WO2018196572A1 - 一株异菌脲单克隆抗体杂交瘤细胞株zxl-2及其应用 - Google Patents

一株异菌脲单克隆抗体杂交瘤细胞株zxl-2及其应用 Download PDF

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WO2018196572A1
WO2018196572A1 PCT/CN2018/081834 CN2018081834W WO2018196572A1 WO 2018196572 A1 WO2018196572 A1 WO 2018196572A1 CN 2018081834 W CN2018081834 W CN 2018081834W WO 2018196572 A1 WO2018196572 A1 WO 2018196572A1
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iprodione
monoclonal antibody
hybridoma cell
zxl
ipma
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匡华
姚蕾珺
胥传来
徐丽广
马伟
刘丽强
吴晓玲
宋珊珊
胡拥明
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Jiangnan University
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    • 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

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  • the invention relates to a heterozygous monoclonal antibody hybridoma cell line ZXL-2 and application thereof, and belongs to the field of food safety immunoassay.
  • Iprodione also known as chlorhexidine
  • chlorhexidine is a high-efficiency broad-spectrum, contact-killing low-toxic bactericide for diimide. It is suitable for controlling early defoliation diseases of various fruit trees, vegetables, melons and other crops. Diseases such as gray mold and early blight.
  • PMRA Canadian Pest Management Regulatory Authority
  • China's pesticide residue limit standards stipulate that the maximum residue limit (MRL) of iprodione in banana, apple and rapeseed is 10, 5, 2 mg/kg, respectively.
  • MRL maximum residue limit
  • 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 heterozygous monoclonal antibody hybridoma cell line, and the antibody prepared from the cell strain has good specificity and detection sensitivity to iprodione, and can be used for establishing immunological detection of iprodione. method.
  • a heterozygous monoclonal antibody hybridoma cell line ZXL-2 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.13093.
  • the present invention also provides a heterourea monoclonal antibody which is secreted by the isoniazid monoclonal antibody hybridoma cell line ZXL-2 having the accession number CGMCC No. 13093.
  • the invention also provides the use of the iprodione monoclonal antibody or the cross-linked cell line ZXL-2 for the analysis of the detection of the residue of iprodione in a food safety test. For example, it is used to prepare a tool for detecting iprodione or directly for detecting iprodione. For example, a kit based on enzyme-linked immunosorbent technology or a test strip based on immunochromatography is developed.
  • the invention also provides a preparation method of the heterourea monoclonal antibody hybridoma cell line ZXL-2, comprising the following steps:
  • IPMA-KLH Preparation of immunogen IPMA-KLH: Weigh 4.2 mg of IPMA, 7 mg of 1-ethylcarbodiimide hydrochloride, 4 mg of N-hydroxysuccinimide, and dissolve with 300 ⁇ L of anhydrous N,N-dimethylformamide. (referred to as liquid A), the reaction was stirred at room temperature for 4 to 5 hours.
  • IPMA-BSA Preparation of the original IPMA-BSA: weigh 2.5 mg of IPMA, 4 mg of 1-ethylcarbodiimide hydrochloride, 2.5 mg of N-hydroxysuccinimide, and 300 ⁇ L of anhydrous N,N-dimethylmethyl The amide is dissolved (referred to as liquid A), and the reaction is stirred at room temperature for 4 to 5 hours. Weigh 10mg bovine serum albumin BSA (IPMA and BSA molar ratio is 30..1), dissolve in 2mL boric acid buffer solution, add A solution to B solution at room temperature, react at room temperature overnight, that is, even The complex IPMA-BSA mixture was separated and the intact antigen and unconjugated small molecule hapten were separated by dialysis.
  • IPMA and BSA molar ratio 10mg bovine serum albumin BSA
  • IPMA-KLH complete antigen was mixed and emulsified with an equal amount of Freund's adjuvant, and BALB/c mice were immunized by subcutaneous injection in the back.
  • complete Freund's adjuvant was used, and multiple boosts were used for incomplete Freund's adjuvant.
  • 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 ZXL-2.
  • the monoclonal antibody secreted by the cell line ZXL-2 provided by the invention has good specificity and detection sensitivity (IC 50 value: 5 ng/mL) to the different ureazides, and can realize water, fruits and vegetables
  • IC 50 value 5 ng/mL
  • the detection of the residual amount of iridone in the grain provides a raw material for the immunoassay of the residual urea residue in the food, and has practical application value.
  • CGMCC General Microbiology Center of the China Microbial Culture Collection Management Committee
  • the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China. Institute of Microbiology, Academy of Sciences, date of deposit, October 31, 2016, the deposit number is CGMCC No. 13093, classified as a monoclonal cell line.
  • Figure 1 is a standard curve of inhibition of iprodione by a monoclonal antibody secreted by ZXL-2.
  • the invention immunizes mice with the complete antigen of iprodione, cultures through cell fusion, HAT selective medium, screens the cell supernatant by ic-ELISA, and finally obtains a monoclonal antibody with good specificity and sensitivity to iprodione.
  • Antibody hybridoma cell line The invention immunizes mice with the complete antigen of iprodione, cultures through cell fusion, HAT selective medium, screens the cell supernatant by ic-ELISA, and finally obtains a monoclonal antibody with good specificity and sensitivity to iprodione.
  • Antibody hybridoma cell line Antibody hybridoma cell line.
  • IPMA-KLH Preparation of immunogen IPMA-KLH: Weigh 4.2 mg of IPMA, 7 mg of 1-ethylcarbodiimide hydrochloride, 4 mg of N-hydroxysuccinimide, and dissolve with 300 ⁇ L of anhydrous N,N-dimethylformamide. (referred to as liquid A), the reaction was stirred at room temperature for 4 to 5 hours.
  • liquid A anhydrous N,N-dimethylformamide
  • B solution boric acid buffer solution
  • 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 1 ⁇ 4 ⁇ 10 7 , which ensured that the SP2/0 tumor cells were in logarithmic growth phase before fusion. 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 1500 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 dropwise 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.
  • IPMA-BSA 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 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 stop solution 2M H 2 SO 4 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 iprodione was determined by ic-ELISA to be 5 ng/mL, indicating that it has good sensitivity to iprodione and can be used for immunoassay of iprodione.
  • 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.

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Abstract

提供了一株异菌脲单克隆抗体杂交瘤细胞株ZXL-2，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，保藏编号为CGMCC No.13093。该杂交瘤细胞株ZXL-2分泌的单克隆抗体对异菌脲具有较好的特异性和检测灵敏度(IC50值为5ng/mL)。该杂交瘤细胞株ZXL-2用于食品中异菌脲残留的免疫检测。

Description

一株异菌脲单克隆抗体杂交瘤细胞株ZXL-2及其应用 技术领域
本发明涉及一株异菌脲单克隆抗体杂交瘤细胞株ZXL-2及其应用，属于食品安全免疫检测领域。
背景技术
异菌脲(iprodione，又称扑海因)是一种二甲酰亚胺类高效广谱、触杀型低毒杀菌剂，适用于防治多种果树、蔬菜、瓜果类等作物早期落叶病、灰霉病、早疫病等病害。科学研究信息表明，若根据现有标签使用，异菌脲对人类健康造成的风险已不符合标准，因此加拿大有害生物管理局(PMRA)于2016年建议取消杀菌剂异菌脲的所有用途。我国农药残留限量标准规定香蕉、苹果、油菜籽中异菌脲的最大残留限量值(MRL)分别为10、5、2mg/kg。CAC、日本、美国和欧盟也制定了其在水果、蔬菜和粮食中的MRL值，并对其残留量进行监测。
为了有效监督监测食品中使用异菌脲的情况，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，加上食品中干扰物多，难以获得准确结果。因此建立快速简便的异菌脲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异菌脲单克隆抗体杂交瘤细胞株，由该细胞株制备的抗体对异菌脲具有较好特异性和检测灵敏度，可以用来建立异菌脲的免疫学检测方法。
本发明的技术方案：一株异菌脲单克隆抗体杂交瘤细胞株ZXL-2，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.13093。
本发明还提供异菌脲单克隆抗体，它由所述保藏编号为CGMCC No.13093的异菌脲单克隆抗体杂交瘤细胞株ZXL-2分泌产生。
本发明还提供所述异菌脲单克隆抗体或交瘤细胞株ZXL-2在食品安全检测中用于分析检测异菌脲残留的用途。例如，用于制备检测异菌脲的工具或者直接用于检测异菌脲。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。
本发明还提供异菌脲单克隆抗体杂交瘤细胞株ZXL-2的制备方法，包括以下步骤：
1)半抗原的合成：
将异菌脲(15.0g，45.45mmol),三乙胺(4.59g，45.45mmol)，3-巯基丙酸(4.82g，45.45mmol)和四三苯基磷钯(5.24g，4.545mmol)加入到乙二醇二甲醚(50.0mL)中，氮气保护下升温至100℃过夜。向反应液中加纯水(100mL)，二氯甲烷萃取(100mL×3次)，饱和食盐水洗一次，无水硫酸钠干燥，浓缩得粗品，制备得半抗原IPMA。
2)完全抗原的制备：
免疫原IPMA-KLH的制备：称取4.2mg IPMA，1-乙基碳二亚胺盐酸盐7mg，N-羟基琥珀酰亚胺4mg，用300μL无水N,N-二甲基甲酰胺溶解(称为A液)，室温搅拌反应4～5h。取匙孔血蓝蛋白KLH 1.47mL(6.8mg/mL,IPMA与KLH摩尔比为4500︰1)，加入等体积硼酸缓冲溶液(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IPMA-KLH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IPMA-BSA的制备：称取2.5mg IPMA，1-乙基碳二亚胺盐酸盐4mg，N-羟基琥珀酰亚胺2.5mg，用300μL无水N,N-二甲基甲酰胺溶解(称为A液)，室温搅拌反应4～5h。称取10mg牛血清蛋白BSA(IPMA与BSA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IPMA-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。
3)小鼠的免疫：IPMA-KLH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。首次免疫用完全弗氏佐剂，多次加强免疫用不完全弗氏佐剂。首次免疫与第二次加强免疫之间间隔一个月，多次加强免疫之间间隔21天。最后一次用IPMA-KLH完全抗原(不含佐剂)冲击免疫；通过间接竞争酶联免疫法(ic-ELISA)检测血清效价和抑制；
4)细胞融合与细胞株建立：通过聚乙二醇(PEG4000)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，最终筛选获得异菌脲单克隆抗体杂交瘤细胞株ZXL-2；
5)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ZXL-2。
本发明的有益效果：本发明提供的细胞株ZXL-2分泌的单克隆抗体，对异菌脲具有较好的特异性和检测灵敏度(IC 50值为5ng/mL)，可实现对水、果蔬、谷物中异菌脲残留量的检 测，为食品中异菌脲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
一株异菌脲单克隆抗体杂交瘤细胞株ZXL-2，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，保藏日期2016年10月31日，保藏编号为CGMCC No.13093，分类学命名为单克隆细胞株。
附图说明
图1 ZXL-2分泌的单克隆抗体对异菌脲的抑制标准曲线。
具体实施方式
本发明通过将异菌脲完全抗原免疫小鼠，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，最终得到了对异菌脲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ZXL-2的制备
(1)半抗原的合成：
将异菌脲(15.0g，45.45mmol),三乙胺(4.59g，45.45mmol)，3-巯基丙酸(4.82g，45.45mmol)和四三苯基磷钯(5.24g，4.545mmol)加入到乙二醇二甲醚(50.0mL)中，氮气保护下升温至100℃过夜。向反应液中加纯水(100mL)，二氯甲烷萃取(100mL×3次)，饱和食盐水洗一次，无水硫酸钠干燥，浓缩得粗品，制备得半抗原IPMA。
(2)完全抗原的合成：
免疫原IPMA-KLH的制备：称取4.2mg IPMA，1-乙基碳二亚胺盐酸盐7mg，N-羟基琥珀酰亚胺4mg，用300μL无水N,N-二甲基甲酰胺溶解(称为A液)，室温搅拌反应4～5h。取匙孔血蓝蛋白KLH 1.47mL(6.8mg/mL,IPMA与KLH摩尔比为4500︰1)，加入等体积硼酸缓冲溶液(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IPMA-KLH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IPMA-BSA的制备：称取2.5mg IPMA，1-乙基碳二亚胺盐酸盐4mg，N-羟基琥珀酰亚胺2.5mg，用300μL无水N,N-二甲基甲酰胺溶解(称为A液)，室温搅拌反应4～5h。称取10mg牛血清蛋白BSA(IPMA与BSA摩尔比为30︰1)，溶解于2mL硼酸缓冲溶液中(称为B液)，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IPMA-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。
(3)动物免疫：选择健康的6～8周龄的BALB/c小鼠进行免疫。取异菌脲完全抗原与 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一个月，多次加强免疫之间间隔21天。第3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5次免疫后21天冲击免疫，腹腔注射，要求冲免剂量减半且不含任何佐剂。
(4)细胞融合：在冲击免疫三天后，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3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 1500由慢到快滴加到细胞中；第2min，静置。第3min和第4min，在1min内滴加1mLRPMI-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。
(5)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，第二步选用异菌脲为标准品，用ic-ELISA对阳性细胞进行抑制效果测定。选择对异菌脲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ZXL-2。
(6)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。
6.1包被：将包被原IPMA-BSA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；
6.2洗涤：将板内溶液倾去，并用洗涤液洗涤3次，每次3min；
6.3封闭：拍干后，加入200μL/孔封闭液，37℃反应2h。洗涤后烘干备用；
6.4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；
6.5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；
6.6终止和测定：每孔加入50μL终止液2M H 2SO 4以终止反应，然后用酶标仪测定各孔的OD 450值。
用ic-ELISA测定单克隆抗体异菌脲的IC 50为5ng/mL，说明对异菌脲有很好的灵敏度，可用于异菌脲免疫分析检测。
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. 一株异菌脲单克隆抗体杂交瘤细胞株ZXL-2，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.13093。
  2. 异菌脲单克隆抗体，其特征在于：它由权利要求1所述保藏编号为CGMCC No.13093的异菌脲单克隆抗体杂交瘤细胞株ZXL-2分泌产生。
  3. 权利要求2所述异菌脲单克隆抗体的应用，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. 根据权利要求3所述的应用，其特征在于，用于食品安全中异菌脲的残留检测。
  5. 一种检测异菌脲的工具，其特征在于，含有权利要求2所述的异菌脲单克隆抗体或者权利要求1所述的异菌脲单克隆抗体杂交瘤细胞株ZXL-2。
  6. 根据权利要求5所述的一种检测异菌脲的工具，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异菌脲含量中的应用。
  8. 一种制备异菌脲抗原的方法，其特征在于，包括以下步骤：
    1)半抗原的合成：
    将异菌脲、三乙胺、3-巯基丙酸和四三苯基磷钯加入到乙二醇二甲醚中，氮气保护下升温至100℃过夜；向反应液中加纯水、二氯甲烷萃取，饱和食盐水洗一次，无水硫酸钠干燥，浓缩得粗品，制备得半抗原IPMA；
    2)完全抗原的制备：
    免疫原IPMA-KLH的制备：称取IPMA，1-乙基碳二亚胺盐酸盐，N-羟基琥珀酰亚胺，用无水N,N-二甲基甲酰胺溶解得到A液，室温搅拌反应4～5h；取匙孔血蓝蛋白KLH与KLH，加入等体积硼酸缓冲溶液得到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IPMA-KLH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异菌脲抗原。
  10. 权利要求8所述抗原在制备异菌脲抗体中的应用。
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