CN113717285A - 抗人d-二聚体抗体及其应用 - Google Patents
抗人d-二聚体抗体及其应用 Download PDFInfo
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- CN113717285A CN113717285A CN202111297289.4A CN202111297289A CN113717285A CN 113717285 A CN113717285 A CN 113717285A CN 202111297289 A CN202111297289 A CN 202111297289A CN 113717285 A CN113717285 A CN 113717285A
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Abstract
本发明涉及生物技术领域,具体涉及抗人D‑二聚体抗体及其应用,公开了一组可以配合使用的抗人D‑二聚体抗体,抗体性能稳定,且能避免其他杂质的干扰,高特异性与D‑二聚体结合,实现D‑二聚体的高亲和性、高准确性、高精密的测量,对辅助医疗诊断试剂的研发意义重大。
Description
技术领域
本发明属于生物技术领域,具体的涉及抗人D-二聚体抗体及其应用。
背景技术
D-二聚体(D-Dimer)是纤维蛋白降解的最终产物(分子量约180kDa),其含有三个多肽链的降解产物,通过二硫键交联。二聚体结构通过γ链C端之间的两个异肽键形成交联结构而维持。正常状态下,纤溶酶和抑制酶之间保持了动态平衡,使血液循环能正常进行。人体内的纤溶***,对保持血管壁的正常通透性及维持血液的流动状态和组织修复发挥着重要的作用。病理状态下,机体发生凝集时,凝血酶作用于纤维蛋白,转变为交联纤维蛋白,同时纤溶***被激活,降解纤维蛋白形成各种碎片。γ链可将两个含D片段的碎片连接起来,形成D-二聚体。D-二聚体的升高,表明体内有纤维蛋白血栓形成和纤溶发生,所以临床上可作为体内高凝状态和纤溶亢进的分子标志物。
因此,D-二聚体的检测对多种疾病的诊断及治疗有重要的价值。目前在临床实践使用的D-二聚体检测方法多种多样,包括免疫比浊法试、胶乳凝集法、ELISA、胶体金检测和荧光免疫法等,它们检测的灵敏度和线性宽度往往是由使用的抗体或抗体对决定的,因而抗体的特异性、灵敏度和检测线性宽度就尤为重要。
发明内容
本发明的目的在于提供抗人D-二聚体抗体,解决现有抗体活性低、亲和力差的问题,实现高精度、高准确性的D-二聚体检测。
抗人D-二聚体抗体,包括:
如SEQ ID NO.1所列的重链可变区CDR1;
如SEQ ID NO.2所列的重链可变区CDR2;
如SEQ ID NO.3所列的重链可变区CDR3;
如SEQ ID NO.4所列的轻链可变区CDR1;
如SEQ ID NO.5所列的轻链可变区CDR2;
如SEQ ID NO.6所列的轻链可变区CDR3;
命名为DD-1D10
或
如SEQ ID NO.7所列的重链可变区CDR1;
如SEQ ID NO.8所列的重链可变区CDR2;
如SEQ ID NO.9所列的重链可变区CDR3;
如SEQ ID NO.10所列的轻链可变区CDR1;
如SEQ ID NO.11所列的轻链可变区CDR2;
如SEQ ID NO.12所列的轻链可变区CDR3;
命名为DD-12B4。
所述DD-1D10重链可变区的氨基酸序列如SEQ ID NO.13所示,轻链可变区的氨基酸序列如SEQ ID NO.14所示。
所述DD-1D10编码重链可变区的核苷酸序列如SEQ ID NO.15,所述DD-1D10编码轻链可变区的核苷酸序列如SEQ ID NO.16。
所述DD-12B4重链可变区的氨基酸序列如SEQ ID NO.17所示,轻链可变区的氨基酸序列如SEQ ID NO.18所示。
所述DD-12B4编码重链可变区的核苷酸序列如SEQ ID NO.19,所述DD-1D10编码轻链可变区的核苷酸序列如SEQ ID NO.20。
本发明所述DD-1D10和DD-12B4抗体能够配合使用,实现对D-二聚体的检测。
所述“配合使用”:两株抗体能够同时识别D-二聚体抗原,在检测时同时发挥作用。
另一方面,本发明提供了一种抗原抗体混合物,所述抗原抗体混合物含有所述DD-1D10或DD-12B4。
另一方面,本发明提供了一种检测D-二聚体的试剂盒,所述试剂盒含有所述抗人D-二聚体抗体或所述抗原抗体混合物。
另一方面,本发明提供了一种结合物所述结合物包含与化学标记或生物标记共价连接的所述的抗人D-二聚体抗体。
另一方面,本发明提供了一种偶联物,所述偶联物由所述的抗人D-二聚体抗体、和/或所述的结合物与固体介质或半固体介质偶联形成。
另一方面,本发明提供了所述的抗人D-二聚体抗体和/或所述的结合物和/或所述的偶联物在制备检测D-二聚体表达的产品中的应用。
有益效果
本发明公开的抗体序列,抗体性能稳定,能避免其他杂质的干扰,高特异性与D-二聚体结合,实现D-二聚体的高亲和性、高准确性、高精密的测量。
附图说明
图1:纯化后的D-二聚体抗体纯度凝胶图。
具体实施方式
实施例1: 重组D-二聚体的表达纯化
将人D-二聚体基因序列克隆至原核表达载体构建原核表达质粒,该表达质粒转化大肠杆菌BL21后用含有抗生素的LB平板培养。挑取单克隆接种至LB培养基。培养至菌液OD值达到0.6后,加入IPTG诱导蛋白的表达。诱导16小时之后离心收集菌液。菌体经破碎以后离心收集上清,并采用离子交换以及分子筛纯化得到抗原蛋白。
实施例2:小鼠免疫及抗体检测
挑选5只6-8周龄SPF级雌性BALB/c小鼠,将弗氏完全佐剂与浓度为2 mg/ml的D-二聚体蛋白按等体积混合并乳化。乳化好的抗原免疫6-8周龄SPF级雌性BALB/c小鼠,采用脚底注射或背部皮下注射每只小鼠注射40μg抗原蛋白。初次免疫完成两周后,将抗原蛋白与弗氏不完全佐剂混合乳化,再次采用脚底注射或背部皮下注射每只小鼠注射40μg抗原蛋白。两周以后通过尾静脉采血,离心收集上清并用ELISA检测血清效价。每两周免疫一次并检测血清效价。2次免疫后,百万倍稀释后血清效价已高达2.0以上。筛选血清效价106以上的小鼠,取淋巴分离淋巴细胞用于细胞融合。
实施例3:单克隆抗体的生产纯化
选择两组6-8周BALB/c小鼠,腹腔注射500μL石蜡油以抑制小鼠免疫反应。于注射一周之后向一组小鼠腹腔内注入0 .5 ml的D-二聚体杂交瘤细胞,细胞数量约1×106数量。两周之后开始腹水搜集。搜集的腹水经硫酸铵沉淀和蛋白A的亲和纯化得到目的抗体。纯化后的抗体纯度如图1所示,其中泳道1、2、3、4和5分别代表非还原的DD-1D10抗体、 还原的DD-1D10抗体、非还原的DD-12B4抗体、还原的DD-12B4抗体和Marker,从图中可以看出,纯化后的抗体条带清晰干净,纯度高。
实施例4:单克隆抗体的亚型鉴定及基因序列克隆
采用SouthernBiothech公司的SBA Clonotyping System-HRP试剂盒,按照说明书的操作来鉴定单克隆抗体重链和轻链的亚型。具体操作为:
1、用包被液(0 .05M pH9 .5的碳酸盐和碳酸氢盐缓冲液)将捕获抗体稀释至1μg/mL,按照100μL/孔加入酶标板,于4℃包被过夜。用含有0 .05%Tween-20的PBS缓冲液(洗板液)洗板3次。
2、用稀释液(1%BSA ,0 .1%PBST)按照1:1稀释待检杂交瘤细胞的培养上清,按照100μL/孔加入酶标板,于37℃孵育30分钟。用稀释液将对应的酶标抗体1:3000稀释。
3、用洗板液3次洗板后每孔加入100μL稀释的酶标抗体,于37℃孵育30分钟。再次洗板3次以后加入显色液,约5分钟(视反应强弱而定)之后加入2M硫酸终止反应,读取OD450吸光值。经过鉴定,DD-1D10的重链亚型为IgG2b,轻链为Kappa;DD-12B4抗体重链亚型为IgG1,轻链为Kappa。根据抗体亚型结果,采用基于RACE技术路线的方法克隆抗体基因序列。收集生长状态良好的杂交瘤细胞,使用总RNA提取试剂盒获得杂交瘤细胞总RNA ,按照Takara公司的SMARTer RACE说明书的操作方法将mRNA逆转录为cDNA,并扩增目的抗体全长序列。
实施例5:抗体稳定性评价
(1)DD-1D10稳定性评价
从表1可以看出,DD-1D10抗体加速和冻融前后偏差均小5%,说明抗体性能非常稳定。
从表2可以看出,DD-12B4抗体加速和冻融前后偏差均小5%,说明抗体性能非常稳定。
综上,DD-1D10和DD-12B4的稳定性良好,适用于应用到试剂盒的研发中,可以从原料端提高试剂盒的检测性能。
实施例6:抗人D-二聚体抗体应用到凝血检测中
1、试剂盒的制备
本试剂盒包含R1和R2两种试剂。
R1:一水合柠檬酸 20g/L,牛血清白蛋白1%,叠氮钠0.1%;
R2:DD-1D10抗体包被的微球 1mg/ml,DD-12B4抗体包被的微球1mg/ml,氯化钠0.9%,牛血清白蛋白1%,叠氮钠0.1%。
2、精密度和准确度试验
(1)精密度检测
检测低值、中值和高值样本,每份检测10次,计算批内精密度,结果如下表3所示,精密度小于5%,证明本发明的DD-1D10和DD-12B4应用到免疫比浊试剂盒中,能够准确的检测D-二聚体的浓度。
(2)准确度检测
检测低值、高值质控品,每个质控检测3次,计算测试结果平均值与靶值的相对偏差,结果如表4所示:
所述质控品为DD抗原与牛血清配制而成。
相对偏差小于1%,说明检测的准确度很高,抗体应用到试剂盒中性能良好。
3、抗干扰试验
检测胆红素、血红蛋白和乳糜对试剂盒的干扰,实验结果如下表5所示:
添加干扰物质的相对偏差小于5%,说明本发明抗人D-二聚体抗体制备的试剂盒能够抵抗胆红素、血红蛋白和乳糜的干扰,可以在临床环境中实现对D-二聚体的稳定检测。
综上,本发明提供的两株抗体能够被应用到凝血试剂盒中,实现高准确性、高精密度、抗干扰的检测,性能良好。
序列表
<110> 北京沃森赛瑟生物技术有限公司
<120> 抗人D-二聚体抗体及其应用
<130> 2021.10.25
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Claims (6)
1.抗人D-二聚体抗体,其特征在于,包括:
如SEQ ID NO.1所列的重链可变区CDR1;
如SEQ ID NO.2所列的重链可变区CDR2;
如SEQ ID NO.3所列的重链可变区CDR3;
如SEQ ID NO.4所列的轻链可变区CDR1;
如SEQ ID NO.5所列的轻链可变区CDR2;
如SEQ ID NO.6所列的轻链可变区CDR3;
命名为DD-1D10
或
如SEQ ID NO.7所列的重链可变区CDR1;
如SEQ ID NO.8所列的重链可变区CDR2;
如SEQ ID NO.9所列的重链可变区CDR3;
如SEQ ID NO.10所列的轻链可变区CDR1;
如SEQ ID NO.11所列的轻链可变区CDR2;
如SEQ ID NO.12所列的轻链可变区CDR3;
命名为DD-12B4。
2.一种抗原抗体混合物,其特征在于,所述抗原抗体混合物含有权利要求1中所述DD-1D10或DD-12B4。
3.一种检测D-二聚体的试剂盒,其特征在于,所述试剂盒含有权利要求1中所述抗人D-二聚体抗体或权利要求2所述抗原抗体混合物。
4.一种结合物,其特征在于,所述结合物包含与化学标记或生物标记共价连接的权利要求1中所述的抗人D-二聚体抗体。
5.一种偶联物,其特征在于,所述偶联物由权利要求1所述的抗人D-二聚体抗体、和/或权利要求4中所述的结合物与固体介质或半固体介质偶联形成。
6.权利要求1中所述的抗人D-二聚体抗体和/或权利要求4中所述的结合物和/或权利要求5中所述的偶联物在制备检测D-二聚体表达的产品中的应用。
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