CN113045666B - 胃蛋白酶原ii单克隆抗体及其应用 - Google Patents
胃蛋白酶原ii单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域，具体涉及胃蛋白酶原II单克隆抗体及其应用，公开了一组可以配合使用的胃蛋白酶原II单克隆抗体，能够应用到不同方法学中，实现对PGII的准确测量，且能避免其他杂质的干扰，实现高特异性结合，本发明提供的单克隆抗体能够辅助医疗诊断试剂的研发，意义重大。
Description
技术领域
本发明涉及生物技术领域，具体涉及胃蛋白酶原II单克隆抗体及其应用。
背景技术
胃蛋白酶原(Pepsinogen或者PG)是胃蛋白酶的前体，根据理化性质及免疫原性的差异分为胃蛋白酶原I(PGI)和胃蛋白酶原II(PGII)，主要由胃黏膜分泌。胃蛋白酶原无消化酶活性，分泌至胃腔后在胃酸作用下活化成具有消化活性的胃蛋白酶。除了主要在消化道行使功能之外，少量胃蛋白酶原会透过胃粘膜而进入血液循环。血清中胃蛋白酶原的含量能够反应胃液分泌的水平以及胃部粘膜的病理变化，正常人血清中PGII的含量为11.9±4.2ng/ml。研究指出，PGII与胃底粘膜病变的相关性较大，其升高与胃底腺管萎缩、肠上皮化生或假幽门腺化生、异型增生有关。测定PGII含量有助于检测出十二指肠溃疡、萎缩性胃炎、胃癌等其他消化道疾病，供临床检测和体检筛查需求。
同时，PGII检测作为非侵入性方法，可降低患者痛苦，简便、经济，具有普查价值。现有的抗人PGII抗体由于活性低、亲和力差，不容易实现应用于PGII蛋白的检测中，且PGII抗体应用到检测试剂中，很容易受到脂类的干扰，导致检测结果不准确，因此本领域对于有效且特异性结合检测PG II并对其进行检测的抗体存在着强烈需求。
发明内容
本发明的目的在于提供抗PGII单克隆抗体，解决现有抗体活性低、亲和力差且易受脂蛋白干扰的问题，实现高精度、高灵敏的PGII检测。
抗PGII单克隆抗体，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；
上述抗PGII单克隆抗体，命名为抗体A
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；
上述抗PGII单克隆抗体，命名为抗体B
或
如SEQ ID NO：13所列的重链可变区CDR1；
如SEQ ID NO：14所列的重链可变区CDR2；
如SEQ ID NO：15所列的重链可变区CDR3；
如SEQ ID NO：16所列的轻链可变区CDR1；
如SEQ ID NO：17所列的轻链可变区CDR2；
如SEQ ID NO：18所列的轻链可变区CDR3；
上述抗PGII单克隆抗体，命名为抗体C。
优选的，所述抗体A、抗体B和抗体C结合胃蛋白酶原II的不同表位。
所述抗体A、抗体B和抗体C可以配合使用，用来检测PGII，所述配合使用为利用其中任意两种或三种抗体结合PGII，构建免疫检测系统。
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上述任何一种或多种抗PGII单克隆抗体。
优选地，所述抗原抗体混合物为：抗体A-PGII-抗体B、抗体A-PGII-抗体C或
另一方面，本发明公开了含有上述抗体、抗体B或抗体C的试剂盒。
另一方面，本发明公开了一种结合物，包含与化学标记或生物标记共价链接的所述抗PGII单克隆抗体。
另一方面，本发明公开了一种偶练物，由所述抗PGII单克隆抗体、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，本发明公开了所述的抗PGII单克隆抗体和/或所述的结合物和/或所述的偶联物在制备检测PGII表达产品中的应用。
有益效果
本发明公开的抗体序列，能够应用到不同方法学中，实现对PGII的准确测量，抗体本身具有抗干扰能力，能够和抗原高特异性结合，避免其他杂质的干扰，能够同时检测血清和血浆样本，对样本的要求较低。
附图说明
图1：血清和血浆样本相关性实验图。
具体实施例
实施例1：重组PGII的表达纯化
将人PGII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2mg/ml的PGII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0.5ml的PGII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。
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100uL/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(Ig-HRP，IgG1-HRP，IgG2a-HRP，IgG2b-HRP，IgG3-HRP，IgM-HRP，kappa-HRP，lamda-HRP)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，抗体A的重链亚型为IgG1，轻链为Kappa；抗体B抗体重链亚型为IgG2b，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：PGII抗体应用到乳胶免疫比浊法中
1、试剂盒的制备
本试剂盒包含R1和R2两种试剂，R1为胶乳反应缓冲液，R2为包被了抗PGII单克隆抗体的胶乳颗粒。其中R1试剂为pH值为7.5的磷酸盐缓冲液，包含浓度为1.6％的PEG6000作为促凝剂。R2试剂的制备方法为用孔径大小为120nm的羧基化胶乳与1-乙基-3-(3-二甲基氨基丙基)-3-乙基碳二亚胺于室温摇床上反应30分钟。反应完成后，将反应混合液分成两份，分别加入抗体A、抗体B震荡混匀，于室温下旋转反应2小时。抗体反应完成之后再同时向两份混合液之中加入6-氨基己酸和BSA并混匀，反应2小时。之后，采用16000g离心力条件离心30分钟，去掉上清。用pH 7.2的Tris缓冲液重悬清洗胶乳，并再次离心。用pH7.2的Tris缓冲液再次重选胶乳，将包被了抗体A和抗体B的胶乳按照等体积混合。使用超声处理使得胶乳足够分散而得到R2试剂。
2、灵敏度和精密度试验
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1所示，精密度小于5％，证明本发明的抗体A和抗体B应用到免疫比浊试剂盒中，能够准确的检测PGII的浓度。
表1精密度试验
| 测试 | 低值(ng/mL) | 中值(ng/mL) | 高值(ng/mL) |
| 1 | 8.61 | 44.34 | 100.43 |
| 2 | 9.01 | 43.73 | 101.37 |
| 3 | 9.08 | 44.79 | 98.12 |
| 4 | 8.91 | 44.60 | 101.32 |
| 5 | 9.09 | 44.96 | 101.33 |
| 6 | 8.77 | 44.88 | 99.50 |
| 7 | 8.86 | 45.17 | 99.66 |
| 8 | 8.75 | 44.64 | 100.09 |
| 9 | 8.86 | 44.81 | 99.48 |
| 10 | 8.75 | 44.40 | 98.62 |
| Mean | 8.87 | 44.63 | 99.99 |
| SD | 0.16 | 0.40 | 1.14 |
| CV | 1.76％ | 0.91％ | 1.14％ |
3、抗干扰试验
检测血红蛋白、乳糜和胆红素对试剂盒的干扰，实验结果如下表2所示：
表2抗干扰试验
添加干扰物质，的相对偏差小于5％，说明本发明PGII单克隆抗体制备的试剂盒能够抵抗血红蛋白、乳糜和胆红素的干扰，可以在临床环境中实现对PGII的稳定检测。
4、血清样本和血浆样本检测相关性
选取30份临床样本，分别在血浆状态和血清状态测量样本中PGII的含量，所述血清样本由血浆样本制得，实验结果如图1所示，结果表明R2＝0.998，血浆和血清的样本检测结果相关性较高。
实施例6：抗体A和抗体B抗体应用到化学发光中
1、试剂盒的准备
本发明试剂盒含有3种试剂组分，分别是2μg/mL的链霉亲和素-碱性磷酸酶偶联物，1μg/mL的生物素-抗体A偶联物，1mg/mL的磁微粒复合物。
所述链霉亲和素-碱性磷酸酶偶联物是将碱性磷酸酶和链霉亲和素偶联得到的。
所述生物素-抗体A偶联物是通过以下的方法制备：在100mM的碳酸盐溶液中将抗体A脱盐并浓缩至浓度为2mg/mL；生物素用DMSO溶解，按照10∶1的比例将生物素加入抗体A中，37℃偶联2小时，用脱盐柱除去未偶联的生物素，得到生物素抗试剂。
所述磁微粒复合物通过以下方法制备：将羧基磁珠用EDC活化后清洗四次，将抗体B以TCEP活化后、超滤管浓缩后更换PBS缓冲液中；将羧基磁珠PGII单克隆抗体按1∶50的质量比混合偶联4小时，偶联结束以后以BSA、PEG20000作为封闭剂，封闭2小时后进行磁分离清洗，即得磁微粒复合物，利用合适的稀释液调节浓度到1mg/mL。
所述PGII单克隆抗体为抗体B和抗体C按1∶1混合的混合物。
a)灵敏度实验
用重庆中元汇吉生物技术有限公司的EXI1800进行检测，测空白样本20次，浓度为5ng/ml的PGII样本5次，计算出检出限为0.01ng/mL，证明本发明抗体应用到化学发光试剂盒中，具有很高的灵敏度，适合用于PGII的检测。
b)精密度试验
检测低值和高值的样本，每份检测20次，计算批间精密度，结果如下表3所示，精密度小于4％，证明本发明的抗体A、抗体B和抗体C应用到化学发光试剂盒中，能够准确的检测PGII的浓度。
表3精密度试验
序列表
<110> 重庆中元生物技术有限公司
<120> 胃蛋白酶原II单克隆抗体及其应用
<130> 2021.3.12
<160> 18
<170> SIPOSequenceListing 1.0
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Claims (7)
1.抗PGII单克隆抗体，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；
上述抗PGII单克隆抗体，命名为抗体A
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；
上述抗PGII单克隆抗体，命名为抗体B
或
如SEQ ID NO:13所列的重链可变区CDR1；
如SEQ ID NO:14所列的重链可变区CDR2；
如SEQ ID NO:15所列的重链可变区CDR3；
如SEQ ID NO:16所列的轻链可变区CDR1；
如SEQ ID NO:17所列的轻链可变区CDR2；
如SEQ ID NO:18所列的轻链可变区CDR3；
上述抗PGII单克隆抗体，命名为抗体C。
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中所述的任何一种或多种抗PGII单克隆抗体。
3.如权利要求2所述的抗原抗体混合物，其特征在于，所述抗原抗体混合物的结构如下：抗体A-PGII-抗体B、抗体A-PGII-抗体C或
。
4.一种检测PGII的试剂盒，其特征在于，所述试剂盒含有权利要求1中所述的抗体A、抗体B或抗体C。
5.一种结合物，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PGII单克隆抗体。
6.一种偶联物，其特征在于，所述偶联物由权利要求1中所述的抗PGII单克隆抗体、和/或权利要求5中所述的结合物与固体介质或半固体介质偶联形成。
7.权利要求1中所述的抗PGII单克隆抗体和/或权利要求5中所述的结合物和/或权利要求6中所述的偶联物在制备检测PGII表达的产品中的应用。
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