶酶-α2抗纤溶酶复合物单克隆抗体的轻链氨基酸序列如SEQ ID NO:7所示,重链氨基酸序列如SEQ ID NO:9所示。
2.根据权利要求1所述的纤溶酶-α2抗纤溶酶复合物单克隆抗体在制备免疫比浊体外诊断试剂盒中的应用。
3.用于稳定表达如权利要求1所述的纤溶酶-α2抗纤溶酶复合物单克隆抗体的CHO细胞株,其特征在于:所述CHO细胞株的保藏编号为CCTCC NO:C202203。
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Citations (4)
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US5888753A (en) * | 1991-05-16 | 1999-03-30 | Behring Diagnostics Gmbh | Monoclonal antibodies against the plasmin-antiplasmin complex, a method for the preparation thereof and the use thereof |
US5888749A (en) * | 1990-12-20 | 1999-03-30 | Iatron Laboratories, Inc. | Anti-human plasmin-α2 -plasmin inhibitor complex antibodies, hybridomas, and immunological determination method |
CN114686441A (zh) * | 2020-12-31 | 2022-07-01 | 广州万孚生物技术股份有限公司 | 一种能分泌tPAI-C单克隆抗体的杂交瘤细胞株及其应用 |
CN115304677A (zh) * | 2022-06-19 | 2022-11-08 | 湖北真福医药有限公司 | 一种抗枯草芽孢杆菌纤溶酶的单克隆抗体qkma-9a10及应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
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US5888749A (en) * | 1990-12-20 | 1999-03-30 | Iatron Laboratories, Inc. | Anti-human plasmin-α2 -plasmin inhibitor complex antibodies, hybridomas, and immunological determination method |
US5888753A (en) * | 1991-05-16 | 1999-03-30 | Behring Diagnostics Gmbh | Monoclonal antibodies against the plasmin-antiplasmin complex, a method for the preparation thereof and the use thereof |
CN114686441A (zh) * | 2020-12-31 | 2022-07-01 | 广州万孚生物技术股份有限公司 | 一种能分泌tPAI-C单克隆抗体的杂交瘤细胞株及其应用 |
CN115304677A (zh) * | 2022-06-19 | 2022-11-08 | 湖北真福医药有限公司 | 一种抗枯草芽孢杆菌纤溶酶的单克隆抗体qkma-9a10及应用 |
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