CN105385660A - Hybridoma cell strain, antibody excreted by hybridoma cell strain, preparation method and application - Google Patents

Hybridoma cell strain, antibody excreted by hybridoma cell strain, preparation method and application Download PDF

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CN105385660A
CN105385660A CN201510737167.0A CN201510737167A CN105385660A CN 105385660 A CN105385660 A CN 105385660A CN 201510737167 A CN201510737167 A CN 201510737167A CN 105385660 A CN105385660 A CN 105385660A
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methamphetamine
anti
hybridoma cell
c2013160
antibody
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CN201510737167.0A
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郑曙剑
陈国琴
殷秀飞
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杭州隆基生物技术有限公司
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Abstract

The invention provides a hybridoma cell strain MET1.0 producing a methylamphetamine-resistant monoclonal antibody, the methylamphetamine-resistant monoclonal antibody, a method for preparing the methylamphetamine-resistant monoclonal antibody excreted and produced by the methylamphetamine-resistant hybridoma cell strain MET1.0 with the preservation number being CCTCC NO:C2013160, a reagent kit for detecting methylamphetamine in saliva, and application of the methylamphetamine-resistant monoclonal antibody excreted and produced by the methylamphetamine-resistant hybridoma cell strain MET1.0 with the preservation number being CCTCC NO:C2013160 to detecting methylamphetamine. The high-affinity methylamphetamine-resistant monoclonal antibody can be easily prepared and obtained, and the effective yield of methylamphetamine artificial antigens is also increased. Only 5 minutes are needed for detection of methylamphetamine, detection sensitivity can reach 50 ng/ml, and specificity is high.

Description

一种杂交瘤细胞株及其分泌的抗体和制备方法、应用 And one kind of hybridoma cell lines secreting antibodies and preparation of application

技术领域 FIELD

[0001] 本发明属于生物技术领域,具体涉及一种抗甲基安非他明杂交瘤细胞株和抗甲基安非他明单克隆抗体。 [0001] The present invention belongs to the field of biotechnology, particularly relates to an anti-methamphetamine A hybridoma cell line anti-methamphetamine and amphetamine monoclonal antibody.

[0002] 还涉及抗甲基安非他明单克隆抗体作为检测甲基安非他明的应用,以及制备该抗甲基安非他明杂交瘤细胞株并分泌抗甲基安非他明单克隆抗体的方法。 [0002] A further relates to an anti-methamphetamine monoclonal antibodies as detector A methamphetamine applications, and methods of making the anti-methamphetamine and amphetamine hybridoma cell lines secreting anti-methamphetamine alone A the method of cloning antibodies.

背景技术 Background technique

[0003] 甲基安非他明(Methamphetamine,MET),化学名为N-甲基-1-苯基-丙烷-2-胺, 其结构式为: [0003] A methamphetamine (Methamphetamine, MET), chemical name N- methyl-1-phenyl - propane-2-amine, having the formula:

Figure CN105385660AD00031

[0005] 甲基安非他明(Methamphetamine),又称甲基苯丙胺,俗称"冰毒",它属于精神类药品,是一种拟交感胺药,医疗上用于治疗注意力不足过动症(ADHD)、嗜睡症、极端的肥胖症、后脑炎、帕金森综合症等,过度服用极易导致精神病状态,表现出活动过度,情感冲动, 野蛮,妄想,偏执狂,幻觉甚至有杀人倾向。 [0005] A methamphetamine (Methamphetamine), also known as methamphetamine, commonly known as "ice", it belongs to psychotropic drugs, is a sympathomimetic amine drugs, medical treatment for attention deficit hyperactivity disorder ( ADHD), narcolepsy, extreme obesity, after encephalitis, Parkinson's syndrome, excessive taking easily lead to a psychotic state, showing hyperactivity, emotional impulses, brutal, delusions, paranoia, hallucinations and even killing tendencies. 此种状态过后,出现一种极度衰竭和抑郁状态, 也有因严重抑郁而自杀者。 After this state, the emergence of a state of extreme exhaustion and depression, but also due to severe depression and suicide. 长期滥用可造成慢性中毒、体重下降、消瘦、溃疡、脓肿、指甲脆化和夜间磨牙。 Long-term abuse can cause chronic poisoning, weight loss, weight loss, ulcers, abscesses, brittle nails and teeth at night. 甲基安非他明耐受性随长期使用而增加,对于未产生耐受的人,使用甲基安非他明30毫克便会引起中毒。 A amphetamine tolerance increases with long-term use, not for human tolerance, the use of methyl amphetamine 30 mg will cause poisoning. 而有报道,长期滥用者,为了达到初期使用时的欣快效应,竟将剂量增至2000毫克,这极易引起急性中毒,可造成惊阙、昏迷甚至死亡。 And there have been reports of long-term abusers, in order to achieve euphoric effects upon initial use, trying to shift the dose increased to 2000 mg, which can easily cause acute poisoning, can cause convulsants, coma and even death. 具有极大的社会危害性。 Great social harm.

[0006] 在甲基安非他明的检测中,国内外主要采取免疫层析法、酶联免疫吸附法(ELISA)、气相色谱-质谱联用、高效液相色谱法、放射性免疫对尿液、血液、组织以及毛发等样本进行检测。 [0006] A methamphetamine in the detection, at home and abroad to take the main immunochromatography, an enzyme-linked immunosorbent assay (ELISA), gas chromatography - mass spectrometry, high performance liquid chromatography, radioimmunoassay urine , blood, tissue and hair and other samples for testing. 尿液样本涉及到伦理与隐私,对于采样存在一定困难和样本的保真性存在一定怀疑;血液样本涉及伦理和创伤,对于供样人有一定的感染风险;组织和毛发等样本需要进行前处理,检测需要大型仪器设备,技术要求高,不适合现场快速检测。 Urine samples related to ethics and privacy, there are some doubts about the existence of certain difficulties sampling and sample fidelity; blood samples involving ethics and trauma, for the kind of people for a certain degree of risk of infection; tissue and hair samples such as the need for pre-treatment, detection requires large equipment, high technical requirements, are not suitable for on-site rapid testing.

[0007] 其中唾液是唯一已成功的用来替代血液进行动力学和一些药物毒理学研究的体液,其中包括滥用药物的研究。 [0007] Saliva is where the only alternative has been successfully used for blood and body fluid dynamics of some of toxicology studies, including the study of drug abuse. 药物的唾液浓度与血液浓度的比值(S/P比值)相对恒定,变异率小,对估计药物在体内的代谢水平具有重要的意义。 Saliva concentration ratio of the drug concentration in the blood (S / P ratio) is relatively constant, low mutation rate, is significant in vivo metabolism of the drug is estimated. 甲基安非他明唾液检测机制:甲基安非他明从血液进入到唾液,原药比代谢物更具有亲脂性,更易通过脂肪膜屏障进入唾液, 因此唾液的毒品检测物为毒品原体;而甲基安非他明的尿液检测机制是通过血液通过肝肾代谢,其中40%仍为原药甲基安非他明,其它通常以安非他明衍生物从尿液中排泄出来。 A amphetamine saliva test mechanism: A methamphetamine entering from the blood into the saliva, the original drug having more lipophilic metabolites, more accessible by saliva fat membrane barrier, thus the drug saliva was detected as the original drug form ; amphetamine and methamphetamine urine detection mechanism by the blood through the liver and kidney metabolism, still 40% of the original drug a methamphetamine, typically other amphetamine derivatives excreted in the urine . 因此唾液检测的是甲基安非他明原药,而尿液检测的是甲基安非他明以及安非他明衍生物, 唾液检测更优于尿液检测。 A saliva test so that the original drug methamphetamine, and methamphetamine urine detection of amphetamine and amphetamine derivative, more than saliva test urine detection.

[0008] 目前国际通用的甲基安非他明诊断阈值为l〇〇〇ng/ml,主要用于尿液检测。 [0008] A current internationally accepted diagnostic methamphetamine threshold l〇〇〇ng / ml, mainly for urine testing. 而唾液中甲基安非他明的含量较低,应用于唾液检测的抗甲基安非他明单克隆抗体要求更高的灵敏度和亲和力,本发明甲基安非他明唾液检测试剂盒利用胶体金免疫层析技术定性检测唾液中是否含有甲基安非他明,其检测阈值可达到50ng/ml,完全能够满足唾液检测的需要。 The saliva out methamphetamine low content of saliva applied to the detection of anti-methamphetamine monoclonal antibody of claim next higher sensitivity and affinity, the present invention is A methamphetamine assay kit using saliva GICA qualitative technique if it contains a saliva methamphetamine, which may reach the detection threshold of 50ng / ml, can fully meet the needs of the saliva test.

发明内容 SUMMARY

[0009] 本发明的一个目的是提供一种产生抗甲基安非他明单克隆抗体的杂交瘤细胞株METL 0,保藏于中国典型培养物保藏中心,保藏编号为CCTCC N0:C2013160,已于2013年11 月12号交中国典型培养物保藏中心(CCTCC)保藏,保藏地址:湖北省武汉市武昌区武汉大学内,保藏编号:CCTCC N0:C2013160。 [0009] An object of the present invention is to provide a producing anti-methamphetamine monoclonal antibody hybridoma cell lines out METL 0, deposited in China Center for Type Culture Collection, accession number CCTCC N0: C2013160, was November 2013 No. 12 post China Type culture Collection (CCTCC) preservation, preservation address: Wuchang District, Wuhan City, Hubei Province in the Wuhan University, accession number: CCTCC N0: C2013160.

[0010] 本发明的第二个目的是提供一种抗甲基安非他明单克隆抗体,它是由保藏编号为CCTCC NO: C2013160的抗甲基安非他明杂交瘤细胞株MET 1. 0分泌产生,能特异性结合甲基安非他明,分泌出的抗甲基安非他明单克隆抗体属于IgGl亚型,轻链是kappa链。 [0010] A second object of the present invention is to provide an anti-methamphetamine A monoclonal antibody which is the accession number CCTCC NO: C2013160 anti-methamphetamine A hybridoma cell line MET 1. 0 secreted, that specifically binds to a methamphetamine, secreted anti-methamphetamine monoclonal antibody is of the IgGl subtype bright light chain is a kappa chain.

[0011] 本发明的第三个目的是提供一种制备所述的由保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METLO分泌产生的抗甲基安非他明单克隆抗体的方法,在制备甲基安非他明人工抗原的过程中利用三氟乙酸酐保护氨基基团,使戊二酸酐能准确结合在其间位上,增强了抗原的免疫原性,有利于制备高亲和力的抗甲基安非他明单克隆抗体。 [0011] A third object of the present invention is to provide a process by the accession number CCTCC N0: C2013160 anti-methamphetamine Amphetamine hybridoma cell lines generated METLO antisecretory A single methamphetamine cloning of antibody in the manufacture of methamphetamine a process using artificial antigen trifluoroacetic anhydride protected amino group, so that accurate binding glutaric anhydride in place therebetween, to enhance the immunogenicity of antigens, favor a preparation of high affinity anti-methamphetamine monoclonal antibody.

[0012] 其中,甲基安非他明人工抗原是这样制备的: [0012] wherein A methamphetamine artificial antigen is prepared:

[0013] 50mL单口瓶中,500mg甲基苯丙胺,用氨水碱化至PH = 9,用乙酸乙酯提取得到390mg淡黄色油状物B,冰浴下加入400ul三氟乙酸酐,搅拌2h后升温至80°C回流过夜,次日减压蒸干后用乙酸乙酯提取,浓缩得到600mg黄色油状物C,再加入等质量的戊二酸酐, 萃取剂200mg,升温至120°C反应48h,再用乙酸乙酯提取得到750mgD,用38mLDMF下溶解, 加入380mgNHS和675mgDCC,搅拌过夜,离心取上清活化液,得到200ml的BSA中(浓度为20mg/ml)搅拌过夜,用PH = 12的似20)3透析三天后再用PH = 7. 4的PBS透析三天,离心后的上清液即为人工抗原。 [0013] 50mL single-neck flask, 500 mg of methamphetamine, was basified with aqueous ammonia to PH = 9, extracted with ethyl acetate to give a pale yellow oil B 390mg, 400ul of trifluoroacetic anhydride was added under ice-cooling, stirred for 2h after warming to 80 ° C reflux overnight, the following day after extraction with ethyl acetate and evaporated to dryness under reduced pressure, and concentrated to give 600mg yellow oil C, then add equal mass glutaric anhydride, extractant 200mg, the reaction temperature was raised to 120 ° C 48h, then extracted with ethyl acetate to give 750mgD, by the following 38mLDMF dissolved, and 380mgNHS 675mgDCC, stirred overnight, centrifuged supernatant was activated, the resulting BSA 200ml (at a concentration of 20mg / ml) was stirred overnight, the PH = 20 12 like) 3 dialyzed three days later with PH = PBS 7.4 is dialyzed three days, the supernatant is the artificial antigen. 用高效液相色谱仪纯化甲基安非他明人工抗原,抗原纯度可达95%以上。 A was purified by high performance liquid chromatography methamphetamine artificial, antigen purity of more than 95%.

[0014] 进一步的,在制备由保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METLO分泌产生的抗甲基安非他明单克隆抗体的方法中,免疫过程中采用新型免疫佐剂联合抗原免疫小鼠,具有无需乳化、抗原量少、免疫时间短(仅需5周)、不破坏抗原构象、无毒、促使B淋巴细胞增多,有利于融合等优点。 [0014] Further, the preparation in the accession number CCTCC N0: C2013160 resistance resisting to methamphetamine produced by hybridoma cell line secreting METLO A method methamphetamine monoclonal antibodies, immune processes employed new immunoadjuvant combined immune mice, with no need to emulsification, a small amount of antigen, immune short time (only 5 weeks), does not destroy the conformation of the antigen, non-toxic, induce lymphocytosis B, advantages facilitate fusion.

[0015] 其中,小鼠免疫过程是这样的: [0015] wherein, mice immunized process is as follows:

[0016] 选择8-12周龄的BALB/c雄鼠用甲基安非他明人工抗原注射靠近淋巴结皮下部位进行免疫,本实验具体步骤是取8周龄的BALB/c雄鼠,甲基安非他明人工抗原(以MET-BSA 为免疫抗原)注射靠近淋巴结皮下部位,注射四点,共四次免疫,每次免疫间隔两周:第一次用PBS缓冲液将甲基安非他明人工抗原20ug/只稀释至150ul,得到稀释抗原与等体积的新型免疫佐剂(主要成分为细菌DNA);第二次用PBS缓冲液将甲基安非他明IOug/只人工抗原免疫量稀释至150ul,得到稀释抗原与等体积的新型免疫佐剂;第三次加强免疫,用PBS缓冲液将甲基安非他明20ug/只人工抗原免疫稀释至150ul,联合等体积的新型免疫佐剂免疫小鼠,饲养一周后处死小鼠,处死前3天用甲基安非他明人工抗原20ug/只做一次加强免疫。 [0016] 8-12 weeks old selection BALB / c male mice were immunized with A methamphetamine artificial lymph nodes near the site beneath the skin injection of antigen, specific steps are taken in this experiment is 8 weeks old BALB / c male mice, methyl artificial antigen amphetamine (in MET-BSA antigen for immunization) lymph nodes near the site beneath the skin injection, four-point injection, a total of four immunization, each immunization two weeks interval: for the first time with PBS buffer methamphetamine Ming artificial antigen 20ug / only diluted to 150ul, be diluted with an equal volume of new antigens immunoadjuvant (the main component of the DNA of bacteria); a second time with PBS buffer a methamphetamine IOug / artificial antigen amount only diluted to 150ul, be diluted with an equal volume of new antigens immunoadjuvant; third booster, methanesulfonic methamphetamine 20ug / only artificial antigen was diluted to 150ul, combined with an equal volume of PBS buffer new immunoadjuvant agent immunized mice, the mice were sacrificed one week after the feeding, a 3 days before sacrifice with artificial antigen methamphetamine 20ug / boosted only once.

[0017] 本发明的第四个目的是提供用于检测唾液中甲基安非他明的试剂盒,它包括支持物、样品垫、胶体金纸片、硝酸纤维素膜和吸水垫,支持物上从加样端开始依次粘贴有样品垫、胶体金纸片、硝酸纤维素膜和吸水垫,硝酸纤维素膜上有检测区和对照区,检测区包被有由保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METL 0分泌产生的抗甲基安非他明单克隆抗体,对照区包被羊抗鼠多克隆抗体。 [0017] A fourth object of the present invention to provide a saliva for detecting methamphetamine A kit, comprising a support, a sample pad, the colloidal gold paper, nitrocellulose membrane, and an absorbent pad, the support from the end of the loaded sequentially affixed a sample pad, the colloidal gold sheet, an absorbent pad and a nitrocellulose membrane, a nitrocellulose membrane with a test area and control area, the detection area is coated with a deposit number of CCTCC N0: C2013160 the anti-a anti-methamphetamine methamphetamine monoclonal antibody, the control area coated with goat anti-mouse polyclonal antibody amphetamine METL 0 hybridoma cell lines secreting generated. 该试剂盒最低检出限为50ng/ ml,通过与40多种常用滥用药物和分子结构类似物,以及常见的戒毒药物、安眠药物、麻醉药物、止咳药物、感冒药物的交叉反应实验表明,本发明对这些物质无交叉反应,特异性强。 The kit detection limit of 50ng / ml, by 40 kinds of molecular structures commonly drug abuse and the like, as well as common pharmaceutical drug, hypnotics, anesthetic drugs, cough medicines, cold medicines cross-reactivity experiments show that the the invention no cross-reactivity of these species specificity.

[0018] 本发明的第五个目的是提供一种由保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METL 0分泌产生的抗甲基安非他明单克隆抗体在检测甲基安非他明中的应用。 [0018] A fifth object of the present invention is to provide a deposit number of CCTCC N0: C2013160 of anti-anti-methamphetamine Amphetamine secreting hybridoma cell lines generated METL 0 A methamphetamine monoclonal antibody detecting methamphetamine application of amphetamine.

[0019] 本发明所具有的有益效果: [0019] The present invention has advantageous effects:

[0020] 1、甲基安非他明是不具有免疫原性的小分子物质,本发明在甲基安非他明人工抗原制备过程中,利用三氟乙酸酐保护甲基安非他明的氨基,选择支链较长的戊二酸酐作为交联剂,使戊二酸酐能准确的结合在甲基安非他明的间位位点上,既提高甲基安非他明的免疫原性,有利于制备得到高亲和力的抗甲基安非他明单克隆抗体,又提高了甲基安非他明人工抗原的有效产量。 [0020] 1, A methamphetamine is not immunogenic small molecules, in the present invention A methamphetamine artificial antigen preparation using trifluoroacetic anhydride A protection of methamphetamine amino, selected longer chain branched glutaric anhydride as the crosslinking agent, so that accurate binding glutaric anhydride in methanesulfonic methamphetamine meta site, both to improve a methamphetamine immunogenic , favorable to the preparation of high affinity anti-methamphetamine monoclonal antibody Ming, but also improves the effective yield of methyl amphetamine artificial antigen.

[0021] 2、本发明在制备抗甲基安非他明单克隆抗体过程中,采用新型免疫佐剂联合抗原免疫小鼠,制备得到高亲和力的单克隆抗体,与常规免疫方法相比,具有无需乳化、抗原量少、免疫时间短(仅需5周)、不破坏抗原构象、无毒、促使B淋巴细胞增多,有利于融合等优点。 [0021] 2, the present invention is in the preparation of anti-methamphetamine monoclonal antibody next process, using the new immunoadjuvant combined immune mice, prepared monoclonal antibodies with high affinity, as compared to conventional immunization methods, having without emulsification, a small amount of antigen, immune short time (only 5 weeks), does not destroy the conformation of the antigen, non-toxic, induce lymphocytosis B, advantages facilitate fusion.

[0022] 3、本发明在纯化甲基安非他明人工抗原采用高效液相色谱仪纯化甲基安非他明人工抗原,抗原纯度可达95%,纯化抗甲基安非他明单克隆抗体的过程中,采用正辛酸-饱和硫酸铵法和G-蛋白亲和层析法结合纯化抗甲基安非他明单克隆抗体,大大提高了抗甲基安非他明抗体纯度,抗体纯度达到95%以上,通过对抗原和抗体的高效纯化,有利于得到高亲和力的抗甲基安非他明单克隆抗体。 [0022] 3, the present invention is employed in the purification of methyl amphetamine HPLC purified artificial antigen A methamphetamine artificial, antigen purity of 95%, purified anti-A monoclonal methamphetamine process antibody, using n-octanoic acid - saturated ammonium sulfate and G- protein purified by affinity chromatography in conjunction with anti-methamphetamine monoclonal antibody out, greatly improving the anti-methamphetamine antibody a purity of antibody purity more than 95% efficient for the antigen and the antibody purified by affinity conducive to high anti-a methamphetamine monoclonal antibody.

[0023] 4、本发明对甲基安非他明的检测仅需5分钟,检测灵敏度可达50ng/ml,本发明在对最低检出量的设定中采取了适当的方法,减少了假阴性和假阳性率的发生,通过抗甲基安非他明单克隆抗体的发明,制备出适合唾液检测的胶体金类产品,通过与40多种常用滥用药物和分子结构类似物,以及常见的戒毒药物、安眠药物、麻醉药物、感冒药物的交叉反应实验表明,本发明对这些物质无交叉反应,特异性强。 [0023] 4, the present invention is the detection of amphetamine methylamphetamine 5 minutes, the detection sensitivity of up to 50ng / ml, the present invention takes the appropriate method of setting the limit of detection, reduced false occurrence of negative and false positive rate, by an anti-methamphetamine monoclonal antibody of the present invention clear, colloidal gold prepared for saliva products detected, by more than 40 kinds of molecular structures commonly drug abuse and the like, as well as common detoxification of drugs, hypnotics, anesthetic drugs, cross-reactivity experiments show that the cold drugs, the present invention is not cross-reactive to these species specificity.

附图说明 BRIEF DESCRIPTION

[0024] 图1甲基安非他明人工抗原制备的紫外扫描图。 [0024] FIG 1 FIG methamphetamine UV scanning his prepared bright artificial antigen.

[0025] 图2抗甲基安非他明单克隆抗体的反应活性图。 [0025] FIG. 2 reactive anti-methamphetamine monoclonal antibody amphetamine FIG.

[0026] 图3抗甲基安非他明单克隆抗体的相对亲和力测定结果图。 [0026] FIG 3 A anti-methamphetamine monoclonal antibody relative affinity of the measurement result in FIG.

[0027] 图4甲基安非他明唾液检测试纸条组装图。 [0027] FIG 4 A saliva methamphetamine test strip assembly of FIG.

具体实施方式 Detailed ways

[0028] 下面结合实施例1对本发明做进一步的分析(下面实施例中PBS缓冲液为含有0. 008M十二水磷酸氢二钠、0. 15M氯化钠、0. 002M二水磷酸二氢钠的水溶液,pH为7. 4 ;免疫小鼠选择BALB/c雄性小鼠,雄性小鼠免疫力强,腹水产生量多;靠近淋巴结皮下部位为四肢腋下、四肢前臂或颈部;I XHAT的DMEM培养液含有体积分数15 %的FBS ;1 XHT的DMEM培养液含有体积分数为15 %的FBS)。 [0028] Example 1 below with further analysis of the present invention (in the following examples PBS buffer containing disodium hydrogen phosphate aqueous 0. 008M twelve, 0. 15M sodium chloride, 0. 002M dihydrogen phosphate dihydrate aqueous solution of sodium, pH of 7.4; selected mice immunized BALB / c male mice, male mice strong immunity, ascites amount; lymph nodes near the site beneath the skin extremities arms, legs forearm or neck; I XHAT in DMEM medium containing 15% volume fraction of FBS; 1 XHT DMEM medium containing 15% volume fraction of FBS).

[0029] 本发明抗甲基安非他明杂交瘤细胞株命名为杂交瘤细胞株METL 0,已于2013年11月12号交中国典型培养物保藏中心(CCTCC)保藏,保藏地址:湖北省武汉市武汉大学内,保藏编号:CCTCC N0:C2013160。 [0029] The present invention is an anti-methamphetamine A hybridoma cell line hybridoma cell line designated METL 0, was 12 November 2013 China post Type Culture Collection (CCTCC) deposited under accession Address: Hubei within Wuhan University in Wuhan, accession number: CCTCC N0: C2013160.

[0030] 本发明涉及的保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METL 0可分泌一种抗甲基安非他明单克隆抗体。 [0030] The present invention relates to a deposit number of CCTCC N0: C2013160 anti-methamphetamine A hybridoma cell line secreting an anti METL 0 A methamphetamine monoclonal antibody.

[0031] 制备本发明涉及的保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METL 0并分泌抗甲基安非他明单克隆抗体的方法,实施例1包括步骤如下: [0031] The present invention relates to the preparation of accession numbers are CCTCC N0: C2013160 anti-methamphetamine A METL 0 and hybridoma cell lines secreting anti-methamphetamine monoclonal antibody out method, comprising the steps of Example 1 :

[0032] 1.甲基安非他明人工抗原的制备: [0032] 1. Preparation of amphetamine methylamphetamine artificial antigen:

Figure CN105385660AD00061

[0034] 50mL单口瓶中,500mg甲基苯丙胺A',用氨水碱化至PH = 9,用乙酸乙酯提取得到390mg淡黄色油状物B',冰浴下加入400ul三氟乙酸酐,搅拌2h后升温至80°C回流过夜, 次日减压蒸干后用乙酸乙酯提取,浓缩得到600mg黄色油状物C',再加入等质量的戊二酸酐,萃取剂200mg,升温至120°C反应48h,再用乙酸乙酯提取得到750mgD',用38mLDMF下溶解,加入380mgNHS和675mgDCC,搅拌过夜,离心取上清活化液,得到200ml的BSA中(浓度为20mg/ml)搅拌过夜,用PH = 12的似20)3透析三天后再用PH = 7. 4的PBS透析三天, 离心后的上清液即为人工抗原。 [0034] 50mL single-neck flask, 500 mg of methamphetamine A ', basified with aqueous ammonia to PH = 9, extracted with ethyl acetate to give 390mg as a pale yellow oil B', 400ul of trifluoroacetic anhydride was added under ice-cooling, stirred for 2h after raising the temperature to 80 ° C to reflux overnight and evaporated to dryness under reduced pressure and extracted with ethyl acetate day, and concentrated to give 600mg yellow oil C ', the added mass of glutaric anhydride and the like, the extraction agent is 200mg, the reaction temperature was raised to 120 ° C 48h, extracted with ethyl acetate to give 750mgD ', by the following 38mLDMF dissolved, and 380mgNHS 675mgDCC, stirred overnight, centrifuged supernatant was activated, the resulting BSA 200ml (at a concentration of 20mg / ml) was stirred overnight, PH = like 20) 12 3 dialyzed three days later with PH = PBS 7.4 dialyzed three days after the artificial antigen is the supernatant. 用高效液相色谱仪纯化甲基安非他明人工抗原,抗原纯度可达95%以上。 A was purified by high performance liquid chromatography methamphetamine artificial, antigen purity of more than 95%. 其中,三氟乙酸酐的加入确保戊二酸酐能准确结合在甲基安非他明间位位点上,避免戊二酸酐与其氨基基团反应,既提高了甲基安非他明人工抗原的有效产量,也增强了甲基安非他明的免疫原性。 Wherein, trifluoroacetic anhydride glutaric anhydride was added to ensure that accurate binding on methamphetamine Ming alleles, glutaric anhydride to avoid reaction therewith amino groups, both to improve the A methamphetamine artificial antigen effective production, and increased the methamphetamine out of immunogenicity.

[0035] 图1是甲基安非他明人工抗原制备嵌合的紫外扫描图,从图中可以看出牛血清白蛋白最大吸收峰所在的紫外波长为278nm,甲基安非他明半抗原最大吸收峰所在的紫外波长为295nm,甲基安非他明人工抗原最大吸收峰所在的紫外波长为281nm,甲基安非他明人工抗原在最大吸收峰的波长与甲基安非他明人工抗原及牛血清白蛋白有明显不同,从而可以认为成功合成了甲基安非他明人工抗原。 [0035] FIG. 1 is a methamphetamine artificial antigens prepared fitted out UV scans, it can be seen from the figure bovine serum albumin ultraviolet wavelengths where maximum absorption peak of 278nm, A hapten methamphetamine ultraviolet wavelength of maximum absorption peak is located 295nm, methamphetamine amphetamine artificial antigen ultraviolet wavelength maximum absorption peak is located is 281nm, a methamphetamine artificial antigen in peak wavelength of maximum absorption of amphetamine and methamphetamine artificial antigen and bovine serum albumin are significantly different, and thus can be considered successful synthesis of methyl amphetamine artificial antigen.

[0036] 2.小鼠免疫: [0036] 2. The mice were immunized:

[0037] 选择8 -12周龄的BALB/c雄鼠用甲基安非他明人工抗原注射靠近淋巴结皮下部位进行免疫,本实验具体步骤是取8周龄的BALB/c雄鼠,甲基安非他明人工抗原(以MET-BSA为免疫抗原)注射靠近淋巴结皮下部位,注射四点,共四次免疫,每次免疫间隔两周:第一次用PBS缓冲液将甲基安非他明人工抗原20ug稀释至150ul,得到稀释抗原与等体积的新型免疫佐剂(主要成分为细菌DNA);第二次用PBS缓冲液将甲基安非他明IOug/ 只人工抗原免疫量稀释至150ul,得到稀释抗原与等体积的新型免疫佐剂;第三次加强免疫,用PBS缓冲液将甲基安非他明20ug/只人工抗原免疫稀释至150ul,联合等体积的新型免疫佐剂免疫小鼠,饲养一周后处死小鼠;处死前3天用甲基安非他明人工抗原20ug/只做一次加强免疫。 [0037] 8-12 weeks old select BALB / c male mice were immunized with A methamphetamine artificial lymph nodes near the site beneath the skin injection of antigen, specific steps are taken in this experiment is 8 weeks old BALB / c male mice, methyl artificial antigen amphetamine (in MET-BSA antigen for immunization) were injected subcutaneously near the site of lymph nodes, injection four points, a total of four immunization, each immunization two weeks interval: for the first time with PBS buffer methamphetamine antigen was diluted to 20ug bright artificial 150ul, new immunoadjuvant obtained was diluted with an equal volume of the antigen (the main component of the DNA of bacteria); a second time with PBS buffer diluted amphetamine methylamphetamine IOug / artificial antigen amounts to only 150ul, be diluted with an equal volume of new antigens immunoadjuvant; third booster, methanesulfonic methamphetamine 20ug / only artificial antigen was diluted to 150ul, an equal volume of new combined adjuvant immunization with PBS buffer mice, the mice were sacrificed for one week; 3 days before sacrifice a methamphetamine artificial antigen 20ug / boosted only once. 整个免疫过程共用甲基安非他明人工抗原量70ug,抗原用量比普通免疫佐剂联合抗原使用,其中抗原量节约一半。 A common procedure entire immune methamphetamine amount 70ug artificial antigen, the antigen combined with an immune adjuvant antigen than ordinary use, wherein the amount of the antigen save half.

[0038] 3.脾细胞与骨髓瘤细胞的融合: [0038] 3. The spleen cells with a myeloma cell fusion:

[0039] 3. 1饲养细胞制备:小鼠摘眼球处死,浸泡于75 %酒精中消毒,撕开小鼠腹外皮肤,暴露其腹膜,用5ml的无菌注射器注入5ml 37°C预热的DMEM无血清培养基,轻揉小鼠腹腔1分钟,悬浮腹腔细胞,吸出腹腔液;剪开小鼠胸腔取胸腺,用无血清培养基清洗后用注射器柄研磨收集悬液,与腹腔液合并1500r/min离心3min,沉淀物用IX HAT的DMEM培养液重悬至80ml,得到饲养细胞悬液。 Preparation of [0039] 3.1 feeder cells: Mice were sacrificed eyeball, soaked in 75% alcohol for disinfection, tearing mouse abdominal skin, which is exposed intraperitoneally injected with 5ml 37 ° C in a preheated sterile syringe 5ml DMEM serum-free medium, mouse peritoneal gently for 1 minute, suspended peritoneal cells, peritoneal fluid aspirated; cut mice after thoracic thymus, washed with serum-free medium was collected suspension was triturated with a syringe handle, combined with peritoneal fluid 1500r / min centrifugation 3min, the precipitate was resuspended to 80ml with DMEM medium IX HAT obtain a feeder cell suspension.

[0040] 3. 2小鼠骨髓瘤细胞SP2/0的培养:把小鼠骨髓瘤细胞SP2/0用含体积分数为10 % FBS的DMEM培养基进行传代培养,细胞融合前一天进行传代以保证小鼠骨髓瘤细胞SP2/0生长状态良好处于对数期,细胞融合时将骨髓瘤细胞SP2/0以1500r/min离心3min, 弃上清液后用无血清培养液重复离心一次,待用。 [0040] 3.2 cells were cultured mouse myeloma SP2 / 0 in: DMEM medium to mouse myeloma cells SP2 / 0 containing a volume fraction of 10% FBS and subcultured subcultured one day prior to cell fusion to ensure mouse myeloma cells SP2 / 0 grew well in log phase, when cell fusion the myeloma cells SP2 / 0 at 1500r / min centrifugal 3min, the supernatant using serum-free medium by centrifugation was repeated once, and set aside.

[0041] 3. 3脾细胞制备:取经步骤(2)免疫的BALB/c雄鼠处死,泡75%的酒精中消毒后剖腹,无菌取出脾脏。 [0041] 3.3 Preparation of spleen cells: learn step (2) Immunization of BALB / c male mice were sacrificed, the bubble 75% alcohol after disinfection laparotomy, spleen was aseptically removed. 用无血清培养基清洗,再将脾脏放在连接着50ml离心管的不锈钢滤网上用手术镊子除去多余的组织,加入5ml无血清的DMEM培养液,用眼科剪剪碎脾脏,然后用无菌的注射器柄轻磨脾脏,使脾脏细胞经过网孔滤入离心管内,加无血清培养液至30ml, 1500r/min离心3min,弃上清和大块的脾脏组织用无血清培养液重复离心一次,待用。 Washed with serum-free medium, and then connected on the spleen 50ml centrifuge tube with surgical tweezers stainless steel screen to remove the excess tissue, was added 5ml DMEM culture medium without serum, cut into pieces with ophthalmic spleen, then with sterile syringe handle spleen light grinding the spleen cells through a mesh filter into the centrifuge tube, added serum-free medium to 30ml, 1500r / min centrifugal 3min, the supernatant was discarded and spleen tissue chunks with serum-free medium by centrifugation was repeated once, inactive .

[0042] 3. 4脾细胞与骨髓瘤细胞融合:采用聚乙二醇融合法。 [0042] 3.4 spleen cells fused with myeloma cells: fusion using polyethylene glycol. 将脾细胞与小鼠骨髓瘤细胞SP2/0计数后,按照脾细胞:SP2/0 = 4 :1混合均匀,lOOOr/min离心6min洗涤细胞,去除上清液,用手指轻弹管底,使两种细胞混匀成糊状,置于37°C中水浴。 After the spleen cells and mouse myeloma cells SP2 / 0 count according splenocytes: SP2 / 0 = 4: 1 mixed, lOOOr / min 6min cells were washed by centrifugation, the supernatant was removed, finger flicking bottom of the tube, so that Both cell mixing into a paste, placed in 37 ° C water bath. 缓慢加入0.7mL 37°C 预温的PEG 4000同时轻轻转动离心管,一分钟内加完,然后静止30秒。 Was slowly added 0.7mL 37 ° C pre-warmed PEG 4000 while slightly rotating of the centrifuge tube, addition was completed within one minute, and then rest for 30 seconds. 再以先慢后快的方法加入35ml 37°C预温的DMEM无血清培养液终止PEG作用,第1分钟加lml,第2分钟加3ml,依次5ml、7ml ;1500r/min离心3min,取沉淀;然后在沉淀中加入步骤3-1的饲养细胞悬液,接种到96孔细胞培养板中,置于37°C,5 % 0)2培养箱中培养。 And then at a faster after the first slow method of adding 35ml 37 ° C pre-warmed DMEM serum-free medium terminated PEG effect, the first minute was added lml of, 2 minutes plus 3ml, sequentially 5ml, 7ml; 1500r / min centrifugal 3min, the precipitate ; feeder cell suspension was then added in the precipitation step 3-1, the seeded into 96-well cell culture plate, placed in 37 ° C, 5% 0) 2 incubator.

[0043] 4.杂交瘤细胞的融合筛选: [0043] 4. Filter hybridoma fusion:

[0044] 融合细胞在5 % CO2培养箱中培养3天后,用IX HAT的DMEM培养液半换液,7天后停止使用HAT培养液,改用HT培养液,观察96孔细胞培养板里的融合细胞生长情况,待细胞生长到细胞团簇(在16倍物镜和10倍目镜下观察,细胞大小以占满1/3视野为宜) 时,吸取融合细胞培养上清液(约在第10-11天),采用间接ELISA方法筛选阳性克隆(同时设置阳性对照孔和阴性对照孔),以融合细胞培养上清液0D450值/阴性对照0D450值>3为阳性克隆,将筛选到的阳性克隆进一步的用间接竞争抑制ELISA方法进行复筛,选择强阳性、强特异性、细胞生长良好的孔进行亚克隆。 [0044] The fused cells were cultured in 5% CO2 incubator for 3 days, the DMEM culture medium was changed half IX HAT, 7 days after the stop using HAT medium, HT medium instead observed in the 96-well cell culture plate fusion when cell growth, cells were grown to until the cell clusters (observed at 16-fold the objective lens and the eyepiece 10, the size of the cells to fill the appropriate field of view 1/3), a fusion draw cell culture supernatants (of about 10- 11 days), using indirect ELISA positive clones (positive control wells and simultaneously provided negative control wells) to the value of the fusion cell culture supernatant 0D450 / 0D450 negative control value> 3 positive clones, positive clones will be screened to further inhibiting the indirect competitive ELISA method for screening, selecting strong positive, strong specific, cell growth subcloned well bore.

[0045] 5.杂交瘤细胞株MET的克隆化: [0045] The cloning of hybridoma cell lines MET:

[0046] 杂交瘤细胞株MET的克隆化培养5倍稀释法,用含体积分数为15 % FBS的IX HT 培养基稀释,首先将复筛后的96孔中的细胞全部吸出,加入6ml培养基,然后以每孔200ul 稀释后的细胞悬液接种到96孔细胞培养板中,加三排后补5ml I XHT培养基,混合后以每孔200ul加三排,继续补5ml I XHT培养基,混合后以每孔200ul加三排,最后再补5ml IXHT培养基,加三排。 Cloning [0046] The hybridoma cell line culture MET 5 dilution method, with volume fraction of 15% FBS diluted IX HT medium, the first aperture 96 in the screening of all the cells was aspirated, media was added 6ml and then the cell suspension diluted with 200ul per well into 96-well cell culture plate, plus three rows of the complement 5ml I XHT medium, mixed 200ul per well plus three rows, continue to make 5ml I XHT medium, after mixing 200ul per well plus three rows, and finally 5ml IXHT supplemented medium, add three rows. 4、5天后观察细胞生长情况(在16倍物镜和10倍目镜下观察,细胞大小占满1/3视野)时,检测细胞培养板中细胞培养上清液的抗体水平,选择3个抗体效价最高的单克隆,再次做有限稀释法克隆化培养,准确计数细胞,用含体积分数为15 % FBS 的IMDM培养基稀释成4个/mL的细胞悬液,然后以每孔200ul稀释后的细胞悬液接种到96 孔细胞培养板中,7天后观察细胞生长情况,并检测细胞培养板中细胞培养上清液的抗体水平,直至单克隆孔抗体检测阳性率达到100 % ;用ELISA法挑选其中效价最高、亲和力最强的杂交瘤细胞株,命名为MET 1. 0,扩大培养后进行抗甲基安非他明单克隆抗体纯化。 When cell growth was observed 4,5 days (observed at 16-fold the objective lens and the eyepiece 10, cell size occupy 1/3 of the field of view), the detection antibody levels in cell culture plates in cell culture supernatants, antibody titers choose 3 monoclonal highest price, do again cloned by limiting dilution of culture, cells were accurately counted, diluted with IMDM medium containing a volume fraction of 15% FBS into 4 / mL of cell suspension, and then diluted with 200ul per well after cells were seeded into 96-well cell culture plate, cell growth was observed after 7 days, and the detection antibody levels in the cell culture supernatant of cell culture plates until a hole monoclonal antibody positive rate of 100%; selection by ELISA wherein the highest titer, affinity, most hybridoma cell lines designated MET 1. 0, non-antibody purified monoclonal anti-methamphetamine him out after the expansion.

[0047] 6.抗甲基安非他明单克隆抗体制备与纯化 [0047] 6. Anti-A methamphetamine monoclonal antibody preparation and purification

[0048] 6. 1抗甲基安非他明单克隆抗体制备 [0048] A 6.1 anti-methamphetamine monoclonal antibody

[0049] 采用动物体内诱生法-腹水制备法。 [0049] The in vivo method of inducing - preparation of ascites method. 将筛选到的单克隆细胞株MET I. 0用含体积分数为10 % FBS的DMEM培养基接种到24孔细胞培养板中,培养1-2天后接种到小号细胞培养瓶中,在16倍物镜和10倍目镜下观察,细胞大小占满1/2视野时,接种到大号细胞培养瓶中,培养1-2天后,观察细胞生长在对数期,离心收集杂交瘤细胞。 The DMEM medium monoclonal cell lines were screened MET I. 0 volume fraction containing 10% FBS were seeded into 24-well cell culture plate, culture was inoculated into cell culture flasks trumpet 1-2 days, in 16-fold observed by the objective lens and the eyepiece 10, when the cell size 1/2 filled field of view, large seeded into cell culture flasks, culture 1-2 days, cell growth was observed in log phase hybridoma cells were collected by centrifugation. 用0. 9%的生理盐水稀释,注射到已注射液体石蜡1-2周的小鼠腹腔内,每只注射IX IO6个杂交瘤细胞。 Diluted with 0.9% saline, was injected into the mice by intraperitoneal injection of liquid paraffin, 1-2 weeks, each injection IX IO6 hybridoma. 约7-10天后,小鼠腹部明显涨大,此时用75%的乙醇消毒腹部,用注射针头收集腹水。 Approximately 7-10 days later, mice were significantly swollen abdomen, then disinfected with 75% ethanol abdomen, the ascites was collected with an injection needle.

[0050] 6. 2抗甲基安非他明单克隆抗体纯化 [0050] 6.2 anti-methamphetamine monoclonal antibody purification Ming

[0051 ] 正辛酸-饱和硫酸铵纯化法和G-蛋白亲和层析柱纯化法 [0051] octanoic - saturated ammonium sulfate method and purified by affinity chromatography G- protein purification

[0052] 1)取IOml 腹水,用0· 06mol/L ρΗ4· 8 的HAc-NaAc 缓冲液稀释2 倍; [0052] 1) Take IOml ascites, HAc-NaAc 0 · 06mol / L ρΗ4 · 8 to 2-fold with dilution buffer;

[0053] 2)逐滴加入所需的正辛酸(按稀释前溶液的体积计算),并搅拌30min ; [0053] 2) was added dropwise to the desired n-octanoic acid (solution prior to dilution by volume) and stirred for 30 min;

[0054] 3)室温下,12000r/min,离心30min,取上清; The [0054] 3) at room temperature, 12000r / min, centrifuged 30min, supernatant;

[0055] 4)加入体积为上清10 %的0· lmol/L ρΗ7· 4的PBS缓冲液,并用1.0 mol/L的NaOH 调pH至7. 4 ; [0055] 4) a volume of the supernatant was added 10% 0 · lmol / L ρΗ7 · PBS 4 buffer, and washed with 1.0 mol / L of NaOH to adjust pH to 7.4;

[0056] 5)搅拌下,滴加等体积pH7. 4的饱和硫酸铵溶液,静置2h ; [0056] 5) was added dropwise under an equal volume of saturated ammonium sulfate solution pH7 4, left 2h.;

[0057] 6)低温离心12000r/min离心30min,弃上清; [0057] 6) Low-temperature centrifuge 12000r / min centrifugal 30min, the supernatant was discarded;

[0058] 7)将I. 5g Protein G-Sepharose CL-4B 干粉用6_7ml 三蒸水溶解,再用0· 02M, PH7. 4的磷酸盐缓冲液(上样缓冲液)浸泡15min,然后装入层析柱中,用10倍柱床体积的上样缓冲液过柱,流速为lml/min,试纸测出流出液体PH为7. 4 ; [0058] 7) The I. 5g Protein G-Sepharose CL-4B powder dissolved 6_7ml triple-distilled water, and then 0 · 02M, phosphate buffer (loading buffer) PH7. 4 soak 15min, then charged column, washed with 10 bed volumes of column loading buffer through the column at a flow rate of lml / min, the effluent liquid PH test paper is 7.4;

[0059] 8)用上样缓冲液进行流洗,10倍柱床体积,流速为lml/min,随后用0. 02M,PH4. 0 的柠檬酸缓冲液洗脱抗体,同时应用A KTA explorer进行监测,当观察到基线开始上升,即出现洗脱峰时,取干净的4ml离心管收集,每收集3ml后,立即用1M,PH9. 0的Tris-HCl缓冲液调整PH至7. 0 ; [0059] 8) with loading buffer solution for the elution, 10 column volumes, flow rate lml / min, followed by 0. 02M, PH4. 0 citrate buffer eluted antibody, for simultaneous application A KTA explorer monitoring, when the baseline is observed to rise, i.e., when the elution peak, taking clean 4ml centrifuge tube was collected after each collecting 3ml, immediately neutralized with 1M, PH9 0 of Tris-HCl buffer solution adjusted to PH 7.0.;

[0060] 9)收集洗脱液至洗脱峰回到基线后,继续用上样缓冲液平和5-10倍柱床体积, 流速调整至lml/min。 [0060] 9) to collect the eluate returned to baseline after elution peak, level and continues with loading buffer 5-10 bed volumes, flow rate was adjusted to lml / min. 再用三蒸水平衡10倍柱床体积; Triple-distilled water and then equilibrated for 10 column volumes;

[0061] 10)透析液过滤。 [0061] 10) the dialysis was filtered. 测试蛋白浓度并送检.蛋白浓度(mg/ml) = OD28。 Inspection and testing protein concentration Protein concentration (mg / ml) = OD28. 值*稀释倍数/1.35 =蛋白浓度。 /1.35 * value = dilution factor protein concentration.

[0062] 实施例1所制得的抗甲基安非他明单克隆抗体即为由保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METL 0分泌产生的抗甲基安非他明单克隆抗体。 [0062] Example 1 A prepared anti-methamphetamine monoclonal antibody that is the accession number CCTCC N0: C2013160 anti-methamphetamine A hybridoma cell line anti-secretory production METL 0 methyl amphetamine monoclonal antibody.

[0063] 实施例2抗甲基安非他明单克隆抗体的性能检测 [0063] Example 2 Anti-A methamphetamine monoclonal antibodies embodiment performance testing

[0064] 1.抗甲基安非他明单克隆抗体Ig类型的鉴定 [0064] 1. Anti-methamphetamine antibody Ig monoclonal clear identification of the type of

[0065] 通过琼脂双扩散免疫沉淀试验对实施例1制备的抗甲基安非他明单克隆抗体进行亚型鉴定。 [0065] By double diffusion immunoprecipitation agar Example A methamphetamine monoclonal antibodies prepared in 1 subtype anti identified.

[0066] 1)制备琼脂糖板:将1 %琼脂糖凝胶置于沸水浴中使其融化。 [0066] 1) Preparation of agar plates: The 1% agarose gel in a boiling water bath to melt. 在水平台上灌制琼脂糖板,待凝固后,用凝胶打孔器打孔,制成中心有一孔、周围6个孔(呈梅花形排列)的琼脂糖板。 A horizontal table mastering agar plates, to be solidified, gel puncher punch, formed with a central hole, 6 holes around (quincunx arrangement) agar plates.

[0067] 2)加样:将单克隆抗体的腹水(经过PBS稀释40倍)加入到中心孔中,四周孔分别加入抗小鼠IgM、IgGl、IgG2a、IgG2b、IgG3a、IgA等不同类和亚类的抗血清。 [0067] 2) loading: A monoclonal antibody ascites fluid (diluted 40-fold through PBS) was added to the center hole surrounded by holes of different classes and subclasses were added anti-mouse IgM, IgGl, IgG2a, IgG2b, IgG3a, IgA etc. class antisera.

[0068] 3)温育:加样后将琼脂糖板移至湿盒内,室温下静置24h至48h后观察结果。 [0068] 3) incubation: After loading agar plates to move in a humid chamber, after left to stand at room temperature for 24h to 48h observations.

[0069] 4)若见沉淀线出现,即可依据相对应孔抗血清的类型鉴定出单克隆抗体的类型。 [0069] 4) See if precipitation line appeared, a monoclonal antibody can be identified according to the type of the corresponding hole type antiserum.

[0070] 经检测结果显示:抗甲基安非他明单克隆抗体属于IgGl亚型,轻链是kappa链。 [0070] The results show tested: anti-methamphetamine monoclonal antibody is of the IgGl subtype bright light chain is a kappa chain.

[0071] 2.抗甲基安非他明单克隆抗体反应活性测定 [0071] 2. Anti-methamphetamine monoclonal antibody reactive assay Ming

[0072] 间接ELISA方法检测实施例1制备的抗甲基安非他明单克隆抗体:①用pH9. 6浓度为0. 05M的碳酸盐缓冲液稀释甲基安非他明人工抗原(MET-BSA)至lug/mL,然后在96 孔酶标板每孔中分别加入IOOul稀释后的甲基安非他明人工抗原,4°C包被过夜;然后用含0. 1 % tween-20的PBS缓冲液洗板3次拍干;②每孔中加入200 μ 1含1 %牛血清蛋白的0· Olmol/L ρΗ7· 4磷酸盐缓冲液,置37°C 60分钟,再用0· 1 % tween-20的PBS缓冲液洗板3次拍干;③加入IOOul浓度为lug/ml的抗甲基安非他明单克隆抗体溶液,做倍比稀释,同时设立阴性对照,37°C反应60分钟后,用含0.1 % tween-20的PBS缓冲液洗板1次拍干; ④将PBS缓冲液加入到羊抗鼠IgG-HRP稀释到2000倍体积,然后在96孔细胞培养板中每孔加入100 μ 1稀释后的羊抗鼠IgG-HRP,37°C反应30分钟,用含0. 1 % tween-20的PBS缓冲液洗板3次拍干;⑤每孔加入IOOul底物TMB 37 [0072] Indirect ELISA for detection of anti embodiments Example 1 Preparation of methanesulfonic methamphetamine monoclonal antibodies:. ① carbonate buffer 0. 05M A diluted artificial antigen methamphetamine (MET with 6 concentration was pH9 -BSA) to lug / mL, and then in each well of 96 well microtiter plate was added diluted IOOul methamphetamine amphetamine artificial antigen, respectively, 4 ° C were coated overnight; then containing 0. 1% tween-20 the plate was washed three times with PBS buffer and pat dry; ② each well was added 200 μ 1 containing 1% bovine serum albumin 0 · Olmol / L ρΗ7 · 4 phosphate buffer, 37 ° C 60 opposing minutes, then 0.5 PBS 1% tween-20 plates were washed three times with buffer and pat dry; ③ IOOul added at a concentration of lug / ml anti-methamphetamine monoclonal antibody solution was clear, do dilution, while the establishment of a negative control, 37 ° C after 60 minutes, the plate was washed with PBS buffer containing 0.1% tween-20 in 1 pat dry; ④ PBS buffer was added to the goat anti-mouse IgG-HRP diluted 2000 times by volume, then a 96-well cell culture plate each well was added 100 μ 1 after dilution of sheep anti-mouse IgG-HRP, 37 ° C for 30 minutes, the plate was washed with PBS buffer containing 0. 1% tween-20 three times and pat dry; ⑤ substrate was added per well IOOul TMB 37 °C反应10分钟后,浓度为2M的H2SOgI 止反应,450nm测定其OD值,以抗甲基安非他命单克隆抗体溶液0D450值/阴性对照0D450 值>3为阳性值。 After ° C for 10 minutes, 2M in H2SOgI stop the reaction, measured OD values ​​of 450 nm, an anti-methamphetamine monoclonal antibody value was 0D450 / 0D450 negative control value> 3 is a positive value. 从图2可以看到,实施例1制备的抗甲基安非他明单克隆抗体反应活性可以到达〇· 〇〇lug/ml。 As seen in Figure 2, prepared in Embodiment Example 1 A anti-methamphetamine monoclonal antibodies reactive to reach square-〇〇lug / ml.

[0073] 3.抗甲基安非他明单克隆抗体亲和力测定 [0073] 3. Anti-methamphetamine monoclonal antibody avidity Ming

[0074] 如图3所示,将本发明所述的由保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METL 0分泌产生的抗甲基安非他明单克隆抗体(用A表示)和市场上其他公司B、C生产的抗甲基安非他明单克隆抗体进行亲和力的定性比较,根据ELISA法,具体操作可参考曾大地等人发表的"一种定性比较抗体相对亲和力的酶联免疫吸附测定方法", 抗原包被、封闭、洗涤、显色操作与一般ELISA方法相同,用PBS溶液按梯度稀释抗体,稀释8个浓度。 [0074] As shown in FIG. 3, the accession number CCTCC N0 of the present invention: C2013160 anti-methamphetamine Amphetamine hybridoma cell line anti-secretory production of A METL 0 methamphetamine monoclonal antibody (indicated by a) and B others on the market, "a qualitative C produced anti-a methamphetamine monoclonal antibody affinity qualitative comparison, according to the ELISA method, reference may be had specific operation earth et al comparison ELISA method for measuring the relative affinity of the antibody ", antigen-coated, blocked, washed, and the general operation of the same color ELISA method, antibody diluted in PBS with a gradient, diluted with 8 concentrations. 一半抗体作为抗原用直接ELISA法进行反应,测定D 492ninZD63am,一半作为抗体用间接ELISA方法进行反应,测定D492m/D 63ftmi,测试结果见图3。 Half as antigen antibody reaction by direct ELISA method, determination of D 492ninZD63am, half of the reaction by indirect ELISA antibody as determined D492m / D 63ftmi, the test results shown in Figure 3. 图3以吸光度为X轴,反映的是抗体浓度,而Y轴的吸光度则反映的是抗体抗原反应强度,相同浓度下抗体与等量抗原的ELISA反应的显色值越高,则抗体亲和力程度越高。 Figure 3 absorbance of the X-axis, reflects the antibody concentration, and the absorbance of the Y-axis reflects the intensity of the antibody-antigen reaction, the higher the value of the color ELISA antibody reactive with an equal amount of the antigen at the same concentration, the degree of affinity of the antibody higher.

[0075] 从图3中可以看出由保藏编号为CCTCC N0:C2013160的抗甲基安非他明杂交瘤细胞株METL 0分泌产生的抗甲基安非他明单克隆抗体比市场上其他公司生产的同类产品亲和力要高。 [0075] As can be seen in FIG. 3 by the Accession number CCTCC N0: C2013160 anti-methamphetamine Amphetamine hybridoma cell line anti-secretory production of A METL 0 methamphetamine monoclonal antibody than others on the market production of similar products to high affinity.

[0076] 4.抗甲基安非他明单克隆抗体特异性、交叉反应性测定 [0076] 4. Anti-methamphetamine monoclonal antibody specific for amphetamine, cross-reactivity assay

[0077] 通过对以下44种常见的可能影响本试剂检测结果的干扰物质进行检测,当药物浓度等于或低于100 μ g/ml时,显示阴性结果,故不发生交叉反应: [0077] by the following 44 kinds of interfering substances that could affect the common detection result of the detection reagent, when the drug concentration equal to or less than 100 μ g / ml, showed negative results, it is not cross-react:

Figure CN105385660AD00101

[0080] 实施例3甲基安非他明胶体金试纸条方法的建立和评估 Establishment and assessment of his bright gold strip colloid method [0080] Example 3 methylamphetamine

[0081] 一、胶体金的制备 I. Preparation of colloidal gold [0081]

[0082] 取Iml 1%氯金酸(HAuCl4)溶液,加入到100mL水中,加热至沸,再加入I. 5ml 1% 柠檬酸三钠,混合煮沸5分钟,直到颜色不再发生变化。 [0082] Take Iml 1% chloroauric acid (HAuCl 4) was added to 100mL of water, heated to boiling, then add I. 5ml 1% trisodium citrate, mixed boiled for 5 minutes, until the color no longer changes. 此时制得的胶体金颗粒为30nm。 At this time, the resulting colloidal gold particles of 30nm.

[0083] 二、金标制备 [0083] Second, the preparation of gold-labeled

[0084] 1、抗体纯化 [0084] 1, antibody purification

[0085] 取一定量的吗啡单克隆抗体(MET-Mab),经过透析、离心处理后,用pH 8.0 IM PB 稀释至lmg/mL,4°C保存备用。 [0085] A certain amount of morphine monoclonal antibody (MET-Mab), dialyzed, centrifuged, diluted to lmg / mL, 4 ° C were stored with 8.0 IM PB pH.

[0086] 2、抗体偶联 [0086] 2, the antibody conjugated

[0087] 取100mL制备好的30nm胶体金,用IM NaOH调节pH至8. 5,然后加入上述抗体lmL,磁力搅拌器上搅拌1小时,然后加入一定量的BSA至终浓度1 %,继续搅拌0. 5小时。 [0087] Take 100mL prepared 30nm colloidal gold, with IM NaOH pH adjusted to 8.5, then added to the antibody lmL, stirred on a magnetic stirrer for 1 hour and then a certain amount of BSA was added to a final concentration of 1%, and stirring was continued 0.5 hours.

[0088] 3、金标工作液的制备 [0088] Preparation of 3, the gold standard working solution

[0089] 将上述已经偶联好抗体的胶体金溶液离心(4000rpm,15分钟),弃去沉淀,保留溶液。 [0089] The colloidal gold solution was centrifuged have good antibody conjugated above (4000rpm, 15 minutes), the pellet is discarded, the solution reserved. 然后进行二次离心(12000rpm,30分钟),弃去上清液,保留沉淀。 Then secondary centrifugation (12000rpm, 30 minutes), the supernatant was discarded, the precipitate retained. 沉淀用含20%的蔗糖的金标抗体稀释液(PH8. 0 0. 02M Tris+1% BSA)复溶至10mL,转入棕色瓶内,4°C保存备用。 Precipitated with gold-labeled antibody diluent containing 20% ​​sucrose (PH8. 0 0. 02M Tris + 1% BSA) reconstituted to 10mL, transferred to a brown bottle, 4 ° C for use.

[0090] 4、金标条的制备 [0090] Preparation of 4, Gold Label strip

[0091] 取上述金标工作液10mL,上样到喷金机,设定参数选取喷量为I. 5ul/cm。 [0091] Take said gold standard working solution 10mL, loaded onto a spraying machine parameters are set to select an amount of discharge I. 5ul / cm. 打开气栗,待气压稳定后,启动喷金机,将金标工作液均勾喷到30cm*6. 5cm的金标垫上。 Li open air, until the pressure stabilized, start spraying machine, gold standard working solution are sprayed onto the hook 30cm * 6. 5cm gold conjugate pad. 将喷好金标工作液的金标垫放入37°C恒温箱内干燥12小时后取出,切成8_宽的条放入自封袋中, 然后封入内装干燥剂的铝箱袋,20°C保存备用。 The good spray gold standard working solution of gold labeling pad into 37 ° C incubator dried for 12 hours after taken out, cut into wide strips 8_ ziplock bag and then the bag is sealed interior desiccant Aluminum, 20 ° C for use.

[0092] 三、硝酸纤维素膜的选择 [0092] Third, the choice of the nitrocellulose membrane

[0093] 选取市场上使用率较高的三家公司的硝酸纤维素膜:PALL Vivid 170、Millipore 135、Sartorius CN 140,规格为2. 5*30cm,带背衬,孔径为8um进行对比,要求膜各点的宽度与厚度均一;跑水性能符合要求:层析4cm的时间在130s-140s,且无倾斜和空白;性能测试符合要求:将三种硝酸纤维素膜分别包被〇. 5mg/mL的抗原并配对相应的金标条,测试一份阴性样本、一份临界阴性样本(-50% cut off,要求3-5分钟显色)以及一份临界阳性样本(cut off,要求5分钟之内不显色) [0093] Select the higher usage three companies market nitrocellulose membrane: PALL Vivid 170, Millipore 135, Sartorius CN 140, specifications for 2. 5 * 30cm, tape backing, a pore diameter of 8um comparison, the film is required each point a uniform width and thickness; running water satisfactory performance: 4cm time chromatographed 130s-140s, and the inclination and no gaps; performance test requirements: the three kinds of cellulose nitrate membranes were wrapped square 5mg / mL. antigens and the corresponding mating gold standard item, a negative test sample, a critical negative samples (-50% cut off, the color required 3-5 min) and a critical positive samples (cut off, it requires 5 minutes without color)

Figure CN105385660AD00111

[0095](注:_表示阴性结果,+表示阳性结果,+/_表示不明显阴性结果)结果分析,我们选择Millipore 135作为吗啡唾液检测试剂盒的生产用硝酸纤维素膜。 [0095] (Note: _ indicates a negative result, + indicates a positive result, + / _ represents obvious negative result) analysis results, we chose Millipore 135 as morphine in saliva production by the test kit nitrocellulose membrane.

[0096] 四、硝酸纤维素膜的包被 [0096] Fourth, the packets are nitrocellulose membrane

[0097] 1、抗原及多抗的纯化 [0097] a purified polyclonal antibody and antigen

[0098] 将一定量的甲基安非他明抗原偶联物(MET-BSA),经过透析、离心处理后,用pH7. 4 0.0 lM PBS稀释至终浓度0. 5mg/mL,4°C保存备用。 After [0098] the amount of A antigen amphetamine conjugate (MET-BSA), dialyzed, centrifuged and diluted with pH7. 4 0.0 lM PBS to a final concentration of 0. 5mg / mL, 4 ° C save spare.

[0099] 将一定量的羊抗鼠多克隆抗体(GAM),经过透析、离心处理后,用pH7. 4 0.0 lM PBS 稀释至终浓度2. Omg/mL,4°C保存备用。 [0099] The amount of goat anti-mouse polyclonal antibody (the GAM), dialyzed, centrifuged, diluted to a final concentration of 2. Omg / mL, 4 ° C were stored with pH7. 4 0.0 lM PBS.

[0100] 2、抗原包被 [0100] 2, antigen-coated

[0101] 将上述抗原及多抗分别上样到点膜机的T、C柱中,设定参数选择喷量为LOul/ cm。 [0101] The above-mentioned polyclonal antibody and antigen, respectively, on the point-like film machine T, C column, setting preferences ejection amount LOul / cm. 调整划膜头的位置,使T、C线在NC中部,且间距5mm。 Adjust the position of the head zoned film, so that T, C in the middle of the line NC, pitch and 5mm. 启动点膜机,将T、C线溶液包被到硝酸纤维素膜上。 Start point film machine, a T, C line was coated onto a nitrocellulose membrane. 将喷好金标工作液的金标垫放入37°C恒温箱内干燥24小时后取出,放入自封袋中,然后封入内装干燥剂的铝箱袋,20°C保存备用。 The good spray gold standard working solution of gold labeling pad into 37 ° C thermostatic tank after 24 hours of drying out and put in a ziplock bag and the bag is sealed interior desiccant Aluminum, 20 ° C for use.

[0102] 五、玻纤的处理 [0102] Five glass processing

[0103] 将玻璃纤维素膜切割成17*300mm条,放入玻纤处理液中浸泡1小时后取出,放入37°C恒温箱内干燥12小时后取出,放入自封袋中,然后封入内装干燥剂的铝箱袋,20°C保存备用。 [0103] The glass was cut into 17 * 300mm cellulose membrane strip into glass after 1 hour soaking in the processing solution, into 37 ° C incubator dried for 12 hours after taken out, placed in ziplock bag and then sealed Aluminum built desiccant bags, 20 ° C for use.

[0104] 玻纤处理液配方 [0104] glass fiber treating liquid formulation

Figure CN105385660AD00121

[0107] 六、试剂条的组装 [0107] Sixth, the test strip assembly

[0108] 如图4所示,用于检测唾液中甲基安非他明的试剂盒包括支持物1、样品垫5、胶体金纸片4、硝酸纤维素膜2和吸水垫,硝酸纤维素膜2为上述准备好的硝酸纤维素膜,支持物1为PVC板,支持物1上从加样端开始依次粘贴有样品垫5、胶体金纸片1、硝酸纤维素膜2和吸水垫3,硝酸纤维素膜2上有检测区和对照区,检测区包被有由保藏编号为CCTCC C2013160的杂交瘤细胞株METL 0分泌产生的抗甲基安非他明单克隆抗体,对照区包被羊抗鼠多克隆抗体。 [0108] As shown in FIG. 4, in saliva A for detecting methamphetamine kit includes a support 1, a sample pad 5, colloidal gold sheet 4, 2 and pad, nitrocellulose nitrocellulose membrane 2 is a film of nitrocellulose membranes prepared as described above, the support is a PVC plate, a support was sequentially starting from the loading end of the sample pad 5 is attached, colloidal gold sheet 1, a nitrocellulose membrane, and an absorbent pad 3 2 , 2 nitrocellulose membrane detecting area and control area, the detection area is coated with an anti accession number CCTCC C2013160 by a hybridoma cell line secreting generated METL 0 a methamphetamine monoclonal antibody, the control area coated goat anti-mouse polyclonal antibody.

[0109] 七、灵敏度测定 [0109] VII assay sensitivity

[0110] 用PBS缓冲液将甲基安非他明标准品配制成300ng/ml、150ng/ml、100ng/ml、 5〇]^/1111、251^/1111、〇1^/1111,将金标检测试纸条分别插入上述配制好的溶液中,5111;[11后观察结果。 [0110] with PBS buffer A amphetamine standards formulated to 300ng / ml, 150ng / ml, 100ng / ml, 5〇] ^ / ^ 1111,251 / 1111, 〇1 ^ / 1111, gold standard test strip are inserted into the solution prepared above, 5111; after [11 observations.

[0111] 表一甲基安非他明试纸条检测不同浓度标准品的结果判定 [0111] Table A methamphetamine test strip detection of different standard concentration determination results

Figure CN105385660AD00122

Claims (6)

1. 一种能产生抗甲基安非他明单克隆抗体的抗甲基安非他明杂交瘤细胞株MET1. 0, 保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:C2013160。 An anti-A to produce monoclonal anti-methamphetamine antibody A hybridoma cell line methamphetamine MET1 0, deposited in China Center for Type Culture Collection, accession number CCTCCNO:. C2013160.
2. 抗甲基安非他明单克隆抗体,其特征在于,它是由保藏编号为CCTCCN0:C2013160 的抗甲基安非他明杂交瘤细胞株MET1. 0分泌产生,能特异性结合甲基安非他明。 2. Anti-A methamphetamine monoclonal antibody, characterized in that it is made under the accession number CCTCCN0:. C2013160 anti-methamphetamine Amphetamine MET1 0 secreted hybridoma cell line, capable of binding specifically methyl amphetamines.
3. 制备如权利要求2所述的由保藏编号为CCTCCNO:C2013160的抗甲基安非他明杂交瘤细胞株MET1.0分泌产生的抗甲基安非他明单克隆抗体的方法,其特征在于在制备甲基安非他明人工抗原的过程中利用三氟乙酸酐保护氨基基团。 3. Preparation as claimed in claim 2 by the accession number CCTCCNO: C2013160 anti-methamphetamine anti-methamphetamine monoclonal antibodies out method amphetamine produced by hybridoma cell line secreting MET1.0, characterized in in that during the preparation of methamphetamine out using artificial antigen trifluoroacetic anhydride protected amino group.
4. 如权利要求3所述的制备由保藏编号为CCTCCN0:C2013160的抗甲基安非他明杂交瘤细胞株MET1.0分泌产生的抗甲基安非他明单克隆抗体的方法,其特征在于免疫过程中采用新型免疫佐剂联合抗原免疫小鼠。 Wherein C2013160 anti-methamphetamine Amphetamine anti secretory production of hybridoma cell line MET1.0 methamphetamine monoclonal antibodies out method,: as claimed in claim 3 prepared by the accession number CCTCCN0 wherein during immunization using new immunoadjuvant combined immune mice.
5. 用于检测唾液中甲基安非他明的试剂盒,其特征在于它包括支持物、样品垫、胶体金纸片、硝酸纤维素膜和吸水垫,支持物上从加样端开始依次粘贴有样品垫、胶体金纸片、 硝酸纤维素膜和吸水垫,硝酸纤维素膜上有检测区和对照区,检测区包被有由保藏编号为CCTCCN0:C2013160的抗甲基安非他明杂交瘤细胞株MET1. 0分泌产生的抗甲基安非他明单克隆抗体,对照区包被羊抗鼠多克隆抗体。 5. A saliva for detecting methamphetamine kit, characterized in that it comprises a support, a sample pad, the colloidal gold paper, nitrocellulose membrane, and an absorbent pad, the support was sequentially starting from the loading end affixed a sample pad, the colloidal gold sheet, an absorbent pad and a nitrocellulose membrane, a nitrocellulose membrane with a test area and control area, the detection area is coated with a deposit number CCTCCN0: C2013160 anti-a methamphetamine hybridoma cell line MET1. 0 secreted anti-methamphetamine a monoclonal antibody, the control area coated with goat anti-mouse polyclonal antibody.
6. 由保藏编号为由保藏编号为CCTCCN0:C2013160的抗甲基安非他明杂交瘤细胞株MET1. 0分泌产生的抗甲基安非他明单克隆抗体在检测甲基安非他明中的应用。 6 by the Accession number Accession number CCTCCN0:. C2013160 anti-methamphetamine Amphetamine hybridoma cell line anti-secretory production of MET1 0 A methamphetamine monoclonal antibodies in the detection of methamphetamine Ming Applications.
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