WO2018196573A1 - 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用 - Google Patents

一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用 Download PDF

Info

Publication number
WO2018196573A1
WO2018196573A1 PCT/CN2018/081847 CN2018081847W WO2018196573A1 WO 2018196573 A1 WO2018196573 A1 WO 2018196573A1 CN 2018081847 W CN2018081847 W CN 2018081847W WO 2018196573 A1 WO2018196573 A1 WO 2018196573A1
Authority
WO
WIPO (PCT)
Prior art keywords
flunixin meglumine
monoclonal antibody
meglumine
flunixin
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2018/081847
Other languages
English (en)
French (fr)
Inventor
胥传来
林璐
匡华
徐丽广
马伟
刘丽强
吴晓玲
宋珊珊
胡拥明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Publication of WO2018196573A1 publication Critical patent/WO2018196573A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9486—Analgesics, e.g. opiates, aspirine

Definitions

  • the invention relates to a flunixin meglumine monoclonal antibody hybridoma cell line YY and application thereof, and belongs to the field of food safety immunoassay.
  • Flunixin meglumine is a new type of antipyretic and analgesic drug for non-steroidal animals.
  • flunixin fluoroamine nicotinic acid, molecular formula C 14 H 11 F 3 N 2 O 2
  • Meglumine molecular amine, molecular formula C 7 H 17 NO 5
  • Meglumine mainly acts as a stabilizer, and the two are fixed at a ratio (1:1).
  • a fluoroamine niacin molecule binds to a meglumine molecule), which is FM.
  • Veterinary clinics are often used to relieve pain and anti-inflammatory caused by visceral colic and musculoskeletal disorders in horses; control of acute inflammation caused by infection of various diseases of cattle, such as laminitis, arthritis, etc.
  • the flunixin meglumine independently developed and declared by Qilu Animal Health Products Co., Ltd. was approved by the Ministry of Agriculture as the national three new veterinary drugs on June 1, 2007.
  • the certificate number is (2007) New Veterinary Drugs Certificate No. 23, 24 .
  • EMEA European Drug Administration
  • flunixin meglumine limits are 20, 30, 300 and 100 ⁇ g/kg respectively; livestock approved by the US FDA in April 2002 Residue limits in bovine liver and muscle were specified in the flunixin meglumine file at 125 and 25 ⁇ g/kg, respectively.
  • the current detection methods such as thin layer chromatography, gas chromatography, liquid chromatography Etc., the separation and purification process is lengthy, the sensitivity is low, and the interference is large, and it is difficult to obtain accurate results.
  • Enzyme-linked immunosorbent assay is an extremely efficient, sensitive and rapid detection method. It requires low purity and easy operation, and is suitable for rapid on-site detection of a large number of samples. It is an important prerequisite to establish a highly efficient immunological assay to screen highly specific monoclonal antibodies.
  • the object of the present invention is to provide a flunixin meglumine monoclonal antibody hybridoma cell strain, and the antibody prepared from the cell strain has good specificity and detection sensitivity to flunixin meglumine, and can be used for establishing fluorine. Immunological detection method of nisin meglumine.
  • a flunixin meglumine monoclonal antibody hybridoma cell line YY has been deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on October 31, 2016, referred to as CGMCC, and the preservation number is CGMCC No. 13096.
  • the present invention also provides a monoclonal antibody to flunixin meglumine secreted by the flunixin meglumine monoclonal antibody hybridoma cell line YY deposited under the accession number CGMCC No. 13096.
  • the invention also provides the application of the flunixin meglumine monoclonal antibody for the analysis and detection of the flunixin meglumine residue in the food safety test. For example, it is used to prepare a tool for detecting flunixin meglumine or directly for detecting flunixin meglumine. For example, a kit based on enzyme-linked immunosorbent technology or a test strip based on immunochromatography is developed.
  • the preparation method of the flunixin meglumine monoclonal antibody hybridoma cell line YY comprises the following steps:
  • mice FM-BSA complete antigen was mixed with an equal amount of Freund's adjuvant and emulsified, and BALB/c mice were immunized by subcutaneous injection in the back.
  • the first immunization 100 ⁇ g/only was performed with complete Freund's adjuvant, multiple boosts (50 ⁇ g/only) with incomplete Freund's adjuvant. There was a 28-day interval between the first immunization and the second booster, and 21 days between multiple immunizations.
  • mice The spleen cells of low-cost IC 50 mice were fused with mouse myeloma cells by PEG method, and screened by indirect competitive enzyme-linked immunosorbent assay and three subclones to obtain a hybridoma cell line YY.
  • the monoclonal antibody secreted by the cell line YY provided by the invention has good specificity and detection sensitivity to the flunixin meglumine (IC 50 value is 0.24 ng/mL), and is a veterinary fluoride.
  • the immunoassay of nising meglumine residues provides raw materials with practical application value.
  • a monoclonal cell line YY has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, referred to as CGMCC.
  • the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number CGMCC No.13096, date of deposit, October 31, 2016.
  • Figure 1 is a standard curve of inhibition of flunixin meglumine by a monoclonal antibody secreted by 1YY.
  • the mouse is immunized with the complete antigen of flunixin meglumine, and the cell supernatant is cultured by cell fusion, HAT selective medium, and the cell supernatant is screened by ic-ELISA, and finally the specificity of the flunixin meglumine is obtained. Sex and sensitivity of monoclonal antibody hybridoma cell lines.
  • SP2/0 tumor cells 7 to 10 days before fusion, SP2/0 tumor cells were cultured in a 5% CO 2 incubator containing 10% FBS (fetal calf serum) RPMI-1640 medium. Before the fusion, the number of SP2/0 tumor cells was required to reach (1 ⁇ 4) ⁇ 10 7 , and the SP2/0 tumor cells before the fusion were in the logarithmic growth phase. When fused, the tumor cells were collected and suspended in RPMI-1640 basal medium for cell counting;
  • FBS fetal calf serum
  • the fusion process is 7 min. At 1 min, 1 mL of PEG 4000 was added to the cells from slow to fast; at 2 min, it was allowed to stand. At 3 min and 4 min, 1 mL of RPMI-1640 medium was added dropwise in 1 min; at 5 min and 6 min, 2 mL of RPMI-1640 medium was added dropwise in 1 min; at 7 min, 1 mL of RPMI-1640 medium was added every 10 s. . Then warm at 37 ° C for 5 min.
  • mice BALB/c mice aged 8-10 weeks were used, and each mouse was intraperitoneally injected with 1 mL of sterile paraffin oil; after 7 days, each mouse was intraperitoneally injected with 1 ⁇ 10 6 hybridoma cells. Ascites was collected from the 7th day, and the ascites was purified by the octanoic acid-ammonium sulfate method.
  • n-octanoic acid can precipitate other heterologous proteins other than IgG immunoglobulin in ascites, then centrifuge and discard the precipitate; then precipitate the IgG monoclonal antibody with an equal amount of ammonium sulfate solution, centrifuge, and discard The supernatant was dissolved in 0.01 M PBS solution (pH 7.4), dialyzed against dialysis, and finally the purified monoclonal antibody was stored at -20 °C.
  • the original FM-OVA was diluted with 0.05M pH 9.6 carbonate buffer from 1 ⁇ g / mL, 100 ⁇ L / well, 37 ° C reaction for 2h;
  • the antiserum was diluted from 1..1000 and added to the coated wells of each dilution, 100 ⁇ L/well, and reacted at 37 ° C for 30 min; after thorough washing, add 1..3000 diluted HRP- ⁇ Anti-mouse IgG, 100 ⁇ L/well, reacted at 37 ° C for 30 min;
  • Termination and assay 50 ⁇ L of 2M H 2 SO 4 stop solution was added to each well to stop the reaction, and then the OD 450 value of each well was measured with a microplate reader.
  • the IC 50 of the monoclonal antibody flunixin meglumine was determined by ic-ELISA to be 0.24 ng/mL, indicating that it has good sensitivity to flunixin meglumine and can be used for immunoassay of flunixin meglumine.
  • CBS carbonate buffer
  • PBST PBS containing 0.05% Tween-20
  • TMB color developing solution liquid A: Na 2 HPO 4 ⁇ 12H 2 O 18.43 g, citric acid 9.33 g, pure water to 100 mL; B liquid: 60 mg TMB dissolved in 100 mL of ethylene glycol. A and B liquids are mixed with a volume ratio of 1..5 to be a TMB coloring liquid, which is currently used.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Pain & Pain Management (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Emergency Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY，该菌株已保藏于中国微生物菌种保藏管理委员会普通微生物中心（简称CGMCC），保藏编号为CGMCC No.13096。还提供了所述杂交瘤细胞株YY分泌的单克隆抗体及其应用。还提供了一种制备氟尼辛葡甲胺抗原的方法和该方法制备的抗原及其应用。

Description

一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY及其应用 技术领域
本发明涉及一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY及其应用，属于食品安全免疫检测领域。
背景技术
氟尼辛葡甲胺(flunixin meglumine，FM)是一种新型的、非甾体类动物专用的解热镇痛药物，其中,flunixin(氟胺烟酸，分子式C 14H 11F 3N 2O 2)是结构特殊的一类非甾体类抗炎镇痛解热药,meglumine(葡甲胺，分子式C 7H 17NO 5)主要起稳定剂的作用，二者以固定比例(1∶1，即一个氟胺烟酸分子结合一个葡甲胺分子)出现，即为FM。
兽医临床上常用于缓解马的内脏绞痛、肌肉与骨骼紊乱引起的疼痛及抗炎；牛的各种疾病感染引起的急性炎症的控制，如蹄叶炎、关节炎等，另外也可用于母猪乳房炎、子宫炎及无乳综合症的辅助治疗。齐鲁动物保健品有限公司独立研制并申报的氟尼辛葡甲胺已于2007年6月1日被农业部批准为国家三类新兽药，证书编号为(2007)新兽药证字23、24号。
EMEA(欧洲药品管理局)规定了氟尼辛葡甲胺在牛肌肉、脂肪、肝脏和肾脏组织中的限量分别为20、30、300和100μg/kg；美国FDA于2002年4月批准的家畜用氟尼辛葡甲胺文件中规定其在牛肝脏和肌肉中的残留限量分别为125及25μg/kg。为了有效监督监测兽医临床上使用氟尼辛葡甲胺的情况，需要寻找一种特异性好、灵敏度高的测定方法，而目前检测方法如薄层层析法、气相色谱法、液相色谱法等，分离纯化过程冗长，灵敏度低，加上干扰物多，难以获得准确结果。因此建立快速简便的氟尼辛葡甲胺检测方法具有重要意义。酶联免疫法(ELISA)是一种极为高效、敏感、快速的检测方法，检测时对样本的纯度要求不高而且操作简便，适用于大量样本的现场快速检测。建立高效的免疫学检测方法，筛选高特异性的单克隆抗体是重要前提。
发明内容
本发明的目的是提供一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株，由该细胞株制备的抗体对氟尼辛葡甲胺具有较好特异性和检测灵敏度，可以用来建立氟尼辛葡甲胺的免疫学检测方法。
本发明的技术方案：一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY，已于2016年10月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，保藏编号为CGMCC No.13096。
本发明还提供氟尼辛葡甲胺单克隆抗体，它由所述保藏编号为CGMCC No.13096的氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY分泌产生。
本发明还提供所述氟尼辛葡甲胺单克隆抗体的应用，用于食品安全检测中氟尼辛葡甲胺残留的分析检测。例如，用于制备检测氟尼辛葡甲胺的工具或者直接用于检测氟尼辛葡甲胺。例如开发基于酶联免疫技术的试剂盒或者基于免疫层析的试纸条。
本发明提供的氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY的制备方法，包括以下步骤为：
1)完全抗原的制备：
免疫原FM-BSA的制备：称取2.2mg FM，1-乙基碳二亚胺盐酸盐2.6mg，N-羟基琥珀酰亚胺1.6mg，用400μL无水N,N-二甲基甲酰胺溶解，室温搅拌反6-8h(称为A液)。取牛血清蛋白BSA 10mg(FM与BSA摩尔比为30︰1)，用4mL硼酸缓冲溶液溶解(称为B液)。在室温条件，逐滴将A液加入到B液中，室温反应过夜，即得偶联物FM-BSA混合液，通过透析分离完全抗原和未偶联的小分子半抗原。
包被原FM-OVA的制备：称取1.6mg FM，1-乙基碳二亚胺盐酸盐1.9mg，N-羟基琥珀酰亚胺1.2mg，用300μL无水N,N-二甲基甲酰胺溶解，室温搅拌反6～8h(称为A液)。称取5mg鸡卵清白蛋白OVA(FM与OVA摩尔比为30︰1)，溶解于2mL硼酸缓冲溶液中，在室温条件下，逐滴将A液加入到B液中，室温反应过夜，即得偶联物FM-OVA混合液，通过透析分离包被原和未偶联的小分子半抗原；
2)小鼠的免疫：FM-BSA完全抗原与等量弗氏佐剂混合乳化后，通过背部皮下注射免疫BALB/c小鼠。首次免疫(100μg/只)用完全弗氏佐剂，多次加强免疫(50μg/只)用不完全弗氏佐剂。首次免疫与第二次加强免疫之间间隔28天，多次加强免疫之间间隔21天。最后一次用FM-BSA完全抗原冲刺免疫(25μg/只，不含佐剂)；通过间接竞争酶联免疫法(ic-ELISA)检测血清效价和抑制；取高效价低IC 50小鼠的脾细胞，通过PEG方法与小鼠骨髓瘤细胞融合，经过间接竞争酶联免疫法筛选和三次亚克隆，得到一株杂交瘤细胞株；
3)细胞融合与细胞株建立：通过聚乙二醇(PEG 4000)法将小鼠脾细胞和小鼠骨髓瘤细胞融合，通过HAT培养基培养，利用间接竞争酶联免疫法(ic-ELISA)检测阳性细胞孔，并进一步利用ic-ELISA测定阳性细胞孔的抑制效果，通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆，最终筛选获得氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY；
4)杂交瘤细胞株性质的鉴定：通过ic-ELISA测定灵敏度和特异性。
取高效价低IC 50小鼠的脾细胞，通过PEG方法与小鼠骨髓瘤细胞融合，经过间接竞争酶联免疫法筛选和三次亚克隆，得到一株杂交瘤细胞株YY。
本发明的有益效果：本发明提供的细胞株YY分泌的单克隆抗体，对氟尼辛葡甲胺具有较好的特异性和检测灵敏度(IC 50值为0.24ng/mL)，为兽用氟尼辛葡甲胺残留的免疫检测提供了原料，具有实际应用价值。
生物材料保藏
一株单克隆细胞株YY，已保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为CGMCC No.13096，保藏日期2016年10月31日。
附图说明
图1YY分泌的单克隆抗体对氟尼辛葡甲胺的抑制标准曲线。
具体实施方式
本发明通过将氟尼辛葡甲胺完全抗原免疫小鼠，通过细胞融合，HAT选择性培养基培养，通过ic-ELISA筛选细胞上清，最终得到了对氟尼辛葡甲胺有较好特异性和灵敏度的单克隆抗体杂交瘤细胞株。
实施例1 杂交瘤细胞株YY的制备
(1)完全抗原的制备：称取2.2mg FM，1-乙基碳二亚胺盐酸盐2.6mg，N-羟基琥珀酰亚胺1.6mg，用400μL无水N,N-二甲基甲酰胺溶解，室温搅拌反6-8h(称为A液)。取牛血清蛋白BSA 10mg(FM与BSA摩尔比为30︰1)，用4mL硼酸缓冲溶液溶解(称为B液)。在室温条件，逐滴将A液加入到B液中，室温反应过夜，即得偶联物FM-BSA混合液，通过透析分离完全抗原和未偶联的小分子半抗原。
包被原FM-OVA的制备：称取1.6mg FM，1-乙基碳二亚胺盐酸盐1.9mg，N-羟基琥珀酰亚胺1.2mg，用300μL无水N,N-二甲基甲酰胺溶解，室温搅拌反6～8h(称为A液)。称取5mg鸡卵清白蛋白OVA(FM与OVA摩尔比为30︰1)，溶解于2mL硼酸缓冲溶液中，在室温条件下，逐滴将A液加入到B液中，室温反应过夜，即得偶联物FM-OVA混合液，通过透析分离包被原和未偶联的小分子半抗原。
(2)动物免疫：选择健康的6～8周龄的BALB/c小鼠进行免疫。取氟尼辛葡甲胺完全抗原与等量弗氏佐剂混合乳化后，通过背部皮下注射免疫BALB/c小鼠。第一次免疫用完全弗氏佐剂，之后都用不完全弗氏佐剂。首次免疫与第二次加强免疫之间间隔28天，多次加强免疫之间间隔21天。第三次免疫后7天采血，使用ic-ELISA测定小鼠血清效价和抑制，选择效价高抑制好的小鼠，在第五次免疫后21天冲刺免疫，腹腔注射，要求冲免剂量减半且不含任何佐剂。
(3)细胞融合：在冲刺免疫三天后，按照常规PEG(聚乙二醇，分子量为4000)方法进行细胞融合，具体步骤如下：
a、摘眼球取血，颈椎脱臼法处死小鼠后，立即放入75％酒精中消毒，浸泡5min左右，无菌操作取出小鼠的脾脏，用注射器的胶头适度研磨并通过200目细胞筛网得到脾细胞悬液，收集，离心(1200rpm，8min)，用RPMI-1640培养基洗涤脾细胞三次，最后一次离心后，将脾细胞稀释到一定体积，计数，备用；
b、收集SP2/0细胞：融合前7～10天，将SP2/0瘤细胞用含10％FBS(胎牛血清)RPMI-1640培养基在5％CO 2培养箱中。融合前要求SP2/0瘤细胞数量达到(1～4)×10 7，保证融合前SP2/0瘤细胞处于对数生长期。融合时，收集瘤细胞，悬浮于RPMI-1640基础培养液中，进行细胞计数；
c、融合过程7min。第1min，将1mL的PEG 4000由慢到快滴加到细胞中；第2min，静置。第3min和第4min，在1min内滴加1mL RPMI-1640培养基；第5min和第6min，在1min内滴加2mL RPMI-1640培养基；第7min，每10s滴加1mL的RPMI-1640培养基。然后37℃温浴5min。离心(800rpm，8min)，弃上清，重悬入含20％胎牛血清、2％的50×HAT的RPMI-1640筛选培养液中，按照200μL/孔加到96孔细胞板，置于37℃、5％CO 2培养箱中培养。
(4)细胞筛选与细胞株建立：在细胞融合的第3天对融合细胞进行RPMI-1640筛选培养液半换液，第5天进行用含20％胎牛血清、1％的100×HT的RPMI-1640过渡培养液进行全换液，在第7天取细胞上清进行筛选。筛选分两步：第一步先用ic-ELISA筛选出阳性细胞孔，第二步选用氟尼辛葡甲胺为标准品，用ic-ELISA对阳性细胞进行抑制效果测定。选择对氟尼辛葡甲胺标准品均有较好抑制的细胞孔，采用有限稀释法进行亚克隆，用同样的方法进行检测。重复三次，获得细胞株YY。
(5)单克隆抗体的制备与鉴定：取8～10周龄BALB/c小鼠，每只小鼠腹腔注射无菌石蜡油1mL；7天后每只小鼠腹腔注射1×10 6杂交瘤细胞，从第7天开始收集腹水，将腹水通过辛酸-硫酸铵法纯化。在偏酸条件下，正辛酸可以沉淀腹水中除IgG免疫球蛋白外的其他杂蛋白，然后离心，弃沉淀；再用等量饱和度的硫酸铵溶液沉淀IgG型的单克隆抗体，离心，弃上清，用0.01M PBS溶液(pH7.4)溶解后，透析脱盐，最终得到纯化后的单克隆抗体置于-20℃保存。
5.1包被：将包被原FM-OVA用0.05M pH9.6碳酸盐缓冲液从1μg/mL开始倍比稀释，100μL/孔，37℃反应2h；
5.2洗涤：将板内溶液倾去，并用洗涤液洗涤3次，每次3min；
5.3封闭：拍干后，加入200μL/孔封闭液，37℃反应2h。洗涤后烘干备用；
5.4加样：将抗血清从1︰1000开始倍比稀释，并加入到各稀释度的包被孔中，100μL/孔，37℃反应30min；充分洗涤后，加入1︰3000稀释的HRP-羊抗鼠IgG，100μL/孔，37℃反应30min；
5.5显色：将酶标板取出，充分洗涤后，每孔加入100μL的TMB显色液，37℃避光反应15min；
5.6终止和测定：每孔加入50μL2M H 2SO 4终止液以终止反应，然后用酶标仪测定各孔的OD 450值。
用ic-ELISA测定单克隆抗体氟尼辛葡甲胺的IC 50为0.24ng/mL，说明对氟尼辛葡甲胺有很好的灵敏度，可用于氟尼辛葡甲胺免疫分析检测。
上述实施例中：
溶液的配置：碳酸盐缓冲液(CBS)：称取Na 2CO 3 1.59g，NaHCO 3 2.93g，分别溶于少量双蒸水后混合，加双蒸水至约800mL混匀，调pH值至9.6，加双蒸水定容至1000mL，4℃贮存备用；
磷酸盐缓冲液(PBS)：8.00g NaCl，0.2g KCl，0.2g KH 2PO 4，2.9g Na 2HPO 4·12H 2O，溶于800mL纯水中，用NaOH或HCl调pH到7.2～7.4，定容至1000mL；
PBST：含0.05％吐温-20的PBS；
TMB显色液：A液：Na 2HPO 4·12H 2O 18.43g，柠檬酸9.33g，纯水定容至1000mL；B液：60mg TMB溶于100mL乙二醇中。A、B液按体积比1︰5混合即为TMB显色液，现用现混。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY，已于2016年10月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，保藏编号为CGMCC No.13096。
  2. 氟尼辛葡甲胺单克隆抗体，其特征在于：它由权利要求1所述保藏编号为CGMCC No.13096的氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY分泌产生。
  3. 权利要求2所述氟尼辛葡甲胺单克隆抗体的应用，其特征在于：用于制备检测异菌脲的工具或者直接用于检测异菌脲。
  4. 权利要求3所述氟尼辛葡甲胺单克隆抗体的应用，其特征在于：用于食品安全检测中氟尼辛葡甲胺残留的分析检测。
  5. 一种检测氟尼辛葡甲胺的工具，其特征在于，含有权利要求2所述的氟尼辛葡甲胺单克隆抗体或者权利要求1所述的氟尼辛葡甲胺单克隆抗体杂交瘤细胞株YY。
  6. 根据权利要求5所述的一种检测氟尼辛葡甲胺的工具，其特征在于，所述工具是基于酶联免疫技术的试剂盒或者基于免疫层析的试纸条。
  7. 权利要求5或6所述工具在检测食品中氟尼辛葡甲胺含量中的应用。
  8. 一种制备氟尼辛葡甲胺抗原的方法，其特征在于，包括以下步骤：
    免疫原FM-BSA的制备：称取FM，1-乙基碳二亚胺盐酸盐，N-羟基琥珀酰亚胺，用无水N,N-二甲基甲酰胺溶解，室温搅拌反6-8h得到A液；取牛血清蛋白BSA，用硼酸缓冲溶液溶解得到B液，在室温条件，逐滴将A液加入到B液中，室温反应过夜，即得偶联物FM-BSA混合液，通过透析分离完全抗原和未偶联的小分子半抗原。
  9. 根据权利要求8所述方法制备得到的氟尼辛葡甲胺抗原。
  10. 权利要求8所述抗原在制备氟尼辛葡甲胺抗体中的应用。
PCT/CN2018/081847 2017-04-27 2018-04-04 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用 Ceased WO2018196573A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710285176.XA CN106867971B (zh) 2017-04-27 2017-04-27 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用
CN201710285176.X 2017-04-27

Publications (1)

Publication Number Publication Date
WO2018196573A1 true WO2018196573A1 (zh) 2018-11-01

Family

ID=59161787

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/081847 Ceased WO2018196573A1 (zh) 2017-04-27 2018-04-04 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用

Country Status (2)

Country Link
CN (1) CN106867971B (zh)
WO (1) WO2018196573A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118373916A (zh) * 2024-06-26 2024-07-23 北京纳百生物科技有限公司 一种氟尼辛葡甲胺单克隆抗体、检测试纸条及其应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106867971B (zh) * 2017-04-27 2020-05-05 江南大学 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用
CN108866009B (zh) * 2018-08-16 2019-10-01 江南大学 一株甲霜灵单克隆抗体杂交瘤细胞株及其应用
CN114316027B (zh) * 2020-10-10 2023-09-05 中国农业大学 一种氟尼辛人工抗原及其制备方法与应用
CN114774368B (zh) * 2022-05-16 2023-06-06 江南大学 一株分泌抗丙炔氟草胺单克隆抗体的杂交瘤细胞株及其应用
CN118465258B (zh) * 2024-05-11 2025-12-09 北京勤邦科技股份有限公司 一种检测氟尼辛葡甲胺及其代谢物的试纸条及方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105754955A (zh) * 2016-05-17 2016-07-13 江南大学 一株噻虫啉、啶虫脒单克隆抗体杂交瘤细胞株gw及其应用
CN106867971A (zh) * 2017-04-27 2017-06-20 江南大学 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105754955A (zh) * 2016-05-17 2016-07-13 江南大学 一株噻虫啉、啶虫脒单克隆抗体杂交瘤细胞株gw及其应用
CN106867971A (zh) * 2017-04-27 2017-06-20 江南大学 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LU , LIN ET AL.: "Development of IC-ELISA and Immunochromatographic Strip Assay for the Detection of Flunixin Meglumine in Milk", FOOD AND AGRICULTURAL IMMUNOLOGY, vol. 29, no. 1, 31 August 2017 (2017-08-31), pages 193 - 203, XP055527086, ISSN: 0954-0105 *
SONG, JINCUI ET AL.: "Optimization for Determination Condition of Flunixin Meglumine by LC-MS/MS", FOOD ENGINEERING, 30 June 2014 (2014-06-30), pages 55 - 57, 64, ISSN: 1673-6044 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118373916A (zh) * 2024-06-26 2024-07-23 北京纳百生物科技有限公司 一种氟尼辛葡甲胺单克隆抗体、检测试纸条及其应用

Also Published As

Publication number Publication date
CN106867971A (zh) 2017-06-20
CN106867971B (zh) 2020-05-05

Similar Documents

Publication Publication Date Title
WO2018196573A1 (zh) 一株氟尼辛葡甲胺单克隆抗体杂交瘤细胞株yy及其应用
CN102827076B (zh) 一种氟喹诺酮类药物通用半抗原、人工抗原、广谱单克隆抗体及其制备方法与应用
CN111454912B (zh) 一株赛拉嗪单克隆抗体杂交瘤细胞株及其应用
CN112877296A (zh) 一种抗非那西丁单克隆抗体杂交瘤细胞株ad及其制备方法与应用
CN113046325B (zh) 一株维生素k3单克隆抗体杂交瘤细胞株及其应用
CN108251381B (zh) 一种百草枯单克隆抗体杂交瘤细胞株及其应用
CN110713986B (zh) 一株维生素b1单克隆抗体杂交瘤细胞株cbdd及其应用
CN110616195A (zh) 一株二甲双胍单克隆抗体杂交瘤细胞株及其应用
CN111334479A (zh) 一株氯羟吡啶单克隆抗体杂交瘤细胞株tyl及其应用
CN105838681A (zh) 一株抗地塞米松特异性单克隆抗体杂交瘤细胞株c3及其应用
WO2018196572A1 (zh) 一株异菌脲单克隆抗体杂交瘤细胞株zxl-2及其应用
CN102168072A (zh) 一种抗盐酸克伦特罗单克隆抗体及检测盐酸克伦特罗的试剂盒
CN109824604B (zh) 磺胺类药物半抗原和人工抗原及其制备方法与应用
CN110950962B (zh) 一株分泌双咪苯脲单克隆抗体的杂交瘤细胞株a11s及其应用
CN110724192A (zh) 一株分泌蛇形菌素单克隆抗体的杂交瘤细胞株及其应用
CN105754954B (zh) 一株吡虫啉单克隆抗体杂交瘤细胞株yh5及其应用
CN112375744A (zh) 一株二氢吡啶单克隆抗体杂交瘤细胞株及其应用
CN108866009B (zh) 一株甲霜灵单克隆抗体杂交瘤细胞株及其应用
CN112813036B (zh) 一株曲安奈德单克隆抗体杂交瘤细胞株及其应用
CN108949699B (zh) 一种分泌美洛昔康单克隆抗体的杂交瘤细胞株及其应用
CN110669736A (zh) 一株安乃近单克隆抗体杂交瘤细胞株cbd及其应用
CN105112376A (zh) 一株金刚烷胺单克隆抗体杂交瘤细胞株及其应用
CN105087500A (zh) 一株利巴韦林单克隆抗体杂交瘤细胞株及其应用
CN110760484B (zh) 一株分泌抗氯苯那敏单克隆抗体的杂交瘤细胞株cbg及其应用
CN106929479B (zh) 一株维生素b2单克隆抗体杂交瘤细胞株gz-4及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18790691

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18790691

Country of ref document: EP

Kind code of ref document: A1