CN116143920A - 胃泌素17的单克隆抗体及其细胞株的制备方法和应用 - Google Patents
胃泌素17的单克隆抗体及其细胞株的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种胃泌素17的单克隆抗体及其细胞株的制备方法和运用,S1制备抗原:合成胃泌素17多肽,将偶联到BSA载体蛋白上,透析后获得胃泌素17抗原;S2免疫:抗原乳化,免疫小鼠;S3细胞融合:小鼠脾细胞悬液依次与小鼠骨髓瘤细胞sp2/0和混和10xHAT的胸腺细胞混合;S4将HAT培养基换成HT培养基,ELISA筛选;S5亚克隆:筛选出的阳性克隆用梯度稀释法进行亚克隆,直至筛选出纯合的阳性细胞株;S6腹水制备,收集于离心管中,4℃或‑20℃保存;S7单克隆抗体的纯化;S8双抗夹心法筛选抗体配对:筛选出一对抗体2C5和8B10。再应用胃泌素17抗体制备免疫层检测试纸条。
Description
技术领域
本发明属于生物医学工程技术领域,尤其是涉及一种胃泌素17的单克隆抗体及其细胞株的制备方法和应用。
背景技术
胃泌素是一种主要由胃窦和十二指肠的G细胞分泌的胃肠激素,对调节消化道功能和维持其结构完整有重要作用。人体中,95%以上有生物活性的胃泌素是α-酰胺化胃泌素,主要含两种异构体G-17和G-34,其中80%~90%是G-17。G-17仅由胃窦部G细胞分泌,因此G-17是反映胃黏膜损伤情况的重要指标。
以胃泌素17作为核心指标的人体血清胃功能检测,属于一项无创、无痛且安全、经济的人体胃病常用检测方法。这种检测项目可以有效地减少胃癌患病风险,大幅度地提高胃癌疾病的早诊以及早治概率,对于各种胃病的预防和治疗都具有重要的积极意义。当人体内胃泌素17处于低值状态时,通常提示患者的胃部存在一些高酸的状况。而当胃泌素17处于高值状态时,则提示患者的胃内部存在一些炎症,患者可能表现出一些高胃泌素血症的发病症状。这种情况可能跟幽门螺旋杆菌感染、胃溃疡以及药物影响有关系。另外,胃泌素17的变化与患者的胃部受到饮食刺激以及患上其他疾病也有一定的联系。
免疫分析技术是利用微量抗原与相应的高特异性抗体之间的免疫反应,来检测如激素、药物、蛋白质、多肽、酶、肿瘤相关抗原、微生素、病毒、细菌及金属元素等生物体内活性物质。免疫分析技术包括标记免疫分析、非标记免疫分析和仪器免疫分析。
荧光免疫分析(FIA)和放射免疫分析(RIA)自问世以来,经历了几十年的发展,但是人们越来越感觉到FIA因自然本地太高,干扰检测结果;RIA采用同位素标记,对人体有极大危害并给实验带来不便。酶免疫分析(EIA)也因酶本身不稳定,受其他影响因素较大,推广应用受到限制。80年代初,人们开始研究用稀土元素代替荧光物质和同位素标记蛋白质或抗体,将时间分辨技术引入到生物检测领域,建立了新型的时间分辨荧光免疫分析技术。该技术采用多学科先进技术,集结了其他免疫分析的特点,在免疫学、分子生物学、细胞学和医学等领域,取得长足的发展和广泛应用。但目前仍存在一些问题:
1)目前胃泌素17的检验在临床上使用越来越广泛,市场上的免疫法检测用抗体一般以来进口,价格昂贵,供货周期长且不稳定。
2)目前临床上检测胃泌素17的常用方法为酶联免疫法(Elisa),加入样本经过一个小时的孵育,然后洗板,加底物,半个小时避光反应后加终止液即完成反应部分,然后读数。由数值来判断结果的浓度值。该方法操作步骤繁琐,检测时间长。
3)化学发光法是新兴发展起来的检测胃泌素17的方法,该方法需要大型的专业设备,价格昂贵,操作较复杂,不适用于基层医院。
发明内容
本发明要解决的技术问题是提供一种胃泌素17的单克隆抗体及其细胞株的制备方法,将一种胃泌素17多肽与载体蛋白进行偶联反应,得到胃泌素17抗原,所得抗原免疫小鼠制备单克隆抗体,所得抗体运用到免疫层析技术上,具有高灵敏度、高特异性、简单快捷和无需大型仪器设备等优点,可用于胃泌素17的检测。
为解决上述技术问题,本发明采用的技术方案是,该胃泌素17的单克隆抗体及其细胞株的制备方法,包括以下步骤:
S1制备抗原:合成胃泌素17多肽,再将胃泌素17多肽偶联到BSA载体蛋白上,透析后获得胃泌素17抗原;
S2免疫:将步骤S1中制得的胃泌素17抗原进行乳化,免疫雌性小鼠;
S3细胞融合:无菌制备免疫达标的小鼠脾细胞悬液,依次与小鼠骨髓瘤细胞sp2/0和混合10xHAT的胸腺细胞混合,然后均匀的分装到若干块铺有饲养细胞的孔板中,放入37℃的5%CO2培养箱中培养;
S4酶联免疫吸附剂测定ELISA筛选阳性杂交瘤细胞:将HAT培养基换成HT培养基,取上清蛋白进行分别ELISA筛选;具体为:7天后将HAT培养基换成HT培养基,1天后取100μL上清蛋白分别ELISA筛选;
S5亚克隆:筛选出的阳性克隆用梯度稀释法进行亚克隆,直至筛选出纯合的阳性细胞株;
S6腹水制备:将对数生长期细胞用无血清培养基洗涤并悬起,其计数约106个/mL,对小鼠腹腔注射悬浮的细胞,7天后开始收集腹水;取出的腹水于4℃离心,吸出中间的腹水,收集于离心管中,4℃或-20℃保存;
S7单克隆抗体的纯化:用HiTrap rProtein A FF(GE公司)亲和层析法从腹水中纯化抗体,纯化后的抗体保存于-20℃;SDS-PAGE胶鉴定纯度,Bradford法测定浓度;
S8双抗夹心法筛选抗体配对:将一株抗体包被在酶标板上,清洗封闭后加入样本在37℃温度下反应70~100min,清洗后加入HRP标记的二抗在37℃进行孵育20~40min,显色终止后读数,筛选出一对抗体2C5和8B10。
采用上述技术方案,将一种胃泌素17与载体蛋白进行偶联反应,得到胃泌素17抗原,所得抗原免疫小鼠制备单克隆抗体,制备了一对胃泌素17单克隆抗体,将所得抗体运用到免疫层析技术上,具有高灵敏度、高特异性、简单快捷和无需大型仪器设备等优点,可用于胃泌素17的检测。
作为本发明的进一步改进在于,所述步骤S1中的胃泌素17多肽的序列为SEQ IDNO:1。SEQ ID NO:1:
pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Cys。
作为本发明的进一步改进在于,所述步骤S2进行乳化的具体步骤为:
S21:将步骤S1中制得的胃泌素17抗原与弗氏完全佐剂(Sigma公司)进行混合乳化,即抗原与弗氏完全佐剂按1:1的比例混合;
S22:选免疫4~6周龄雌性Balb/c小鼠,腹部皮下注射每只小鼠5点,剂量为100μg/只,进行第一次免疫;
S23:每14天给每只小鼠进行加强免疫一次,每次加强免疫的剂量为50μg/只;
S24:在第三次加强免疫后的第7天以间接ELISA波长450nm检测小鼠血清中抗免疫原的多抗效价,将效价最高的小鼠以尾静脉注射冲击免疫,尾静脉注射冲击免疫采用抗原与生理盐水混匀,免疫的剂量为50μg/只,完成免疫。
作为本发明的进一步改进在于,所述步骤S3细胞融合的具体步骤为:将无菌制备免疫达标的小鼠脾细胞悬液,与小鼠骨髓瘤细胞sp2/0以5:1比例混合,离心1200rpm,5min,弃上清后离心管放入37℃水浴中,在1分钟内缓慢加入1mL的PEG1500(Sigma公司),并搅动细胞;在37℃温水中静置1min后,加入10mL无血清的DMEM(Sigma公司),混匀,离心1000rpm,5min;弃上清后,加入10mL血清,小心的将细胞吹打起来,并加入5mL混合10xHAT(Sigma公司)的胸腺细胞,混匀;然后均匀的分装到10块铺有饲养细胞的96孔板中,放入37℃5%CO2培养箱中培养。
本发明要解决的技术问题是提供一种胃泌素17的单克隆抗体的应用,使用胃泌素17的单克隆抗体制备胃泌素17免疫层检测试纸条。
为了解决上述技术问题,该胃泌素17免疫层检测试纸条包括样本垫、结合垫、硝酸纤维素膜、吸水纸和底板;所述硝酸纤维素膜包括被上述胃泌素17抗体包被的检测线和被羊抗鸡抗体包被的质控线。
作为本发明的进一步改进在于,该胃泌素17免疫层检测试纸条的制备方法,包括以下步骤:从加样端开始依次将样品垫、结合垫、包被好的硝酸纤维素膜、吸水纸四个部分粘贴在底板上,然后切割成所要求的宽度,形成试纸条,装入塑料卡壳中,形成试剂卡。
作为本发明的进一步改进在于,所述样品垫在使用前先采用样品垫处理液进行处理,后再将玻璃纤维浸泡在样品垫处理液中,然后烘干。
作为本发明的进一步改进在于,其中所述结合垫的制备方法,包括以下步骤:
S1-1:荧光微球与EDC在磷酸盐缓冲液中进行活化处理;
S1-2:将胃泌素17抗体加入上述荧光微球溶液中进行反应;
S1-3:将偶联后的微球-抗体复合物进行封闭反应,经离心后制得免疫荧光微球标记物;
S1-4:将调配好浓度免疫荧光微球标记物溶液包被到玻璃纤维上,烘干,制得结合垫。
作为本发明的进一步改进在于,所述硝酸纤维素膜的制备方法为:采用8B10抗体配制T线溶液,羊抗鸡IgY配制C线溶液,然后包被在硝酸纤维素膜上的T先即检测线和C线即质控线区域,烘干。
作为本发明的进一步改进在于,所述样品垫处理液包括pH为8~10的缓冲液、表面活性剂、海藻糖和红细胞单抗。
与现有技术相比,本发明具有的有益效果为:将一种胃泌素17与载体蛋白进行偶联反应,得到胃泌素17抗原,所得抗原免疫小鼠制备单克隆抗体,制得一对胃泌素17单克隆抗体,所得抗体运用到免疫层析技术上,具有高灵敏度、高特异性、简单快捷和无需大型仪器设备等优点,可用于胃泌素17的检测。
具体实施方式
本说明书中描述的实施例仅仅是为了解释本发明,并非为了限定本发明,实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。
该胃泌素17的单克隆抗体及其细胞株的制备方法,包括以下步骤:
S1制备抗原:合成胃泌素17多肽,再将胃泌素17多肽偶联到BSA载体蛋白上,透析后获得胃泌素17抗原;所述步骤S1中的胃泌素17多肽的序列为SEQ ID NO:1:
pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Cys;
S2免疫:将步骤S1中制得的胃泌素17抗原进行乳化,免疫雌性小鼠;所述步骤S2进行乳化的具体步骤为:
S21:将步骤S1中制得的胃泌素17抗原与弗氏完全佐剂(Sigma公司)进行混合乳化,即抗原与弗氏完全佐剂按1:1的比例混合;
S22:选免疫4~6周龄雌性Balb/c小鼠,腹部皮下注射每只小鼠5点,剂量为100μg/只,进行第一次免疫;
S23:每14天给每只小鼠进行加强免疫一次,每次加强免疫的剂量为50μg/只;
S24:在第三次加强免疫后的第7天以间接ELISA波长450nm检测小鼠血清中抗免疫原的多抗效价,将效价最高的小鼠以尾静脉注射冲击免疫,尾静脉注射冲击免疫采用抗原与生理盐水混匀,免疫的剂量为50μg/只,完成免疫;
S3细胞融合:将步骤S2中获得的小鼠脾细胞悬液依次与小鼠骨髓瘤细胞sp2/0和混和10xHAT的胸腺细胞混合,然后均匀的分装到若干块铺有饲养细胞的96孔板中,放入37℃的5%CO2培养箱中培养;所述步骤S3细胞融合的具体步骤为:将无菌制备免疫达标的小鼠脾细胞悬液,与小鼠骨髓瘤细胞sp2/0以5:1比例混合,离心1200rpm,5min,弃上清后离心管放入37℃水浴中,在1分钟内缓慢加入1mL的PEG1500(Sigma公司),并搅动细胞;在37℃温水中静置1min后,加入10mL无血清的DMEM(Sigma公司),混匀,离心1000rpm,5min;弃上清后,加入10mL血清,小心的将细胞吹打起来,并加入5mL混合10xHAT(Sigma公司)的胸腺细胞,混匀;然后均匀的分装到10块铺有饲养细胞的96孔板中,放入37℃5%CO2培养箱中培养;
S4酶联免疫吸附剂测定ELISA筛选阳性杂交瘤细胞:将HAT培养基换成HT培养基,取培养基中的上清蛋白进行分别ELISA筛选;具体为:7天后将HAT培养基换成HT培养基,1天后取100μL上清蛋白分别ELISA筛选;
S5亚克隆:筛选出的阳性克隆用梯度稀释法进行亚克隆,直至筛选出纯合的阳性细胞株;
S6腹水制备:将对数生长期细胞用无血清培养基洗涤并悬起,其计数约106个/mL,对小鼠腹腔注射悬浮的细胞,7天后开始收集腹水;取出的腹水于4℃离心,吸出中间的腹水,收集于离心管中,4℃或-20℃保存;
S7单克隆抗体的纯化:用HiTrap rProtein A FF(GE公司)亲和层析法从腹水中纯化抗体,纯化后的抗体保存于-20℃;SDS-PAGE胶鉴定纯度,Bradford法测定浓度;
S8双抗夹心法筛选抗体配对:将一株抗体包被在酶标板上,清洗封闭后加入样本在37℃温度下反应90min,清洗后加入HRP标记的二抗在37℃进行孵育30min,显色终止后读数,筛选出一对抗体2C5和8B10。
该胃泌素17免疫层检测试纸条包括样本垫、结合垫、硝酸纤维素膜、吸水纸和底板;所述硝酸纤维素膜包括被上述胃泌素17抗体包被的检测线和被羊抗鸡抗体包被的质控线。
该胃泌素17免疫层检测试纸条的制备方法,包括以下步骤:从加样端开始依次将样品垫、结合垫、包被好的硝酸纤维素膜、吸水纸四个部分粘贴在底板上,然后切割成所要求的宽度,形成试纸条,装入塑料卡壳中,形成试剂卡;其中该胃泌素17免疫层析试剂条整荧光微球标记2C5抗体度为10~20ug/mL;所述8B10抗体的包被浓度为0.5~1.0mg/mL;所述羊抗鸡IgY抗体的浓度为0.5~1.0mg/mL;
制备方法具体为:
其中所述样品垫在使用前先采用样品垫处理液进行处理,后再将玻璃纤维浸泡在样品垫处理液中,然后烘干;所述样品垫处理液包括pH为8~10的缓冲液、表面活性剂、海藻糖和红细胞单抗;
所述结合垫的制备方法,包括以下步骤:
S1-1:荧光微球与EDC在磷酸盐缓冲液中进行活化处理;
S1-2:将胃泌素17抗体加入上述荧光微球溶液中进行反应;
S1-3:将偶联后的微球-抗体复合物进行封闭反应,经离心后制得免疫荧光微球标记物;
S1-4:将调配好浓度免疫荧光微球标记物溶液包被到玻璃纤维上,烘干,制得结合垫;
所述硝酸纤维素膜的制备方法为:采用8B10抗体配制T线溶液,羊抗鸡IgY配制C线溶液,然后包被在硝酸纤维素膜上的T先即检测线和C线即质控线区域,烘干。
试剂盒的性能分析:
1)胃泌素17检测试剂盒与对照检测试剂盒测定结果比较
选择15例样品,浓度范围1.2~47.8pmol/L,分别用胃泌素17检测试剂盒和对照检测试剂盒进行检测,在比较数据的基础上进行相关分析:相关系数(r)≥0.95,偏差≤15%;测定结果数据比较表如表1所示;
表1胃泌素17检测试剂盒与对照检测试剂盒测定结果比较
序号 | 对照测定 | 自制试剂盒 | 偏差 |
1 | 1.4 | 1.5 | 7.1% |
2 | 2.6 | 2.5 | -3.8% |
3 | 3.3 | 3.8 | 15.2% |
4 | 4.1 | 5.2 | 26.8% |
5 | 5.3 | 4.9 | -7.5% |
6 | 6.5 | 6.8 | 4.6% |
7 | 8.8 | 9 | 2.3% |
8 | 12.6 | 10.8 | -14.3% |
9 | 19.7 | 20.9 | 6.1% |
10 | 22.3 | 26.8 | 20.2% |
11 | 29.1 | 28.2 | -3.1% |
12 | 32.2 | 33.4 | 3.7% |
13 | 36.6 | 38.1 | 4.1% |
14 | 42.7 | 39.5 | -7.5% |
15 | 46 | 45.8 | -0.4% |
2)灵敏度检测
对样本进行浓度梯度稀释,以稀释液作为检测样本,平行测定10次,检测结果的平均值减去两倍标准偏差SD值对应的浓度,结果不高于1.2pmol/L。
以上显示和描述了本发明的基本原理、主要特征及优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列1:
胃泌素17多肽序列:
pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Cys。
Claims (10)
1.一种胃泌素17的单克隆抗体及其细胞株的制备方法,其特征在于,包括以下步骤:
S1制备抗原:合成胃泌素17多肽,再将胃泌素17多肽偶联到BSA载体蛋白上,透析后获得胃泌素17抗原;
S2免疫:将步骤S1中制得的胃泌素17抗原进行乳化,免疫雌性小鼠;
S3细胞融合:无菌制备免疫达标的小鼠脾细胞悬液,依次与小鼠骨髓瘤细胞sp2/0和混合10xHAT的胸腺细胞混合,然后均匀的分装到若干块铺有饲养细胞的孔板中,放入37℃的5%CO2培养箱中培养;
S4酶联免疫吸附剂测定ELISA筛选阳性杂交瘤细胞:将HAT培养基换成HT培养基,取上清蛋白进行分别ELISA筛选;
S5亚克隆:筛选出的阳性克隆用梯度稀释法进行亚克隆,直至筛选出纯合的阳性细胞株;
S6腹水制备:将对数生长期细胞用无血清培养基洗涤并悬起,其计数约106个/mL,对小鼠腹腔注射悬浮的细胞,7天后开始收集腹水;取出的腹水于4℃离心,吸出中间的腹水,收集于离心管中,4℃或-20℃保存;
S7单克隆抗体的纯化:用HiTrap rProtein A FF亲和层析法从腹水中纯化抗体,纯化后的抗体保存于-20℃;
S8双抗夹心法筛选抗体配对:将一株抗体包被在酶标板上,清洗封闭后加入样本在37℃温度下反应70~100min,清洗后加入HRP标记的二抗在37℃进行孵育20~40min,显色终止后读数,筛选出一对抗体2C5和8B10。
2.根据权利要求1所述的胃泌素17的单克隆抗体及其细胞株的制备方法,其特征在于,所述步骤S1中的胃泌素17多肽的序列为SEQ ID NO:1。
3.根据权利要求1所述的胃泌素17的单克隆抗体及其细胞株的制备方法,其特征在于,所述步骤S2进行乳化的具体步骤为:
S21:将步骤S1中制得的胃泌素17抗原与弗氏完全佐剂进行混合乳化,即抗原与弗氏完全佐剂按1:1的比例混合;
S22:选免疫4~6周龄雌性Balb/c小鼠,腹部皮下注射每只小鼠5点,剂量为100μg/只,进行第一次免疫;
S23:每14天给每只小鼠进行加强免疫一次,每次加强免疫的剂量为50μg/只;
S24:在第三次加强免疫后的第7天以间接ELISA检测小鼠血清中抗免疫原的多抗效价,将效价最高的小鼠以尾静脉注射冲击免疫,尾静脉注射冲击免疫采用抗原与生理盐水混匀,免疫的剂量为50μg/只,完成免疫。
4.根据权利要求1所述的胃泌素17的单克隆抗体及其细胞株的制备方法,其特征在于,所述步骤S3细胞融合的具体步骤为:将无菌制备免疫达标的小鼠脾细胞悬液,与小鼠骨髓瘤细胞sp2/0以5:1比例混合,离心1200rpm,5min,弃上清后离心管放入37℃水浴中,在1分钟内缓慢加入1mL的PEG1500,并搅动细胞;在37℃温水中静置1min后,加入10mL无血清的DMEM,混匀,离心1000rpm,5min;弃上清后,加入10mL血清,小心的将细胞吹打起来,并加入5mL混合10xHAT的胸腺细胞,混匀;然后均匀的分装到10块铺有饲养细胞的96孔板中,放入37℃5%CO2培养箱中培养。
5.一种胃泌素17免疫层检测试纸条,其特征在于,该胃泌素17免疫层检测试纸条包括样本垫、结合垫、硝酸纤维素膜、吸水纸和底板;所述硝酸纤维素膜包括被上述胃泌素17抗体包被的检测线和被羊抗鸡抗体包被的质控线。
6.根据权利要求5所述的胃泌素17免疫层检测试纸条,其特征在于,该胃泌素17免疫层检测试纸条的制备方法,包括以下步骤:从加样端开始依次将样品垫、结合垫、包被好的硝酸纤维素膜、吸水纸四个部分粘贴在底板上,然后切割成所要求的宽度,形成试纸条,装入塑料卡壳中,形成试剂卡。
7.根据权利要求6所述的胃泌素17免疫层检测试纸条,其特征在于,所述样品垫在使用前先采用样品垫处理液进行处理,后再将玻璃纤维浸泡在样品垫处理液中,然后烘干。
8.根据权利要求6所述的胃泌素17免疫层检测试纸条,其特征在于,其中所述结合垫的制备方法,包括以下步骤:
S1-1:荧光微球与EDC在磷酸盐缓冲液中进行活化处理;
S1-2:将胃泌素17抗体加入上述荧光微球溶液中进行反应;
S1-3:将偶联后的微球-抗体复合物进行封闭反应,经离心后制得免疫荧光微球标记物;
S1-4:将调配好浓度免疫荧光微球标记物溶液包被到玻璃纤维上,烘干,制得结合垫。
9.根据权利要求6所述的胃泌素17免疫层检测试纸条,其特征在于,所述硝酸纤维素膜的制备方法为:采用8B10抗体配制T线溶液,羊抗鸡IgY配制C线溶液,然后包被在硝酸纤维素膜上的T先即检测线和C线即质控线区域,烘干。
10.根据权利要求7所述的胃泌素17免疫层检测试纸条,其特征在于,所述样品垫处理液包括pH为8~10的缓冲液、表面活性剂、海藻糖和红细胞单抗。
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