CN116284425A - 一种抗壬基酚聚氧乙烯醚的单克隆抗体、试剂盒及应用 - Google Patents
一种抗壬基酚聚氧乙烯醚的单克隆抗体、试剂盒及应用 Download PDFInfo
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Abstract
本发明提供一种抗壬基酚聚氧乙烯醚的单克隆抗体、试剂盒及应用,抗壬基酚聚氧乙烯醚的单克隆抗体,包括轻链和重链,轻链可变区包括CDR1、CDR2、CDR3;重链可变区包括CDR1、CDR2、CDR3;轻链CDR1的氨基酸序列如SEQ ID NO.1所示,轻链CDR2的氨基酸序列如SEQ ID NO.2所示,轻链CDR3的氨基酸残基为LKI;重链CDR1的氨基酸序列如SEQ ID NO.3所示,重链CDR2的氨基酸序列如SEQ ID NO.4所示,重链CDR3的氨基酸序列如SEQ ID NO.5所示。该单克隆抗体与壬基酚聚氧乙烯醚结合力强,灵敏度好;该试剂盒的检测限更低。
Description
技术领域
本发明涉及快速生物技术检测领域,具体涉及一种抗壬基酚聚氧乙烯醚的单克隆抗体、试剂盒及应用。
背景技术
壬基酚聚氧乙烯醚(NPE)为壬基酚和环氧乙烷在催化剂作用下缩合反应的非离子表面活性剂。由于其具有良好的渗透、乳化、分散、抗酸、抗碱、抗硬水、抗还原、抗氧化能力,被广泛应用于洗涤剂、印染、化工等领域。研究表明,壬基酚聚氧乙烯醚被排放到环境中会迅速分解成壬基酚(NP),而壬基酚是一种环境激素,具有较强的生理学毒性,严重危害生物体繁殖系统,被动物摄取后可通过食物链进入人体。
目前壬基酚聚氧乙烯醚的常规检测方法有液相色谱-质谱和气相色谱-质谱法。液相色谱-质谱分辨率高,灵敏度高,分析速度快,重复性好。适合分析像壬基酚聚氧乙烯醚分子结构复杂,分子量分布范围宽的化合物。但液相色谱-质谱的价格昂贵,而且测试成本较高,目前国内难以普及。同时由于液相色谱柱的分离能力不强,测试过程易受杂质干扰,不能准确定量。与液相色谱-质谱相比,气相色谱-质谱仪具有分离能力强,灵敏度高的特点,能够准确的定性,而且该设备的成本远低于液相色谱-质谱。但其很重要的缺点是对很多异构体,尤其是位置异构无法分辨。仪器法进行定量分析具有较低的检测限,但是仪器操作复杂,费用昂贵,无法达到现场规模快速检测的要求。
发明内容
本发明解决的技术问题是提供一种抗壬基酚聚氧乙烯醚的单克隆抗体、试剂盒及应用,该单克隆抗体与壬基酚聚氧乙烯醚结合力强,灵敏度好;该试剂盒的检测限更低。
为了解决上述问题,本发明的一个方面提供一种抗壬基酚聚氧乙烯醚的单克隆抗体,包括轻链和重链,轻链可变区包括CDR1、CDR2、CDR3;重链可变区包括CDR1、CDR2、CDR3;
轻链CDR1的氨基酸序列如SEQ ID NO.1所示,轻链CDR2的氨基酸序列如SEQ IDNO.2所示,轻链CDR3的氨基酸残基为LKI;
重链CDR1的氨基酸序列如SEQ ID NO.3所示,重链CDR2的氨基酸序列如SEQ IDNO.4所示,重链CDR3的氨基酸序列如SEQ ID NO.5所示。
所述SEQ ID NO.1的序列结构为:RFSKSLGHSKVITYLY。
所述SEQ ID NO.2的序列结构为:QLSNLAS。
所述SEQ ID NO.3的序列结构为:SHLMH。
所述SEQ ID NO.4的序列结构为:ATSPGNSHTSSNPKFKG。
所述SEQ ID NO.5的序列结构为:TGYMLILYWYYAV。
优选地,所述轻链可变区的氨基酸序列如SEQ ID NO.6所示。
所述SEQ ID NO.6的序列结构为:
QLLGVMGFWIAGFTTDFDWRQAAFSNSVTVGSSAYISHRFSKSLGHSKVITYLYWYEQKAGQSPQLLIYQLSNLASGVADRFSNSGSGTDFTLRINRVEAEDVGVYYVLKIYFRTRSEGGPSWKYGLMLRQLYPSSHHPVSSNICPQSGART。
优选地,所述重链可变区的氨基酸序列如SEQ ID NO.7所示。
所述SEQ ID NO.7的序列结构为:
TWGQSLKTLTLIMECSWTLPFILSDTSGVYSKFQLLQSGNVLARPEASVKMTWKGSDYTLYSHLMHWEKQRPGQGLEWIDATSPGNSHTSSNPKFKGKGKLTADISTSSAYMRLSSLTNKYSAAYYCTRTGYMLILYWYYAVWCPGTTFTISSAQTTPPSVYPLPPGSAAPSTSMVSLGWLVKAYLP。
优选地,编码轻链可变区的基因序列如SEQ ID NO.8所示。
所述SEQ ID NO.8的序列结构为:
CAGCTTCTTGGGGTTATGGGTTTTTGGATTGCTGGATTCACAACAGATTTTGATTGGAGGCAGGCGGCATTCTCCAATTCAGTCACTGTTGGATCTTCAGCTTACATCTCCCACAGGTTTAGTAAGAGTCTCGGACATAGTAAAGTCATCACTTATTTGTATTGGTATGAGCAGAAAGCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGTTGTCCAACCTTGCCTCAGGAGTGGCAGACAGGTTCAGTAACAGTGGGTCAGGAACTGATTTCACATTGAGAATCAACAGAGTGGAGGCTGAGGATGTGGGTGTCTACTATGTTCTCAAAATCTACTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATACGGGCTGATGCTGCGCCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTAACATCTGTCCTCAGTCGGGCGCGAGAACG。
优选地,编码重链可变区的基因序列如SEQ ID NO.9所示。
所述SEQ ID NO.9的序列结构为:
ACATGGGGCCAGTCACTGAAAACATTGACTCTAATCATGGAATGTAGCTGGACACTTCCTTTTATTCTGTCGGATACTTCAGGGGTCTACTCAAAGTTTCAGCTCCTGCAGTCTGGGAATGTGCTGGCAAGGCCTGAGGCTTCCGTGAAGATGACCTGGAAGGGTTCTGACTACACCTTATACAGCCATTTGATGCACTGGGAAAAACAGAGGCCTGGACAGGGCCTGGAATGGATTGACGCTACTTCTCCTGGAAATAGTCATACTAGCTCCAACCCCAAGTTCAAGGGCAAGGGCAAACTGACTGCAGACATATCCACCAGCAGTGCCTACATGAGGCTCAGCAGCCTGACAAATAAGTACTCTGCGGCATATTACTGTACAAGAACAGGCTATATGTTAATTCTTTACTGGTACTACGCTGTCTGGTGCCCAGGGACCACGTTCACCATCTCCTCAGCCCAAACGACCCCCCCATCAGTCTATCCACTGCCCCCTGGATCAGCTGCCCCAAGTACCTCCATGGTGAGCCTGGGATGGCTGGTCAAGGCCTATTTGCCT。
本发明的另一个方面提供上述的抗壬基酚聚氧乙烯醚的单克隆抗体在制备检测壬基酚聚氧乙烯醚的试剂或试剂盒中的应用。
本发明的再一方面提供一种壬基酚聚氧乙烯醚检测试剂盒,所述试剂盒为胶体金检测试剂盒,所述胶体金检测试剂盒的微孔试剂为胶体金标记的抗壬基酚聚氧乙烯醚的单克隆抗体。
本发明与现有技术相比,具有以下有益效果:
本发明通过壬基酚聚氧乙烯醚人工抗原免疫小鼠,通过杂交瘤技术筛选得到了对壬基酚聚氧乙烯醚具有较高亲和力和检测灵敏度的单克隆抗体,为间接竞争ELISA试剂盒以及胶体金试纸条的研发推广奠定基础。试验证明:该单克隆抗体具有特异性强、灵敏度高等特点,可作为酶联免疫检测和胶体金免疫检测的原料。
附图说明
图1是本发明实施例2中采用间接竞争ELISA方法检测抗壬基酚聚氧乙烯醚单克隆抗体灵敏度的RIDA SOFT四参数法R2曲线图;
图2是本发明实施例2中采用间接竞争ELISA方法检测抗壬基酚聚氧乙烯醚单克隆抗体灵敏度的RIDA SOFT四参数法IC50曲线图;
图3是本发明实施例4中抗壬基酚聚氧乙烯醚单克隆抗体轻链基因序列同源性比对结果图;
图4是本发明实施例4中抗壬基酚聚氧乙烯醚单克隆抗体轻链氨基酸序列同源性比对结果图;
图5是本发明实施例4中抗壬基酚聚氧乙烯醚单克隆抗体重链基因序列同源性比对结果图;
图6是本发明实施例4中抗壬基酚聚氧乙烯醚单克隆抗体重链氨基酸序列同源性比对结果图;
图7是本发明实施例5的壬基酚聚氧乙烯醚检测试纸条的横截面图;
图8是本发明实施例5的壬基酚聚氧乙烯醚检测试纸条的俯视图;
图9是本发明实施例5的微孔试剂的结构示意图。
其中:1-吸水纸;2-硝酸纤维素膜;3-样品垫;4-质控线;5-检测线;6-底板;7-试纸条;8-微孔条;9-微孔试剂;10-微孔塞。
具体实施方式
下面将结合本发明的实施例,对本发明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 抗壬基酚聚氧乙烯醚的单克隆抗体的制备
1.1 免疫小鼠
初次免疫:将壬基酚聚氧乙烯醚人工抗原与弗氏完全佐剂乳化(1:1),皮下注射6-8周龄的Balb/c小鼠,免疫剂量为500μg/只。从首次免疫开始,每4周加强免疫一次,共加强免疫2次,用弗氏不完全佐剂代替弗氏完全佐剂,方法与剂量同首次免疫。每次免疫7-10天后,可眼底静脉采血检测效价和抑制,验证免疫效果。第三次免疫后,有抑制且效价达到1:10000以上时,可进行1次冲击免疫,即腹腔直接注射免疫原溶液0.5mL,三天后取脾细胞与骨髓瘤细胞融合,筛选阳性孔。利用有限稀释法对阳性孔进行克隆,得到并建立稳定分泌抗壬基酚聚氧乙烯醚单克隆抗体的杂交瘤细胞株。
1.2 抗壬基酚聚氧乙烯醚单克隆抗体的制备
细胞复苏:取出抗壬基酚聚氧乙烯醚单克隆抗体杂交瘤细胞株冻存管,立即放入37℃水浴锅中速融,提前用15mL离心管加入10mL DMEM,将融后的细胞放入DMEM中,1000rpm离心5min,弃去上清,将细胞移入培养瓶内培养。
制备腹水:采用小鼠体内诱生法,取健康的Balb/C小鼠,注入无菌液体石蜡0.8mL/只,一周左右使用。将扩大培养的阳性单克隆杂交瘤细胞用无菌1×PBS制备成细胞悬液,细胞计数后,细胞总量为2×106个,注射小鼠腹腔,0.5mL/只。7-10天后收集腹水,一只小鼠可获得2-5mL腹水。10000rpm离心7min,收取澄清液体后放入-20℃冻存,准备纯化。
抗体纯化:取腹水(mL)记录腹水名称,离心,记体积。加3mL pH=4.0 0.06M 乙酸乙酸钠缓冲液,混匀5min,加入10uL辛酸,4℃搅拌15min。用注射器通过脱脂棉过滤一次。12000rpm、15min 4℃离心。取上清,加入等上清液体积的饱和硫酸铵至终浓度为50%,边加边搅拌,4℃静置3h。12000rpm、15min 4℃离心。弃上清,向离心管中加入1.8mL 0.01M PBS(pH:7.2)至沉淀完全溶解。用枪确定总体积,加1/2体积的饱和硫酸铵至浓度为33%,边加边搅拌,4℃过夜。12000rpm、15min 4℃离心。弃上清,加0.45mL 0.01M PBS溶解沉淀。处理透析袋(煮沸5min,用纯水洗净捡漏)。将溶解后溶液加入透析袋置于0.01M PBS过夜透析。换透析液,一般换2-3次后,收集抗体,测浓度并分装。
实施例2 抗壬基酚聚氧乙烯醚单克隆抗体的灵敏度
采用间接竞争ELISA方法检测单克隆抗体的灵敏度。
灵敏度测试方法:如下表1,以壬基酚聚氧乙烯醚各浓度(0,0.5,2,4,8,12ng/ml)为横坐标,以壬基酚聚氧乙烯醚各浓度对应的OD450nm值为纵坐标,使用RIDA SOFT四参数法绘制标准曲线,计算半数抑制浓度(IC50)。结果表明,曲线R2=0.9990,IC50=1.9ng/ml,如图1和图2。灵敏度高于专利CN103308685A报道的IC50=8.9ng/ml。
表1
实施例3 抗壬基酚聚氧乙烯醚的单克隆抗体轻链和重链可变区基因克隆
3.1杂交瘤细胞培养及总RNA提取:
用RPMI 1640完全培养基在37℃、5%二氧化碳培养杂交瘤细胞1×107。培养细胞总RNA提取试剂盒(购自天根)提取细胞总RNA。
3.2 cDNA第一链的合成
TIANScriptⅡcDNA第一链合成试剂盒(购自天根)合成cDNA。
3.3 基因扩增
设计Lambda链、Kappa链、Heavy链下游引物及上游通用引物。
引物:F:AAGCAGTGGTATCAACGCAGA
Rκ:AACATTGATGTCTTTGGGGTAGAA
Rλ:AATCGTACACACCAGTGTGGG
RH:AGGGATCCAGAGTTCCAGGT
以cDNA第一链为模板进行PCR,反应体系50μL。
PCR反应体系:模板3μL,上下游引物(10μM)各2.5μL,Green Taq Mix 25μL,ddH2O17μL。
PCR反应条件为:95℃ 5min;95℃ 30s,60℃ 30s,72℃ 1min,循环12次;95℃30s,56℃ 30s,72℃ 1min,循环28次;72℃ 7min。
3.4 PCR扩增产物的克隆和筛选
PCR产物经2%琼脂糖凝胶电泳,用琼脂糖凝胶DNA回收试剂盒(北京天根)回收抗体Kappa链,Lambda链和Heavy链片段,用pLB零背景快速克隆试剂盒(北京天根)将该片段插入pLB载体中,转化至DH5α感受态细胞(氨苄抗性)中,筛选重组阳性克隆测序。
PCR产物经2%琼脂糖凝胶电泳,结果显示Lambda链未见条带,Kappa链可变区基因序列如SEQ ID NO.8所示,氨基酸序列如SEQ ID NO.6所示;Heavy链可变区基因序列如SEQID NO.9所示,氨基酸序列如SEQ ID NO.7所示。
SEQ ID NO.6的序列结构为:
QLLGVMGFWIAGFTTDFDWRQAAFSNSVTVGSSAYISHRFSKSLGHSKVITYLYWYEQKAGQSPQLLIYQLSNLASGVADRFSNSGSGTDFTLRINRVEAEDVGVYYVLKIYFRTRSEGGPSWKYGLMLRQLYPSSHHPVSSNICPQSGART。
SEQ ID NO.7的序列结构为:
TWGQSLKTLTLIMECSWTLPFILSDTSGVYSKFQLLQSGNVLARPEASVKMTWKGSDYTLYSHLMHWEKQRPGQGLEWIDATSPGNSHTSSNPKFKGKGKLTADISTSSAYMRLSSLTNKYSAAYYCTRTGYMLILYWYYAVWCPGTTFTISSAQTTPPSVYPLPPGSAAPSTSMVSLGWLVKAYLP。
SEQ ID NO.8的序列结构为:
CAGCTTCTTGGGGTTATGGGTTTTTGGATTGCTGGATTCACAACAGATTTTGATTGGAGGCAGGCGGCATTCTCCAATTCAGTCACTGTTGGATCTTCAGCTTACATCTCCCACAGGTTTAGTAAGAGTCTCGGACATAGTAAAGTCATCACTTATTTGTATTGGTATGAGCAGAAAGCAGGCCAGTCTCCTCAGCTCCTGATTTATCAGTTGTCCAACCTTGCCTCAGGAGTGGCAGACAGGTTCAGTAACAGTGGGTCAGGAACTGATTTCACATTGAGAATCAACAGAGTGGAGGCTGAGGATGTGGGTGTCTACTATGTTCTCAAAATCTACTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATACGGGCTGATGCTGCGCCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTAACATCTGTCCTCAGTCGGGCGCGAGAACG。
SEQ ID NO.9的序列结构为:
ACATGGGGCCAGTCACTGAAAACATTGACTCTAATCATGGAATGTAGCTGGACACTTCCTTTTATTCTGTCGGATACTTCAGGGGTCTACTCAAAGTTTCAGCTCCTGCAGTCTGGGAATGTGCTGGCAAGGCCTGAGGCTTCCGTGAAGATGACCTGGAAGGGTTCTGACTACACCTTATACAGCCATTTGATGCACTGGGAAAAACAGAGGCCTGGACAGGGCCTGGAATGGATTGACGCTACTTCTCCTGGAAATAGTCATACTAGCTCCAACCCCAAGTTCAAGGGCAAGGGCAAACTGACTGCAGACATATCCACCAGCAGTGCCTACATGAGGCTCAGCAGCCTGACAAATAAGTACTCTGCGGCATATTACTGTACAAGAACAGGCTATATGTTAATTCTTTACTGGTACTACGCTGTCTGGTGCCCAGGGACCACGTTCACCATCTCCTCAGCCCAAACGACCCCCCCATCAGTCTATCCACTGCCCCCTGGATCAGCTGCCCCAAGTACCTCCATGGTGAGCCTGGGATGGCTGGTCAAGGCCTATTTGCCT。
实施例4 可变区基因和氨基酸序列同源性分析
在NCBI数据库中进行比对分析,分析结果表明单克隆抗体轻链可变区基因序列与小鼠免疫球蛋白Kappa链可变区mRNA(Sequence ID:EU159569.1)同源性最高,同源性为349/396,同源性百分比为88%,如图3;单克隆抗体轻链可变区氨基酸序列与鼠源Ig KappaV-region 24B(Sequence ID:AAA39053.1)同源性最高,同源性为81/108,同源性百分比为75%,如图4;单克隆抗体重链可变区基因序列与小鼠免疫球蛋白重链可变区mRNA(SequenceID:JF412709.1)同源性最高,同源性为330/378,同源性百分比为87%,如图5;单克隆抗体重链可变区氨基酸序列与小鼠免疫球蛋白(Sequence ID:UVK70143.1)同源性最高,同源性为105/175,同源性百分比为60%,如图6。编码抗壬基酚聚氧乙烯醚单克隆抗体的轻链和重链可变区的基因序列和氨基酸序列同源性分析结果表明,未发现与本发明相同的序列。
将轻链可变区和重链可变区序列进行分析,得出其CDR区。
抗壬基酚聚氧乙烯醚单克隆抗体轻链可变区的3个互补决定区(CDR)的序列分别为:
CDR1,序列为SEQ ID NO.1:RFSKSLGHSKVITYLY。
CDR2,序列为SEQ ID NO.2:QLSNLAS。
CDR3,氨基酸残基为:LKI。
抗壬基酚聚氧乙烯醚单克隆抗体重链可变区的3个互补决定区(CDR)的序列分别为:
CDR1,序列为SEQ ID NO.3:SHLMH。
CDR2,序列为SEQ ID NO.4:ATSPGNSHTSSNPKFKG。
CDR3,序列为SEQ ID NO.5:TGYMLILYWYYAV。
实施例5 壬基酚聚氧乙烯醚检测试剂盒的制备
如图7、图8、图9所示,本实施例的检测壬基酚聚氧乙烯醚的试剂盒,包括试纸条、微孔条、微孔塞和微孔试剂。
其中,试纸条7包括底板6,底板6上沿底板6的长度方向依次排列设置有样品垫3、硝酸纤维素膜2、吸水纸1;硝酸纤维素膜2上靠近吸水纸1的一端设有质控线4,靠近样品垫3的一端设有检测线5;质控线4上喷涂有羊抗鼠IgG抗体和色素;检测线5上喷涂有壬基酚聚氧乙烯醚人工抗原。
其中,微孔条8中冻干有所述微孔试剂9,所述微孔塞10设于所述微孔条8上,所述微孔试剂9为壬基酚聚氧乙烯醚单克隆抗体-胶体金标记物。
该试剂盒的制备方法包括:在硝酸纤维素膜2(NC膜)上用划膜仪划两条平行带,条带间隔6.5mm,条带宽度约为1mm。其中第一条为质控线4,接近吸水纸1端,第二条为检测线5,接近样品垫3端。质控线上喷涂羊抗鼠IgG抗体和万分之一蓝色色素,浓度为0.5~1mg/mL;检测线上喷涂壬基酚聚氧乙烯醚人工抗原,浓度为0.1~0.5mg/mL。得层析膜,40℃干燥16h后密封,常温下保存。将吸水纸1、NC膜2和样品垫3依次按顺序黏贴在底板6上,样品垫3的始端与NC膜2末端相连,NC膜2的始端与吸水垫1相连,样品垫3的末端与底板6的末端对齐,吸水垫1的始端与底板6的始端对齐。用切条机将粘贴好的塑料板纵向切成4.5mm宽的试纸条7。微孔条8中冻干有壬基酚聚氧乙烯醚单克隆抗体-胶体金标记物,上方设置微孔塞10。取8条裁切好的试纸条7与1条微孔条8放入试剂桶中密封,2-8℃下保存。
实施例6 壬基酚聚氧乙烯醚检测试剂盒检出限的界定
6.1检测方法
使用完全不含有壬基酚聚氧乙烯醚的牛奶为阴性样本,配制待测样本1(壬基酚聚氧乙烯醚3ppb),待测样本2(壬基酚聚氧乙烯醚1.5ppb),待测样本3(壬基酚聚氧乙烯醚0ppb)。用微量移液器吸取100µL待测样本溶液于微孔试剂中,缓慢抽吸至样品溶液与微孔试剂充分混匀,置于孵育器上,40℃条件下3min孵育反应;孵育结束后,立即将试纸条插入微孔试剂中,使吸水纸端向上,另一端向下并充分浸入溶液中,反应5min;反应结束后,取出试纸条,并在1min内读取结果。
6.2结果判定
阴性(—):质控线(C线)和检测线(T线)均显色,检测线(T线)显色远比质控线(C线)强,表示样本中不含壬基酚聚氧乙烯醚或远低于检出限。
阳性(+):质控线(C线)显色;检测线(T线)显色与质控线(C线)相同、检测线(T线)显色比质控线(C线)弱或者检测线(T线)不显色,均表示样本壬基酚聚氧乙烯醚浓度等于或高于检出限。
无效:未出现质控线(C线),表明不正确的操作过程或试纸卡已变质失效。
6.3检出限界定
待测样本1(壬基酚聚氧乙烯醚3ppb)为阳性,待测样本2(壬基酚聚氧乙烯醚1.5ppb)为阴性,待测样本3(壬基酚聚氧乙烯醚0ppb)为阴性。此结果表明试纸条检测限为3ppb。本试纸条的灵敏度优于CN10684188A报道的5ppb。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (7)
1.一种抗壬基酚聚氧乙烯醚的单克隆抗体,包括轻链和重链,其特征在于,轻链可变区包括CDR1、CDR2、CDR3;重链可变区包括CDR1、CDR2、CDR3;
轻链CDR1的氨基酸序列如SEQ ID NO.1所示,轻链CDR2的氨基酸序列如SEQ ID NO.2所示,轻链CDR3的氨基酸残基为LKI;
重链CDR1的氨基酸序列如SEQ ID NO.3所示,重链CDR2的氨基酸序列如SEQ ID NO.4所示,重链CDR3的氨基酸序列如SEQ ID NO.5所示。
2.根据权利要求1所述的抗壬基酚聚氧乙烯醚的单克隆抗体,其特征在于:
所述轻链可变区的氨基酸序列如SEQ ID NO.6所示。
3.根据权利要求1所述的抗壬基酚聚氧乙烯醚的单克隆抗体,其特征在于:
所述重链可变区的氨基酸序列如SEQ ID NO.7所示。
4.根据权利要求2所述的抗壬基酚聚氧乙烯醚的单克隆抗体,其特征在于:
编码轻链可变区的基因序列如SEQ ID NO.8所示。
5.根据权利要求3所述的抗壬基酚聚氧乙烯醚的单克隆抗体,其特征在于:
编码重链可变区的基因序列如SEQ ID NO.9所示。
6.权利要求1-5中任一项所述的抗壬基酚聚氧乙烯醚的单克隆抗体在制备检测壬基酚聚氧乙烯醚的试剂或试剂盒中的应用。
7.一种壬基酚聚氧乙烯醚检测试剂盒,其特征在于,所述试剂盒为胶体金检测试剂盒,所述胶体金检测试剂盒的微孔试剂为胶体金标记的权利要求1-5中任一项所述的抗壬基酚聚氧乙烯醚的单克隆抗体。
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CN103308685A (zh) * | 2013-05-17 | 2013-09-18 | 广东产品质量监督检验研究院 | 壬基酚聚氧乙烯醚检测试剂盒及其制备和使用方法 |
CN110950953A (zh) * | 2018-09-26 | 2020-04-03 | 福建医科大学 | 抗b7-h3的单克隆抗体及其在细胞治疗中的应用 |
CN110684188A (zh) * | 2019-10-31 | 2020-01-14 | 北京纳百生物科技有限公司 | 壬基酚聚氧乙烯醚半抗原和全抗原及其制备方法与应用 |
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