CN105372419A - Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit - Google Patents

Preparing method for chloramphenicol hapten and antigen and application of chloramphenicol hapten and antigen in quantum dot immunofluorescence reagent kit Download PDF

Info

Publication number
CN105372419A
CN105372419A CN 201410420843 CN201410420843A CN105372419A CN 105372419 A CN105372419 A CN 105372419A CN 201410420843 CN201410420843 CN 201410420843 CN 201410420843 A CN201410420843 A CN 201410420843A CN 105372419 A CN105372419 A CN 105372419A
Authority
CN
Grant status
Application
Patent type
Prior art keywords
chloramphenicol
antigen
hapten
quantum
preparing
Prior art date
Application number
CN 201410420843
Other languages
Chinese (zh)
Other versions
CN105372419B (en )
Inventor
江海洋
沈建忠
吴小平
王战辉
丁双阳
李建成
史为民
温凯
王照鹏
王世恩
王文珺
韩京朋
Original Assignee
中国农业大学
北京维德维康生物技术有限公司
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

Links

Abstract

The invention discloses chloramphenicol hapten, corresponding artificial antigen and a monoclonal antibody marked by a quantum dot and further discloses a preparing method and application of the chloramphenicol hapten, the corresponding artificial antigen and the monoclonal antibody marked by the quantum dot. The chloramphenicol antigen can be obtained when the chloramphenicol hapten and carrier protein are connected. The chloramphenicol antigen can be applied to preparing a chloramphenicol specific antibody. The preparing method is easy and convenient to implement and low in cost, and the yield of the hapten is high. The artificial antigen and the chloramphenicol specific antibody marked by the quantum dot can be used for preparing a quantum dot immunofluorescence reagent kit for detecting chloramphenicol residues and have the advantages of being simple, fast, large in quantity of treated samples, high in sensitivity, high in specificity and the like.

Description

氯霉素半抗原和抗原的制备方法及其在量子点免疫荧光试剂盒中的应用 Preparation chloramphenicol haptens and antigens and its application in the quantum dot immunofluorescence kit

技术领域 FIELD

[0001] 本发明涉及一种半抗原、抗原及其制备方法,具体涉及一种氯霉素半抗原和抗原的制备方法及其在量子点免疫荧光试剂盒中的应用。 [0001] The present invention relates to a hapten, antigen and its preparation method, particularly relates to a method for preparing haptens and antigens chloramphenicol and its application in the quantum dot immunofluorescence kit.

背景技术 Background technique

[0002] 氯霉素(Chloramphenicol,CAP)是一种对革兰氏阳性和阴性细菌都有很好的抑制作用的廉价高效广谱抗生素,一度被广泛应用于农牧业中。 [0002] chloramphenicol (Chloramphenicol, CAP) is a Gram-positive and negative bacteria have a very good inhibition of a cheap and efficient broad-spectrum antibiotic, was once widely used in agriculture and animal husbandry. 但是动物源性食品中残留的氯霉素随着食物链被人体长期摄入,可引发多种疾病。 However, residues in food of animal origin chloramphenicol as the food chain is a long-term intake of the human body, can cause a variety of diseases. 轻者破坏人体内正常菌群的平衡状态,菌群失调,使人体产生耐药菌珠,给今后患病使用抗生素治疗带来不良影响;抗生素过敏体质的人会出现过敏反应,危及健康。 Light damage the body's normal flora balance, flora, makes the body produce drug-resistant beads, sick to be treated with antibiotics in the future adversely affected; antibiotic allergies occur allergic reactions, endanger health. 严重时可干扰骨髓细胞蛋白质的合成,并抑制幼稚细胞DNA合成,导致粒细胞减少,引发再生障碍性贫血、溶血、紫癜等恶性疾病。 Severe bone marrow may interfere with cellular protein synthesis, and inhibition of DNA synthesis naive cells, resulting in neutropenia, aplastic anemia induced, hemolytic, purpura and other malignant diseases.

[0003] 鉴于氯霉素的毒副作用,国际食品学界将其列为禁药,欧盟、美国等均在法规中规定氯霉素残留限量标准为"零容许量" (Zerotolerance),即不得检出,根据欧盟"2002/657/ EC"标准规定,动物源性食物中氯霉素的最大要求检出极限是0. 3 μ g/kg。 [0003] In view of the toxicity of chloramphenicol, international food circles will be listed as banned substances, the European Union, the United States etc. prescribed chloramphenicol residue limits of "zero tolerance" (Zerotolerance) in the regulations, that is not detected , according to EU standards "2002/657 / EC", chloramphenicol in food of animal origin requires maximum detection limit is 0. 3 μ g / kg. 不久美国FDA也做出相应的规定。 Soon the US FDA also make provisions. 我国农业部规定了CAP在所有食品动物的可食用组织中不得检出,并将其从《中国兽药典》中删除,列为禁药。 Ministry of Agriculture of the CAP may not be detected in all edible tissues of food animals, and remove it from the "China Veterinary Pharmacopoeia" in as doping. 并相应制定了SN0219-93和SCT3018-2004行业标准,与国际接轨。 And instituted SN0219-93 and SCT3018-2004 industry standards with international standards.

[0004] 氯霉素的检测方法分为物理化学法和免疫化学法,前者有液相色谱法(LC)、高效液相色谱法(HPLC)、质谱法(MS)等,后者主要是酶免法(ELISA),检测的灵敏度均达到ppb(yg/kg)级别。 [0004] The physical method for detecting chloramphenicol divided into chemical and immunochemical methods, the former liquid chromatography (the LC), high performance liquid chromatography (HPLC), mass spectrometry (MS), the latter enzyme mainly Free method (ELISA), the detection sensitivity reached ppb (yg / kg) level. 近几年,国内外学者又研发出液相色谱串联质谱法(LC-MS/MS)、液相色谱电喷雾电离质谱法(LC-EITMS)、微生物分析等物理检测新方法,对ELISA方法的应用也有了进一步研究。 In recent years, research and development scholars and liquid chromatography tandem mass spectrometry (LC-MS / MS), liquid chromatography physical testing electrospray ionization mass spectrometry (LC-EITMS), microbiological analysis and other new methods, ELISA methods application has also been further research.

[0005] 量子点免疫荧光检测方法具有特异性强、稳定快速、检测范围宽、操作简单自动化程度高、试剂稳定且有效期长出~18个月)等优点,其检测限比ELISA和理化检测方法高。 [0005] Immunofluorescence detection of quantum dots having a specific, stable and fast, wide detection range, a high degree of automation simple, valid and stable reagent grow to 18 months), etc., the detection method detection limit than the ELISA and physicochemical high.

发明内容 SUMMARY

[0006] 本发明的目的是提供一种氯霉素半抗原、抗原的制备方法及其在量子点免疫荧光试剂盒中的应用。 [0006] The object of the present invention is to provide a chloramphenicol hapten, an antigen preparation and its application in the quantum dot immunofluorescence kit.

[0007] 本发明提供的氯霉素半抗原,为式I所示的化合物; [0007] The present invention provides chloramphenicol hapten compound of Formula I;

[0008] [0008]

Figure CN105372419AD00031

[0009] 本发明还公开了式I所示化合物的制备方法,包括如下步骤: [0009] The present invention also discloses a method for preparing a compound of formula I, comprising the steps of:

[0010] ①10ml单口瓶中投入氯霉素原料药323mg,5ml批陡溶解; [0010] ①10ml single-neck flask into drug chloramphenicol 323mg, 5ml dissolved batch steep;

[0011] ②冰浴降温至0-5度,分批加入对羧基苯磺酰氯230mg,1小时后升温至室外,搅拌反应18小时; [0011] ② ice bath cooling to 0-5 °, was added 230 mg of benzenesulfonyl chloride carboxyl, after 1 hour warmed to outdoor, and the reaction stirred for 18 hours;

[0012] ③TLC检测,原料药氯霉素反应完全后处理,将反应液倾倒入25ml冰水中,析出白色沉淀,过滤,水洗滤饼,干燥得氯霉素半抗原450mg,mass检测。 [0012] ③TLC detection, chloramphenicol drug treatment after completion of the reaction, the reaction solution was poured into 25ml of ice water, the white precipitate was filtered, the filter cake was washed with water, and dried to give chloramphenicol hapten 450mg, mass detection.

[0013] 本发明提供的氯霉素抗原,是将式I所示化合物和载体蛋白偶联得到的偶联物。 [0013] The present invention provides chloramphenicol antigen, is a compound of formula I and a carrier protein conjugate resulting conjugate.

[0014]常用载体蛋白均可采用,如牛血清白蛋白(BSA),卵清蛋白(0VA),人血清白蛋白(HSA),鼠血清白蛋白(MSA),甲状腺蛋白(TG)或血蓝蛋白(KLH)等。 [0014] Common carrier proteins can be used, such as bovine serum albumin (BSA), ovalbumin (0VA), human serum albumin (HSA), rat serum albumin (the MSA), transthyretin (TG) or hemocyanin hemocyanin (KLH) and the like.

[0015] 所述式I所示化合物与BSA偶联得到的氯霉素抗原的结构见图1。 [0015] The structure of the compound of formula I obtained and conjugated to BSA antigen chloramphenicol Figure 1.

[0016] 本发明还公开了所述氯霉素抗原制备的方法,包括如下步骤: [0016] The present invention also discloses a method for the preparation of chloramphenicol antigen, comprising the steps of:

[0017] ①称取BSA50mg,溶解在3. 5mlCB 液中,称取半抗原50/67000*60*507 = 22. 7mg 溶解在1. 5mlDMF 中,加入NHS50/67000*120*115. 09 = 10. 3mg,EDC50/67000*120*191. 7 = 17. 2mg,室温活化2小时; [0017] ① weighed BSA50mg, dissolved in the liquid 3. 5mlCB, weighed 50/67000 * 60 * 507 = 22. 7mg hapten dissolved in 1. 5mlDMF, added NHS50 / 67000 * 120 * 115. 09 = 10 .. 3mg, EDC50 / 67000 * 120 * 191 7 = 17. 2mg, activated 2 hours at room temperature;

[0018] ②冰浴下将活化液滴加到BSA的CB溶液中,4度反应过夜,PBS透析。 The [0018] ② an ice bath was added dropwise to the activated BSA solution in CB, the reaction overnight at 4 degrees, dialyzed PBS.

[0019] 所述氯霉素抗原可以作为免疫原制备氯霉素特异性抗体,也可以作为包被原制备量子点免疫荧光微孔板。 [0019] The chloramphenicol chloramphenicol antigen specific antibodies may be prepared as an immunogen, may be prepared as a quantum dot coating antigen immunofluorescence microplate.

[0020] 应用氯霉素抗原制备得到的特异性抗体具体可为单克隆抗体或多克隆抗体。 Specific antibodies [0020] Preparation of chloramphenicol application specific antigens may be obtained as a monoclonal or polyclonal antibody.

[0021 ] 所述氯霉素抗原、所述特异性抗体均可应用于检测氯霉素。 [0021] The chloramphenicol antigen-specific antibody can be applied to the detection of chloramphenicol.

[0022] 本发明还公开了应用氯霉素抗原和氯霉素特异性抗体制备得到的量子点免疫荧光酶联免疫试剂盒,包括量子点标记的所述抗体。 [0022] The present invention also discloses a quantum dot immunofluorescence ELISA kits chloramphenicol, and chloramphenicol antigen specific antibody preparation obtained, including the quantum dot-labeled antibody.

[0023] "量子点标记的所述抗体"的制备方法具体如下: [0023] "the quantum dot labeled antibody" is prepared as follows:

[0024] ①取2. 5mg量子点,用ρΗ4· 7、0· 1M的MES缓冲液洗涤,然后用1ml ρΗ4· 7、0· 1M的MES 缓冲液重悬,加入0· 96mg EDC 和1. 15mg NHS,37°C反应30 分钟,20000rpm 离心5min, 收集沉淀,即为活化后的量子点; [0024] ① quantum dot 2. 5mg taken, washed with MES buffer ρΗ4 · 7,0 · 1M used, then buffered with 1ml ρΗ4 · 7,0 · 1M resuspended in MES, and the added 0 · 96mg EDC 1. 15mg NHS, 37 ° C for 30 minutes, centrifuged 20000rpm 5min, the precipitate was collected, after activation is the quantum dot;

[0025] ②取步骤①得到的活化后的量子点,用pH8. 5、50mM的硼砂缓冲液洗涤,然后将2. 5mg活化后量子点、所述抗体(蛋白质含量为0. 15mg)和pH8. 5、50mM的硼砂缓冲液混匀(总体积为〇· 8ml),25°C反应3. 5小时; [0025] ② ① obtained from step after activation quantum dots. PH8 5,50mM was washed with borax buffer, then the quantum dot 2. 5mg activation, the antibody (protein content of 0. 15mg) and pH8 . 5,50mM borax buffer mix (total volume square · 8ml), 25 ° C 3.5 hours the reaction;

[0026] ③取完成步骤②的液相,加入BSA并使其质量百分含量为5%,37°C反应30分钟, 20000rpm离心5min,收集沉淀,用pH7. 4、0. 02M的PBS缓冲液洗涤,用lml pH7. 4、0. 02M的PBS缓冲液重悬; [0026] ③ ② steps taken to complete a liquid phase, BSA was added and allowed mass percentage of 5%, 37 ° C for 30 minutes, centrifuged at 20000 rpm 5min, the precipitate was collected, dried pH7. 4,0. 02M PBS buffer . was washed with PBS lml pH7 4,0 02M resuspended in buffer;

[0027] ④取步骤③得到的液相,用ρΗ7· 4、0· 02M的PBS缓冲液稀释至64000倍体积。 [0027] ④ liquid phase obtained from step ③, diluted to 64000 times by volume with ρΗ7 · 4,0 · 02M PBS buffer.

[0028] 本发明还保护以上任一所述试剂盒在检测待测样本中是否含有氯霉素中的应用。 [0028] The present invention also protects a kit according to any of the above a test sample in the detection of whether or not containing chloramphenicol.

[0029]本发明依靠免疫学、免疫化学基本原理和残留分析技术手段,设计、合成小分子目标分析物半抗原,并与载体蛋白偶联,制备有效人工抗原,免疫动物制备针对小分子分析物的特异性抗体。 [0029] The present invention relies on immunology, chemistry and basic principles residue analysis techniques, design, synthetic small molecule target analyte hapten, and, artificial preparation effective to immunize an animal for the preparation of small molecular analytes conjugated to carrier protein specific antibodies. 利用抗原抗体的特异性免疫学反应,定量的检测样品中微量小分子目标分析物。 Specific antigen-antibody immunological response, detection of trace amount of a small molecule target analyte. 本发明制备方法简便可行、成本较低,半抗原产率较高。 The method of preparation of the present invention is simple and feasible, lower cost, higher yield hapten. 本发明克服了现有检测技术中对氯霉素样品预处理复杂、耗时、且需要大量有机溶剂萃取,以及在检测过程中要用到精密昂贵的检测仪器而不适于推广使用等缺点。 The present invention overcomes the prior art detection chloramphenicol sample pretreatment complexity, time consuming, and requires large amounts of organic solvent extraction, and the detection process to use sophisticated and expensive detection equipment is not suitable for promoting the use of other shortcomings. 本发明的氯霉素抗原,通过免疫动物可产生了针对氯霉素的特异性抗体,用于快速检测食品中的氯霉素残留,具有操作简单、快速,处理样品量大,灵敏度高,特异性强等诸多优点。 Chloramphenicol antigens of the invention, can be produced by immunizing an animal against chloramphenicol specific antibodies for the rapid detection of chloramphenicol residues in food, simple, rapid, large sample processing, high sensitivity, specificity and strong advantages.

附图说明 BRIEF DESCRIPTION

[0030] 图1为氯霉素抗原的结构示意图。 [0030] FIG. 1 is a schematic view of chloramphenicol antigen.

[0031] 图2为氯霉素量子点免疫荧光检测试剂盒标准曲线图。 [0031] FIG. 2 is a quantum dot chloramphenicol immunofluorescence detection kit standard curve.

具体实施方式 detailed description

[0032] 以下的实施例便于更好地理解本发明,但并不限定本发明。 [0032] The following examples facilitate a better understanding of the invention, but not limit the invention. 下述实施例中的实验方法,如无特殊说明,均为常规方法。 The experimental methods in the following examples, Unless otherwise specified, all conventional methods. 下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。 Experimental Examples Materials used in the following examples, Unless otherwise specified, were purchased from conventional stores Biochemicals obtained.

[0033] 实施例1、氯霉素半抗原的制备 , Chloramphenicol hapten Preparation Example 1 [0033] Embodiment

[0034] 氯霉素半抗原的制备方法,具体操作步骤包括: [0034] chloramphenicol hapten preparation method, the specific steps include:

[0035] 10ml单口瓶中投入氯霉素原料药323mg,5ml吡啶溶解,冰浴降温至0-5度,分批加入对羧基苯磺酰氯230mg,1小时后升温至室外,搅拌反应18小时,TLC检测,原料药氯霉素反应完全后处理,将反应液倾倒入25ml冰水中,析出白色沉淀,过滤,水洗滤饼,干燥得半抗原450mg,mass检测。 [0035] 10ml single neck flask into drug chloramphenicol 323mg, 5ml of pyridine were dissolved, the ice bath was cooled to 0-5 °, was added 230 mg of benzenesulfonyl chloride carboxyl, after 1 hour warmed to outdoor, and the reaction stirred for 18 hours, by TLC, the reaction was complete drug chloramphenicol workup, the reaction mixture was poured into 25ml of ice water, the white precipitate was filtered, the filter cake was washed with water, and dried to obtain a hapten 450mg, mass detection.

[0036] 半抗原的结构式见式I : [0036] structure, see formula hapten of formula I:

[0037] [0037]

Figure CN105372419AD00051

[0038] 实施例2、氯霉素人工抗原的制备 Preparation Example 2, chloramphenicol artificial antigen [0038] Embodiment

[0039] 一、氯霉素免疫抗原(CAP-BSA)的合成 [0039] First, an immunizing antigen chloramphenicol (CAP-BSA) Synthesis of

[0040] ①称取BSA50mg,溶解在3. 5mlCB 液中,称取半抗原50/67000*60*507 = 22. 7mg 溶解在1. 5mlDMF 中,加入NHS50/67000*120*115. 09 = 10. 3mg,EDC50/67000*120*191. 7 = 17. 2mg,室温活化2小时; [0040] ① weighed BSA50mg, dissolved in the liquid 3. 5mlCB, weighed 50/67000 * 60 * 507 = 22. 7mg hapten dissolved in 1. 5mlDMF, added NHS50 / 67000 * 120 * 115. 09 = 10 .. 3mg, EDC50 / 67000 * 120 * 191 7 = 17. 2mg, activated 2 hours at room temperature;

[0041] ②冰浴下将活化液滴加到BSA的CB溶液中,4 °C反应过夜,PBS透析。 [0041] Under an ice bath ② activated BSA was added dropwise a solution of CB, 4 ° C overnight, PBS dialysis.

[0042] 免疫原的结构式如下。 [0042] structural formula immunogen.

[0043] [0043]

Figure CN105372419AD00061

[0044] 二、氯霉素包被抗原(CAP-OVA)的合成 [0044] Second, the coating antigen chloramphenicol (CAP-OVA) Synthesis of

[0045] ①称取0VA33. 5mg,溶解在3. 5mlCB 液中,称取半抗原33. 5/45000*60*507 =22. 5mg 溶解在1. 5mlDMF 中,加入NHS33. 5/45000*120*115. 09 = 10. 3mg, EDC33. 5/45000*120*191. 7 = 17. 17mg,室温活化2 小时; [0045] ① weighed 0VA33. 5mg, was dissolved in 3. 5mlCB, hapten weighed 33. 5/45000 * 60 * 507 = 22. 5mg dissolved in 1. 5mlDMF, added NHS33. 5/45000 * 120 . * 115 09 = 10. 3mg, EDC33 5/45000 * 120 * 191 7 = 17. 17mg, activation room temperature for 2 hours.;

[0046] ②冰浴下将活化液滴加到0VA的CB溶液中,4 °C反应过夜,PBS透析。 [0046] ② activated ice bath was added dropwise a solution of CB of 0VA, 4 ° C overnight, PBS dialysis.

[0047] 包被原的结构式见式II。 [0047] Formula II were coated See the original formula.

[0048] [0048]

Figure CN105372419AD00062

[0049] 实施例3、氯霉素特异性抗体制备 Preparation Example 3 chloramphenicol-specific antibodies [0049] Embodiment

[0050] -、氯霉素多克隆抗体的制备 [0050] -, chloramphenicol polyclonal antibody

[0051 ] 取实施例1制备的免疫原(CAP-BSA)溶液,用pH7. 4、0. 01M的PBS缓冲液稀释,得到免疫原稀释液,用于多克隆抗体的制备。 [0051] Take an immunogen (CAP-BSA) prepared in Example 1 was diluted pH7. 4,0. 01M PBS buffer to give immunogen dilutions for preparing polyclonal antibodies. 采用新西兰大白兔作为免疫动物。 Using rabbits as an immune animal.

[0052] 免疫过程如下: [0052] The immune process is as follows:

[0053] 首次免疫:将免疫原稀释液与等体积的弗氏完全佐剂混合制成乳化剂,颈背部皮下多点注射,免疫剂量为2. 5mg/只; [0053] First immunization: The immunogen was diluted with an equal volume of Freund's complete adjuvant is prepared by mixing an emulsifier, neck subcutaneous injection at multiple sites, immunization dose of 2. 5mg / only;

[0054] 加强免疫:首次免疫4周后、8周后和12周后,各进彳丁一次加强免疫,将免疫原手布释液与等体积的弗氏不完全佐剂混合乳化,颈背部皮下多点注射,单次免疫剂量为2. 5mg/ 只; [0054] boosting: 4 weeks after the first immunization, 8 weeks and 12 weeks, each of the left foot butoxy into the first vaccination, the immunogen hand release liquid fabric with an equal volume of Freund's incomplete adjuvant emulsified, back of the neck subcutaneous multi-point injection, a single immunization dose of 2. 5mg / only;

[0055] 末次免疫:首次免疫16周后进行末次免疫,直接颈背部皮下多点注射免疫原稀释液,免疫剂量为2. 5mg/只。 [0055] the last immunization: the first immunization 16 weeks after the last immunization, the neck directly subcutaneous injection at multiple sites immunogen diluent, immunization dose of 2. 5mg / only.

[0056] 末次免疫1周后,采血并分离血清,即为免疫原对应的多克隆抗体。 [0056] One week after the last immunization, blood was collected and serum was separated, namely an immunogen corresponding to polyclonal antibodies.

[0057] 二、氯霉素单克隆抗体的制备 [0057] Second, Monoclonal Antibody Preparation

[0058] ①取实施例1制备的免疫原(CAP-BSA)溶液,按100 μ g/只,以生理盐水溶解免疫原与弗氏完全佐剂等体积混匀,颈背部皮下注射免疫6~8周龄Balb/c雌鼠,初次免疫后第7、14、28天以免疫原与弗氏不完全佐剂等体积混匀,各追加免疫一次,融合前3天以免疫复合物100 μ g/只,不加弗氏佐剂再追加免疫一次。 [0058] ① fetch immunogen (CAP-BSA) solution prepared in Example 1, by 100 μ g / only to the immunogen dissolved in physiological saline and mix an equal volume of complete Freund's adjuvant, subcutaneous injection of immune 6 ~ 8-week-old Balb / c female mice, 14, 28 days after the initial immunization with an immunogen in incomplete Freund's adjuvant mixing equal volumes of each booster immunization once, 3 days before fusion of immune complexes 100 μ g / only, no Freund's adjuvant booster immunization once again.

[0059] ②按常规方法进行,取免疫小鼠的脾细胞与处于对数生长期的小鼠骨髓瘤细胞(SP2/0)混合,然后在45s内缓慢加入预热的融合剂(PEG4000)进行融合,用HAT培养基悬浮均匀,再加入适量的饲养细胞,培养于96孔培养板,于37°C,5 % C02培养箱中培养,5天后用HT培养基半换液,9天时候进行全换液。 [0059] ② according to a conventional method, spleen cells of immunized mice with mouse myeloma cells in the logarithmic growth phase (SP2 / 0) were mixed and then slowly added to the 45s pre-heated fluxing agent (PEG4000) for fusion with HAT medium were suspended uniformly, and then appropriate amount of feeder cells, cultured at 37 ° C, 5% C02 incubator in 96-well culture plate, 5 days later with HT medium was changed half, the time for 9 days full medium was changed.

[0060] ③细胞融合后,待细胞长到培养孔面积的1/4时,采用分步筛选法筛选杂交瘤细胞。 After [0060] ③ cell fusion, the cells grow to be 1/4 of the area of ​​the culture wells, hybridoma screening using the screening step. 初选采用间接ELISA方法,以包被抗原(预先用方阵法常规滴定其最佳包被浓度和阳性血清稀释度)包被量子点免疫荧光微孔板,加入被测孔培养上清,孵育,清洗后加入羊抗鼠IgG-HRP和IgM-HRP,0PD进行显色反应。 Primary indirect ELISA method, to the coating antigen (previously titrated by a conventional method phalanx optimal coating concentration and positive serum dilution) were coated microplate immunofluorescence quantum dots, measured hole culture supernatant was added and incubated after washing goat anti-mouse was added IgG-HRP and IgM-HRP, 0PD color reaction. 筛选出的阳性孔再用间接竞争ELISA方法筛选, 先将细胞上清与1〇〇μ g/mL的氯霉素等体积混合,37°C水浴作用30min,再加入到包被好的量子点免疫荧光微孔板中。 Positive wells then screened indirect competitive ELISA screening method, cell supernatants were first mixed with a volume 1〇〇μ g / mL of chloramphenicol, 37 ° C water bath 30min, then was added to a well coated quantum dot immunofluorescence microplates. 同时用PBS取代氯霉素作对照,其余步骤同上。 Meanwhile chloramphenicol substituted with PBS as control, the remaining steps above. 若经氯霉素阻断后的〇D 45Qnm值下降到对照孔的50%以下,则判为阳性,经2~3次检测都为阳性的孔,立即用有限稀释法进行亚克隆化。 If dropped to 50% of the control wells was blocked 45Qnm 〇D the chloramphenicol value is judged as positive, by 2 to 3 times detected are positive holes, immediately subcloned by limiting dilution.

[0061] ④将2~3次亚克隆建株后的杂交瘤细胞扩大培养,收集上清液用间接ELISA测定效价,冻存;并取8~10周龄Balb/c小鼠腹腔注射液体石蜡0· 5mL/只,7~10日后腹腔注射杂交瘤细胞1~2 X 106/只,7~10日后抽取小鼠腹水,离心取上清,测定效价,并冻存备用。 [0061] ④ hybridoma cells after 2 to 3 times to build subclone strain culture expansion, the supernatant was collected by indirect ELISA titer, frozen; and takes 8 to 10 weeks old Balb / c mice by intraperitoneal injection of liquid Paraffin 0 · 5mL / only 7 to 10 days by intraperitoneal injection of hybridoma cells 1 ~ 2 X 106 / only 7 to 10 days mouse ascites fluid extracted, centrifuged supernatant, titer, and frozen for use.

[0062] 三、量子点标记的特异性抗体的制备 Three Preparation of [0062], specific antibodies labeled quantum dots

[0063] ①取2. 5mg量子点,用ρΗ4· 7、0· 1M的MES缓冲液洗涤,然后用1ml ρΗ4· 7、0· 1M的MES 缓冲液重悬,加入0· 96mg EDC 和1. 15mg NHS,37°C反应30 分钟,20000rpm 离心5min, 收集沉淀,即为活化后的量子点。 [0063] ① quantum dot 2. 5mg taken, washed with MES buffer ρΗ4 · 7,0 · 1M used, then buffered with 1ml ρΗ4 · 7,0 · 1M resuspended in MES, and the added 0 · 96mg EDC 1. 15mg NHS, 37 ° C for 30 minutes, centrifuged at 20000 rpm 5min, the precipitate was collected, that is activated after a quantum dot.

[0064] ②取步骤①得到的活化后的量子点,用pH8. 5、50mM的硼砂缓冲液洗涤,然后将2. 5mg活化后量子点、单克隆抗体(蛋白质含量为0. 15mg)和pH8. 5、50mM的硼砂缓冲液混匀(总体积为〇.8ml),25°C反应3. 5小时(单克隆抗体和量子点形成稳定的肽键共价结合)。 [0064] ② ① obtained from step after activation quantum dots. PH8 5,50mM was washed with borax buffer, then after activation 2. 5mg quantum dot, a monoclonal antibody (protein content of 0. 15mg) and pH8 borax buffer. 5,50mM the mix (total volume 〇.8ml), 25 ° C 3.5 hours the reaction (formation of a monoclonal antibody and quantum dots stable peptides covalently bound).

[0065] ③取完成步骤②的液相,加入BSA并使其质量百分含量为5% (目的是对剩余活性氨基位点进行封闭),37°C反应30分钟,20000rpm离心5min,收集沉淀,用pH7. 4、0. 02M的PBS缓冲液洗涤,用lmlpH7. 4、0. 02M的PBS缓冲液重悬,4°C保存待用。 [0065] ③ ② steps taken to complete a liquid phase, BSA was added and allowed mass percentage of 5% (to the remaining active site amino be closed), 37 ° C for 30 minutes, centrifuged at 20000 rpm 5min, the precipitate was collected , washed with pH7. 4,0. 02M with PBS buffer, with PBS lmlpH7. 4,0. 02M resuspension buffer, 4 ° C storage stand.

[0066] ④取步骤③得到的液相,用pH7. 4、0. 02M的PBS缓冲液稀释至64000倍体积,得到一抗工作液。 [0066] ④ liquid phase obtained from step ③, diluted with PBS buffer pH7. 4,0. 02M to 64000 times the volume, to obtain an anti-working fluid.

[0067] 四、量子点标记的氯霉素抗体效价的测定 [0067] Fourth, the antibody titer was measured chloramphenicol quantum dot mark

[0068] 氯霉素标准品购自Sigma公司。 [0068] chloramphenicol standards were purchased from Sigma.

[0069] 用方阵滴定法确定氯霉素包被抗原和制备的氯霉素特异性抗体的工作浓度,氯霉素包被抗原的工作浓度为2 μ g/L,量子点标记的多克隆抗体的工作浓度为1:4000,量子点标记的单克隆抗体的工作浓度为1:64000。 [0069] The specific antibody is determined working concentration of chloramphenicol chloramphenicol and coating antigen matrix prepared by titration, the working concentration of coating antigen chloramphenicol 2 μ g / L, quantum dot labeled polyclonal working concentration of antibody was 1: 4000, the working concentration of the quantum dot-labeled monoclonal antibody is 1: 64,000.

[0070] 实施例4、检测氯霉素的量子点免疫荧光试剂盒及其制备方法 [0070] Example 4, the quantum dot detecting chloramphenicol immunofluorescence kit and its preparation method

[0071] 量子点购自深圳市泰勒斯科技有限公司,产品目录号为TLS®LumiQDTM20。 [0071] quantum dots were purchased from Thales Shenzhen Science and Technology Co., Ltd., catalog number TLS®LumiQDTM20. 该量子点的表征如下:粒径为20nm、粒径的CV为15%,量子产率为60%,表面羧基含量为5X l〇-3mmol/mg,水溶性,CdSe/ZnS核壳结构,激发光波长为345nm,发射波长是620nm ;红色荧光量子点。 Characterizing the quantum dots as follows: particle diameter of 20nm, CV particle diameter of 15% quantum yield of 60%, the surface carboxyl group content 5X l〇-3mmol / mg, a water-soluble, CdSe / ZnS core-shell structure, the excitation light having a wavelength of 345nm, emission wavelength of 620 nm; red fluorescent quantum dots. 量子点上的羧基与蛋白质上的氨基形成肽键并连接起来。 Carboxyl and amino groups on the protein on the quantum dots forming a peptide bond and connected.

[0072] 量子点免疫突光检测试剂盒包括如下组件: [0072] The quantum dot light projecting immunoassay test kit comprising the following components:

[0073] -、包被了包被原的微孔板 [0073] - the coating antigen coated microplate

[0074] 取实施例2制备的包被原溶液,用包被缓冲液稀释(包被缓冲液即pH9. 6、0. 05M 的碳酸盐缓冲液),得到蛋白浓度为5ng/mL的包被原稀释液。 [0074] Get_packet Example 2 was prepared in the original solution was diluted with coating buffer (coating buffer i.e. pH9. 6,0. 05M carbonate buffer) to give a protein concentration of 5ng / mL package the original diluent. 将10g牛血清白蛋白、0. lmL proclin 300和1000mL pH7. 4、0. 01M的磷酸盐缓冲液混合,得到封闭液。 10g of bovine serum albumin, 0. phosphate mixed solution lmL proclin 300 and 1000mL pH7. 4,0. 01M buffer, blocking solution obtained.

[0075] (1)将包被原稀释液加入微孔板(100 μ L/孔),37°C孵育16小时。 [0075] (1) the packet is added to the original dilution microtiter plate (100 μ L / hole), 37 ° C for 16 h.

[0076] (2)完成步骤⑴后,倾去孔内的液体,洗涤,拍干。 [0076] (2) After completion of step ⑴, bore liquid decanted, washed and patted dry.

[0077] (3)完成步骤⑵后,每孔加入200 μ L封闭液,37°C温育2h。 After [0077] (3) completion of step ⑵, each well was added 200 μ L of blocking solution, 37 ° C and incubated 2h.

[0078] (4)完成步骤(3)后,倾去孔内的液体,干燥后用铝膜真空密封保存。 After [0078] (4) completion of step (3), the liquid decanted bore, sealed by an aluminum film and dried in vacuo.

[0079] 二、量子点标记的抗体工作液 [0079] Second, the quantum dot-labeled antibody solution

[0080] 将实施例3制备的量子点标记的多克隆抗体用抗体稀释液稀释4000倍或将实施例3制备的单克隆抗体用抗体稀释液稀释64000倍,得到氯霉素抗体工作液。 Polyclonal antibodies [0080] The quantum dots prepared in Example 3 embodiment labeled antibody dilutions was diluted with 4000-fold or Preparation Example 3 The monoclonal antibodies were diluted 64000-fold with antibody dilution to give chloramphenicol antibody solution.

[0081] 抗体稀释液:取BSAlOmg,用ρΗ7· 4、0· 02M的PBS缓冲液溶解并定容至1000mL,得到抗体稀释液。 [0081] Antibody dilution: Take BSAlOmg, dissolved ρΗ7 · 4,0 · 02M PBS buffer and volume to 1000mL, to obtain antibody dilutions. 三、标准品溶液 Third, the standard solution

[0082] 氯霉素标准溶液:将氯霉素溶于pH7. 4、0. 05M的磷酸盐缓冲液,分别得到浓度为0· 02 μ g/L、0. 06 μ g/L、0. 18 μ g/L、0. 54 μ g/L 和1. 62 μ g/L 的标准溶液。 [0082] chloramphenicol Standard solution: chloramphenicol dissolved in phosphate buffer pH7 4,0 05M respectively give concentrations of 0 · 02 μ g / L, 0 06 μ g / L, 0.... 18 μ g / L, 0. 54 μ g / L and 1. 62 μ g / L standard solution. 将ρΗ7· 4、0· 05M 的磷酸盐缓冲液作为标准溶液的阴性对照溶液,称为0溶液。 The ρΗ7 · 4,0 · 05M phosphate buffered saline solution as a negative control standard solution, called the 0 solution.

[0083] 四、稀释液 [0083] IV diluent

[0084] pHL 4、0· 0观的PBS 缓冲液。 [0084] PBS pHL 4,0 · 0 View buffer.

[0085] 五、浓缩洗涤液:将10mL吐温-20、5g叠氮化钠和990mL磷酸盐缓冲液混合,得到所述洗涤液;所述磷酸盐缓冲液的浓度为〇. 01M pH值为7. 4 ; [0085] Fifth, the concentrated detergent solution: mixing 10mL of sodium azide and Tween -20,5g 990mL phosphate buffer, to obtain a washing liquid; phosphate concentration of the buffer is square 01M pH value. 7.4;

[0086] 实施例5、检测氯霉素的量子点免疫荧光试剂盒使用方法[0087] 一、组织(猪肉、鸡肉、鱼肉、虾肉)前处理方法 5, quantum dots chloramphenicol detection kit immunofluorescence method using [0087] a pretreatment tissue (meat, chicken, fish, shrimp) Example [0086] Embodiment

[0088] ①准确称取3±0· 03g均质后的样品于50mL离心管中; [0088] ① sample was accurately weighed and homogenized 3 ± 0 · 03g in 50mL centrifuge tube;

[0089] ②加入6mL乙酸乙酯,充分润动lmin; [0089] ② 6mL of ethyl acetate was added, run full dynamic Lmin;

[0090] ③ 4000r/min 以上,离心10min ; [0090] ③ 4000r / min or more, centrifuged 10min;

[0091] ④取4mL上层乙酸乙酯于新的离心管中; [0091] ④ upper take 4mL ethyl acetate in the new centrifuge tube;

[0092] ⑤50~60°C水浴中,氮气吹干; [0092] ⑤50 ~ 60 ° C water bath, blown dry with nitrogen;

[0093] ⑥加入2mL正己烷,充分涡动10s,再加入lmL复溶液,充分涡动30s; [0093] ⑥ 2mL of n-hexane was added, sufficient vortex 10s, lmL complex solution was added, vortexed sufficiently 30s;

[0094] ⑦4000r/min以上,离心5min,完全弃去上层正己烧及中间层杂质; [0094] ⑦4000r / min or more, centrifuged 5min, n-hexyl supernatant is discarded completely burning the impurities and the intermediate layer;

[0095] ⑧取50 μ L进行检测。 [0095] ⑧ take 50 μ L detected.

[0096] 组织(猪肉、鸡肉、鱼肉、虾肉)样品稀释系数:0· 5 [0096] tissue sample dilution factor (pork, chicken, fish, shrimp): 0 · 5

[0097] 二、应用量子点免疫荧光试剂盒检测 [0097] Second, the quantum dot immunofluorescence kit

[0098]向包被了包被原的微孔板中加入标准品溶液或待测样本溶液50 μ L,一抗工作液50 μ L,室温反应30min,弃上清,洗涤3次(每次洗涤过程均如下:每孔中加入250 μ L洗涤液,30秒后弃上清),用吸水纸拍干,用荧光酶标仪检测其荧光强度数值。 [0098] coated with the coating antigen and standard microtiter plate was added test solution or the sample solution 50 μ L, an anti-working fluid 50 μ L, room temperature for 30min, the supernatant was discarded, washed three times ( washing process are as follows: 250 μ L were added to each well was washed, the supernatant was discarded after 30 seconds), patted dry with absorbent paper, using a fluorescence plate reader detecting the fluorescence intensity value. 荧光酶标仪设置为激发波长345nm,发射波长620nm。 A fluorescence plate reader is set to the excitation wavelength 345nm, emission wavelength 620nm. 每个浓度的标准品溶液的发光强度的平均值(Β)除以0 溶液的发光强度值(B0),再乘以100%,即结合率。 The average emission intensity of a standard solution of each concentration (Beta) divided by the light emission intensity value 0 solution of (B0), and then multiplied by 100%, i.e. binding rate. 计算公式:结合率(%) =B/B0X100%。 Calculated: binding ratio (%) = B / B0X100%. 以标准品溶液中的氯霉素浓度(ng/mL)的半对数值为X轴,Β/Β0为Y轴,绘制标准曲线图(见图2)。 Chloramphenicol at a concentration of standard solution (ng / mL) of the half-value of the X-axis, Β / Β0 Y-axis, the standard curve (see Figure 2). 根据标准曲线的回归方程可以求出待测样本溶液中氯霉素的浓度。 The regression equation of the standard curve can be determined in the test sample solution concentrations of chloramphenicol. 本发明中检测结果的分析可以利用专业软件,可以实现大量样本的快速分析,整个检测过程只需60分钟就可以完成。 Analysis of results of detection of the present invention may utilize a specialized software, rapid analysis of large numbers of samples can be achieved, the entire testing process takes only 60 minutes to complete. 结合率(Β/Β0)为50%时对应的标准品溶液中的氯霉素浓度即为IC50值。 Association rate (Β / Β0) chloramphenicol corresponding concentration of the standard solution of 50% is the IC50 value. 根据标准曲线图,IC50 = 0· 060ng/mL。 The standard curve, IC50 = 0 · 060ng / mL.

[0099] 三、样品中氯霉素浓度的测定 [0099] Third, the concentration of chloramphenicol in the measurement sample

[0100] 用每个检测样品溶液的荧光强度平均值除以0溶液的发光强度平均值,再乘以100%,得到抑制率。 [0100] dividing the average fluorescence intensity of each sample solution was the average of luminous intensity of 0, multiplied by 100% to obtain the rate of inhibition. 相对应每一个检测样品溶液的抑制率,则可从标准曲线上读出检测样品溶液的半对数值,再根据样品溶液的半对数值换算出样品溶液中氯霉素的残留量,最后再乘以各样品前处理过程的稀释倍数,即可计算出样品中氯霉素的浓度。 Corresponding to the inhibition rate of each test sample solution, can be read out half of the value of the test sample solution from the standard curve, and then converted the residual amount of the sample solution from the half of the value of chloramphenicol sample solution, and finally by in front of each dilution of sample processing, to calculate the sample concentration of chloramphenicol.

[0101] 实施例6、氯霉素量子点免疫荧光试剂盒检测效果评价 [0101] Example 6, chloramphenicol quantum dot immunofluorescence kit Evaluation

[0102] 一、试剂盒灵敏度 [0102] a kit sensitivity

[0103] 以最低检测限作为本发明试剂盒的灵敏度指标。 [0103] In the detection limit as an index of sensitivity of the kit of the present invention. 取20份空白样品进行检测,计算空白样品荧光强度的平均值,并将此平均值带入标准曲线得到对应的样品浓度,计算各对应浓度值的标准差(SD),由平均值加三倍标准差即为该样品的最低检测限(L0D),结果见表1。 20 parts of blank samples taken for testing, calculate the average fluorescence intensity of the blank samples, and the average value into this standard curve corresponding to the sample concentration to calculate concentration values ​​each corresponding standard difference (the SD), by the mean plus three times the It is the standard deviation of the sample is the lowest detection limit (L0D), results shown in Table 1.

[0104] 表1试剂盒在猪肉、鸡肉、鱼肉、虾肉中的最低检测限 [0104] Table 1 Kit pork, chicken, fish, shrimp lowest detection limit

[0105] [0105]

Figure CN105372419AD00091

[0106] 二、准确度和精密度试验 [0106] Second, the accuracy and precision test

[0107] 向不含氯霉素的猪肉、鸡肉、鱼肉、虾肉样品中添加氯霉素标准品,使氯霉素标准品在猪肉、鸡肉样品中的终浓度分别为〇. 015、0. 03、0. 06μ g/kg;在鱼肉、虾肉样品中的终浓度分别为〇. 〇25、0. 05、0. 1 μ g/kg,将添加后的样品分别按照所述方法进行前处理,得到检测样品溶液。 [0107] chloramphenicol was added to the standard pork, chicken, fish, shrimp sample without chloramphenicol, chloramphenicol to a final concentration of standards in pork, chicken samples were square. 015,0. before the fish, shrimp sample square 〇25,0 final concentration of 05,0 1 μ g / kg, after the addition of the sample were carried out according to the method;. 03,0 06μ g / kg... to give a sample solution is detected.

[0108] 从3个不同批次的试剂盒中各抽取3个试剂盒进行检测,每个样品重复5次,分别计算批内批间变异系数。 [0108] from three different lots of kits each extracted three detection kit, repeated 5 times for each sample, were calculated between batch coefficient of variation within the batches. 结果分别见表2~5。 The results are shown in Table 2-5.

[0109] 结果表明:猪肉、鸡肉、鱼肉、虾肉样品的平均添加回收率在75.0~100.0%,说明试剂盒的准确度良好;批内变异系数在4. 2~10. 0%,批间变异系数在4. 5~8. 1 %。 [0109] The results showed that: pork, the average recoveries of chicken, fish, shrimp sample in 75.0 to 100.0%, indicating a good accuracy of the kit; intra-assay coefficient of variation at 4.2 - 100%, inter-assay. coefficient of variation 4.5 ~ 8.1%. 批内批间变异系数均< 10%,说明试剂盒的精密性良好。 The inter-assay coefficient of variation was <10%, indicating good precision kit.

[0110] 表2猪肉中氯霉素检测准确度和精密度试验结果 [0110] Table 2 pork chloramphenicol accuracy and precision of test results

[0111] [0111]

Figure CN105372419AD00101

[0112] 表3鸡肉中氯霉素检测准确度和精密度试验结果 [0112] Table 3 in Chicken chloramphenicol accuracy and precision of test results

[0113] [0113]

Figure CN105372419AD00102

[0115] 表4鱼肉中氯霉素检测准确度和精密度试验结果 [0115] Table 4 fish chloramphenicol accuracy and precision test results

[0116] [0116]

Figure CN105372419AD00111

[0117] 表5虾肉中氯霉素检测准确度和精密度试验结果 [0117] Table 5 shrimp chloramphenicol detection accuracy and precision test results

[0118] [01]

Figure CN105372419AD00112

[0119] 三、试剂盒保存期 [0119] Third, the shelf life of the kit

[0120] 试剂盒保存条件为2_8°C,保存6个月后,测定氯霉素的IC50值。 [0120] The kit of preservation condition 2_8 ° C, after storage for 6 months, chloramphenicol IC50 values ​​are determined. 考虑在运输和使用过程中,会有非正常保存条件出现,将试剂盒在37°C保存条件下放置6天,进行热加速实验。 In consideration of the transport and use, there is an abnormal condition occurs preservation, the kit is placed in 37 ° C for 6 days storage conditions, heat acceleration test. 结果表明该试剂盒各项指标完全符合要求。 The results show that the kit of the indicators in full compliance with the requirements. 考虑到试剂盒冷冻情况发生,将试剂盒放入-20°C冰箱冷冻9天,测定结果也表明试剂盒各项指标完全正常。 Taking into account the freezing occurs kit, the kit will be placed in -20 ° C freezer 9 days, measurement results show that the indicators kit completely normal.

[0121] 表6低温保存实验结果 [0121] The results in Table 6 Cryopreservation

[0122] [0122]

Figure CN105372419AD00113

[0123] 表7热加速实验 [0123] TABLE 7 Thermal acceleration test

[0124] [0124]

Figure CN105372419AD00114

[0125] 四、交叉反应率试验 [0125] Fourth, the cross-reactivity test

[0126] 选择与CAP结构或功能相似的其他药物按照所述方法配制标准溶液,并绘制标准曲线,通过各种药物的标准曲线分别计算其50%抑制浓度。 [0126] Selection and CAP structure or similar functions other drugs formulated according to the method of standard solution, the standard curve and calculate the 50% standard curve inhibitory concentration of each drug, respectively. 用下列公式计算试剂盒对其它类似物的交叉反应率。 Cross-reactivity to other analogs kit with the following formula. 与其他药物的交叉反应率越小,说明氯霉素量子点免疫荧光检测试剂盒对氯霉素的检测特异性越好。 Cross-reactivity with other drugs smaller, the better the quantum dot chloramphenicol immunofluorescence assay kit for specific detection of chloramphenicol. 结果见表8。 The results are shown in Table 8.

[0127] 交叉反应率(% )=(氯霉素的IC5。值/待测药物的IC5。值)X 100% [0127] cross-reactivity rate (%) = (chloramphenicol IC5. Value / test drug IC5. Value) X 100%

[0128] 试验结果表明,本发明试剂盒对氯霉素、琥珀氯霉素交叉反应率较好,可用于同时检测样品中氯霉素和琥珀氯霉素;对红霉素、甲基氯霉素、氟苯尼考、庆大霉素的交叉反应率均小于1%,所以试剂盒对氯霉素的特异性好,即本发明试剂盒可以检测氯霉素。 [0128] The results show that the present invention is a kit to chloramphenicol, chloramphenicol succinate good cross reactivity, may be used to simultaneously test sample chloramphenicol and chloramphenicol succinate; erythromycin, chloramphenicol methyl Su, florfenicol, gentamicin were less than 1% cross-reactivity, it is a good kit specific for chloramphenicol, i.e. kit of the invention can be detected chloramphenicol.

[0129] 表8氯霉素试剂盒交叉反应率 [0129] Table 8 chloramphenicol kit crossreactivity

[0130] [0130]

Figure CN105372419AD00121

Claims (10)

  1. 1. 一种氯霉素半抗原,为式I所示化合物: A chloramphenicol hapten as a compound of formula I:
    Figure CN105372419AC00021
  2. 2. 式I所示化合物的制备方法,包括如下步骤: ①IOml单口瓶中投入氯霉素原料药323mg,5ml化巧溶解; ② 冰浴降温至0-5度,分批加入对駿基苯礙醜氯230mg,1小时后升温至室外,揽拌反应18小时; ⑨TLC检测,原料药氯霉素反应完全后处理,将反应液倾倒入25ml冰水中,析出白色沉淀,过滤,水洗滤饼,干燥得氯霉素半抗原450mg,mass检测。 2. preparation of a compound of formula I, comprising the steps of: ①IOml single-neck flask into drug chloramphenicol 323mg, 5ml of clever dissolved; ② ice bath cooling to 0-5 °, was added portionwise to hinder Chun benzene chlorine 230 mg ugly, after 1 hour warmed to outdoor, embrace stirred for 18 hours; ⑨TLC detection, chloramphenicol drug treatment after completion of the reaction, the reaction solution was poured into 25ml of ice water, the white precipitate was filtered, the filter cake was washed with water, dried obtained chloramphenicol hapten 450mg, mass detection.
  3. 3. -种氯霉素抗原,是将式I所示化合物和载体蛋白偶联得到的偶联物。 3. - Antigen chloramphenicol species, it is a compound of formula I and a carrier protein conjugate resulting conjugate.
  4. 4. 根据权利要求3所述氯霉素抗原,其特征在于,所述载体蛋白为人血清白蛋白、牛血清白蛋白、卵清蛋白、鼠血清蛋白或兔血清蛋白。 4. The method of claim 3 chloramphenicol antigen, wherein said carrier protein is human serum albumin, bovine serum albumin, ovalbumin, mouse serum albumin or rabbit serum albumin.
  5. 5. 权利要求3或4所述的氯霉素抗原的制备方法,包括如下步骤: ① 称取BSASOmg,溶解在3. 5mlCB液中,称取半抗原50/67000*60*507=22. 7mg溶解在1.SmlDMF中,加入NHS50/67000*120*115. 09=10. 3mg,EDC50/67000*120*191. 7=17. 2mg,室温活化2小时; ② 冰浴下将活化液滴加到BSA的CB溶液中,4度反应过夜,PBS透析。 Preparation of 3 or chloramphenicol antigen according to claim 4, comprising the following: ① From weighed BSASOmg, dissolved in the liquid 3. 5mlCB weighed hapten 50/67000 * 60 * 507 = 22 7mg. dissolved in 1.SmlDMF, added NHS50 / 67000 * 120 * 115 09 = 10 3mg, EDC50 / 67000 * 120 * 191 7 = 17 2mg, activated 2 hours at room temperature;.... the activated ② ice bath was added dropwise CB to the BSA solution, the reaction overnight at 4 ° C, dialyzed PBS.
  6. 6. 权利要求3或4所述氯霉素抗原在制备氯霉素特异性抗体中的应用。 Application-specific antibody in the preparation of chloramphenicol, chloramphenicol or 34 antigens claim.
  7. 7. 应用权利要求3或4所述氯霉素抗原制备得到的量子点标记的特异性抗体。 34 specific antibody or the antigen preparation obtained chloramphenicol quantum dot labeled 7. Application claims.
  8. 8. 权利要求3或4所述氯霉素抗原、权利要求7所述特异性抗体在检测氯霉素中的应用。 Chloramphenicol or 34 antigen of claim 1, said application specific antibodies detected 7 chloramphenicol claims.
  9. 9. 应用权利要求3或4所述氯霉素抗原、权利要求7所述特异性抗体制备得到的量子点免疫英光检测试剂盒。 9. The use as claimed in claim 34 or claim chloramphenicol antigen, said immune England 7 quantum dot light detection kit for preparing specific antibodies obtained claims.
  10. 10. 权利要求9所述量子点免疫英光检测试剂盒,其特征在于,它包括;包被有氯霉素抗原的微孔板、量子点标记的抗体工作液、氯霉素标准溶液、浓缩洗涂液。 9 10. The quantum dot optical immunoassay test kit British claim, characterized in that it comprises; chloramphenicol microtiter plate coated with antigen, antibody solution quantum dot labeled chloramphenicol standard solution, washed and concentrated coating solution.
CN 201410420843 2014-08-25 2014-08-25 Preparation chloramphenicol haptens and antigens and its application in the quantum dot immunofluorescence kit CN105372419B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 201410420843 CN105372419B (en) 2014-08-25 2014-08-25 Preparation chloramphenicol haptens and antigens and its application in the quantum dot immunofluorescence kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 201410420843 CN105372419B (en) 2014-08-25 2014-08-25 Preparation chloramphenicol haptens and antigens and its application in the quantum dot immunofluorescence kit

Publications (2)

Publication Number Publication Date
CN105372419A true true CN105372419A (en) 2016-03-02
CN105372419B CN105372419B (en) 2018-05-22

Family

ID=55374799

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 201410420843 CN105372419B (en) 2014-08-25 2014-08-25 Preparation chloramphenicol haptens and antigens and its application in the quantum dot immunofluorescence kit

Country Status (1)

Country Link
CN (1) CN105372419B (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4013004A1 (en) * 1990-04-24 1991-10-31 Elvira Schecklies ELISA determn. of low mol. wt. pesticides, antibiotics or toxins - using enzyme labelled antibody reactant, providing lower detection limit
JPH06220072A (en) * 1992-12-01 1994-08-09 Tanpaku Kogaku Kenkyusho:Kk Activation of prodrug by catalyst antibody
EP0656422A1 (en) * 1993-11-05 1995-06-07 AMERSHAM INTERNATIONAL plc Cat assay
US5968515A (en) * 1996-06-28 1999-10-19 Thaco Research, Ltd. Methods for the quantitative analysis of organic compounds
CN1766629A (en) * 2005-11-03 2006-05-03 北京望尔生物技术有限公司 ELISA kit for detecting chloramphenicols in animal derived food
CN101993487A (en) * 2009-08-27 2011-03-30 深圳市三方圆生物科技有限公司 Immunogen and coatinggen of chloramphenicol and application thereof in collaurum test paper
CN103018450A (en) * 2011-09-20 2013-04-03 北京勤邦生物技术有限公司 Chloramphenicol chemiluminescence enzyme-linked immunodetection kit

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4013004A1 (en) * 1990-04-24 1991-10-31 Elvira Schecklies ELISA determn. of low mol. wt. pesticides, antibiotics or toxins - using enzyme labelled antibody reactant, providing lower detection limit
JPH06220072A (en) * 1992-12-01 1994-08-09 Tanpaku Kogaku Kenkyusho:Kk Activation of prodrug by catalyst antibody
EP0656422A1 (en) * 1993-11-05 1995-06-07 AMERSHAM INTERNATIONAL plc Cat assay
US5968515A (en) * 1996-06-28 1999-10-19 Thaco Research, Ltd. Methods for the quantitative analysis of organic compounds
CN1766629A (en) * 2005-11-03 2006-05-03 北京望尔生物技术有限公司 ELISA kit for detecting chloramphenicols in animal derived food
CN101993487A (en) * 2009-08-27 2011-03-30 深圳市三方圆生物科技有限公司 Immunogen and coatinggen of chloramphenicol and application thereof in collaurum test paper
CN103018450A (en) * 2011-09-20 2013-04-03 北京勤邦生物技术有限公司 Chloramphenicol chemiluminescence enzyme-linked immunodetection kit

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
IAIN A.MURRAY等: "Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogs revealed by site-directed mutagenesis and x-ray crystallography of chloramphenicol acetyltransferase", 《BIOCHEMISTRY》 *
方伟等: "氯霉素人工抗原的合成与鉴定", 《华中农业大学学报》 *
陆蕾等: "氯霉素人工抗原的合成及多克隆抗体的制备", 《中国食品学报》 *

Also Published As

Publication number Publication date Type
CN105372419B (en) 2018-05-22 grant

Similar Documents

Publication Publication Date Title
CN101377490A (en) Magnetic microparticle separating chemiluminescence immune analysis determination reagent kit for detecting related sign object and preparing method thereof
Xie et al. Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk
CN1153908A (en) Reagent kit and detection method of aflatoxins B1
CN1963507A (en) Immune testing method for enzyme linked retained 3-methylquinoxaline-2-carboxylic acid and reagent set
CN1844926A (en) ELISA kit for detecting Sudan red medicines and detection method thereof
CN103018450A (en) Chloramphenicol chemiluminescence enzyme-linked immunodetection kit
CN1807601A (en) Immune colloid gold test paper strip for detecting sulfadiazine residue and its preparation method
WO1996041172A1 (en) Functional surrogates of analytes of interest and methods of obtaining and using same
CN101799472A (en) Diethylstilbestrol detection kit and detection method
US6500629B1 (en) Materials and methods for detection and quantitation of an analyte
CN102079788A (en) Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof
CN1861632A (en) Coupling compound of Norfloxacin, preparation process and application thereof
CN101865924A (en) Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay
CN1766629A (en) ELISA kit for detecting chloramphenicols in animal derived food
CN101046474A (en) Enzyme-linked immunological kit for detecting quinoxaline medicine residue
CN101413955A (en) ELISA test box for detecting zearalenone and preparing and detecting method thereof
CN101915844A (en) Method and special quantum dot fluorescent immunoassay kit for detecting quinolone compounds
CN101962358A (en) Ciprofloxacin hapten, artificial antigen and antibody and preparation method and application thereof
US20040023309A1 (en) Immunoassay and kit for an early and simultaneous detection of biochemical markers in a patient&#39;s sample
CN1766624A (en) ELISA kit for detecting furazolidone metabolites and detection method thereof
CN101482562A (en) Detection reagent kit and detection method for diethyl stilbestrol
US20030077670A1 (en) Homogeneous assay of vancomycin using a stable particle-vancomycin conjugate, a novel rate enhancer, and a novel dose response modulator
CN101013129A (en) Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof
CN101793894A (en) Direct competitive enzyme-linked immunoassay kit for detecting medroxyprogesterone acetate
CN103575890A (en) Chemiluminescence assay kit of ractopamine (RAC) and application thereof

Legal Events

Date Code Title Description
C06 Publication
SE01