CN117384061B - 敌草胺半抗原、抗原、抗体、检测装置及其制备和应用 - Google Patents
敌草胺半抗原、抗原、抗体、检测装置及其制备和应用 Download PDFInfo
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Classifications
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- C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C235/18—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides
- C07C235/20—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/12—Preparation of carboxylic acid amides by reactions not involving the formation of carboxamide groups
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/14—Preparation of carboxylic acid amides by formation of carboxamide groups together with reactions not involving the carboxamide groups
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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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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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
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明公开了敌草胺半抗原、抗原、抗体、检测装置及其制备和应用,涉及敌草胺半抗原、抗原、抗体、胶体金检测装置及其制备和检测农产品中敌草胺残留的应用。本发明提供由敌草胺半抗原偶联载体蛋白制备的人工抗原,以及免疫实验动物后使其机体产生的特异性针对敌草胺的抗体,效价高,对敌草胺的最低检测限为2.4 ng/mL,IC50为11.5 ng/mL。本发明提供的针对敌草胺的检测方法具有灵敏度高、特异性强、成本低、操作简单、检测时间短、可实现对农产品中敌草胺的残留含量现场快速检测等优点。
Description
技术领域
本发明涉及免疫学检测技术领域,更具体地,本发明涉及敌草胺半抗原、抗原、抗体、检测装置及其制备和应用。
背景技术
敌草胺(Napropamide)属芳氧酰胺类除草剂。敌草胺在土壤中持效期长,对杂草的防除效果较好,但使用不合理则易造成土壤和农产品残留超标,或溶入地表水和地下水中,最终对人和动物造成危害。因大鼠体内试验显示酰胺类除草剂可进一步转化成有致癌作用的二烷基醌亚胺,其残留情况受到了广泛关注。现有技术中,对敌草胺残留检测分析主要采用气相色谱法,为了得到较低的检测限,所用的检测器多为高灵敏度的氮磷检测器 (NPD)、电子捕获检测器(ECD)和质谱,其样品预处理以液液萃取和固相萃取分离杂质为主。有关敌草胺高效液相色谱分析的研究报道,采用甲苯萃取,弗罗里硅土柱层析,但此方法中靶标物的色谱峰处干扰物质较多,干扰测定。因此,建立快速、简单的敌草胺残留快速检测方法显得十分重要。
免疫学检测分析方法是一种以抗原抗体的特异性识别、可逆性结合反应为基础的分析方法,具有灵敏度高、特异性高、对仪器的要求不高、快速、操作简便、成本低等优点。然而,建立免疫学检测分析技术并将其应用于检测农产品中的敌草胺,关键技术在于能够获取到特异性强、灵敏度高的抗体,而要实现这一目标,前提条件就是设计并合成出合适的敌草胺半抗原。但是,目前尚未有关于敌草胺半抗原合成的相关报道。
因此,亟需设计和开发出一种合适的敌草胺半抗原,并由此建立相应的敌草胺快速检测方法,实现通过免疫学方法来快速检测农产品中的敌草胺。
发明内容
本发明的目的在于提供了敌草胺半抗原、抗原、抗体、检测装置及其制备和检测方法,针对检测农产品中残留的敌草胺。
根据本发明的一个方面,提供了敌草胺半抗原,其结构如式(Ⅰ)所示:
(Ⅰ)。
根据本发明的另一个方面,提供了制备敌草胺半抗原的方法,包括以下步骤:
S1. 将敌草胺与浓硝酸在冰乙酸中发生硝化反应,得到第一中间体,第一中间体的结构式如式(Ⅱ)所示:
(Ⅱ) ;
S2. 第一中间体与氢气在催化剂催化下发生还原反应,得到氨基敌草胺即第二中间体,第二中间体的结构式如式(III)所示:
(III);
S3. 第二中间体与丁二酸酐在吡啶中发生反应,得到敌草胺半抗原,其结构式如式(Ⅰ)所示。
在一些实施方式中,步骤S1中敌草胺与浓硝酸的摩尔比为1:1,步骤S2中催化剂为醋酸/锌粉混合物或钯碳中的一种。
在一些实施方式中,骤S3中第二中间体与丁二酸酐的摩尔比为1:(1-2)。
根据本发明的再一个方面,敌草胺抗原为敌草胺半抗原与载体蛋白的偶联物,载体蛋白为牛血清白蛋白、人血清白蛋白、鸡卵清白蛋白或血蓝蛋白。
根据本发明的第四个方面,提供了一种用于检测敌草胺的组合物,包括
敌草胺免疫用抗原和敌草胺包被用抗原,敌草胺免疫用抗原为敌草胺半抗原与牛血清蛋白的偶联物,敌草胺包被用抗原为敌草胺半抗原与鸡卵清白蛋白的偶联物。
根据本发明的第五个方面,提供了敌草胺半抗原或敌草胺抗原在敌草胺的免疫学检测中的应用。
根据本发明的第六个方面,提供了敌草胺抗体,敌草胺抗体是由敌草胺抗原经动物免疫制备得到,敌草胺抗体为敌草胺单克隆抗体。
根据本发明的第七个方面,提供了敌草胺检测装置,包括试纸条和反应杯,试纸条包括反应膜,反应膜设有检测区和质控区,检测区包被有敌草胺抗原,反应杯中含有胶体金标记的敌草胺抗体。
根据本发明的第八个方面,提供了一种检测样品中敌草胺的方法,方法为使用敌草胺检测装置对样品中的敌草胺进行检测,样品为农产品。
本发明的有益效果:本发明提供的敌草胺半抗原的制备方法,使用的化学试剂容易得到、操作过程简单、合成步骤简洁有效、产率较高、并且检测成本较低。本发明利用层析式免疫胶体金原理,通过试纸条中检测线与质控线之间的比色来定性检测样品中敌草胺的含量,应用时不需要使用液相色谱或质谱等大型仪器,达到快速检测的目的。与现有技术相比,本发明提供的检测方法具有灵敏度高、特异性强、成本低、操作简单、检测时间短等优点。本发明提供由敌草胺半抗原偶联载体蛋白制备的人工抗原,以及免疫实验动物后使其机体产生的特异性针对抗敌草胺的抗体,效价高,对敌草胺的最低检测限为2.4 ng/mL、IC50值为11.5 ng/mL,符合最新颁布的GB 2763-2021《食品安全国家标准 食品中最大农药残留限量》中敌草胺的限量规定。因此,本发明可应用于对农产品中敌草胺的残留含量的快速检测。
附图说明
图1为本发明的一种实施方案的敌草胺半抗原的合成路线图。
图2为本发明的一种实施方案的敌草胺半抗原的质谱图。
图3为实施例2的敌草胺半抗原、敌草胺免疫用抗原(敌草胺半抗原-BSA)以及BSA紫外扫描图。
图4为实施例2的敌草胺半抗原、敌草胺包被用抗原(敌草胺半抗原-OVA)以及OVA紫外扫描图。
图5为本发明的一种实施方案的基于敌草胺单克隆抗体建立的间接竞争ELISA标准曲线。
图6为本发明的一种实施方案的敌草胺检测装置的试纸条的剖面结构示意图。
图7为本发明的一种实施方案的敌草胺检测装置的微孔反应杯的结构示意图。
具体实施方式
通过具体的实施案例对本发明作进一步详细的描述,应理解这些实施例仅用于说明本发明而不用于限制本发明的保护范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定。若无特殊说明,本发明的所有原料和试剂均为常规市场可购买的原料、试剂。
实施例1 敌草胺半抗原的制备方法,包括以下步骤:
S1.向100mL干净单口瓶中依次加入敌草胺原药5.1g和乙酸20mL,然后滴加浓硝酸2mL,滴毕后反应3~4h,将反应液旋蒸,然后加10mL水,用乙酸乙酯萃取两遍,合并有机相过柱纯化,得黄色油状液体,即第一中间体,第一中间体得率为85.6%,第一中间体的结构式如式(II)所示;
S2. 向100mL干净单口瓶中依次加入第一中间体1 2.3g、甲醇20mL、钯碳0.1g,通入H2,室温反应2~3h,TLC检测反应完成后处理反应液,将反应液过滤,旋蒸过柱,得棕色油状液体产物,即第二中间体,第二中间体得率为95.4%,第二中间体的结构式如式(III)所示;
S3. 向100mL干净单口瓶中依次加入产物2500mg、丁二酸酐150mg、吡啶20mL溶解,100℃搅拌反应过夜,TLC检测反应完成后处理反应液,将反应液旋蒸,然后加10mL水,用乙酸乙酯萃取两遍,合并有机相过柱纯化,得敌草胺半抗原0.55g,得率为81.5%。本实施例中敌草胺半抗原的合成流程图见说明书附图1。
采用质谱法鉴定制备得到的敌草胺半抗原,所得到的质谱图见说明书附图2。从质谱图可以看出,该敌草胺半抗原的分子离子峰为EI-MS(negative)m/z:385.30[M- H]-,且为最高峰,与该敌草胺半抗原的分子量386.18相符,表明成功合成了式(Ⅰ)所示的敌草胺半抗原。EI-MS是一种软离子化手段,它一般不会造成化合物直接得失电子或者碎裂。通常它会使一个化合物M被质子化,故负离子模式下,使得化合物失去一个质子,故出峰为[M-H]-。
实施例2 敌草胺免疫用抗原的制备
2.1 敌草胺免疫用抗原的制备方法
利用实施例1制备得到的敌草胺半抗原,通过活泼酯法偶联牛血清白蛋白(BSA)制备敌草胺免疫用抗原,其方法具体如下:
称量50mg敌草胺半抗原,溶于2.5mLDMF中,加入25mg NHS、30mg EDC.HCl,室温反应6h,制备得到活化液;取45mg BSA溶于3ml 0.1M PH9.0的硼酸缓冲液,加入1mLDMF,0.6mL上述活化液,室温反应4h后用PBS(0.01 mol/L,pH =7.4的磷酸盐缓冲液)透析,每4 h换液1次,换液7~ 8 次,透析后4000转/min离心5min,取上清液,制备得到敌草胺免疫用抗原,即敌草胺半抗原-BSA偶联物,于-20℃保存。
2.2 敌草胺免疫用抗原的鉴定
在190~400 nm的紫外光下,对载体蛋白牛血清白蛋白(BSA)、敌草胺半抗原及敌草胺免疫用抗原进行紫外扫描测定。
2.3鉴定结果
结果如图3所示,从图3可以看出完全抗原的紫外特征吸收峰相对于敌草胺半抗原和载体蛋白(BSA)都有不同程度的偏移,且发现敌草胺免疫用抗原同时具备敌草胺半抗原和BSA的特征吸收峰,说明敌草胺半抗原与BSA偶联成功,成功制备得到敌草胺免疫用抗原,其结构式如下式(V)所示:
式(V)。
实施例3 敌草胺包被用抗原的制备
3.1敌草胺包被用抗原的制备方法
利用实施例1制备得到的敌草胺半抗原,通过重氮化法偶联鸡卵清白蛋白(OVA)制备敌草胺包被用抗原,其方法具体如下:
称取10.5 mg敌草胺第二中间体,加入1mL蒸馏水,0.115mL1N HCl,冰水浴加入38μL 50mg/mL亚硝酸钠,冰水中30min,得到重氮液;将12mg OVA溶于2mL 0.1M pH 9.5 碳酸钠缓冲液,滴入上述重氮液,再反应3h,加入3mL蒸馏水,用PBS透析2至3天,离心,制备得到敌草胺包被用抗原,即敌草胺半抗原-OVA偶联物,于-20℃保存备用。
3.2 敌草胺包被用抗原的鉴定
在190~400 nm的紫外光下,对载体蛋白鸡卵清白蛋白(OVA)、敌草胺半抗原及敌草胺包被用抗原进行紫外扫描测定。
3.3 鉴定结果
鉴定结果如附图4所示,从附图4可以看出敌草胺包被用抗原的紫外特征吸收峰,相对于敌草胺半抗原和载体蛋白(OVA)都有不同程度的偏移,且发现敌草胺包被用抗原同时具备敌草胺半抗原和OVA的特征吸收峰,说明敌草胺半抗原与OVA偶联成功,成功制备得到敌草胺包被用抗原,其结构式如下式(VI)所示:
式(VI)。
实施例4 敌草胺单克隆抗体的制备、纯化、鉴定以及效价和抑制率测定
4.1 小鼠的免疫:
选择健康的6~8周龄的BALB/c小鼠进行免疫,将实施例2中得到的敌草胺免疫用抗原与等量弗氏佐剂混合乳化后,对BALB/c小鼠进行颈背部皮下多点注射免疫(冲刺免疫除外)。首次免疫用完全弗氏佐剂,剂量为180μg/只;时隔4周后进行加强免疫,剂量为90μg/只,用不完全弗氏佐剂混合乳化,之后多次的加强免疫时间间隔3周;冲刺免疫时剂量再减半,为45μg/只,完全抗原用生理盐水稀释,对小鼠进行腹腔注射。小鼠第三次免疫后可进行断尾采血检测,通过间接竞争酶联免疫法(ic-ELISA)检测小鼠血清的效价和IC50,选择效价高、IC50低的小鼠进行融合;
4.2 细胞融合:
取免疫BALB/c小鼠脾细胞,按10:1的数量配比比例与SP2/0骨髓瘤细胞融合,筛选得到稳定分泌敌草胺单克隆抗体的敌草胺单克隆杂交瘤细胞株;
4.3 细胞冻存与复苏:
将敌草胺单克隆杂交瘤细胞用冻存液制成5×106个/mL的细胞悬液,在液氮中长期保存。复苏时取出冻存管,立即放入37℃水浴中速融,离心去除冻存液后,移入培养瓶内培养;
4.4 单克隆抗体的制备与纯化:
增量培养法:将敌草胺单克隆杂交瘤细胞置于细胞培养基中,在37℃条件下进行培养,用辛酸-饱和硫酸铵法将得到的培养液进行纯化,得到敌草胺单克隆抗体,-20℃保存。其中,细胞培养基为向RPMI-1640培养基中添加小牛血清和碳酸氢钠,使小牛血清在细胞培养基中的质量百分含量为20%,使碳酸氢钠在细胞培养基中的质量百分含量为0.2%,细胞培养基的pH为7.4。
4.5 利用间接竞争ELISA测定敌草胺抗体的效价和抑制率
具体方法为:
(1)包被:用包被液稀释包被原到1μg/mL,100μL/孔,37℃水浴中包被过夜;
(2)封闭:弃去包被液,洗板2次,吸水纸上拍干,每孔加120μL封闭液,37℃水浴孵育封闭3 h,甩干,37℃烘箱倒置1 h烘干;
(3)加抗体及药物:敌草胺单克隆抗体E6用PBS稀释1K(1000)、2K、4K、8K、16K、32K、64K等倍数,敌草胺标准品用PBS稀释至10 ng/mL,备用;
效价列:每孔先加入50μL PBS缓冲液,后将倍比稀释好的单克隆抗体E6按50μL/孔依次加入孔内,最后一个孔加PBS作空白对照;
抑制列:每孔先加入50μL经PBS缓冲液稀释的药物,后将倍比稀释好的单克隆抗体E6按50μL/孔依次加入孔内,最后一个孔加PBS作空白对照;
37 ℃温育40 min,洗板5次;
(4)加二抗:加入经PBST缓冲液稀释5000倍的羊抗鼠二抗(100μL/孔),温育37℃30 min,洗板5次;
(5)显色:TMB底物缓冲液A、B液等体积混合得到底物液,加入底物液(100μL/孔),37℃孵育10 min;
(6)终止:在酶标板上加入10% H2SO4的终止液(50μL/孔)终止反应;
(7)读数:在波长450 nm条件下用酶标仪读取吸光值(OD),选择吸光度值在1.0~1.5区间内的抗体稀释倍数为抗体效价,敌草胺抗体的药物识别性能由其抑制率得出,抑制率的计算方式如下式1所示:
(式1)。
表1 抗血清效价和抑制率
从表1可知,在20ng的敌草胺药物浓度下,表现出较高效价为15000,此时抑制率为85%。说明了该血清的灵敏度高,效价好,可以进一步研制高效价,高灵敏,高特异的敌草胺的检测方法。
实施例5 敌草胺抗体的IC50及最低检测限的测定
5.1测定方法包括以下步骤:
(1)将实施例3制备的敌草胺包被用抗原,用碳酸盐缓冲液(CB,0.1M pH=9.8)稀释至4μg/mL,包被96孔酶标板,每孔加入100μL,37℃孵育过夜(12 h),倾去孔中液体,用洗涤液洗涤2次,每次30 s,拍干,然后在每孔中加入200μL封闭液,37℃避光孵育2 h,倾去孔内液体拍干,干燥后用铝膜真空密封保存;
(2)弃去包被液,洗涤两次,拍干;
(3)每孔加入120μL封闭液(即质量比5%脱脂奶粉),37℃封闭3 h;
(4)弃去封闭液,拍板,37℃烘干30 min后取出,用自封袋装好备用;
(5)用磷酸盐缓冲液(PBS,0.01M,pH=7.4)以1:15000倍稀释实施例4制备的敌草胺单克隆抗体,并将待检测的敌草胺药物以4倍梯度稀释至 1000μg/L、250μg/L、62.50μg/L、15.63μg/L、3.91μg/L、0.98μg/L、0.25μg/L;
(6)每行加入50μL待检测敌草胺药物稀释液(三组平行),再按照加入50μL/孔的添加比例,加入敌草胺单克隆抗体的稀释液,37℃孵育40 min,洗涤五次,拍干;
(7)加入经磷酸吐温缓冲液(PBST,0.01M)以5000倍稀释后的羊抗鼠二抗-HRP,100μL/孔,37℃孵育30 min,洗涤五次,拍干;
(8)加入显色液,每孔100μL,显色10 min;
(9)加入50μL 10%的H2SO4溶液终止反应,并在450 nm处读取OD值;
(10)按上述步骤(1)到(9)进行操作,将步骤(6)中待测敌草胺稀释液替换成经过提取的待测样品的稀释液,结合所绘制的标曲,即可测定未知样品中实际敌草胺药物的含量。
5.2 实验结果
用于检测敌草胺药物的抗体间接竞争ELISA标准曲线如附图5所示,从图5可知,敌草胺抗体的半抑制浓度(IC50)为11.5ng/ml,对敌草胺的最低检测限为2.4ng/ml,说明本发明制备得到的敌草胺抗体灵敏度高,可以满足检测要求。
实施例6 敌草胺抗体的特异性实验及交叉反应
本试验利用icELISA程序,在已经优化好的icELISA条件下,选择几种与敌草胺结构相似的类似物,考察实施例4制备的敌草胺单克隆抗体的特异性。依次作敌草胺的类似物竞争抑制曲线,求出各自抑制率为50%时的标准品质量浓度,然后再换算成摩尔浓度后用公式计算出每种结构类似物与抗体的交叉反应率,见下表2,每个处理设置3个重复。
表2 敌草胺抗体与敌草胺结构类似物的交叉反应率结果表
从表2可以看出该敌草胺单克隆抗体与敌草胺结构类似物没有交叉反应,说明该抗体对敌草胺具有高度特异性,能够对敌草胺进行专一检测,避免样品中含有的敌草胺结构类似物产生干扰。
实施例7 敌草胺检测装置的制备
7.1 胶体金溶液的制备
用双蒸去离子水将质量分数1%的氯金酸溶液稀释成0.01%(质量分数),取100mL0.01%的氯金酸溶液置于锥形瓶中,用恒温电磁搅拌器加热至沸腾,在持续高温、持续搅拌下加入2.0 mL1%柠檬酸三钠溶液,继续匀速搅拌加热至溶液呈透亮的红色时停止,冷却至室温后用去离子水恢复到原体积,得到胶体金溶液,4℃保存。制备好的胶体金溶液外观纯净、透亮、无沉淀和漂浮物;
7.2 敌草胺单克隆抗体-胶体金标记物的制备
在磁力搅拌下,用0.2mol/L碳酸钾调胶体金溶液的pH值至7.2,按每毫升胶体金溶液中加入20~60μg敌草胺单克隆抗体的标准,向胶体金溶液中加入实施例4制备得到的敌草胺单克隆抗体,继续搅拌混匀30min,静置10min后,加入10%牛血清白蛋白(BSA)溶液,使其在胶体金溶液中的体积百分含量为1%,静置10min。以12000rpm,在4℃条件下离心40min,弃上清液,用体积为初始胶体金溶液体积1/10的复溶缓冲液将沉淀重悬,制备得到敌草胺单克隆抗体-胶体金标记物,置于4℃保存备用;
复溶缓冲液:含体积百分含量0.3%~0.5%牛血清白蛋白、质量百分含量0.1%~0.3%吐温-20、质量百分含量3%~6%海藻糖,pH= 7.2的0.02mol/L磷酸盐缓冲液;
7.3微孔反应杯的制备
向微孔反应杯中加入100μL敌草胺单克隆抗体-胶体金标记物,放入冷冻干燥机中,在冷阱温度为-50℃条件下,预冻3h后,再真空干燥6h,即可取出,得到冻干有敌草胺单克隆抗体-胶体金标记物的微孔反应杯,密封保存,其中敌草胺单克隆抗体-胶体金标记物冻干量为0.20~0.50μg/ml;
7.4 样品吸收垫的制备
将样品吸收垫置于含牛血清白蛋白的0.02mol/L的磷酸盐缓冲液中浸泡2h,50℃烘干2h备用。0.02mol/L的磷酸盐缓冲液的pH为7.2,其中牛血清白蛋白的体积百分含量为1.0%;
7.5 反应膜的制备
包被过程:用磷酸缓冲液分别将敌草胺包被用抗原稀释到浓度为10mg/ml,用金标喷金点膜仪将其包被于硝酸纤维素膜上的检测区(T区),包被浓度为0.5 mg/ml;用浓度0.01mol/L,pH= 7.4的磷酸盐缓冲液将羊抗鼠抗体浓度稀释到10mg/ml,用金标喷金点膜仪将其包被于硝酸纤维素膜上的质控区(C区),包被浓度为1.0 mg/ml。将包被好的反应膜置于50℃条件下干燥6h,干燥完毕后,放入4℃干燥间作为生产备用。
7.6敌草胺检测装置的组装
7.6.1 试纸条的组装
将样品吸收垫、反应膜、吸水垫依次按顺序粘贴在底板上,其中,底板为PVC底板,样品吸收垫为吸滤纸,吸水垫为吸水滤纸,反应膜为硝酸纤维素膜。样品吸收垫的末端与反应膜的始端相连,反应膜的末端与吸水垫的始端相连,样品吸收垫的始端与底板的始端对齐,吸水垫的末端与底板的末端对齐。
7.6.2 试纸盒的组装
将上述步骤7.6.1得到的试纸条与上述步骤7.3得到的微孔反应杯组装成试纸盒,在2-8℃的环境中贮存。
实施例8 一种检测样品中敌草胺的方法
8.1 样品提取液的配制
准确称取氯化钠1.16g、氯化钾1.49 g、磷酸氢二钠 2.84 g、磷酸二氢钾2.72 g、0.3-0.5ml吐温-20、1-3ml甲醇,用水溶解并定容至100 ml,即为样品提取液,样品提取液为0.2mmol/L磷酸盐缓冲溶液,pH值为9.8。
8.2 样品前处理
称取2.0g±0.01g均质的蔬菜样品或水果样品至50mL聚苯乙稀离心管中,加入上述样品提取液8mL,混匀,涡震荡旋30s以上,室温(20-25℃)3000r/min离心2min,取上清液为待测液。
8.3 测定步骤
吸取200μl上述待测液于微孔反应杯中,上下抽吸5~10次使混合均匀。室温温育3min,将试纸条插入到反应杯中,室温温育3min,取出试纸条,轻轻刮去试纸条下端的样品垫,并进行结果判读。
8.4 结果判定
通过对比控制线(C线)和检测线(T线)的颜色深浅进行结果判定。
阳性:当质控区(C)显示出条带,检测区(T)不显色判为阳性,即样品中有敌草胺,用“ + ”表示;
阴性:当质控区和检测区均显示出条带,判为阴性,即样品中没有敌草胺,用“-”表示;
无效:当质控区(C)不显示条带,试纸失效。
实施例9 敌草胺检测装置的灵敏度和假阴性率
选取经SN/T 5442-2022 《出口植物源食品中敌草胺及其代谢物残留量的测定 液相色谱-质谱/质谱法》测试不含敌草胺的马铃薯、玉米笋和蓝莓作为空白样品,依据《GB2763-2021》中规定马铃薯、玉米笋、蓝莓最高残留限量(MRL)分别为0.02mg/kg,0.02mg/kg和1.5 mg/kg,因此设定马铃薯和玉米笋的方法检出限为0.02 mg/kg,蓝莓的方法检出限为1.5 mg/kg,即关注浓度。添加水平分别为1倍关注浓度、2倍关注浓度,考察灵敏度和假阴性率。两个添加浓度水平的样品,每个浓度水平各50 份试样,按实施例8中检测方法进行检测,检测结果见下表3。
表3 敌草胺检测装置的灵敏度和假阴性率检测结果
由表3可知,本实施例中的检测方法对样品中敌草胺残留的检测灵敏度≥95%,假阴性率为≤5%。
实施例10 敌草胺检测装置的特异性和假阳性
选用马铃薯、玉米笋和蓝莓的空白样品,采用空白基质加标的方式,制备成2个浓度水平(0.5倍检测限、空白基质)的样品各50份。采用实施例8中检测方法对样品进行检测,检测结果见下表4。
表4 敌草胺检测装置的特异性和假阳性检测结果
由表4可知,本实施例中的检测方法的特异性≥90%,假阳性率为≤10%。上述结果表明本发明的检测敌草胺的检测装置具有良好的特异性,能够准确地检测出蔬菜、水果样品中的敌草胺,因此可以对蔬菜、水果样品中的敌草胺残留进行快速检测。
以上所述的仅是本发明的一些实施方式,对于本领域的普通技术人员来说,在不脱离本发明创造构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (7)
1.敌草胺半抗原,其特征在于,其结构如式(Ⅰ)所示:
(Ⅰ)。
2.制备权利要求1所述的敌草胺半抗原的方法,其特征在于,包括以下步骤:
S1. 将敌草胺与浓硝酸在冰乙酸中发生硝化反应,得到第一中间体,所述第一中间体的结构式如式(Ⅱ)所示:
(Ⅱ) ;
S2. 所述第一中间体与氢气在催化剂催化下发生还原反应,得到氨基敌草胺即第二中间体,所述第二中间体的结构式如式(III)所示:
(III);
S3. 所述第二中间体与丁二酸酐在吡啶中发生反应,得到敌草胺半抗原,其结构式如式(Ⅰ)所示。
3.根据权利要求2所述的方法,其特征在于,所述步骤S1中敌草胺与浓硝酸的摩尔比为1:1,所述步骤S2中催化剂为醋酸/锌粉混合物或钯碳中的一种。
4.根据权利要求2所述的方法,其特征在于,所述步骤S3中第二中间体与丁二酸酐的摩尔比为1:(1-2)。
5.敌草胺抗原,其特征在于,所述敌草胺抗原为权利要求1所述的敌草胺半抗原与载体蛋白的偶联物,所述载体蛋白为牛血清白蛋白、人血清白蛋白、鸡卵清白蛋白或血蓝蛋白。
6.一种用于检测敌草胺的组合物,其特征在于,所述组合物包括敌草胺免疫用抗原和敌草胺包被用抗原,所述敌草胺免疫用抗原为权利要求1所述的敌草胺半抗原与牛血清蛋白的偶联物,所述敌草胺包被用抗原为权利要求1所述的敌草胺半抗原与鸡卵清白蛋白的偶联物。
7.权利要求1所述的敌草胺半抗原或权利要求5所述的敌草胺抗原在敌草胺的免疫学检测中的非疾病诊断的应用。
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