CN116589576B - 一种抗hbp单克隆抗体的制备方法、单克隆抗体及应用 - Google Patents
一种抗hbp单克隆抗体的制备方法、单克隆抗体及应用 Download PDFInfo
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Abstract
本发明涉及一种抗HBP单克隆抗体的制备方法、单克隆抗体及应用。本发明以HBP真核重组蛋白作为免疫原,通过免疫兔子,对兔子的PBMC进行细胞分选,获取特异性结合免疫原的细胞,然后对抗体进行基因扩增获取抗体的轻重链基因序列,通过后续的细胞重组表达获取高纯度和高亲和力的单克隆抗体。本发明涉及的单克隆抗体作为诊断用的重要原料,可以特异性结合天然样本中的HBP蛋白,具有较高的临床符合率,对于临床细菌性感染性疾病的早期诊断与治疗具有重要指示意义。
Description
技术领域
本发明属于单克隆抗体制备技术领域,具体是一种抗肝素结合蛋白的单克隆抗体、单克隆抗体及其应用。
背景技术
肝素结合蛋白(HBP)是存在于中性粒细胞中的蛋白质,由Shafer教授在1984年发现并分离成功。由于当时测得的相对分子质量为37000,因此将其命名为CAP37(cationicantimicrobial protein of 37000)。后来学者又从嗜苯胺蓝颗粒中分离出具有杀菌活性的嗜苯胺蓝蛋白,命名为Azurocidin,即天青杀素。再后来发现其具有极强的肝素结合能力,因此命名为肝素结合蛋白(Heparin-bindin protein,HBP)。
肝素结合蛋白是细菌感染最早升高的标志物,当发生感染时,细菌侵入血管内,刺激中性粒细胞释放HBP,血浆中HBP含量升高。与其他传统感染指标如PCT、CRP、IL-6、白细胞数量等相比,HBP的感染变化出现早,灵敏度、特异性、阳性和阴性检出率等综合指征显著优于其他感染指标。动态监测血浆HBP浓度,可用于评估脓毒症的严重程度,预测严重感染患者休克风险、脓毒症患者心搏骤停风险等。对临床细菌感染性疾病尽早诊断和治疗具有重要指示意义。
HBP的检测方法主要为免疫荧光法,定量检测。该方法学具有操作过程简单、报告结果时间短等优势,应用于各个临床科室可有效提高检测效率,特别对于危重症患者,可有效缩短样本流转时间,尽快帮助临床医生了解患者病情。但是目前市场普遍反馈准确度较低,根本原因就是作为试剂开发的原料,单克隆抗体的特异性较差,本底信号较高,导致临床符合率较差。所以,开发高性能的抗HBP单克隆抗体尤为重要。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一;为此,本发明提出了一种抗肝素结合蛋白的单克隆抗体。
一种抗HBP单克隆抗体的制备方法,该方法具体包括如下步骤:
制备抗HBP的单克隆抗体,具体为:
(1)动物免疫:以HBP蛋白作为抗原,与等量的福氏完全佐剂充分乳化后,经皮下注射免疫兔子,以两周时间间隔进行加强免疫3次,末次加强免疫后5-8d 后经兔子静脉取血,用ELISA法检测血清效价,血清效价达到>128K的标准后,进行外周血单个核细胞(PBMC)悬液的制备;
(2)PBMC制备:在离心管中加入淋巴细胞分离液Ficoll,取抗凝外周血(全血)与无菌PBS按照1:1-1:5充分混匀,用移液管沿管壁缓慢叠加于分层液面上,水平离心400g×30分钟,离心后去除部分上层液体,余下约1mL,用移液器插到云雾层,吸取单个核细胞;置入另一离心管中,加入5倍以上体积的PBS,离心300g×10分钟,洗涤细胞两次;末次离心后,弃上清,加入红细胞裂解液,室温孵育2分钟,裂解红细胞,加入10mLPBS,离心300g×10分钟,洗涤细胞两次;弃上清,加入含有10%FBS的RPMI1640,重悬细胞,计数;
(3)获取单个B细胞:利用制备好的PBMC,通过对抗原标记,使用流式分选设备进行细胞分选获得具有抗原特异性的B细胞;通过对目标B细胞的基因扩增,获取抗体的轻重链基因序列。
与现有技术相比,本发明的有益效果是:
本发明以HBP真核重组蛋白作为免疫原,通过免疫兔子,对兔子的PBMC进行细胞分选,获取特异性结合免疫原的细胞,然后对抗体进行基因扩增获取抗体的轻重链基因序列,通过后续的细胞重组表达获取高纯度和高亲和力的兔单克隆抗体,本发明涉及的单克隆抗体作为诊断用的重要原料,可以特异性结合天然样本中的HBP蛋白,具有较高的临床符合率,对于临床细菌性感染性疾病的早期诊断与治疗具有重要指示意义。
附图说明
图1为本发明抗HBP抗体的SDS-PAGE检测结果的图;
图2-a为本发明抗HBP抗体通过HPLC检测的色谱图;
图2-b为本发明抗HBP抗体通过HPLC检测的色谱图;
图2-c为本发明抗HBP抗体通过HPLC检测的色谱图;
图2-d为本发明抗HBP抗体通过HPLC检测的色谱图;
图3-a为本发明为间接ELISA法测定抗HBP抗体与HBP蛋白结合活性结果的图;
图3-b为本发明为间接ELISA法测定抗HBP抗体与HBP蛋白结合活性结果的图;
图3-c为本发明为间接ELISA法测定抗HBP抗体与HBP蛋白结合活性结果的图;
图3-d为本发明为间接ELISA法测定抗HBP抗体与HBP蛋白结合活性结果的图;
图4为时间分辨荧光法HBP检测试剂临床样本相关性的结果的图。
实施方式
下面将结合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它实施例,都属于本发明保护的范围。
请参阅图1,本申请提供了一种抗HBP单克隆抗体的制备方法,该方法具体包括如下步骤:
一、制备抗HBP的单克隆抗体;此处HBP即为肝素结合蛋白,为了便于表示,后文统一用HBP表示;
(1)动物免疫:以HBP蛋白作为抗原,与等量的福氏完全佐剂充分乳化后,经皮下注射免疫兔子,以两周时间间隔进行加强免疫3次,末次加强免疫后5-8d 后经兔子静脉取血,用ELISA法检测血清效价,血清效价达到>128K的标准后,进行外周血单个核细胞(PBMC)悬液的制备;
(2)PBMC制备:在离心管中加入淋巴细胞分离液Ficoll,取抗凝外周血(全血)与无菌PBS按照1:1-1:5充分混匀,用移液管沿管壁缓慢叠加于分层液面上,水平离心400g×30分钟,离心后去除部分上层液体,余下约1mL,用移液器插到云雾层,吸取单个核细胞;置入另一离心管中,加入5倍以上体积的PBS,离心300g×10分钟,洗涤细胞两次;末次离心后,弃上清,加入红细胞裂解液,室温孵育2分钟,裂解红细胞,加入10mLPBS,离心300g×10分钟,洗涤细胞两次;弃上清,加入含有10%FBS的RPMI1640,重悬细胞,计数;
(3)获取单个B细胞:利用制备好的PBMC,通过对抗原标记,使用流式分选设备进行细胞分选获得具有抗原特异性的B细胞;通过对目标B细胞的基因扩增,获取抗体的轻重链基因序列。
二、抗HBP单克隆抗体重轻链基因序列的获得;
(1)cDNA制备:将实施例1中的分选的B细胞进行RNA提取,以RNA为模板进行反转录获得cDNA,并用PCR的方式扩增轻重链的基因;
(2)轻重链基因的获取:利用兔源特异性的引物进行扩增,分别将扩增到的VH+CH1及VL+CL的DNA序列克隆至载体中,然后进行转化涂板,挑取克隆、提取质粒进行测序;
(3)抗体的轻重链可变区基因的分析:根据测序结果对VH及VL以及恒定区进行分析,最终获得重链全长氨基酸序列(SEQIDNO:1)、轻链全长氨基酸序列(SEQIDNO:2)、重链全长氨基酸序列(SEQIDNO:3)和轻链全长氨基酸序列(SEQIDNO:4)、重链全长氨基酸序列(SEQIDNO:5)、轻链全长氨基酸序列(SEQIDNO:6)、重链全长氨基酸序列(SEQIDNO:7)和轻链全长氨基酸序列(SEQIDNO:8)。
SEQIDNO:1
QSVEESGGRVTPGGTTTCTASGFTSSNHMCWVRQAPGKGEYGFTGGRAYYASWAKGRFTSRTSTTVDKMTSTTEDTATYFCARGAYSSWGQGTVTVSSGQPKAPSVFPAPCCGDTPSSTVTGCVKGYPEPVTVTWNSGTTNGVRTFPSVRQSSGYSSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPEGGPSVFFPPKPKDTMSRTPEVTCVVVDVSQDDPEVQFTWYNNEQVRTARPPREQQFNSTRVVSTPTHQDWRGKEFKCKVHNKAPAPEKTSKARGQPEPKVYTMGPPREESSRSVSTCMNGFYPSDSVEWEKNGKAEDNYKTTPAVDSDGSYFYNKSVPTSEWQRGDVFTCSVMHEAHNHYTQKSSRSPGK;
SEQIDNO:2
ADVMTQTPASVSEPVGGTVTKCQASQSSNNAWYQQKPGQPPKYASTASGVPSRFKGSGSGTEYTTSDECADAATYYCQYGSSYGFGGGTGVVVKGDPVAPTVFPPAADQVATGTVTVCVANKYFPDVTVTWEVDGTTQTTGENSKTPQNSADCTYNSSTTTSTQYNSHKEYTCKVTQGTTSVVQSFSRKNC
SEQIDNO:3
QEQKESGGGVTPGTPTTCTVSGFSSSYWSWVRQAPGKGLEWGTSSNTYYATWAKGRFTSKTSSTTVKMTSPTTETATYFCCRTSGGGAFFTWGQGTVTVSSGQPKAPSVFPAPCCGTPSSTVTGCVKGYPEPVTVTWNSGTTNGVRTFPSVRQSSGYSSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPEGGPSVFFPPKPKDTMSRTPEVTCVVVDVSQDDPEVQFTWYNNEQVRTARPPREQQFNSTRVVSTPTHQDWRGK
EFKCKVHNKAPAPEKTSKARGQPLEPKVYTMGPPREESSRSVSTCMNGFYPSDSVEWEKNGKAEDNYKTTPAVDSDGSYFYNKSVPTSEWQRGDVFTCSVMHEAHNHYTQKSSRSPGK
SEQIDNO:4
AIVMTQTPSSVEAAVGGTVTIKCQASQSSSWSWYQQKPGQPPKYQASKPSGVPSRFKGSGSGTEFTTSECAAATYYCQSYSTGSSYGVFGGGTEVVVNGPVAPTVFPPAAQVATGTVTVCVAKYFPVTVTWEVGTTQTTGESKTPQNSACTYNSSTTTSTQYSHKEYTCKVTQGTTSVVQSFNRGC
SEQIDNO:5
QSVEESGGRVTPGTPTCTVSGFSSYPMSWVRQAPEKGEYGYTGSASYASWAKGRFTSKTSTTVDRTSPTTEDTATYFCARGYWVFWGQGTVTVSSGQPKAPSVFPAPCCGDTPSSTVTGCVKGYPEPVTVTWNSGTTNGVRTFPSVRQSSGYSSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPEGGPSVFFPPKPKDTMSRTPEVTCVVVDVSQDDPEVQFTWYNNEQVRTARPPREQQFNSTRVVSTPTHQDWRGKEFKCKVHNKAPAPEKTSKARGQPEPKVYTMGPPREESSRSVSTCMNGFYPSDSVEWEKNGKAEDNYKTTPAVDSDGSYFYNKSVPTSEWQRGDVFTCSVMHEAHNHYTQKSSRSPGK
SEQIDNO:6
QAVTQTPSPVSAAVGGTVTISCQSSQSVYSRSWFQQKPGQPPKYYASTASGVPSRFSGSGSGTQFTTSGVQCDDGAYYCGGYAETFAFGGGTEVVVKGDPVAPTVFPPAADQVATGTVTVCVANKYFPDVTVTWEVDGTTQTTGENSKTPQNSADCTYNSSTTTSTQYNSHKEYTCKVTQGTTSVVQSFSRKSC
SEQIDNO:7
QSVEESGGRVTPGTPTTCKVSGFSSRYAMSWVRQTPGKGEWIGVSSGNTYYASWAKGRFTSKTSTTVDKTSPTTEDTATYFCAREGYDYGCGGNWGQGTVTVSSGQPKAPSVFPAPCCGDTPSSTVTGCVKGYPEPVTVTWNSGTTNGVRTFPSVRQSSGYSSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPEGGPSVFFPPKPKDTMSRTPEVTCVVVDVSQDDPEVQFTWYNNEQVRTARPPREQQFNSTRVVSTPTHQDWRGKEFKCKVHNKAPAPEKTSKARGQPEPKVYTMGPPREESSRSVSTCMNGFYPSDSVEWEKNGKAEDNYKTTPAVDSDGSYFYNKSVPTSEWQRGDVFTCSVMHEAHNHYTQKSSRSPGK
SEQIDNO:8
AAVTQTPSPVSVAVGGTVTNCQSSQSVNYSWYQQKPGQSPRYDASNASGVPDRFSGSGSGTQFTTSGVQCDDAATYYCGGYDATFVFGGGTEVVVKGDPVAPTVFPPAADQVATGTVTVCVANKYFPDVTVTWEVDGTTQTTGENSKTPQNSADCTYNSSTTTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
上述内容中下划线部分为抗体CDR区。
三、抗HBP单克隆抗体的表达、纯化;
(1) 培养N293悬浮细胞,当密度达到1-3×/ml,成活率>80-90%时进行细胞转染;
(2)将实施例2中含抗体轻重链基因的重组质粒转染N293细胞:PEI/质粒的质量比为2:1-5:1,按照1-3μg质粒/1mlN293细胞的比例转染;
(3)转染后5-7天,收集细胞上清,用ProteinA树脂进行亲和纯化获得高纯度的抗体蛋白,并通过透析将洗脱缓冲液置换成PBS,利用Nanodrop测定抗体浓度。
四、抗HBP单克隆抗体的检测
(1)抗HBP的抗体的纯度检测:
① SDS-PAGE检测:取2μg抗体加入适量的上样缓冲液,使总体积为20μl,同时制备还原性SDS-PAGE样品及非还原性SDS-PAGE样品,然后进行上样,首先80V进行电泳30min,然后120V电泳直至条带分隔清晰;染色及脱色:将电泳后的PAGE胶取下,然后进行考马斯亮蓝染色,15min之后将染色液去掉,用清水冲洗干净,然后加入脱色液进行脱色,直至看到清晰的条带为止。
②HPLC检测:取纯化之后的抗体10μl加入到内插管中,排尽气泡,待上机;配制HPLC用150mM的磷酸盐缓冲液,调pH至7.0,并将缓冲液进行真空抽滤处理除菌,然后将其置于超声仪中进行超声除气,备用;仪器开机,打开高效液相色谱各个模块,进行开机预热处理,首先对仪器系统进行排气泡处理,然后用流动相对仪器进行溶液置换,将SEC柱子接入到仪器中,排气泡之后用平衡缓冲液对柱子进行平衡,待用;上样:打开软件,进行方法学编辑,生成序列,然后进行上样;结果分析及处理:打开软件,导入结果,进行分析,生成分析报告。
如图1和图2-a到图2-d所示,显示HBP单克隆抗体均具有较高的纯度;
(2)抗HBP的抗体的活性检测:
①抗原包被: 将抗原稀释至合适的浓度,然后加入空的酶标板中,体积100ul/孔;将酶标板放置4℃冰箱,过夜孵育;
②封闭:用洗板机洗板5次,扣干,将封闭液加入酶标板,每孔150ul,置于37℃恒温箱中,封闭1h;用洗板机洗板5次,扣干;
③孵育一抗:用1xPBS将抗体稀释至合适的浓度梯度;将稀释后的抗体加入酶标板,每孔100ul,反应时间:37度/1h;用洗板机洗板5次,扣干;
④孵育二抗:将酶标二抗按合适比例稀释,然后加入酶标板中,每孔100ul,反应时间:37度/30min;用洗板机洗板5次,扣干;
⑤显色:将显色液加入酶标板,每孔100ul,反应时间:37度/15min;
⑥终止、读数:添加终止液,体积100ul/孔;检测波长设置:检测波长450nm,参比波长620nm,将检测结果导出成Excel表格;
⑦数据分析:利用软件进行数据分析及作图,拟合EC50。
如图3-a到图3-d所示,显示4株HBP单克隆抗体均具有较高的亲和力。
五、制备时间分辨荧光法HBP检测试剂
(1)荧光微球-抗HBP单克隆抗体复合物的制备
①时间分辨荧光微球的预处理
取时间分辨荧光微球25ul,加入400ul且pH6.0的PB缓冲液,混匀后,14000rpm,10℃,离心15min ,去除上清液,沉淀物400ul pH6 .0的PB缓冲液重悬;分别配备2.5mg/ml的碳二亚胺和琥珀酰亚胺,各加入2.5ul,室温反应30min后,14000rpm,10℃,离心15min,沉淀物用200ul且pH6.0的PB溶液重悬,获得活化的微球溶液;
②微球耦连HBP单克隆抗体
上述时间分辨荧光微球溶液中加入10-50ug HBP单克隆抗体,混匀,室温反应 3小时,用1-5%的BSA室温封闭0.5小时后,14000rpm,10℃,离心15min,去上清,沉淀用50ul保存液重悬,获得荧光微球-抗HBP单克隆抗体复合物;所述保存液含有PH7.5-8.5的10-100mMTris缓冲液、1%-5% 蔗糖、1%-5%海藻糖、1%-5% BSA;
(2)喷金
将荧光微球-抗HBP单克隆抗体复合物终浓度稀释至为0.5-2mg/mL,喷金量设置为1-4μL/cm,然后将喷好的标记垫置于37℃烘箱内干燥1-4小时,然后在不高于36%的湿度下将标记垫放入预先加入干燥剂的铝箔袋中封口保存备用;
(3)包被质控线C、检测线T
①质控线C、检测线T线包被液的制备:取羊抗兔抗体,用PH7.4的10-100mM PBS缓冲液稀释成0.5-1.0mg/mL的抗体溶液;取抗HBP单克隆抗体,用PH7.4的10-100mM PBS缓冲液稀释成0.5-1.0mg/mL的抗体溶液;
②包被:将质控线C、检测线T线包被液分别包被至硝酸纤维素膜的质控线和检测线位置,C、T线间隔5-8mm,包被量均为0.8-1.5μL/cm;将包被后的PVC底板置于37℃烘箱中,烘12-24小时,然后在不高于36%的湿度下将PVC底板放入预先加入干燥剂的铝箔袋中封口保存备用;
(4)样品垫的预处理
清洗烘网,将样品垫用样品垫预处理液浸泡润湿后,旋转沥干至没有水滴滴下即可,然后置于37℃烘箱内干燥4-6小时,然后在不高于36%的湿度下将其放入预先加入干燥剂的铝箔袋中封口保存备用;
所述样品垫预处理液含有PH7.5-8.5的10-100mM Tris缓冲液、0.1%-0.5% PVP10、0.1%-0.5% PEG20000、0.1%-0.5% Tween20和0.1%-0.5%酪蛋白钠;
(5)膜材的组装与切割
在不高于36%的湿度下,取出吸水垫、已处理的样品垫和标记垫,按照(15-20mm)*300mm尺寸进行切割,取出包被的PVC底板,按以下流程进行组装:先在PVC底板上贴上标记垫,使标记垫压着硝酸纤维素膜1-2mm,再贴上样品垫,使样品垫部分压着标记垫3-5mm,最后贴上吸水垫,吸水垫压着硝酸纤维素膜1-2mm;组装好的PVC底板切割成3-5mm的用于检测HBP的时间分辨荧光免疫层析试纸条;
(6)试纸条检测
用样本稀释液将临床样本稀释3倍,加样75ul,用荧光免疫分析仪进行检测,并处理数据。
如图4所示,显示HBP检测试剂具有较好的临床相关性。
以上实施例仅用以说明本发明的技术方法而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方法进行修改或等同替换,而不脱离本发明技术方法的精神和范围。
Claims (2)
1.一种抗HBP的单克隆抗体,其特征在于,
选自下列组中任一组:
第一组:重链全长氨基酸序列SEQIDNO:1和轻链全长氨基酸序列SEQIDNO:2;
第二组:重链全长氨基酸序列SEQIDNO:3和轻链全长氨基酸序列SEQIDNO:4;
第三组:重链全长氨基酸序列SEQIDNO:5和轻链全长氨基酸序列SEQIDNO:6;
第四组:重链全长氨基酸序列SEQIDNO:7和轻链全长氨基酸序列SEQIDNO:8。
2.一种包含抗HBP的单克隆抗体的试剂盒,其特征在于,所述试剂盒中含有权利要求1所述的单克隆抗体。
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