CN114672464A - 一种分泌抗牛妊娠相关糖蛋白单克隆抗体的杂交瘤细胞、单克隆抗体及应用 - Google Patents
一种分泌抗牛妊娠相关糖蛋白单克隆抗体的杂交瘤细胞、单克隆抗体及应用 Download PDFInfo
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Abstract
本发明涉及可分泌抗牛妊娠相关糖蛋白(PAGs)单克隆抗体的杂交瘤细胞、单克隆抗体、试剂盒、试纸条及检测方法。具体为可分泌抗牛妊娠相关蛋白(PAG)单克隆抗体的杂交瘤细胞P‑4E11,保藏编号为CCTCC NO:C2021260,以及可分泌抗牛妊娠相关蛋白(PAG)单克隆抗体的杂交瘤细胞P‑5A9,保藏编号为CCTCC NO:C2021261。并由上述细胞株获得了抗牛妊娠相关糖蛋白(PAGs)的特异性抗体P‑4E11和P‑5A9,采用上述单克隆抗体制备了牛妊娠相关糖蛋白(PAGs)检测试剂盒或试纸条。采用本发明提供的牛妊娠相关糖蛋白(PAGs)检测试剂盒或试纸条,操作简单、准确度高,可实现对牛妊娠相关糖蛋白(PAGs)的快速检测。
Description
技术领域:
本发明属于免疫检测领域,具体涉及一种可分泌抗牛妊娠相关糖蛋白(PAGs)单克隆抗体的杂交瘤细胞、单克隆抗体及应用。
背景技术:
妊娠相关糖蛋白(pregnancy associated glycoproteins,PAGs)是反刍动物妊娠后外周血液出现的特异性蛋白,在妊娠过程中发挥着重要的作用,在生产上通过检测PAGs在血清中浓度能够进行反刍动物的妊娠诊断。检测牛血清或EDTA血浆中的早期妊娠相关糖蛋白,在配种后28天就可以检测,且不受上次怀孕干扰,用于分娩后60天及以上的牛,可在配种后尽早精确地识别空怀牛以及在更短的胎间距内产犊。
传统的牛早孕判断主要包括三种方法:直肠触诊法、B超诊断法、孕酮检测法。直肠触诊法通常在配种后35-65天由兽医来进行首次检查,成本低,但对操作人员有较高技术要求,具有侵入性,容易造成流产。B超诊断法在配种后26-33天检测具有较高准确性,所需要的设备昂贵,且在对动物保定过程中使用困难,实践操作不易。孕酮检测方法可在配种后24天测试,需要连续几天测试才能判断妊娠牛,其判断标准很难确定,而且影响因素较多。
随着技术的发展,以PAGs为检测目标的检测方法在国际上广泛应用,如美国IDEXX公司生产的牛妊娠可视化快速检测试剂盒和美国Biotracking公司生产的BioPRYN牛怀孕检测试剂盒,其整体采用双抗体夹心ELISA法,但操作繁琐耗时、不便于使用、并且价格昂贵,限制了在国内的使用。发展操作简便、价格实惠的国产试剂对于国内牧场现场检测需求具有重要意义,而检测试剂所需的核心原料都是特异性抗原或抗体。基于此,制备特异的PAGs抗体,成为本领域技术人员亟待解决的技术问题。
发明内容:
为了解决上述技术问题,本发明将提供一种可分泌抗牛妊娠相关糖蛋白(PAGs,氨基酸序列如序列表SEQ ID NO.1所示)单克隆抗体的杂交瘤细胞、单克隆抗体,用于牛妊娠的快速便捷检测。
本发明提供的技术方案之一,是两株能够分泌抗牛妊娠相关糖蛋白(PAGs)单克隆抗体的杂交瘤细胞,具体为:
可分泌抗牛妊娠相关蛋白(PAG)单克隆抗体的杂交瘤细胞P-4E11,该杂交瘤细胞已于2021年9月29日保藏于中国典型培养物保藏中心(简称CCTCC;地址:中国武汉,武汉大学;邮编:430072),保藏编号为CCTCC NO:C2021260;
可分泌抗牛妊娠相关蛋白(PAG)单克隆抗体的杂交瘤细胞P-5A9,该杂交瘤细胞已于2021年9月29日保藏于中国典型培养物保藏中心(简称CCTCC;地址:中国武汉,武汉大学;邮编:430072),保藏编号为CCTCC NO:C2021261。
本发明所述的牛妊娠相关糖蛋白,本文简称PAGs或PAG,氨基酸序列如下所示:
MKWLVLLGLVAFSECIVKIPLRRLKTMRNVVSGKNMLNNFLKEHAYSLSQISFRGSNLTTHPLRNIKDLVYMGNITIGTPPQEFQVVFDTASSDLWVPSDFCTSPACSTHVRFRHLQSSTFRLTNKTFRITYGSGRMKGVVVHDTVRIGNLVSTDQPFGLSIEEYGFEGRIYDGVLGLNYPNISFSGAIPIFDKLKNQRAISEPVFAFYLSKDEREGSVVMFGGVDHRYYEGELNWVPLIQAGDWSVHMDRISIERKIIACSDGCKALVDTGTSDIVGPRRLVNNIHRLIGAIPRGSEHYVPCSEVNTLPSIVFTINGINYPVPGRAYILKDDRGRCYTTFQENRVSSSTETWYLGDVFLRLYFSVFDRGNDRIGLARAV(SEQ ID NO.1)。
本发明提供的技术方案之二,是杂交瘤细胞P-4E11和杂交瘤细胞P-5A9的应用;
进一步地,所述应用为杂交瘤细胞P-4E11或杂交瘤细胞P-5A9在制备抗牛妊娠相关糖蛋白(PAGs)单克隆抗体中的应用。
本发明提供的技术方案之三,是杂交瘤细胞P-4E11分泌的单克隆抗体P-4E11,或杂交瘤细胞P-5A9分泌的单克隆抗体P-5A9。
本发明提供的技术方案之四,是单克隆抗体P-4E11或单克隆抗体P-5A9的应用;
进一步地,所述应用为单克隆抗体P-4E11或单克隆抗体P-5A9在检测牛妊娠相关糖蛋白中的应用。
本发明提供的技术方案五,是一种含有单克隆抗体P-4E11和单克隆抗体P-5A9的试剂盒;
进一步地,所述试剂盒用于牛妊娠相关糖蛋白的检测。
本发明提供的技术方案之六,是一种胶体金试纸条,所述胶体金试纸条的胶体金垫上包被胶体金标记的单克隆抗体P-5A9,包被膜的检测线(T线)上包被单克隆抗体P-4E11,包被膜的质控线(C线)上包被抗鼠IgG抗体;
进一步地,胶体金试纸条包括底板(又称背衬)、样品垫、结合垫(胶体金)、缓释垫、包被膜和吸水垫;沿样品流动方向,样品垫、结合垫(胶体金)、缓释垫、包被膜和吸水垫依次固定于底板上;包被膜上沿样品流动方向依次设置T线和C线;
更进一步地,所述抗鼠IgG抗体具体可为羊抗鼠IgG抗体;
进一步地,所述胶体金试纸条用于牛妊娠相关糖蛋白的检测。
本发明提供的技术方案之七,是一种荧光试纸条,所述荧光试纸条的荧光垫上包被荧光微球标记的单克隆抗体P-5A9,包被膜的检测线(T线)上包被单克隆抗体P-4E11,包被膜的质控线(C线)上包被抗鼠IgG抗体;
进一步地,所述荧光试纸条包括底板(又称背衬)、样品垫、结合垫(荧光)、包被膜和吸水垫;沿样品流动方向,样品垫、结合垫(荧光)、缓释垫、包被膜和吸水垫依次固定于底板上;包被膜上沿样品流动方向依次设置T线和C线;
更进一步地,所述抗鼠IgG抗体具体可为羊抗鼠IgG抗体。
本发明提供的技术方案之八,是一种ELISA试剂盒,所述试剂盒的微孔板上包被单克隆抗体P-4E11,酶标记单克隆抗体P-5A9;
进一步地,所述ELISA试剂盒包括阳性质控品、阴性质控品、包被微孔板、酶标记抗体、清洗液、显色液、终止液等;
进一步地,所述ELISA试剂用于牛妊娠相关糖蛋白的检测。
有益效果:
灵敏性高且特异性高的抗体是抗原检测技术的开发和实施的关键。本发明基于单克隆抗体技术制备了杂交瘤细胞株P-4E11和杂交瘤细胞株P-5A9,并获得了抗牛妊娠相关糖蛋白(PAGs)的特异性抗体P-4E11和P-5A9,采用上述单克隆抗体制备了牛妊娠相关糖蛋白(PAGs)检测试剂盒或试纸条。采用本发明提供的牛妊娠相关糖蛋白(PAGs)检测试剂或试纸,操作简单、准确度高,可实现对牛妊娠相关糖蛋白(PAGs)的快速检测。
附图说明:
图1为实施例2中单克隆抗体P-4E11,P-5A9与牛妊娠相关糖蛋白的结合曲线。
图2为试纸条的结构示意图
其中,试纸条包括底板(又称为背衬)、样品垫1、结合垫(胶体金或者荧光)2、缓释垫3、包被膜4和吸水垫纸7。沿样品流动方向,样品垫1、结合垫(胶体金或者荧光)2、缓释垫3、包被膜4和吸水垫7依次固定于底板上(相邻垫之间略重叠)。沿样品流动方向,包被膜4上依次设有检测线5、质控线6。检测线5亦称T线,质控线6亦称C线。
图3抗体特异性测试结果。
具体实施方式:
为了使本专利的目的、技术方案及优点更加清楚明白,以下结合具体实施例,对本专利进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本专利,但不用来限制本发明的范围。
实施例1:可分泌抗牛妊娠相关糖蛋白(PAGs)的单克隆抗体的杂交瘤细胞株的获得
根据GenBank提供的PAG全长基因编码序列(SEQ ID NO.1)进行分析,在编码序列两端加上酶切位点EcoRⅠ和XhoⅠ及保护碱基进行全基因合成。双酶切载体pcDNA3.1(+)后与同样酶切过的PAG基因片段连接转化,构建重组质粒并进行测序。将测序结果比对正确的PAG重组质粒瞬时转染HEK293细胞,收集细胞并裂解后纯化重组PAG蛋白。将纯化后的牛妊娠相关糖蛋白(PAG)用生理盐水配制为浓度500μg/mL浓度与Quick Antibody-Mouse 3W佐剂等体积混合,后小腿单点肌肉注射100μL免疫2只6周龄Balb/c小鼠,第14天按同样方式加强免疫。第21天自小鼠尾部取血,分离血清后采用间接ELISA法检测血清效价,效价达到融合要求。在细胞融合前3天,用100μg蛋白进行脾脏加强免疫。
将Balb/c小鼠脾脏细胞与对数生长期的NS1骨髓瘤细胞按9:1比例混合后,在50%的PEG1500作用下进行融合。融合后细胞于HAT半固体培养基培养8-10天,挑取细胞克隆到HT培养基筛选培养2天后,采用间接ELISA法检测细胞培养上清中的抗体特异性。将阳性细胞再用HT半固体培养基克隆1次,获得能稳定分泌抗体的单克隆细胞株,扩大培养后,用含10%DMSO的FBS冻存,细胞密度为106个/mL。采用体内诱生法制备腹水,腹水经Protein A亲和层析纯化得到纯化的抗体,于0.01mol/L磷酸盐缓冲液中透析后,用Nanodrop测定蛋白浓度,获得了12株单克隆细胞株。进行抗体配对实验:将获得的12株单克隆细胞株所分泌的12种抗体分别包被膜和标记胶体金后互相完全搭配组合,检测PAG阴性和阳性血清样品,经过对抗体配对实验,结果见表1(配对抗体筛选的方法是双抗夹心ELISA法。筛选的原理为:在得到了单克隆抗体后,从中取出两种单克隆抗体,一种作为捕获抗体,另一种作为标记抗体,将捕获抗体包被在抗原板上,先加入抗原,孵育后洗去未结合的抗原,再加入标记抗体孵育后洗去未结合的标记抗体,最后加入显色液显色。如果可以显色,说明标记抗体与抗原特异性结合,该捕获抗体与标记抗体为一对配对抗体。如果不能显色,说明标记抗体不能与抗原结合,从而被洗脱,该捕获抗体与标记抗体不是配对抗体。)。
从单克隆细胞株中筛选到最好的两株能稳定分泌单克隆抗体且能配对用于双抗体夹心法测试的细胞株,将其分别命名为可分泌抗牛妊娠相关蛋白(PAG)单克隆抗体的杂交瘤细胞P-4E11,P-5A9。
表1 12株单克隆抗体相互配对实验结果
注:-为无法配对;+为能配对,阳性结果弱;++为能配对,阳性结果强;+++为能配对,阳性结果最强。
杂交瘤细胞P-4E11,P-5A9,均已于2021年9月29日保藏在中国典型培养物保藏中心(简称CCTCC;地址:中国武汉,武汉大学;邮编:430072),保藏编号分别为CCTCC NO:C2021260,CCTCC NO:C2021261。杂交瘤细胞P-4E11,P-5A9可分泌抗牛妊娠相关糖蛋白(PAG)的单克隆抗体,将杂交瘤细胞株P-4E11,P-5A9分泌的单克隆抗体分别命名为单克隆抗体P-4E11,P-5A9。
实施例2、单克隆抗体P-4E11,P-5A9的制备、纯化以及效价
一、单克隆抗体P-4E11,P-5A9的制备
1、增量培养法
细胞培养基的制备方法:向RPMI-1640培养基中添加胎牛血清,胎牛血清的终浓度为20%(质量百分含量)。
将杂交瘤细胞株P-4E11,P-5A9分别置于细胞培养基中,37℃培养3-4天,细胞培养上清液用Protein A亲和层析纯化,得到纯度为95%以上的单克隆抗体P-4E11和P-5A9(-20℃保存)。
2、腹水制备
Balb/c小鼠腹腔注射灭菌石蜡油(0.5mL/只),10天后分别腹腔注射杂交瘤细胞株P-4E11,P-5A9(1×106个细胞/只),8-10天后采集腹水,采用Protein A亲和层析纯化,然后转移到透析袋中在pH7.4、0.01M的PBS缓冲液中进行透析,然后收集透析袋中的液体,分别得到纯度为95%以上的单克隆抗体P-4E11,P-5A9,-20℃保存。
后续步骤以及后续实施例中所用的单克隆抗体P-4E11、P-5A9均为腹水制备得到的。
二、效价检测(间接ELISA法)
1、取酶标板,加入包被液(100μL/孔),4℃包被过夜。包被液:取牛妊娠相关糖蛋白(PAGs),用pH9.6、0.05M的碳酸盐缓冲液稀释至蛋白浓度为2μg/mL,即为包被液。
2、完成步骤1后,取所述酶标板,用PBST溶液洗涤4次,然后加入封闭液(200μL/孔),37℃孵育2小时。封闭液:含1g/100mL BSA的PBST溶液。
3、完成步骤2后,取所述酶标板,用PBST溶液洗涤4次,然后加入抗体稀释液(100μL/孔),37℃孵育1h。抗体稀释液:取单克隆抗体P-4E11,P-5A9腹水,分别用pH7.4、0.02M的PBS缓冲液进行10倍梯度稀释,得到不同浓度的抗体稀释液。
4、完成步骤3后,取所述酶标板,用PBST溶液洗涤4次,然后加入HRP标记二抗工作液,37℃孵育1h。HRP标记二抗工作液:HRP标记二抗的1:4000稀释液。HRP标记二抗:洛阳佰奥通实验材料中心,货号为C030205。
5、完成步骤4后,取所述酶标板,用PBST溶液洗涤4次,然后加入TMB底物反应液显色10分钟,然后加入2M硫酸溶液终止显色,然后用酶标仪测定450nm吸光值(见表2)。
可见,单克隆抗体P-4E11,P-5A9的效价均为1:10000000。
表2
三、EC50值检测(使用纯化后的抗体)
抗体稀释液:取单克隆抗体P-4E11,P-5A9,用pH7.4、0.02M的PBS缓冲液稀释至蛋白浓度为10μg/mL,然后进行10倍梯度稀释。
替换抗体稀释液,其他同步骤二。
结果见图1。单克隆抗体的EC50低于20ng/mL。EC50指的是半数效应浓度,最大OD的50%时对应的抗体浓度,其中,P-4E11,EC50为14.5ng/mL;P-5A9,EC50为19.7ng/mL。单克隆抗体P-4E11,P-5A9对于PAG的检测限均为0.01ng/mL。
四、抗体亚型鉴定:
采用商品化抗体Ig类/亚类/亚型鉴定试剂盒(洛阳佰奥通实验材料中心,货号C060101-L)鉴定抗体亚型,结果如下表所示,结果表明抗体P-4E11为IgG2b亚型,抗体P-5A9为IgG1亚型,抗体轻链均为Kappa链。
亚型 | P-4E11 | P-5A9 |
IgG1 | 0.079 | 1.894 |
IgG2a | 0.093 | 0.087 |
IgG2b | 1.922 | 0.083 |
IgG3 | 0.078 | 0.05 |
IgM | 0.068 | 0.06 |
IgA | 0.078 | 0.056 |
Lamda | 0.087 | 0.062 |
Kappa | 0.932 | 0.88 |
五、抗体特异性测试(替换包被液和抗体稀释液,其他同步骤二):
分别以牛血清白蛋白(BSA)、阴性血清、孕牛血清、重组PAG蛋白(实施例1制备)包板,分别加入0.1μg/mL浓度的纯化PAG抗体P-4E11、P-5A9,测试抗体与这些蛋白的反应。结果如图3所示,表明抗体与PAG反应,与其他蛋白不反应。
实施例3、胶体金快速检测试纸的制备
一、胶体金标记抗体的制备
1、采用柠檬酸三钠还原法制备胶体金
用量筒量取495ml去离子水,倒入玻璃两颈瓶中,加入5ml 1g/100mL氯金酸水溶液;在玻璃两颈瓶中放入一个磁力搅拌子,连接球形冷凝管,用可控温电磁加热搅拌器加热至沸腾;边搅动边加入1g/100mL柠檬酸三钠水溶液5ml,金黄色的氯金酸溶液在2分钟内变为紫红色,继续煮沸5分钟,然后自然冷却,即为胶体金溶液。
2、制备胶体金标记的P-5A9抗体
取20体积份胶体金溶液,用0.1M碳酸钾水溶液调pH至7.5,然后加入单克隆抗体P-5A9(每毫升胶体金溶液配比10微克抗体),室温搅拌反应1小时,然后加入BSA并使其浓度为1g/100mL反应1小时;反应完毕后,置于离心机中4℃、12000rpm离心20分钟,弃除上清,剩余的红色沉淀物用1体积份含1g/100mL BSA的PBS缓冲液(pH7.4、0.02M)重悬,即为胶体金标记的P-5A9抗体溶液,4℃保存。
二、胶体金试纸条的制备
1、制备包被膜
取硝酸纤维素膜,采用BioDot的XYZ3050喷膜系统将C溶液喷至质控线(C线)位置,将T溶液喷至检测线(T线)位置(按1μL/cm量喷涂,平均每个试纸条上3.5mm的喷涂量为0.35μL)于相对湿度为10%以下的干燥车间抽湿4小时,得到包被膜。采用pH7.4、0.02M的PBS缓冲液,将羊抗鼠IgG抗体配制为浓度1mg/ml的溶液,即为C溶液。采用pH7.4、0.02M的PBS缓冲液,将单克隆抗体P-4E11配制为浓度2mg/ml的溶液(以蛋白浓度计),即为T溶液。羊抗鼠IgG抗体:洛阳佰奥通实验材料中心,货号为C020201。
2、制备胶体金垫
(1)取玻璃纤维纸,置于含0.2g/100mL Tween-20、1g/100mL BSA、2g/100mL蔗糖的pH7.4、0.02M的PBS缓冲液中37℃浸泡半小时,然后于相对湿度为10%以下的干燥车间抽湿4小时。
(2)完成步骤(1)后,取所述玻璃纤维纸,采用BioDot的XYZ3050喷膜系统喷涂金标抗体溶液(按12μL/cm量喷涂,平均每个试纸条上3.5mm的喷涂量为4.2μL),然后于相对湿度为10%以下的干燥车间抽湿4小时,得到胶体金垫。金标抗体溶液:取步骤一制备的胶体金标记的P-5A9抗体溶液,用含0.2g/100mL Tween-20、1g/100mL BSA、2g/100mL蔗糖的pH7.4、0.02M的PBS缓冲液稀释,使胶体金标记的P-5A9抗体的浓度为2μg/ml(以蛋白浓度计)。
3、试纸条的组装
在10万级洁净和干燥的车间中进行试纸条的组装。
试纸条包括底板(又称为背衬)、样品垫1、结合垫(胶体金垫)2、缓释垫3、包被膜4和吸水垫纸7。沿样品流动方向,样品垫1、结合垫(胶体金垫)2、缓释垫3、包被膜4和吸水垫7依次固定于底板上(相邻垫之间略重叠)。沿样品流动方向,包被膜上依次设有检测线5、质控线6。检测线5亦称T线,质控线6亦称C线。
沿样品流动方向作为试纸条的长度方向,与长度方向垂直的方向作为试纸条的宽度方向。试纸条的宽度为3.5mm,试纸条的总长度为6cm。其中,样品垫1长度1.5-1.8cm,结合垫(胶体金垫)2的长度为0.5cm,缓释垫3的长度0.4cm,包被膜4的长度为2.5cm,吸水垫7的长度为2cm。C线靠近吸水垫7、远离缓释垫3。T线靠近缓释垫3、远离吸水垫7。
缓释垫3的材质为玻璃纤维膜,用于减慢胶体金标记抗体和样品流到包被膜上的速度,使得胶体金标记抗体和样品接触时间更多,反应更充分。
吸水垫7的材质为吸水纸。
样品垫1的材质为玻璃纤维膜。
底板仅起支撑作用,常规材质均可,例如本实施例具体为聚氯乙烯(PVC)。
三、试纸条的使用方法
将60μL待检样品垂直缓慢滴入胶体金试纸条的加样端,室温水平放置10-15分钟,然后进行如下结果判断:
C线显色,表示结果可信;
如果T线显色,说明待检样品中含有牛妊娠相关糖蛋白(PAGs),从而判断受试者为PAGs阳性;
如果T线不显色,说明待检样品中不含有PAGs,从而判断受试者为PAG阴性。
四、试纸条的灵敏度测试
将重组PAG蛋白(实施例1制备)用0.02M PBS稀释配制为系列浓度,用实施例3的试纸条进行测试,检测灵敏度为1ng/mL。
浓度(ng/mL) | 0 | 0.1 | 1 | 10 | 100 | 1000 |
结果 | 阴性 | 阴性 | 阳性 | 阳性 | 阳性 | 阳性 |
实施例4、荧光快速检测试纸的制备
一、荧光微球标记抗体的制备
1、制备羧基修饰的荧光微球
将苯乙烯和甲基丙烯酸甲脂按1:1的体积比混合,加入稀土络合物Eu(TTA)3Phen并使其在体系中的浓度为1g/100mL,超声混匀,得到a液。将羧基化聚乙烯醇和碳酸氢钠溶于水,使羧基化聚乙烯醇的浓度为0.05g/100mL、碳酸氢钠的浓度为0.05g/100mL,得到b液。将1体积份a液加入10体积份b液中,然后超声15分钟,通氮气30分钟搅拌除氧,加热至80℃,加入过硫酸钾并使其在体系中的浓度为0.1g/100mL(实际情况中,0.01-0.1g/100mL均可),反应12小时,之后依次进行过滤、离心和去离子水清洗,得到羧基修饰的荧光微球。
实际应用中,也可用如下方法制备a液:将苯乙烯和甲基丙烯酸甲脂按1:1的体积比混合,加入CdSe/ZnS量子点并使其在体系中的浓度为0.5g/100mL,超声混匀,得到a液。
2、活化
取10mg羧基修饰的荧光微球,用pH4.7、0.1M的MES缓冲液洗涤并离心,然后用1mlpH4.7、0.1M的MES缓冲液重悬,加入EDC(使其在体系中的浓度为5mM)和NHS(使其在体系中的浓度为10mM),室温避光反应半小时,得到活化后羧基修饰的荧光微球。EDC:1-乙基-(3-二甲基氨基丙基)碳二亚胺。NHS:N-羟基丁二酰亚胺。
3、制备荧光微球标记抗体
(1)取步骤2得到的活化后羧基修饰的荧光微球,用pH8.5、50mM的硼砂缓冲液洗涤。
(2)取单克隆抗体P-5A9(抗体含量为0.7mg)和完成步骤(1)的微球,在pH8.5、50mM的硼砂缓冲液中充分混匀,室温避光反应2小时(抗体和微球形成稳定的肽键共价结合从而得到偶联物),然后加入BSA并使其在体系中的浓度为1g/100mL以封闭剩余活性羧基位点,室温避光反应0.5小时,然后用pH7.4、0.02M的PBS缓冲液洗涤、重悬,得到荧光微球标记的P-5A9抗体溶液(浓度为5mg/ml,以微球浓度计),4℃保存。
二、荧光试纸条的制备
1、制备包被膜
同实施例3的步骤二的1。
2、制备荧光垫
(1)取玻璃纤维纸,置于含0.2g/100mL Tween-20、1g/100mL BSA、2g/100mL蔗糖的pH7.4、0.02M的PBS缓冲液中37℃浸泡半小时,于相对湿度为10%以下的干燥车间抽湿4小时。
(2)完成步骤(1)后,取所述玻璃纤维纸,采用BioDot的XYZ3050喷膜系统喷涂荧光标记抗体溶液(按12μL/cm量喷涂),于相对湿度为10%以下的干燥车间抽湿4小时,得到荧光垫。荧光标记抗体溶液:取步骤一制备的荧光微球标记的P-5A9抗体溶液,用含0.2g/100mL Tween-20、1g/100mL BSA、2g/100mL蔗糖的pH7.4、0.02M的PBS缓冲液稀释,使荧光微球标记的P-5A9抗体的浓度为2μg/ml(以蛋白浓度计)。
3、试纸条的组装
在10万级洁净和干燥的车间中进行试纸条的组装。
试纸条包括底板(又称为背衬)、样品垫1、结合垫(荧光垫)2、缓释垫3、包被膜4和吸水垫纸7。沿样品流动方向,样品垫1、结合垫(荧光垫)2、缓释垫3、包被膜4和吸水垫7依次固定于底板上(相邻垫之间略重叠)。沿样品流动方向,包被膜上依次设有检测线5、质控线6。检测线5亦称T线,质控线6亦称C线。
沿样品流动方向作为试纸条的长度方向,与长度方向垂直的方向作为试纸条的宽度方向。试纸条的宽度为3.5mm。试纸条的总长度为6cm,其中,样品垫1长度1.8cm,结合垫(荧光垫)2的长度为0.5cm,缓释垫3的长度0.4cm,包被膜4的长度为2.5cm,吸水垫7的长度为2cm。C线靠近吸水垫7、远离缓释垫3。T线靠近缓释垫3、远离吸水垫7。
缓释垫3的材质为玻璃纤维膜,用于减慢胶体金标记抗体和样品流到包被膜上的速度,使得胶体金标记抗体和样品接触时间更多,反应更充分。
吸水垫7的材质为吸水纸。
样品垫1的材质为玻璃纤维膜。
底板仅起支撑作用,常规材质均可,例如本实施例具体为聚氯乙烯(PVC)三、试纸条的使用方法
将60μl待检样品垂直缓慢滴入荧光试纸条的加样端,室温水平放置10-15分钟,365nm紫外灯照射或者用荧光检测仪判读,然后进行如下结果判断:
C线显色,表示结果可信;
如果T线显色,说明待检样品中含有牛妊娠相关糖蛋白(PAGs),从而判断受试者为PAG阳性;
如果T线不显色,说明待检样品中不含有PAGs,从而判断受试者为PAGs阴性。
四、试纸条的灵敏度测试
将重组PAG蛋白(实施例1制备)用0.02M PBS稀释配制为系列浓度,用实施例4的试纸条进行测试,检测灵敏度为0.1ng/mL。
浓度(ng/mL) | 0 | 0.1 | 1 | 10 | 100 | 1000 |
结果 | 阴性 | 阳性 | 阳性 | 阳性 | 阳性 | 阳性 |
实施例5、ELISA检测试剂盒的制备
一、HRP标记抗体的制备
称取1mg HRP溶解于去离子水中,浓度为10mg/ml,体积为0.1ml;于配好的HRP溶液中,加入0.1ml新配的12.8mg/ml NaIO4溶液,与酶液等体积,逐滴加入,4℃下避光反应30min,至反应液呈墨绿色;逐滴加入0.1ml浓度0.5%乙二醇,混匀,4℃下避光反应30min,至反应液呈棕色;加入1mg单克隆抗体P-5A9,混匀,将抗体和活化酶装入透析袋中,在50mMPH9.6的CBS缓冲液透析,4℃过夜;取出透析组分,加20μL新配的5mg/ml NaBH4溶液,避光静置2h;再加入等体积的饱和(NH4)2SO4溶液沉淀蛋白,5000rpm离心30min;去除上清,沉淀再用50%(NH4)2SO4溶液重悬,5000rpm离心30min;沉淀用PBS溶解,然后用脱盐柱脱盐,加入等体积的甘油作保护剂,得到酶标记抗体P-5A9,分装后-20℃保存。
二、包被板的制备
将单克隆抗体P-4E11用碳酸盐缓冲液稀释至终浓度2μg/ml,吸取100μL/孔稀释抗体到PVC酶标板中。酶标板上覆盖封口膜,4℃过夜。倒掉包被液,用PBST洗板3次,用含5%脱脂奶粉的PBST缓冲液封闭酶标板上剩余的蛋白结合位点,每孔加200μL。封口膜覆盖酶标板37℃恒温箱温育2小时,PBST洗板3次。干燥后用铝箔袋密封,得到包被板,4℃保存。
三、ELISA检测试剂盒的使用方法
取出包被板,恢复到室温,用移液器吸取100μL阴性对照(阴性牛血清)加入到一个反应孔中,用移液器吸取100μL阳性对照(10ng/mL重组PAG)加入到另一个反应孔中,用移液器吸取100μL待检样品加入到剩余的反应孔中,在每个反应孔中加入50μL酶标记抗体(200ng/mL),酶标板盖上板盖或者覆盖封口膜,轻弹10次混匀。开始计时。15-30度下孵育30分钟。孵育完成后,移除板盖或者封口膜,快速翻转酶标板,将板孔内液体弃入废液缸,并在吸水纸上拍干,PBST洗板3次,加入100μLTMB底物溶液显色10分钟,再加入50μL终止液(2M硫酸)。可用肉眼判读结果或者用酶标仪检测结果。
阳性对照孔显色,阴性对照孔不显色,表示结果可信;
如果样品反应孔显色,且比阴性对照孔深,说明待检样品中含有牛妊娠相关糖蛋白(PAGs),从而判断受试者为PAG阳性;
如果样品反应孔显色比阴性对照孔浅或者相同,说明待检样品中不含有PAGs,从而判断受试者为PAGs阴性。
四、ELISA检测试剂盒的灵敏度测试
将重组PAG蛋白(实施例1制备)用0.02MPBS稀释配制为系列浓度,用实施例5的试剂盒的进行测试,检测灵敏度为0.01ng/mL。
浓度(ng/mL) | 0 | 0.001 | 0.01 | 0.1 | 1 | 10 | 100 | 1000 |
结果 | 阴性 | 阴性 | 阳性 | 阳性 | 阳性 | 阳性 | 阳性 | 阳性 |
实施例6、实施例试剂的性能测试
孕牛血清测试:测试配种后28天的孕牛血清185份,分别用IDEXX可视化试剂盒和实施例3试纸条、实施例4试纸条、实施例5试剂盒测试,实施例3试纸条与IDEXX可视化试剂盒符合率为98.9%,实施例4试纸条与IDEXX可视化试剂盒符合率为99.5%,实施例5试剂盒与IDEXX可视化试剂盒符合率为99.5%。同时,采用实施例3、4试纸条进行检测仅需10min即可完成。采用实施例5试剂盒进行检测需45min即可完成。
虽然上文已经用一般性说明、具体实施方式及实验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 吴峰
<120> 一种分泌抗牛妊娠相关糖蛋白单克隆抗体的杂交瘤细胞、单克隆抗体及应用
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 380
<212> PRT
<213> 牛
<400> 1
Met Lys Trp Leu Val Leu Leu Gly Leu Val Ala Phe Ser Glu Cys Ile
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Val Lys Ile Pro Leu Arg Arg Leu Lys Thr Met Arg Asn Val Val Ser
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Gly Lys Asn Met Leu Asn Asn Phe Leu Lys Glu His Ala Tyr Ser Leu
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Ser Gln Ile Ser Phe Arg Gly Ser Asn Leu Thr Thr His Pro Leu Arg
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Asn Ile Lys Asp Leu Val Tyr Met Gly Asn Ile Thr Ile Gly Thr Pro
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Pro Gln Glu Phe Gln Val Val Phe Asp Thr Ala Ser Ser Asp Leu Trp
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Val Pro Ser Asp Phe Cys Thr Ser Pro Ala Cys Ser Thr His Val Arg
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Phe Arg His Leu Gln Ser Ser Thr Phe Arg Leu Thr Asn Lys Thr Phe
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Arg Ile Thr Tyr Gly Ser Gly Arg Met Lys Gly Val Val Val His Asp
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Thr Val Arg Ile Gly Asn Leu Val Ser Thr Asp Gln Pro Phe Gly Leu
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Ser Ile Glu Glu Tyr Gly Phe Glu Gly Arg Ile Tyr Asp Gly Val Leu
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Gly Leu Asn Tyr Pro Asn Ile Ser Phe Ser Gly Ala Ile Pro Ile Phe
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Asp Lys Leu Lys Asn Gln Arg Ala Ile Ser Glu Pro Val Phe Ala Phe
195 200 205
Tyr Leu Ser Lys Asp Glu Arg Glu Gly Ser Val Val Met Phe Gly Gly
210 215 220
Val Asp His Arg Tyr Tyr Glu Gly Glu Leu Asn Trp Val Pro Leu Ile
225 230 235 240
Gln Ala Gly Asp Trp Ser Val His Met Asp Arg Ile Ser Ile Glu Arg
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Lys Ile Ile Ala Cys Ser Asp Gly Cys Lys Ala Leu Val Asp Thr Gly
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Thr Ser Asp Ile Val Gly Pro Arg Arg Leu Val Asn Asn Ile His Arg
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Leu Ile Gly Ala Ile Pro Arg Gly Ser Glu His Tyr Val Pro Cys Ser
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Tyr Pro Val Pro Gly Arg Ala Tyr Ile Leu Lys Asp Asp Arg Gly Arg
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Claims (10)
1.一种可分泌抗牛妊娠相关蛋白单克隆抗体的杂交瘤细胞株,其特征在于,具体为可分泌抗牛妊娠相关蛋白(PAG)单克隆抗体的杂交瘤细胞P-4E11,保藏编号为CCTCC NO:C2021260。
2.一种可分泌抗牛妊娠相关蛋白单克隆抗体的杂交瘤细胞株,其特征在于,具体为可分泌抗牛妊娠相关蛋白(PAG)单克隆抗体的杂交瘤细胞P-5A9,保藏编号为CCTCC NO:C2021261。
3.权利要求1和,或2所述杂交瘤细胞的应用。
4.一种单克隆抗体,其特征在于,由权利要求1所述的杂交瘤细胞P-4E11产生,具体为单克隆抗体P-4E11。
5.一种单克隆抗体,其特征在于,由权利要求2所述的杂交瘤细胞P-5A9产生,具体为单克隆抗体P-5A9。
6.权利要求4和,或5所述单克隆抗体的应用。
7.一种用于牛妊娠相关糖蛋白检测的试剂盒,其特征在于,所述试剂盒包含权利要求4和5所述单克隆抗体。
8.一种试纸条,其特征在于,所述试纸条为胶体金试纸条,所述胶体金试纸条的胶体金垫上包被胶体金标记的单克隆抗体P-5A9,包被膜的检测线上包被单克隆抗体P-4E11,包被膜的质控线上包被抗鼠IgG抗体;或者,
所述试纸条为荧光试纸条,所述荧光试纸条的荧光垫上包被荧光标记的单克隆抗体P-5A9,包被膜的检测线上包被单克隆抗体P-4E11,包被膜的质控线上包被抗鼠IgG抗体。
9.一种ELISA试剂盒,其特征在于,所述试剂盒的微孔板上包被单克隆抗体P-4E11,酶标记单克隆抗体P-5A9。
10.权利要求1-9任意一项杂交瘤细胞株、单克隆抗体、试纸条或ELISA试剂盒在牛妊娠相关糖蛋白检测中的应用,所述牛妊娠相关糖蛋白如SEQ ID NO.1所示。
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CN101918445A (zh) * | 2007-12-13 | 2010-12-15 | 孟山都技术公司 | 用于早期妊娠诊断的组合物和方法 |
CN105838679A (zh) * | 2016-04-12 | 2016-08-10 | 北京瑞鹰生物技术有限公司 | 牛妊娠相关糖蛋白pag特异性单克隆抗体细胞株及其应用 |
CN106831976A (zh) * | 2017-03-30 | 2017-06-13 | 山西农业大学 | 奶牛pag1多肽、其多克隆抗体及应用 |
CN111587252A (zh) * | 2017-11-15 | 2020-08-25 | Sls生物有限公司 | 与牛妊娠相关糖蛋白1特异性结合的抗体及其用途 |
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CN101918445A (zh) * | 2007-12-13 | 2010-12-15 | 孟山都技术公司 | 用于早期妊娠诊断的组合物和方法 |
CN105838679A (zh) * | 2016-04-12 | 2016-08-10 | 北京瑞鹰生物技术有限公司 | 牛妊娠相关糖蛋白pag特异性单克隆抗体细胞株及其应用 |
CN106831976A (zh) * | 2017-03-30 | 2017-06-13 | 山西农业大学 | 奶牛pag1多肽、其多克隆抗体及应用 |
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