CN113831290B - 一种检测食品中羟基甲硝唑残留量的方法及产品 - Google Patents
一种检测食品中羟基甲硝唑残留量的方法及产品 Download PDFInfo
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- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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- C07D233/94—Nitro radicals attached in position 4 or 5 with hydrocarbon radicals, substituted by oxygen or sulfur atoms, attached to other ring members
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
本发明公开了一种检测食品中羟基甲硝唑残留量的方法及产品。检测所用产品为式I所示羟基甲硝唑半抗原化合物。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。利用抗原抗体的特异性免疫学反应,定性或定量检测食品样本中微量小分子目标分析物,具有特异、灵敏、准确、快速、方便、廉价等特点。
Description
技术领域
本发明属于药物残留快速检测领域,涉及一种检测食品中羟基甲硝唑残留量的方法及产品。
背景技术
羟基甲硝唑为硝基咪唑类药物甲硝唑的代谢物,由于甲硝唑具有抗菌和抗原虫作用,能杀灭和预防厌氧菌和病原虫,因此在农业生产中常被用于预防和治疗家禽的滴虫病和球虫病、猪密螺旋体性痢疾及动物的各种厌氧菌感染。但甲硝唑具有潜在的致癌、致畸、诱变和遗传毒性作用,其残留对动物性食品构成了威胁。我国《食品安全国家标准食品中兽药最大残留限量》(GB 31650—2019)中规定甲硝唑允许作治疗用兽药,但不得在动物性食品中检出。日本规定甲硝唑在食品中不得检出,欧盟和美国均规定了甲硝唑为禁用兽药。甲硝唑在体内吸收快,所以为保障动物源性食品安全,研究动物源食品中甲硝唑代谢物检测的方法具有重要意义。
目前羟基甲硝唑含量测定方法大多采用液相色谱法或液相色谱串联质谱法,过程繁琐,成本较高。免疫化学分析由于在抗原抗体的定性定量方面独特的优势和操作简便快速、成本低、灵敏度较高、分析样本量大的优点弥补了理化分析的不足,但目前针对食品中羟基甲硝唑免疫检测方法研究较少,本发明对该检测方法进行了研究,免疫化学分析法在抗原抗体的定性定量方面具有独特的优势,其操作简便快速、成本低、灵敏度较高、分析样本量大,弥补了理化分析的不足,对于科学使用抗菌药、遏制细菌耐药性、公共健康安全及食品安全具有重大意义。
发明内容
本发明的目的是提供一种检测食品中羟基甲硝唑残留量的方法及产品,具有灵敏度高、准确性强、灵敏度高、操作简便的优点。
本发明的一个目的是提供一种羟基甲硝唑半抗原化合物,其结构式如式I所示:
Chemical Formula:C16H17ClN4O6
Exact Mass:396.08
m/z:396.08(100.0%),398.08(32.0%),397.09(17.3%),399.08(5.5%),397.08(1.5%),398.09(1.4%),398.09(1.2%)
式I
本发明所提供的制备所述羟基甲硝唑半抗原的方法,具体可包括如下步骤:
羟基甲硝唑原料1g,用3ml甲醇钠溶解,加入对苯二甲醛缩二乙醛1.38g,搅拌,TLC检测反应完全时,加入盐酸淬灭反应,加入硅胶,进行柱层析拌样,旋干后进行柱层析,收集产物点,旋干得到中间体1,共725mg。
将725 mg中间体1用盐酸溶液溶解,搅拌,监测反应完全,调PH值到7,旋干溶剂进行柱层析,展开剂比例为二氯甲烷:甲醇=10:1,收集所需溶液,旋干,得到中间体2,共526mg。
称取526 mg中间体2,用乙醇溶解,加入羧甲基羟胺470 mg,搅拌,反应完全后,加入硅胶,进行柱层析,收集产物点,旋干溶剂,加入盐酸配置成羟基甲硝唑盐酸盐,得到488mg羟基甲硝唑半抗原。
在羟基甲硝唑半抗原的基础上构建所得的羟基甲硝唑抗原也属于本发明的保护范围。
所述羟基甲硝唑抗原,为将所述羟基甲硝唑半抗原(式I)与载体蛋白偶联所得的抗原。在本发明的一个实施例中,所述载体蛋白具体为牛血清白蛋白(BSA)或卵清蛋白(OVA)。
所述羟基甲硝唑抗原的制备方法也属于本发明的保护范围。
在本发明中,所述羟基甲硝唑抗原具体是按照包括如下步骤的方法制备获得的:
(1)将16.1 mg羟基甲硝唑半抗原(式I)溶解于1.5 mL二甲基甲酰胺(DMF)中,然后加入25.75 mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和15.46 mg N-羟基琥珀酰亚胺(NHS),20-25℃磁力搅拌反应2-3h,得到溶液I;
(2)将所述载体蛋白置于0.1M碳酸氢钠缓冲液中,200rpm搅拌10min,充分溶解,得到溶液II;
其中,若所述载体蛋白为牛血清白蛋白(BSA),则所述牛血清白蛋白(BSA)与所述0.1M碳酸氢钠缓冲液的配比为50mg:3.5mL;若所述载体蛋白为卵清蛋白(OVA),则所述卵清蛋白(OVA)与所述0.1M碳酸缓冲液的配比为33.6mg:3.5mL;
(3)将所述溶液I和所述溶液II混合,具体为在0-4℃条件下,1000rpm搅拌下,将溶液I逐滴加入到所述溶液II中,500rpm搅拌反应24h,得到溶液III;
(4)用磷酸盐缓冲液(0.01M PBS,pH7.2),于4℃对所述溶液III搅拌透析3天,得到所述羟基甲硝唑抗原。
所述羟基甲硝唑半抗原(式I)或所述羟基甲硝唑抗原在定性或定量检测羟基甲硝唑中的应用也属于本发明的保护范围。
利用所述羟基甲硝唑抗原制备的抗体也属于本发明的保护范围。所述抗体可为多克隆抗体、单克隆抗体或抗血清。
本发明还要求保护一种用于检测食品中羟基甲硝唑的方法,包括:用所述羟基甲硝唑抗原或所述抗体对食品进行检测;
或者,用所述检测羟基甲硝唑的试剂盒对食品进行检测。
所述试剂盒包括含不同梯度浓度羟基甲硝唑标准品工作液、包被羟基甲硝唑抗原的酶标板、酶标抗体工作液、抗体工作液、样品稀释液、洗涤液、底物显色液、终止液。
所述酶标抗体工作液为经辣根过氧化酶标记的羊抗鼠的抗体;规格为1瓶(12mL)。
所述抗体工作液为将获得的羟基甲硝唑单克隆抗体用抗体稀释液稀释90000倍,获得含有羟基甲硝唑的单克隆抗体工作液;
其中,所述抗体稀释液为:1 g环糊精、2 g BSA、35.82 g十二水合磷酸氢二钠、13.6g磷酸二氢钾、5g氯化钠、1mL Tween-20,0.5 mLProclin-300,加入100 mL 0.5 mol/LEDTA,900mL去离子水配制而成,规格为1瓶(7 mL)。
所述样品稀释液为0.01M pH7.4的PBS,规格为1瓶(50 mL)。
所述洗涤液为0.01M pH7.4的PBST溶液,规格为1瓶(20×,25mL)。
所述底物显色液为底物A液和底物B液各1瓶(7mL);具体的,所述底物A为2%过氧化脲水溶液;所述底物B为1%四甲基联苯胺水溶液;所述终止液为2 M H2SO4溶液,规格为1瓶(7mL)。
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。利用抗原抗体的特异性免疫学反应,定量的检测样本中微量小分子目标分析物,具有特异、灵敏、准确、快速、方便、廉价等特点。
附图说明
图1羟基甲硝唑半抗原质谱图。
图2羟基甲硝唑酶联免疫试剂盒标准曲线。
具体实施方式
下面结合具体实施例对本发明作进一步阐述,但本发明并不限于以下实施例。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、羟基甲硝唑半抗原的制备
一、羟基甲硝唑半抗原的制备
将50ml圆底烧瓶冲洗干净,用乙醇吹干,将其固定在搅拌器上,加入搅拌子。称取原料1g,用3ml甲醇钠溶解,搅拌,全部溶解后留样,此时溶液为紫色,称取对苯二甲醛缩二乙醛1.38g,加入其中,搅拌,通过TLC发现反应完全时,处理,加入盐酸淬灭反应,加入硅胶,进行柱层析拌样,旋干后进行柱层析,收集产物点,旋干得到中间体1,共725mg。
称取725mg中间体1,用盐酸溶液溶解,搅拌,监测反应,直到完全反应,处理,调PH值到7,旋干溶剂进行柱层析,展开剂比例为(二氯甲烷:甲醇=10:1)收集所需要的溶液,旋干,得到产物,得到中间体2,共526mg。
称取526mg中间体2,用乙醇溶解,加入羧甲基羟胺350mg,搅拌,监测反应,其中补加120mg,羧甲基羟胺,反应完全后,加入硅胶,进行柱层析,收集产物点,旋干溶剂,加入盐酸配置成羟基甲硝唑盐酸盐,得到约488mg。
反应方程式如下:
二、羟基甲硝唑半抗原结构鉴定
对所得488mg羟基甲硝唑半抗原进行质谱检测(图1),结果显示其化学结构式如式I所示,即为羟基甲硝唑半抗原。
Chemical Formula:C16H17ClN4O6
Exact Mass:396.08
m/z:396.08(100.0%),398.08(32.0%),397.09(17.3%),399.08(5.5%),397.08(1.5%),398.09(1.4%),398.09(1.2%)
式I
实施例2、羟基甲硝唑人工抗原的制备
一、羟基甲硝唑人工抗原的制备
1、免疫原的合成
(1)将16.1mg羟基甲硝唑半抗原用1.5ml DMF溶解,200rpm搅拌10min,加入EDC25.75mg,NHS15.46mg溶解,室温搅拌(500rpm)活化2-3h。
(2)称取BSA 50mg溶于3.5ml 0.1M碳酸氢钠溶液中,200rpm搅拌10min,使其充分溶解,冰浴降温0-4℃,1000rpm搅拌下,将步骤1反应液逐滴加入(1ml/min),500rpm搅拌反应24h.
(3)将反应产物装入蒸馏水冲洗干净透析袋(10cm),1L0.01M PBS(1×,pH7.2)4℃搅拌(100rpm)透析3d,每天换液3次(早中晚各一次),共计换液9次,将透析产物5000rpm离心6min,1.5ml/管分装,将抗原编号,-20℃保存备用。
2、包被原的合成
(1)将16.1mg羟基甲硝唑半抗原用1.5ml DMF溶解,200rpm搅拌10min,加入EDC25.75mg,NHS15.46mg溶解,室温搅拌(500rpm)活化2-3h。
(2)称取OVA 33.6mg溶于3.5ml 0.1M碳酸氢钠溶液中,200rpm搅拌10min,使其充分溶解,冰浴降温0-4℃,1000rpm搅拌下,将步骤1反应液逐滴加入(1ml/min),500rpm搅拌反应24h.
(3)将反应产物装入蒸馏水冲洗干净透析袋(10cm),1L0.01M PBS(1×,pH7.2)4℃搅拌(100rpm)透析3d,每天换液3次(早中晚各一次),共计换液9次,将透析产物5000rpm离心6min,1.5ml/管分装,将抗原编号,-20℃保存备用。
实施例3、羟基甲硝唑人工抗原免疫动物制备单克隆抗体
按照常规方法制备羟基甲硝唑单克隆抗体,具体步骤如下:
一、动物免疫
用实施例2制备出的免疫原(羟基甲硝唑-BSA)按100μg/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100μg/只,不加弗氏佐剂再追加免疫一次。
二、细胞融合与克隆
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℃,5%CO2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。
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。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)包被酶标板,加入被测孔培养上清,孵育,清洗后加入羊抗鼠IgG-HRP和IgM-HRP,OPD进行显色反应。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100μg/mL的羟基甲硝唑等体积混合,37℃水浴作用30min,再加入到包被好的酶标板中。同时用PBS取代羟基甲硝唑作对照,其余步骤同上。若经羟基甲硝唑阻断后的OD450nm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。
三、单克隆抗体的制备及纯化
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1~2×105/只,7~10日后抽取小鼠腹水。收集细胞上清或腹水,采用间接ELISA 法测定其效价(测定效价时以P/N>2.1 的细胞上清或腹水最大稀释倍数表示),结果表明细胞上清的效价为1:10000,腹水的效价为1:50000。接着,用辛酸-饱和硫酸铵法对其进行纯化,纯化后放入-20℃环境保存。
实施例4、羟基甲硝唑酶联免疫试剂盒的检测
一、羟基甲硝唑酶联免疫试剂盒
1、羟基甲硝唑酶联免疫试剂盒的组成包括如下:
(1)羟基甲硝唑标准品工作液:6瓶,1.5mL/瓶,浓度为0µg/L、0.10 µg/L、0.3µg/L、0.9µg/L、2.7µg/L、8.1µg/L。
(2)羟基甲硝唑酶标板:1块(8孔×12条),为包被了实施例2制备得到的“羟基甲硝唑-OVA”的酶标板。
(3)羟基甲硝唑抗体工作液:1瓶(10mL),为抗体稀释液将抗体进行1:90000稀释,所述羟基甲硝唑抗体为实施例3制备所得的单克隆抗体;
所述抗体稀释液为:1 g环糊精、2 g BSA、35.82 g十二水合磷酸氢二钠、13.6g磷酸二氢钾、5g氯化钠、1mLTween-20,0.5 mL Proclin-300,加入100 mL 0.5mol/L EDTA,900mL去离子水配制而成。
(4)酶标记物工作液:经辣根过氧化酶标记的羊抗鼠的抗体。
(5)样品稀释液:0.01M pH7.4的PBS。
(6)洗涤液:0.01M pH7.4的PBST溶液。使用时20倍稀释,配制成洗涤工作液。
(7)底物A液、底物B液各1瓶(7mL)。其中,底物A为2%过氧化脲水溶液。底物B为1%四甲基联苯胺水溶液。
(8)终止液:1瓶(7mL),为2 M H2SO4溶液。
(9)盖板膜;
(10)自封袋。
2、设备和材料
(1)设备
酶标仪(检测波长450nm,参考波长630nm)、天平(精度:0.01g)、漩涡振荡器、离心机(4000g)、水浴锅(可控温度:60℃)、氮吹仪、微量移液器、计时器。
(2)试剂
乙酸乙酯、正己烷、0.3 M NaOH、0.1 M HCl、2 M 硫酸。
3、试剂盒检测原理
样品中的羟基甲硝唑与酶标板上固定的抗原特异性竞争抗体,加入酶标记物,催化底物显色,根据显色的深浅来判断样品中羟基甲硝唑的含量。显色深,含量少,显色浅,含量多。
二、羟基甲硝唑酶联免疫试剂盒的使用方法
1、样品前处理
(1)动物肉类、鸡蛋
a) 称取2±0.05 g均质后的样品于50mL 离心管中;b) 加入2 mL 0.3 M NaOH,充分涡动30 s;c) 加入6 mL 乙酸乙酯,涡动2min;d) 4000 g 以上,离心10 min;e) 取3 mL上清于新的离心管中;f) 50-60℃水浴中,氮气吹干;g) 加入2 mL 正己烷,再加入0.5 mL样品稀释液,充分涡动30 s;h) 4000 g 以上,离心5 min;i) 弃去上层正己烷及中间层杂质;j) 取50 μL 进行检测。
(2)液态奶
a) 量取1 mL 新鲜样品于干净离心管中,加入20 μL 2 M 硫酸,涡动1 min;b)4000 g 以上,离心5 min;c) 去除上层脂肪层,取50 μL 上层清液于450 μL 样品稀释液中,充分涡动30 s;d) 取50 μL 进行检测。
2、检测步骤
(1)将板条插入酶标板架上,并记录下各标准品和样品的位置,建议均做双孔平行,未使用的板条用自封袋密封后,立即保存于2-8℃环境中;
(2)将50 μL各浓度的羟基甲硝唑标准品工作液(或待测样品溶液)分别加入对应的标准品(或待测样品孔)中;
(3)在每孔中加入50μL抗体工作液;
(4)盖好盖板膜,轻轻振荡酶标板10s,充分混匀,4℃避光反应80 min;
(5)揭开盖板膜;
(6)倒掉板孔中液体,在每孔加入260μL洗涤工作液,充分洗涤4次,每次浸泡15-30s;
(7)倒掉板孔中液体,拍干酶标板;
(8)在每孔中加入100 μL酶标记物工作液;
(9)盖好盖板膜,轻轻振荡酶标板10s,充分混匀,室温下(25±2℃),避光反应10min;重复步骤(5)-(7);
(10)立即在每孔中加入100 μL 底物A、B 混合液;盖好盖板膜,轻轻振荡酶标板10s,充分混匀,室温下(25±2℃),避光反应10-15 min;
(11)揭开盖板膜,在每孔中加入50μL终止液,轻轻振荡酶标板10s,充分混匀;终止后5min内用酶标仪在双波长450nm、630nm下读取酶标板吸光度值。
3、结果计算或判定
(1)各标准品(或待测样品)的平均吸光度值,除以零标(浓度为0 µg/L的标准品)吸光度值,乘以100,可以得到各标准品对应的吸光度的百分比,即百分吸光度值。
(2)以各标准品的百分吸光度值为纵坐标,以对应的羟基甲硝唑浓度为横坐标绘制标准曲线。
(3)将待测样品的百分吸光度值代入标准曲线方程,可得出待测样品对应的浓度,再乘以相应样品的稀释倍数,可得待测原样品中羟基甲硝唑的实际含量。
三、羟基甲硝唑酶联免疫试剂盒检测羟基甲硝唑
1、羟基甲硝唑酶联免疫试剂盒灵敏度和特异性检测
羟基甲硝唑酶联免疫试剂盒的特异性是通过与相应的物质进行交叉反应试验来确定的。交叉反应越小,特异性越好。
将羟基甲硝唑及其它类似物(替硝唑,奥硝唑,羟甲基甲硝咪唑,地美硝唑)分别做系列稀释,分别按照如上步骤二2进行操作,以羟基甲硝唑及其它类似物的系列稀释液替代其中的“羟基甲硝唑标准品工作液”,制作标准曲线,并在曲线上找出各自50%抑制浓度(IC50),具体方法如下:得到纵坐标数值等于50%对应的羟基甲硝唑浓度(µg/L),即IC50值。用下式计算试剂盒对羟基甲硝唑和各类似物的交叉反应率。
交叉反应率(%)=(引起50%抑制的羟基甲硝唑浓度/引起50%抑制的羟基甲硝唑类似物浓度)×100%
结果如表1所示,从表1中可以看出,羟基甲硝唑酶联免疫试剂盒对各种类似物的交叉反应率均小于5%。这说明羟基甲硝唑酶联免疫试剂盒对羟基甲硝唑具有极高的特异性,可有效的排除其它类似物的干扰,可专门用于羟基甲硝唑的检测。
表1羟基甲硝唑酶联免疫试剂盒的特异性
药物名称 | 交叉反应率(%) |
羟基甲硝唑 | 100 |
替硝唑 | 1.5 |
奥硝唑 | 0.9 |
羟甲基甲硝咪唑 | 0.6 |
地美硝唑 | <0.1 |
建立羟基甲硝唑ELISA标准曲线,结果见附图2。试剂盒的IC50为0.72,线性范围(IC20~IC80)为0.28~2.31 µg/L,线性相关性R2为0.999。
2、羟基甲硝唑酶联免疫试剂盒最低检测限测定
采用羟基甲硝唑酶联免疫试剂盒对猪肉、鸡蛋和液态奶中羟基甲硝唑进行检测时的最低检测限,分别测定20个空白样品,按照步骤二1中方法进行前处理,根据标准曲线求出测定值,计算出其平均值,再加上3倍标准差,即为最低检测限(LOD)。
表2 空白样品测定结果 µg/L
结果显示,测得羟基甲硝唑在猪肉中的LOD为0.09µg/L、在鸡蛋中为0.25µg/L、在液态奶中为0.93µg/L。为避免产品假阳性情况,设定羟基甲硝唑在猪肉中的LOD为0.1µg/L、在鸡蛋中为0.3µg/L、在液态奶中为1.0µg/L。
3、羟基甲硝唑酶联免疫试剂盒的准确度和精密度
通过添加回收试验和回收率的计算来判定试剂盒的准确性和精密度。分别在猪肉、鸡蛋和液态奶中添加LOD、2LOD的羟基甲硝唑,测定添加回收率,每个样本重复测定5次。
表3 试剂盒准确度和精密度
结果表明,猪肉、鸡蛋、液态奶样品各添加浓度的回收率在85.56~109.4%之间,变异系数小于13.16%。准确性和精密度满足试剂盒的检测要求。
4、羟基甲硝唑ELISA试剂盒的样本测定
在对猪肉的盲样检测能力验证中,羟基甲硝唑含量为0.5 µg/L、1.0 µg/L的盲样均检出阳性(检出率100%),羟基甲硝唑含量为0.05 µg/L的盲样均未检出(检出率0%);在对鸡蛋的盲样检测能力验证中,羟基甲硝唑含量为0.5 µg/L、1.0 µg/L的盲样均检出阳性(检出率100%),羟基甲硝唑含量为0.2 µg/L的盲样均未检出(检出率0%);在对液态奶的盲样检测能力验证中,羟基甲硝唑含量为5 µg/L、10µg/L的盲样均检出阳性(检出率100%),羟基甲硝唑含量为3.0 µg/L的盲样均未检出(检出率0%);羟基甲硝唑阴性样品试剂盒均正确判定。因此,羟基甲硝唑ELISA试剂盒的准确性良好,可用于猪肉、鸡蛋、液态奶等样本中羟基甲硝唑的检测。
表4检测羟基甲硝唑盲样测定结果(猪肉) (µg/L)
表5检测羟基甲硝唑盲样测定结果(鸡蛋) (µg/L)
表6检测羟基甲硝唑盲样测定结果(液态奶) (µg/L)
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Claims (10)
1.一种化合物,其结构如式I所示:
2.制备权利要求1所述化合物的方法,包括步骤为:
羟基甲硝唑原料1g,用3ml甲醇钠溶解,加入对苯二甲醛缩二乙醛1.38g,搅拌,TLC检测反应完全时,加入盐酸淬灭反应,加入硅胶,进行柱层析拌样,旋干后进行柱层析,收集产物点,旋干得到中间体1,共725mg;
将725mg中间体1用盐酸溶液溶解,搅拌,监测反应完全,调pH值到7,旋干溶剂进行柱层析,展开剂比例为二氯甲烷:甲醇=10:1,收集所需溶液,旋干,得到中间体2,共526mg;
称取526mg中间体2,用乙醇溶解,加入羧甲基羟胺470mg,搅拌,反应完全后,加入硅胶,进行柱层析,收集产物点,旋干溶剂,加入盐酸配置成羟基甲硝唑盐酸盐,得到488mg羟基甲硝唑半抗原。
3.权利要求1所述式I所示羟基甲硝唑半抗原在制备羟基甲硝唑抗原中的应用。
4.根据权利要求3所述羟基甲硝唑抗原,其特征在于:由所述式I所示羟基甲硝唑半抗原化合物与载体蛋白偶联所得;所述载体蛋白具体为BSA或OVA。
5.根据权利要求4所述的羟基甲硝唑抗原,其制备方法包括如下步骤:
(1)将16.1mg羟基甲硝唑半抗原用1.5ml DMF溶解,200rpm搅拌10min,加入25.75mgEDC,15.46mg NHS溶解,500rpm室温搅拌活化2-3h,得到溶液I;
(2)将所述载体蛋白置于0.1M碳酸氢钠缓冲液中,200rpm搅拌10min,充分溶解,得到溶液II;所述载体蛋白与所述0.1M碳酸氢钠缓冲液的配比为33.6-50mg:3.5mL;
(3)将所述溶液I和所述溶液II混合,具体为在0-4℃条件下,1000rpm搅拌下,将溶液I逐滴加入到所述溶液II中,500rpm搅拌反应24h,得到溶液III;
(4)用0.01M pH7.2 PBS于4℃对所述溶液III搅拌透析3天,得到所述羟基甲硝唑抗原。
6.权利要求1所述式I所示羟基甲硝唑半抗原或权利要求4所述羟基甲硝唑抗原在制备抗体中的应用;由权利要求1所述式I所示羟基甲硝唑半抗原或由权利要求4所述羟基甲硝唑抗原作为免疫原制得抗体。
7.权利要求1所述式I所示羟基甲硝唑半抗原或权利要求4所述羟基甲硝唑抗原或权利要求6所述抗体在检测羟基甲硝唑中的应用。
8.一种检测羟基甲硝唑的试剂盒,包括权利要求4所述羟基甲硝唑抗原和权利要求6所述抗体。
9.一种用于检测食品中羟基甲硝唑的方法,包括:用权利要求4所述羟基甲硝唑抗原或权利要求6所述抗体对食品进行检测;
或者,用所述检测羟基甲硝唑的试剂盒对食品进行检测。
10.根据权利要求8所述的检测羟基甲硝唑的试剂盒,其特征在于:所述试剂盒包括含不同梯度浓度羟基甲硝唑标准品工作液、包被羟基甲硝唑抗原的酶标板、酶标抗体工作液、抗体工作液、样品稀释液、洗涤液、底物显色液、终止液;
所述抗体工作液为将羟基甲硝唑单克隆抗体用抗体稀释液稀释90000倍获得的;
所述抗体稀释液为:1g环糊精、2g BSA、35.82g十二水合磷酸氢二钠、13.6g磷酸二氢钾、5g氯化钠、1mL Tween-20,0.5mL Proclin-300,加入100mL0.5mol/L EDTA,900mL去离子水配制而成。
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