WO2023165252A1 - 一种鼠抗ndm型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用 - Google Patents

一种鼠抗ndm型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用 Download PDF

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WO2023165252A1
WO2023165252A1 PCT/CN2022/143804 CN2022143804W WO2023165252A1 WO 2023165252 A1 WO2023165252 A1 WO 2023165252A1 CN 2022143804 W CN2022143804 W CN 2022143804W WO 2023165252 A1 WO2023165252 A1 WO 2023165252A1
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antibody
seq
ndm
variable region
carbapenemase
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French (fr)
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苑庆华
李可可
何永胜
陈晓玲
王亚苗
孔迪
王思怡
周跃辉
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天津一瑞生物科技股份有限公司
北京金山川科技发展有限公司
天津喜诺生物医药有限公司
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Priority to EP22871087.7A priority Critical patent/EP4261278A4/en
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  • Carbapenem antibiotics are one of the most effective drugs for controlling clinical pathogenic bacterial infections.
  • Carbapenem-producing organisms (CPOs) and carbapenem-resistant Enterobacteriaceae have become a global public health issue because of their broad-spectrum drug resistance. Sanitation issues, very limited treatment options for patients.
  • Carbapenemase refers to a class of ⁇ -lactamases that can significantly hydrolyze at least imipenem or meropenem, including Ambler molecular structure classification A, B, and D three types of enzymes, of which, B type is metal ⁇ -lactamase Lactamase (IMP type, VIM type, NDM type, etc.), referred to as metalloenzyme, is mainly found in Pseudomonas aeruginosa, Acinetobacter, and Enterobacteriaceae bacteria; A and D types are serinases, and A type enzymes (KPC type etc.) are mainly found in Enterobacteriaceae; type D enzymes (OXA type) are mainly found in Acinetobacter.
  • B type is metal ⁇ -lactamase Lactamase (IMP type, VIM type, NDM type, etc.), referred to as metalloenzyme
  • IMP type metal ⁇ -lactamase Lactamase
  • NDM Since it was first discovered in a strain of Klebsiella pneumoniae isolated from a Swedish patient who had traveled to India in 2008, NDM has spread worldwide at an alarming rate. So far, NDM has appeared in dozens of countries in Europe, the United States, Canada, and Mexico in North America, and China, Japan, South Korea, and Singapore in Asia. In India, Pakistan and other countries, NDM has become more prevalent, with a detection rate of 38.5%.
  • a mouse anti-NDM type carbapenemase antibody, antibody 1GH10 includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1 as shown in SEQ ID NO: 1, SEQ ID NO: CDRL2 shown in 2 and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5 and shown in SEQ ID NO:6 CDRH3;
  • amino acid sequence of the light chain variable region of antibody 1GH10 is shown in SEQ ID NO: 7
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9;
  • amino acid sequence of the light chain variable region of antibody 12HE11 is shown in SEQ ID NO: 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19;
  • Biological material 1GH10, the preservation date is March 24, 2021, the preservation unit is the General Microbiology Center (CGMCC), and the preservation number is CGMCC No.21963;
  • CGMCC General Microbiology Center
  • the antibody 1GH10 produced by the mouse anti-NDM type carbapenemase hybridoma cell line includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1, SEQ ID NO: 1 CDRL2 shown in ID NO:2 and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5 and SEQ ID NO: CDRH3 indicated in 6;
  • the antibody 12HE11 produced by the mouse anti-NDM type carbapenemase hybridoma cell line
  • the light chain variable region includes CDRL1 shown in SEQ ID NO:11, CDRL2 shown in SEQ ID NO:12 and SEQ ID NO: CDRL3 shown in 13
  • the heavy chain variable region includes CDRH1 shown in SEQ ID NO: 14, CDRH2 shown in SEQ ID NO: 15 and CDRH3 shown in SEQ ID NO: 16;

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Abstract

提供鼠抗NDM型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用，通过小鼠杂交瘤单克隆抗体筛选及RT-PCR法克隆Ig可变区基因，获得稳定分泌鼠抗NDM型碳青霉烯酶抗体的杂交瘤细胞株及其可变区序列；通过系统性评价，鼠抗NDM型碳青霉烯酶抗体在各方面均有较佳表现，效价达到了1:1280000以上，适合作为免疫诊断试剂用于制备碳青霉烯酶耐药菌株体外诊断试剂盒。

Description

一种鼠抗NDM型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用 技术领域
本发明属于抗体制备技术领域，尤其是涉及一种鼠抗NDM型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用。
背景技术
碳青霉烯类抗生素是控制临床致病菌感染最有效的药物之一，产碳青霉烯酶生物(CPO)和耐碳青霉烯肠杆菌因其广谱耐药性成为了一个全球公共卫生问题，患者的治疗方法非常有限。碳青霉烯酶是指能够明显水解至少亚胺培南或美罗培南的一类β-内酰胺酶，包括Ambler分子结构分类的A、B、D三类酶，其中，B类为金属β-内酰胺酶(IMP型、VIM型、NDM型等)，简称金属酶，主要见于铜绿假单孢菌、不动杆菌、肠杆菌科细菌；A、D类为丝氨酸酶，A类酶(KPC型等)主要见于肠杆菌科细菌；D类酶(OXA型)主要见于不动杆菌。
自2008年首次在一位曾在印度旅游的瑞典籍患者体内分离的一株肺炎克雷伯菌中发现以来,NDM便以惊人的速度在世界范围内播散流行。到目前为止，NDM已在欧洲数十个国家、北美洲的美国、加拿大及墨西哥，亚洲的中国、日本、韩国及新加坡等国家出现。在印度、巴基斯坦等国家，NDM更是引起了流行，其检出率达38.5％。
实验室检测碳青霉烯酶的方法众多。主要包括改良Hodge试验、Carba NP试验、改良碳青霉烯灭活试验(modified carbapenem inactivation method，mCIM)、酶抑制剂增强试验、免疫金标试验以及分子生物学方法等。免疫诊断试剂是体外诊断试剂中发展最快的细分领域，利用抗原与抗体之间的特异性结合进行定性或定量检测。碳青霉烯酶快速诊断产品对于耐药菌株的早期分型、指导用药具有突出作用，对碳青霉烯酶快速诊断产品的研发依然迫在眉睫。
发明内容
为解决上述技术问题，本发明提供一种鼠抗NDM型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用。
本发明采用的技术方案是：一种鼠抗NDM型碳青霉烯酶杂交瘤细胞株，命名为1GH10，保藏编号为CGMCCNo.21963；或者命名为12HE11，保藏编号为 CGMCCNo.21964。
一种鼠抗NDM型碳青霉烯酶抗体，抗体1GH10，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；
SEQ ID NO:1RSSQSIVHSDGNTYLD(CDRL1)
SEQ ID NO:2KVSNRFS(CDRL2)
SEQ ID NO:3FQISRVPFT(CDRL3)
SEQ ID NO:4GYAFSNYLIE(CDRH1)
SEQ ID NO:5VINPGRDDTNYNEKFKG(CDRH2)
SEQ ID NO:6FPSILRYDSGSFSYFGLDC(CDRH3)
或者，
抗体12HE11，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3；
SEQ ID NO:11RSSQSLFNSGNQKNYLT(CDRL1)
SEQ ID NO:12WAVTRES(CDRL2)
SEQ ID NO:13QNDYSYPLT(CDRL3)
SEQ ID NO:14GYTLSDYHVK(CDRH1)
SEQ ID NO:15DIRPQNGDIVYNQKFKD(CDRH2)
SEQ ID NO:16HYYAYGKWFPY(CDRH3)
优选地，抗体1GH10轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
SEQ ID NO:7
Figure PCTCN2022143804-appb-000001
SEQ ID NO:9
Figure PCTCN2022143804-appb-000002
抗体12HE11轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示；
SEQ ID NO:17
Figure PCTCN2022143804-appb-000003
SEQ ID NO:19
Figure PCTCN2022143804-appb-000004
优选地，抗体1GH10由保藏编号为CGMCCNo.21963的鼠抗NDM型碳青霉烯酶杂交瘤细胞株产生；
抗体12HE11由保藏编号为CGMCCNo.21964的鼠抗NDM型碳青霉烯酶杂交瘤细胞株产生。
一种核酸分子，包含编码鼠抗NDM型碳青霉烯酶抗体的核苷酸。
优选地，核酸分子编码抗体1GH10的轻链可变区的核苷酸序列如SEQIDNO:8所示，核酸分子编码抗体1GH10的重链可变区的核苷酸序列如SEQIDNO:10所示；
SEQIDNO:8
Figure PCTCN2022143804-appb-000005
SEQIDNO:10
Figure PCTCN2022143804-appb-000006
核酸分子编码抗体12HE11的轻链可变区的核苷酸序列如SEQIDNO:18所示，核酸分子编码抗体12HE11的重链可变区的核苷酸序列如SEQIDNO:20所示；
SEQ ID NO:18
Figure PCTCN2022143804-appb-000007
SEQ ID NO:20
GTGCAGCTTCACGAGTCAGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAACTGTCCTGCACGGCTTCCGGATACACCCTCAGTGACTACCACGTAAAATGGGTGAAACAGAACCATGGACAGAGCCTTGAGTGGATTGGAGATATTAGGCCTCAGAATGGTGATATTGTTTACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCACAACAGCCTACATACGACTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCATTCCATTATTACGCCTACGGGAAGTGGTTTCCTTATTGGGGCCAAGGGACTCTGGTCACCGTCTCC鼠抗NDM型碳青霉烯酶抗体在制备检测NDM型碳青霉烯酶抗原试剂中的应用。
优选地，将鼠抗NDM型碳青霉烯酶抗体用于制备体外诊断试剂盒或微流体芯片，体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。
优选地，制备双抗体夹心法免疫胶体金试纸条，抗体1GH10为包被抗体，抗体12HE11为金标抗体；
或者，抗体12HE11为包被抗体，抗体1GH10为金标抗体。
本发明具有的优点和积极效果是：本发明提供两种鼠抗NDM型碳青霉烯酶杂交瘤细胞株，分别能够产生两种鼠抗NDM型碳青霉烯酶抗体；通过系统性评价，包括对抗体亚型及效价、试剂盒灵敏度、特异性和稳定性的评价，鼠抗NDM型碳青霉烯酶单克隆抗体在各方面均有较佳表现，效价达到了1:1280000以上，从而适合作为免疫诊断试剂用于制备碳青霉烯酶耐药菌株体外诊断试剂盒。
附图说明
图1是鼠抗NDM型碳青霉烯酶蛋白电泳图；
图2是鼠抗NDM型碳青霉烯酶抗体蛋白电泳图；
图3是胶体金法鼠抗NDM型碳青霉烯酶检测卡检测结果。
生物材料：1GH10，保藏日期为2021年3月24日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号是CGMCC No.21963；
生物材料：12HE11，保藏日期为2021年3月24日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号是CGMCC No.21964。
具体实施方式
下面对本发明的实施例做出说明。
本发明涉及鼠抗NDM型碳青霉烯酶杂交瘤细胞株，生物材料命名为1GH10，属杂交瘤细胞，其保藏编号是CGMCC No.21963；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2021年3月24日，检测为存活。另有一株鼠抗NDM型碳青霉烯酶杂交瘤细胞株，生物材料命名为12HE11，属杂交瘤细胞，其保藏编号是CGMCC No.21964；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2021年3月24日，检测为存活。
鼠抗NDM型碳青霉烯酶杂交瘤细胞株生产得到的抗体1GH10，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；
SEQ ID NO:1RSSQSIVHSDGNTYLD(CDRL1)
SEQ ID NO:2KVSNRFS(CDRL2)
SEQ ID NO:3FQISRVPFT(CDRL3)
SEQ ID NO:4GYAFSNYLIE(CDRH1)
SEQ ID NO:5VINPGRDDTNYNEKFKG(CDRH2)
SEQ ID NO:6FPSILRYDSGSFSYFGLDC(CDRH3)
抗体1GH10轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
SEQ ID NO:7
Figure PCTCN2022143804-appb-000008
SEQ ID NO:9
Figure PCTCN2022143804-appb-000009
编码抗体1GH10的轻链可变区的核苷酸序列如SEQIDNO:8所示，编码抗体1GH10的重链可变区的核苷酸序列如SEQIDNO:10所示；
SEQIDNO:8
Figure PCTCN2022143804-appb-000010
Figure PCTCN2022143804-appb-000011
SEQIDNO:10
Figure PCTCN2022143804-appb-000012
鼠抗NDM型碳青霉烯酶杂交瘤细胞株生产得到的抗体12HE11，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3；
SEQ ID NO:11RSSQSLFNSGNQKNYLT(CDRL1)
SEQ ID NO:12WAVTRES(CDRL2)
SEQ ID NO:13QNDYSYPLT(CDRL3)
SEQ ID NO:14GYTLSDYHVK(CDRH1)
SEQ ID NO:15DIRPQNGDIVYNQKFKD(CDRH2)
SEQ ID NO:16HYYAYGKWFPY(CDRH3)
抗体12HE11轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示；
SEQ ID NO:17
Figure PCTCN2022143804-appb-000013
SEQ ID NO:19
VQLHESGPELVKPGASVKLSCTASGYTLSDYHVKWVKQNHGQSLEWIG DIRPQNGDIVYNQKFKDKATLTVDKSSTTAYIRLSSLTSEDSAVYYCAFHYYAYGKWFPYWGQGTLVTVS编码抗体12HE11的轻链可变区的核苷酸序列如SEQIDNO:18所示，编码抗体12HE11的重链可变区的核苷酸序列如SEQIDNO:20所示；
SEQ ID NO:18
Figure PCTCN2022143804-appb-000014
SEQ ID NO:20
Figure PCTCN2022143804-appb-000015
抗NDM型碳青霉烯酶单克隆抗体通过系统性评价，包括对抗体亚型及效价、试剂盒灵敏度、特异性和稳定性的评价，抗NDM型碳青霉烯酶单克隆抗体在各方面均有较佳表现，从而适合作为免疫诊断试剂用于制备体外诊断试剂盒。可以制作成胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒，或者制作为微流体芯片；制作的试剂盒能够检测NDM型碳青霉烯酶抗原。本方案所涉及的两种抗体尤其适合搭配制成双抗体夹心法免疫胶体金试纸条，其中抗体1GH10为包被抗体，抗体12HE11为金标抗体，制得的双抗体夹心法免疫胶体金试纸条灵敏性更高，同时，也可将抗体1GH10做为金标抗体，抗体12HE11为包被抗体。下面通过具体实施例对本发明做出进一步说 明。其中，未具体说明操作步骤的实验方法，均按照相应商品说明书进行，实施例中所用到的仪器、试剂、耗材如无特殊说明，均可从商业公司购买得到。
实施例1：鼠抗NDM型碳青霉烯酶单克隆抗体的制备
1.1抗原制备
获得重组质粒：将NDM型基因序列构建到Pet-28a(+)载体上并进行全基因合成。
转化到宿主菌：将含有目的基因的载体Pet-28a(+)转化到克隆宿主大肠杆菌。
扩大培养：将验证正确的转化子进行扩大培养，并转接到含有卡那霉素抗性的LB液体培养基中，37℃摇床震荡培养。
诱导：当菌液OD值在0.4～0.6之间时，加入IPTG终浓度为0.1mM，30℃培养4小时，离心收集菌体。
超声破碎菌体：约1g加入30ml PBS重悬菌体后超声破碎，功率为400W，超声3s，间隔5s，约30分钟后，菌液不黏稠且澄清后，离心去掉菌体碎片，上清过0.45μm滤膜。
纯化：将上清液过镍柱纯化后，透析到PBS中，获得纯度较高目的蛋白，分子量和预计28.25kD相符，SDS-PAGE见图1，用BCA法定量后分装进行后续实验。
1.2小鼠免疫
用纯化的NDM型碳青霉烯酶免疫6周龄左右的雌性Balb/c小鼠，进行抗体制备，蛋白含量为0.1mg/ml，按照免疫剂量分为2组，每组5只小鼠；按照蛋白含量计算，第一组免疫剂量为25ug/只，第二组免疫剂量为50ug/只，首免，取适量NDM型碳青霉烯酶经蒸馏水稀释至300ul，加入等量弗氏完全佐剂300ul乳化均匀，皮下多点注射免疫小鼠；两周后，取相同剂量进行二免，二免为腹腔注射免疫小鼠，两周后再追加免疫一次，亦为腹腔注射免疫小鼠，7天后鼠尾采血，ELISA测定小鼠血清效价。
具体步骤为：NDM 0.2ug/ml，100ul/孔，4℃过夜包被ELISA板，甩干，PBST洗涤3次。5％脱脂乳粉，200ul/孔，37度封闭2h。小鼠鼠尾采血，3000转/min，离心后收集血清，从1:1000开始用PBS进行倍比稀释至1:512000，备用。甩干， PBST洗涤3次，1:1000倍起加入PBS稀释的一抗，100ul/孔，37度，1h。甩干，PBST洗涤3次,加PBS 1:6000倍稀释的羊抗鼠二抗，100ul/孔，37度，45min。甩干，PBST洗涤5次，加100ulTMB/孔，37℃，显色10min，终止，读值。
1.3细胞融合
融合前三天进行小鼠加强免疫，接种量同前次免疫，不加佐剂，腹腔注射。融合前一天准备饲养层细胞，取6-8周龄小鼠Balb/c 1只，取眼球放血后颈椎脱位致死，放于75％酒精中消毒5min，固定于盘上，在超净台中无菌剪开腹部皮肤。用无菌注射器吸取HAT选择培养液10ml注入小鼠腹腔，用酒精棉球轻揉腹部，抽回培养基。加入40ml HAT培养液中，铺入到4块96孔细胞培养板中，100μL/孔，37℃，5％CO 2细胞培养箱中培养。融合前一周复苏骨髓瘤细胞(Sp2/0细胞)，用含10％胎牛血清的PRMI-1640培养基培养，37℃，5％CO 2培养箱中传代培养。将处于对数生长期的细胞收集至离心管中，细胞计数，把细胞稀释为10 7个/ml备用。取加强免疫3天的Balb/c小鼠，摘眼球放血制备阳性血清，脱颈椎处死，75％酒精消毒5min，在超净工作台无菌取出脾脏，在无菌平皿中冼涤数次，剥离结缔组织。将脾脏放在微孔铜网上，加入新鲜的RPMI-1640培养液，先用注射器吸取培养液由脾脏一段注入，吹下脾细胞，反复数次之后，用注射器的内塞轻轻将剩余脾脏研磨，直到无明显的红色组织块。将平皿中脾细胞悬液轻轻吹打后转移到50ml离心管中，1000r/min离心5min，收集脾细胞，计数后备用。将免疫鼠脾细胞与Sp2/0细胞按细胞数量10:1混合，加入50ml的离心管内，1000r/min离心5min，弃上清，在手心轻轻摩擦使两种细胞充分混匀，将离心管至于100ml蓝盖瓶内，蓝盖瓶内装有37℃热水，将预热好的1ml DMSO/PEG在1min内逐滴加入融合管内，先慢后快，边加边轻轻旋转离心管。然后立即加入无抗无血RPMI-1640培养液终止反应，第一分钟加1ml，第二分钟加2ml，第三分钟加3ml，第四分钟加4ml。37℃水浴5min，后800r/min离心5min，弃上清，将沉淀以HAT悬起，混匀到40ml含37℃预热的20％小牛血清的HAT选择培养液中，铺入已加有饲养细胞的96孔细胞板中，100μL/孔，将培养板放入37℃，5％CO 2培养箱培养。7d后将用新鲜的HAT培养基对细胞板半换液，10天后用HT培养基全换液。将96孔板中检测阳性的细胞采用有限稀释法进行亚克隆：首先按照上述方法制备饲养层细胞，取待克隆杂交瘤细胞进行细 胞计数，用HT培养基将细胞稀释至5-8个细胞/ml，加入到已铺饲养细胞的96孔细胞板中100μL/孔，每株杂交瘤细胞克隆一块96孔细胞板，37℃、5％CO 2细胞培养箱中培养。约5天后数出细胞孔里的克隆数，标记，7天时并换新的培养基，待细胞铺满整个孔底的1/3～1/2时检测。经过2-3次克隆化，待96孔板所有细胞孔均为阳性时，即可进行扩大培养，定株，冻存。将检测阳性确定定株的杂交瘤细胞扩大培养并冻存。具体过程如下：将生长旺盛、状态良好的杂交瘤细胞用无抗无血DMEM轻轻从细胞瓶上吹下，1000r/min离心5min，弃去上清。加入冻存液(含40％RPMI-1640培养液、50％胎牛血清、10％DMSO)，将细胞吹散后分装到细胞冻存管中。将冻存管放入冻存盒置于-70℃冰箱中，一天后将冻存管转移入液氮中，做好记录。
1.4腹水制备
取10-12周龄雌性Balb/c小鼠，腹腔注射无菌液体石蜡，0.5ml/只，7d后腹腔注射培养至对数期的杂交瘤细胞，5×10 6个细胞/只。每天注意观察，约7-10天，待小鼠腹部出现明显隆起后，用75％酒精棉球消毒下腹部皮肤，用16号针头刺入腹腔，收集腹水。待腹水再生积聚后，再次收集。将收集的腹水3000r/min离心10min，取中间澄清部分，用滤纸过滤后，分装，-70℃保存。
1.5抗体纯化
用Protein-G柱子进行腹水纯化，步骤如下：取腹水2ml(n)，10000g离心，取澄清部分，加入2ml(1:1)洗涤缓冲液，混匀，柱子用20％乙醇流尽后用8ml洗涤液平衡，样本过柱子，流速为8S/滴沉，反复上样3次，然后用15ml洗涤缓冲液进行洗涤淀，流速为8S/滴沉，洗涤完毕后用10ml的洗脱缓冲液进行洗脱，洗脱完毕会用1M Tris PH＝9调PH至7.4，然后用浓缩注进行浓缩，于50kd透析袋，PBS，4℃透析过夜。
实施例2：鼠抗NDM型碳青霉烯酶单克隆抗体的鉴定
2.1抗体亚类鉴定
按照SIGMA试剂盒说明书，以捕获ELISA的方法进行单抗的亚类鉴定，具体如下：将单抗亚类鉴定试剂1:1000稀释后，加入酶标孔中，100μL/孔，37℃孵育1h；PBST洗三次，拍干；将抗体1:1000倍稀释后加样，100μL/孔，37℃孵育1h； PBST洗三次，拍干；HRP酶标羊抗鼠IgG二抗以1:6000稀释后加样，100μL/孔，室温孵育30min；显色10～20min。以OD 450读值明显高于其他孔所加亚类试剂为单抗所属亚类类型。抗体12HE11、1GH10的抗体亚型为IgG1。
2.2抗体效价测定
采用间接ELISA法进行纯化后抗体效价测定，步骤如下：NDM碳青霉烯酶分别稀释至0.2ug/ml，100ul/孔，同时设立不包被对照，4℃过夜包被，甩干，PBST洗涤3次；5％脱脂乳粉，200ul/孔，37度封闭2h；甩干，PBST洗涤3次，加入从1:1000倍开始进行倍比稀释的抗体(浓度为1mg/ml)，共计12个梯度，同时设立不包被对照100ul/孔，37℃，1h。甩干，PBST洗涤3次,加PBS 1:6000倍稀释的羊抗鼠二抗，100ul/孔，37℃，45min。甩干，PBST洗涤5次，加100ulTMB/孔，37℃，显色10min，终止，读值。纯化后抗体稀释至1mg/ml，效价达到了1:1280000以上。
2.3抗体纯度及分子量鉴定
采用SDS-PAGE法进行抗体分子量及纯度鉴定；制胶，分离胶为12％，浓缩胶为5％；制样，20ul样品+20ul buffer，混匀，煮沸3min；每孔上样20ul，同时设立蛋白预染Marker对照；80伏30min，120伏2h；电泳完毕后，放入考马斯亮蓝溶液进行染色；脱色，去离子水煮沸脱色，每次5min，共计3次；lgG抗体重链的分子质量一般为50-75KDa，lgG抗体轻链的分子量约为25KDa，通过SDS-PAGE对纯化得到的单克隆抗体进行鉴定；如图2所示，抗体1GH10和抗体12HE11在50-75KDa和约25KDa处均各有清晰的条带。
实施例3：鼠抗NDM型碳青霉烯酶单克隆抗体的基因验证
RT-PCR法克隆Ig可变区基因。提取总RNA，合成单链cDNA，用Trizol法(试剂盒购自Invitrogen)提12HE11、1GH10杂交瘤细胞株的总RNA，用M-MLV逆转录酶(购自Invitrogen)将总RNA逆转为cDNA文库。
重链骨架区上游引物
P1：5’SAGGTGMAGCTKCASSARTCWGG3’
重链可变区下游引物
P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’
轻链前导肽上游引物
P3：5’ATGGATTTTCAAGTGCAGATTTTCAG3’
轻链可变区下游引物
P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’
配制PCR反应体系(50μl)如下：
cDNA:2μl；上游引物(10μM):2μl；下游引物(10μM):2μl；dNTP mixture:2μl；pfu DNA聚合酶(5U/μl):1μl；10×pfu BufferⅡ:5μl；ddH 2O补足至50μl。
反应条件：95℃预变性5min；重复如下循环35次：95℃30s，58℃30s，72℃1min；最后，72℃延伸10min。
琼脂糖凝胶电泳分离并回收VL、VH片段。将回收后的VL、VH片段分别与pMD19-T(simple)载体(Takara公司)进行连接，连接体系如下：
VL PCR产物/VH PCR产物各70ng，pMD19-T(simple)载体1μl，Solution I连接反应液5μl；ddH 2O补足至10μl，4℃连接过夜。
连接产物转化入E.coli DH5α感受态细菌中，37℃过夜培养后，挑取单个菌落，37℃震摇2小时后进行菌液PCR鉴定，以对应抗体的cDNA为阳性对照。配制反应体系(25μl)如下：
菌液：1μl，上游引物(10μM)：1μl；下游引物(10μM)：1μl；dNTP Mixture(各2.5Mm)2μl；Taq DNA聚合酶(5U/μl)：0.5μl；10×Taq Buffer(Mg 2+plus)：2.5μl；补水至25μl。反应条件同前。
选取菌PCR阳性的克隆扩大培养，用质粒提取试剂盒(Takara公司)提取阳性克隆质粒，送检测序。每个抗体的每条链至少送检5个克隆样品，至少三个样品测序结果相同为止。成功克隆得到抗体12HE11、1GH10的重链、轻链可变区序列，经比对符合典型抗体可变区序列特征。
实施例4：胶体金法制备NDM型碳青霉烯酶测卡
使用胶体金法制备NDM型碳青霉烯酶测卡，制备双抗体夹心法免疫胶体金试纸条，制备方法为：
步骤一向胶体金溶液中边搅拌边加入0.1M K 2CO 3溶液，调节pH值后加入抗NDM型碳青霉烯酶单克隆抗体12HE11，搅拌后加入10％的牛血清白蛋白溶 液，2％PEG20000，搅拌后低速离心取上清，再高速离心后取沉淀，用胶体金重悬液定容形成金标抗体；
步骤二将金标抗体喷于玻璃纤维素膜，烘干制成金标垫；
步骤三向抗NDM型碳青霉烯酶单克隆抗体1GH10中加入1％的硫柳汞钠溶液，混匀后形成检测线包被液，再向羊抗鼠IgG中加入PBS和1％的硫柳汞钠溶液，混匀后形成质控线包被液，将质控线包被液和检测线包被液划在硝酸纤维素膜上，烘干后获得包被膜；
步骤四将包被膜贴在底板上，将金标垫和吸水纸搭上包被膜，层压后切割获得胶体金法NDM型碳青霉烯酶检测卡。
取通过上述方法制备得到的胶体金法NDM型碳青霉烯酶检测卡，分别取空白样、NDM型碳青霉烯酶和含有NDM型碳青霉烯酶阳性样本点样于检测卡。结果如图3所示，从左到右依次为空白样、NDM型碳青霉烯酶和含有NDM型碳青霉烯酶阳性样本，能够看出，空白样点样的检测卡检测结果呈阴性，NDM型碳青霉烯酶和含有NDM型碳青霉烯酶阳性样本点样结果均为阳性，证明通过本方案制备得到的胶体金法NDM型碳青霉烯酶检测卡能够检测NDM型碳青霉烯酶及相应阳性样本。
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。

Claims (9)

  1. 一种鼠抗NDM型碳青霉烯酶杂交瘤细胞株，其特征在于：命名为1GH10，保藏编号为CGMCCNo.21963；或者命名为12HE11，保藏编号为CGMCCNo.21964。
  2. 一种鼠抗NDM型碳青霉烯酶抗体，其特征在于：抗体1GH10，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；
    或者，
    抗体12HE11，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3。
  3. 根据权利要求2所述的鼠抗NDM型碳青霉烯酶抗体，其特征在于：抗体1GH10轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
    或者，
    抗体12HE11轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示。
  4. 根据权利要求2或3所述的鼠抗NDM型碳青霉烯酶抗体，其特征在于：抗体1GH10由保藏编号为CGMCCNo.21963的鼠抗NDM型碳青霉烯酶杂交瘤细胞株产生；
    抗体12HE11由保藏编号为CGMCCNo.21964的鼠抗NDM型碳青霉烯酶杂交瘤细胞株产生。
  5. 一种核酸分子，其特征在于：包含编码权利要求2或3所述的鼠抗NDM型碳青霉烯酶抗体的核苷酸。
  6. 根据权利要求5所述的核酸分子，其特征在于：所述核酸分子编码抗体1GH10的轻链可变区的核苷酸序列如SEQIDNO:8所示，所述核酸分子编码抗体1GH10的重链可变区的核苷酸序列如SEQIDNO:10所示；
    所述核酸分子编码抗体12HE11的轻链可变区的核苷酸序列如SEQIDNO:18所示，所述核酸分子编码抗体12HE11的重链可变区的核苷酸序列如SEQIDNO:20所示。
  7. 权利要求2-4中任一所述的鼠抗NDM型碳青霉烯酶抗体在制备检测NDM型碳青霉烯酶抗原试剂中的应用。
  8. 根据权利要求7所述的鼠抗NDM型碳青霉烯酶抗体在制备检测NDM型碳青霉烯酶抗原试剂中的应用，其特征在于：将鼠抗NDM型碳青霉烯酶抗体用于制备体外诊断试剂盒或微流体芯片，所述体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。
  9. 根据权利要求7所述的鼠抗NDM型碳青霉烯酶抗体在制备检测NDM型碳青霉烯酶抗原试剂中的应用，其特征在于：制备双抗体夹心法免疫胶体金试纸条，抗体1GH10为包被抗体，抗体12HE11为金标抗体；
    或者，抗体12HE11为包被抗体，抗体1GH10为金标抗体。
PCT/CN2022/143804 2022-03-02 2022-12-30 一种鼠抗ndm型碳青霉烯酶杂交瘤细胞株，单克隆抗体及应用 WO2023165252A1 (zh)

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