技术领域technical field
本发明涉及一株吡虫啉单克隆抗体杂交瘤细胞株YH5及其应用，属于食品安全免疫检测领域。The invention relates to an imidacloprid monoclonal antibody hybridoma cell line YH5 and its application, and belongs to the field of food safety immune detection.
背景技术Background technique
吡虫啉是一种新型的硝基亚甲基类的高效内吸性广谱杀虫剂，属新烟碱类药物，是烟碱型乙酰胆碱受体的作用体，目前在农业生产中已广泛使用，属低毒类杀虫剂，对蜜蜂的毒性为高毒。2002年，意大利北部和法国一些地区在使用吡虫啉过程中出现蜜蜂大量消失的现象，新烟碱类药物属于神经毒素，可以阻碍蜜蜂及其他昆虫大脑中的感受器接收神经信号。Imidacloprid is a new type of high-efficiency systemic broad-spectrum insecticide of nitromethylene. It is a neonicotinoid drug and acts on the nicotinic acetylcholine receptor. It is a low-toxicity insecticide with high toxicity to bees. In 2002, a large number of bees disappeared during the use of imidacloprid in northern Italy and some areas of France. Neonicotinoids are neurotoxins that can block receptors in the brains of bees and other insects from receiving nerve signals.
2012年木村黑田（Kimura-Kuroda）等人在PLoS One杂志上发表的一篇科学论文，提出吡虫啉的暴露可能会对哺乳动物神经系统的发育产生影响。欧盟食品安全局（EFSA）针对此论文分析发现，吡虫啉可能会对人类发育中的神经系统造成影响。在EFSA审查发现吡虫啉对蜜蜂有害之后，近两年欧盟已限制该药在一些作物上的使用（《Agrow》No.665，p6）。建立针对吡虫啉的快速、准确和灵敏的检测方法很有必要。A 2012 scientific paper by Kimura-Kuroda et al. in the journal PLoS One suggested that imidacloprid exposure may have effects on the development of the mammalian nervous system. The European Food Safety Authority (EFSA) analyzed the paper and found that imidacloprid may have effects on the developing nervous system in humans. After the EFSA review found that imidacloprid was harmful to bees, the EU has restricted the use of the drug on some crops for the past two years ("Agrow" No.665, p6). It is necessary to establish a rapid, accurate and sensitive detection method for imidacloprid.
目前检测吡虫啉色谱法应用最为广泛，包括气相色谱法(GC)、高效液相色谱法(HPLC)、气质联用(GC-MS)、液质联用 (LC-MS)、超临界流体色谱(SFC)、毛细管电泳(CE)等的仪器方法，但这些方法需要昂贵的仪器、专业的操作人员，且样品前处理复杂，成本高，时间长，不能实现大量样品的快速检测，因此建立快速简便的吡虫啉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。At present, the most widely used chromatographic methods for the detection of imidacloprid, including gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), supercritical fluid chromatography ( SFC), capillary electrophoresis (CE) and other instrument methods, but these methods require expensive instruments, professional operators, and complex sample pretreatment, high cost, long time, can not achieve rapid detection of a large number of samples, so the establishment of fast and easy The imidacloprid detection method is of great significance. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive and rapid detection method. It does not require high sample purity and is easy to operate. It is suitable for on-site rapid detection of a large number of samples. The establishment of efficient immunological detection methods and the screening of highly specific monoclonal antibodies are important prerequisites.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一株吡虫啉单克隆抗体杂交瘤细胞株，由该细胞株制备的抗体对吡虫啉具有较好特异性和检测灵敏度，可以用来建立吡虫啉的免疫学检测方法。The purpose of the present invention is to provide an imidacloprid monoclonal antibody hybridoma cell line, the antibody prepared from the cell line has good specificity and detection sensitivity to imidacloprid, and can be used to establish an immunological detection method for imidacloprid.
本发明的技术方案，一株吡虫啉单克隆抗体杂交瘤细胞株YH5，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.12015。The technical solution of the present invention, an imidacloprid monoclonal antibody hybridoma cell line YH5, has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, referred to as CGMCC, and the deposit number is CGMCC No.12015.
吡虫啉单克隆抗体，它由所述保藏编号为CGMCC No.12015的吡虫啉单克隆抗体杂交瘤细胞株YH5分泌产生。The imidacloprid monoclonal antibody is secreted and produced by the imidacloprid monoclonal antibody hybridoma cell line YH5 with the deposit number of CGMCC No. 12015.
所述吡虫啉单克隆抗体的应用，用于食品安全检测中吡虫啉残留的分析检测。The application of the imidacloprid monoclonal antibody is used for the analysis and detection of imidacloprid residues in food safety detection.
本发明提供的吡虫啉单克隆抗体杂交瘤细胞株YH5的制备基本步骤为：The basic steps for preparing the imidacloprid monoclonal antibody hybridoma cell line YH5 provided by the present invention are:
1）半抗原的合成：1) Synthesis of hapten:
将吡虫啉原药1(2 g，7.82 mmol），巯基丙酸（1.66 g，2 eq），Cs2CO3（25.5g，10 eq）混悬于己二酸二甲酯（30 mL）中，120℃下反应16 h，冷至室温，加入四氢呋喃（50 mL）后过滤，所得固体粗品（30g）加入少量水中，prep-HPLC纯化，最后得到100mg半抗原IMPA。用液质联用分析法进行鉴定与分析；Imidacloprid technical drug 1 (2 g, 7.82 mmol), mercaptopropionic acid (1.66 g, 2 eq), Cs2 CO3 (25.5 g, 10 eq) were suspended in dimethyl adipate (30 mL), The reaction was carried out at 120 °C for 16 h, cooled to room temperature, tetrahydrofuran (50 mL) was added, and then filtered. The obtained crude solid product (30 g) was added to a small amount of water, and purified by prep-HPLC to obtain 100 mg of the hapten IMPA. Identification and analysis by LC-MS analysis;
2）完全抗原IMPA-KLH的制备：称取2.5mg IMPA，1-乙基-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）5mg，N-羟基琥珀酰亚胺（NHS）3mg，用300μL无水N,N-二甲基甲酰胺（DMF）溶解(称为A液)，室温搅拌反应8 h。取匙孔血蓝蛋白KLH 1mL(5mg/mL)，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IMPA-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，并通过紫外吸收扫描方法鉴定；2) Preparation of complete antigen IMPA-KLH: Weigh 2.5mg IMPA, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) 5mg, N-hydroxysuccinimide (NHS) 3 mg, dissolved in 300 μL of anhydrous N,N-dimethylformamide (DMF) (referred to as solution A), and stirred for 8 h at room temperature. Take 1mL of keyhole limpet hemocyanin KLH (5mg/mL), add an equal volume of boric acid buffer solution (called B solution), add solution A to solution B dropwise at room temperature, and react overnight at room temperature to obtain coupling Complete antigen and unconjugated small molecule hapten were separated by dialysis and identified by UV absorption scanning method;
3）小鼠的免疫：吡虫啉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。最后一次用吡虫啉完全抗原（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；3) Immunization of mice: BALB/c mice were immunized by subcutaneous injection on the back after emulsification of complete imidacloprid antigen and equal amount of Freund's adjuvant. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for multiple booster immunizations. The interval between the first immunization and the second booster immunization was one month, and the interval between multiple booster immunizations was 21 days. The last pulse immunization with imidacloprid complete antigen (without adjuvant); serum titer and inhibition were detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA);
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，最终筛选获得吡虫啉单克隆抗体杂交瘤细胞株YH5；4) Cell fusion and cell line establishment: mouse splenocytes and mouse myeloma cells were fused by polyethylene glycol (PEG4000) method, cultured in HAT medium, and detected by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) The positive cell wells were further determined by ic-ELISA, and the positive cell wells with the best inhibition were subcloned three times by the limiting dilution method, and finally the imidacloprid monoclonal antibody hybridoma cell line YH5 was obtained by screening;
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。5) Characterization of hybridoma cell lines: Sensitivity and specificity were determined by ic-ELISA.
吡虫啉完全抗原与等量弗氏佐剂混合乳化完全，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，最后一次用吡虫啉完全抗原（25μg/只，不含佐剂）冲击免疫。取高效价低IC50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。此细胞株分泌的单克隆抗体，对吡虫啉具有较好的特异性和检测灵敏度（IC50值为0.3 ng/mL），可实现对水、果蔬、谷物中吡虫啉残留量的检测，为食品中吡虫啉残留的免疫检测提供了原料，具有实际应用价值。The complete imidacloprid antigen was mixed with an equal amount of Freund's adjuvant and emulsified completely, and BALB/c mice were immunized by subcutaneous injection on the back. Complete Freund's adjuvant was used for the first immunization (100μg/a), incomplete Freund's adjuvant was used for multiple booster immunizations (50μg/a), and the last time was imidacloprid complete antigen (25μg/b), without adjuvant). Spleen cells from mice with high titer and low IC50 were taken, fused with mouse myeloma cells by PEG method, screened by indirect competitive enzyme-linked immunosorbent assay and subcloned three times to obtain a hybridoma cell line. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity for imidacloprid (IC50 value is 0.3 ng/mL), which can realize the detection of imidacloprid residues in water, fruits and vegetables, and grains. Residual immunodetection provides raw materials and has practical application value.
本发明的有益效果：本发明提供的细胞株YH5分泌的单克隆抗体，对吡虫啉具有较好的特异性和检测灵敏度（IC50值为0.3 ng/mL），可实现对水、果蔬、谷物中吡虫啉残留量的检测，为食品中吡虫啉残留的免疫检测提供了原料，具有实际应用价值。Beneficial effects of the present invention: the monoclonal antibody secreted by the cell line YH5 provided by the present invention has good specificity and detection sensitivity for imidacloprid (IC50 value is 0.3 ng/mL), and can realize the detection of water, fruits, vegetables and grains. The detection of imidacloprid residues provides raw materials for the immunodetection of imidacloprid residues in food, and has practical application value.
生物材料样品保藏：一株吡虫啉单克隆抗体杂交瘤细胞株YH5，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1月20日，保藏编号为CGMCC No.12015。Preservation of biological material samples: an imidacloprid monoclonal antibody hybridoma cell line YH5, which has been preserved in the General Microbiology Center of the China Microorganism Culture Collection Management Committee, referred to as CGMCC, at the address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, deposited on January 20, 2016, with a deposit number of CGMCC No.12015.
附图说明Description of drawings
图1 YH5单克隆抗体对吡虫啉的抑制标准曲线。Figure 1 Inhibition standard curve of YH5 monoclonal antibody to imidacloprid.
具体实施方式Detailed ways
本发明下面的实施例仅作为本发明内容的进一步说明，不能作为本发明的限定内容或范围。下面通过实施例对本发明作进一步说明。The following embodiments of the present invention are only used as a further description of the content of the present invention, and cannot be used as a limitation or scope of the present invention. The present invention will be further described below through examples.
本发明通过将吡虫啉完全抗原免疫小鼠，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，最终得到了对吡虫啉有较好特异性和检测灵敏度的单克隆抗体杂交瘤细胞株。In the present invention, by immunizing mice with imidacloprid complete antigen, culturing in HAT selective medium through cell fusion, and screening cell supernatant through ic-ELISA, the monoclonal antibody hybridoma with better specificity and detection sensitivity to imidacloprid is finally obtained. cell line.
实施例1 杂交瘤细胞株YH5的制备Example 1 Preparation of hybridoma cell line YH5
（1）半抗原的合成：将吡虫啉原药1 (2g，7.82mmol），巯基丙酸（1.66g，2eq），Cs2CO3（25.5g，10eq）混悬于DMA（30mL）中，120℃下反应16 h，冷至室温，加入四氢呋喃（50mL）后过滤，所得固体粗品（30g）加入少量水中，prep-HPLC纯化，最后得到100mg半抗原IMPA，用液质联用分析法进行鉴定。(1) Synthesis of hapten: Imidacloprid technical drug 1 (2g, 7.82mmol), mercaptopropionic acid (1.66g, 2eq), Cs2 CO3 (25.5g, 10eq) were suspended in DMA (30mL), 120 The reaction was carried out at ℃ for 16 h, cooled to room temperature, tetrahydrofuran (50 mL) was added, and then filtered. The obtained crude solid product (30 g) was added to a small amount of water, purified by prep-HPLC, and finally 100 mg of the hapten IMPA was obtained, which was identified by liquid chromatography-mass spectrometry.
（2）完全抗原的合成：称取2.5mg IMPA，EDC 5mg，NHS 3mg，用300μL无水DMF溶解(称为A液)，室温搅拌反应8h。取KLH 1mL(5 mg/mL)，加入等体积BB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IMPA-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，并通过紫外吸收扫描方法鉴定。(2) Synthesis of complete antigen: Weigh 2.5 mg of IMPA, 5 mg of EDC, and 3 mg of NHS, dissolve in 300 μL of anhydrous DMF (referred to as solution A), and stir at room temperature for 8 h. Take 1 mL of KLH (5 mg/mL), add an equal volume of BB solution (referred to as solution B), add solution A to solution B dropwise at room temperature, and react overnight at room temperature to obtain the conjugate IMPA-KLH mixture Complete antigen and unconjugated small molecule hapten were separated by dialysis and identified by UV absorption scanning method.
（3）动物免疫：选择健康的6～8周龄的BALB/c小鼠进行免疫。取吡虫啉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天冲击免疫，腹腔注射，要求冲免剂量减半且不含任何佐剂。(3) Animal immunization: healthy BALB/c mice aged 6-8 weeks were selected for immunization. The complete imidacloprid antigen was mixed and emulsified with equal amount of Freund's adjuvant, and then BALB/c mice were immunized by subcutaneous injection on the back. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second booster immunization was one month, and the interval between multiple booster immunizations was 21 days. Blood was collected 7 days after the third immunization, and ic-ELISA was used to determine the serum titer and inhibition of mice. Mice with high titers and good inhibition were selected. 21 days after the fifth immunization, the immunization was performed by intraperitoneal injection, and the immune dose was required. Halved and without any adjuvants.
（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：(4) Cell fusion: After three days of shock immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight of 4000) method. The specific steps are as follows:
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 5 min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，8 min），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；a. Remove the eyeballs and take blood. After the mice were killed by cervical dislocation, they were immediately put into 75% alcohol for disinfection, soaked for about 5 minutes, and the spleen of the mice was taken out aseptically. The spleen cell suspension was obtained by sieving the mesh, collected, centrifuged (1200 rpm, 8 min), and the spleen cells were washed three times with RPMI-1640 medium. After the last centrifugation, the spleen cells were diluted to a certain volume, counted, and used for later use;
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% CO2培养箱中。融合前要求SP2/0瘤细胞数量达到1-4×107，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；b. Collection of SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were treated with RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2/0 tumor cells was required to reach 1-4×107 to ensure that the SP2/0 tumor cells were in the logarithmic growth phase before fusion. During fusion, tumor cells were collected, suspended in RPMI-1640 basal medium, and counted;
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5 min。离心（800 rpm，8 min），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% CO2培养箱中培养。c. The fusion process is 7 minutes. The first minute, 1 mL of PEG 4000 was added dropwise to the cells from slow to fast; the second minute, let stand. On the 3rd and 4th minutes, 1mL of RPMI-1640 medium was added dropwise within 1min; on the 5th and 6th minutes, 2mL of RPMI-1640 medium was added dropwise within 1min; on the 7th minute, 1mL of RPMI-1640 medium was added dropwise every 10s . Then incubate at 37°C for 5 min. Centrifuge (800 rpm, 8 min), discard the supernatant, resuspend it in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50×HAT, add 200 μL/well to a 96-well cell plate, set Incubate in a 37°C, 5% CO2 incubator.
（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，第二步选用吡虫啉为标准品，用ic-ELISA对阳性细胞孔进行抑制效果测定。选择对吡虫啉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YH5。(5) Cell selection and cell line establishment: On the 3rd day of cell fusion, the fusion cells were screened with RPMI-1640 and the culture medium was half-changed, and on the 5th day, the cells were treated with 20% fetal bovine serum and 1% 100×HT. The RPMI-1640 transition medium was completely replaced, and the cell supernatant was taken for screening on the 7th day. The screening is divided into two steps: in the first step, ic-ELISA is used to screen out the positive cell wells, and in the second step, imidacloprid is used as the standard substance, and the inhibitory effect of the positive cell wells is determined by ic-ELISA. Cell wells with better inhibition of imidacloprid standard were selected, subcloned by limiting dilution method, and detected by the same method. Repeat three times to obtain cell line YH5.
（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1×106杂交瘤细胞，从第七天开始收集腹水，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0.01 M 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) Preparation and identification of monoclonal antibodies: Take 8-10-week-old BALB/c mice, and inject 1 mL of sterile paraffin oil into each mouse intraperitoneally; 7 days later, inject 1×106 hybridoma cells into each mouse intraperitoneally , the ascites was collected from the seventh day, and the ascites was purified by the caprylic acid-ammonium sulfate method. Under partial acid conditions, n-octanoic acid can precipitate other impurity proteins except IgG immunoglobulin in ascites, then centrifuge and discard the precipitate; then use ammonium sulfate solution of equal saturation to precipitate IgG-type monoclonal antibody, centrifuge, discard The supernatant was dissolved in 0.01 M PBS solution (pH 7.4), dialysis and desalted, and the purified monoclonal antibody was finally obtained and stored at -20°C.
6.1包被：将包被原IMPA-KLH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；6.1 Coating: Dilute the original IMPA-KLH with 0.05M pH9.6 carbonate buffer starting from 1µg/mL, 100µL/well, and react at 37°C for 2h;
6.2洗涤：将板内溶液倾去，并用洗涤液洗涤3次，每次3min；6.2 Washing: Pour off the solution in the plate, and wash with washing solution 3 times, 3min each time;
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；6.3 Blocking: After patting dry, add 200μL/well blocking solution, react at 37°C for 2h, wash and dry for later use;
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1h；充分洗涤后，加入1:3000稀释的HRP-羊抗鼠IgG，100μL/孔，37℃反应1h；6.4 Sample loading: Dilute the antiserum from 1:1000 to the coated wells of each dilution, 100 μL/well, and react at 37°C for 1 h; after thorough washing, add HRP-goat diluted 1:3000. Anti-mouse IgG, 100 μL/well, reacted at 37°C for 1 h;
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；6.5 Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color developing solution to each well, and react at 37 °C for 15 minutes in the dark;
6.6终止和测定：每孔加入50μL终止液以终止反应，然后用酶标仪测定各孔的OD450值。6.6 Stop and measure: Add 50 μL stop solution to each well to stop the reaction, and then measure the OD450 value of each well with a microplate reader.
用ic-ELISA测定单克隆抗体吡虫啉的IC50为：0.3 ng/mL，说明对吡虫啉有很好的灵敏度，可用于吡虫啉免疫分析检测。The IC50 of the monoclonal antibody imidacloprid was determined by ic-ELISA: 0.3 ng/mL, indicating that it has a good sensitivity to imidacloprid and can be used for imidacloprid immunoassay detection.
溶液的配置：碳酸盐缓冲液（CBS）：称取Na2CO3 1.59 g，NaHCO3 2.93 g，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；Solution configuration: Carbonate buffer solution (CBS): Weigh Na2 CO3 1.59 g, NaHCO3 2.93 g, respectively dissolve in a small amount of double distilled water and mix, add double distilled water to about 800 mL and mix well, adjust pH value To 9.6, add double distilled water to make up to 1000mL, and store at 4°C for later use;
磷酸盐缓冲液（PBS）：8.00g NaCl，0.2g KCl，0.2g KH2PO4，2.9g Na2HPO4·12 H2O，溶于800mL纯水中，用NaOH或HCl调pH到7.2～7.4，定容至1000mL；Phosphate Buffered Saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2 PO4 , 2.9g Na2 HPO4 ·12 H2 O, dissolved in 800 mL of pure water, adjusted to pH 7.2 with NaOH or HCl ~7.4, dilute to 1000mL;
PBST：含0.05 % 吐温20的PBS；PBST: PBS containing 0.05% Tween 20;
TMB显色液：A液：Na2HPO4.12H2O 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。TMB color developing solution: A solution: Na2 HPO4. 12H2 O 18.43g, citric acid 9.33g, and purified water to 1000mL; B solution: 60mg TMB was dissolved in 100mL ethylene glycol. A and B liquids are mixed according to the volume ratio of 1:5, which is TMB, and the color developing liquid is mixed now.
表1.YH5单克隆抗体的交叉反应率Table 1. Cross-reactivity rates of YH5 monoclonal antibodies
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610324646.4ACN105754954B (en) | 2016-05-17 | 2016-05-17 | An imidacloprid monoclonal antibody hybridoma cell line YH5 and its application |
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| Application Number | Title | Priority Date | Filing Date |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109536457A (en)* | 2018-11-28 | 2019-03-29 | 北京勤邦生物技术有限公司 | A kind of hybridoma cell strain that secreting anti-imidacloprid monoclonal antibody and its application |
| CN113502272B (en)* | 2021-08-08 | 2023-08-04 | 江南大学 | Amaranth and carmine monoclonal antibody hybridoma cell strain and application thereof |
| CN114591916B (en)* | 2022-03-23 | 2023-05-16 | 扬州大学 | Cell strain secreting anti-imidacloprid monoclonal antibody, monoclonal antibody thereof and application thereof |
| Title |
|---|
| Evaluation and Validation of a Commercially Available Enzyme-Linked Immunosorbent Assay for the Neonicotinoid Insecticide Imidacloprid in Agricultural Samples;EIKI WATANABE et al;《J. Agric. Food Chem》;20040414;2756-2762* |
| Monoclonal antibody-based enzyme-linked immunosorbent assay for the insecticide imidacloprid;Hee-Joo Kim et al;《Analytica Chimica Acta》;20041231;111-118* |
| 抗吡虫啉单克隆抗体的制备及鉴定;李刚等;《生物工程学报》;20110625;第27卷(第6期);943-951* |
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