CN111825713A - 一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原、完全抗原制备方法及其应用 - Google Patents
一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原、完全抗原制备方法及其应用 Download PDFInfo
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Abstract
本发明提供一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原、完全抗原制备方法及其应用。以1份5‑羟基‑2‑硝基苯甲酸甲酯溶解在3‑4份乙腈中,加入2‑3份无水碳酸钠和1.2份氯硫代磷酸二乙酯制得中间体;将中间体溶于3‑4份四氢呋喃,加入等体积的6mol/L盐酸,制得化合物,即二乙氧基硫代磷酸酯类有机磷农药半抗原。将半抗原与载体蛋白偶联,制备得到完全抗原。本发明的制备方法简单易行,制备得到的二乙氧基硫代磷酸酯类有机磷农药半抗原、二乙氧基硫代磷酸酯类有机磷农完全抗原,可有效地刺激被免疫动物产生灵敏度高、广谱性强的抗体,可用于免疫分析中,满足国内对部分二乙氧基硫代磷酸酯类有机磷农药残留的检测。
Description
技术领域
本发明属于生物技术领域,具体涉及一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原、完全抗原制备方法及其应用。
背景技术
二乙氧基硫代磷酸酯类有机磷农药(DDPs)是通过抑制乙酰胆碱酯酶活性,干扰昆虫神经冲动进而达到杀虫的目的是一类杀虫剂,其具有高效、广谱、成本低廉的特点,曾被广泛使用于农业生产中。尽管现在已经明令禁止使用该类农药的一些剧毒产品,如对硫磷、甲基对硫磷等,此外,蔬菜、果树、茶叶、中草药材上不得使用和限用甲拌磷、治螟磷、蝇毒磷等农药,但高毒农药因过去长期使用及现在低毒农药的普遍,导致环境中二乙氧基硫代磷酸酯类有机磷农药仍有残留。因此,建立针对该类农药的快速检测方法对于监测环境、检测食用蔬果、粮食、中草药中残留的二乙氧基硫代磷酸酯类有机磷农药具有重要意义。
目前,对二乙氧基硫代磷酸酯类有机磷农药的检测方法以配置不同检测器的气相色谱(GC)和高效液相色谱(HPLC)等为主,但色谱方法操作复杂、费时、成本高且通常需要进行繁琐的样品前处理,难以满足快速经济现场实时检测和样品大批量筛选检测的要求,酶抑制检测法是市场上检测二乙氧基硫代磷酸酯类有机磷农药普遍使用的快速检验方法,但该方法准确度大约为30%~40%之间,而酶联免疫检测法是基于抗原-抗体特异反应的免疫分析方法,具有特异性强、灵敏度高、通常不需复杂的样品前处理、操作简便且成本低廉的特点,同时,基于该方法研发的胶体金试纸条可用于现场快速筛选检测。农民使用农药时通常混合使用,造成一个样品中可能残留多种农药,因此制备具有高交叉反应率的抗体,可以实现多个农药的广谱特异性免疫检测。而优秀的半抗原、抗原可以激发被免疫动物产生灵敏度高、识别能力强的抗体,因此本发明设计一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原、完全抗原,并提供其制备方法及相应应用,旨在为快速检测环境、食品中的二乙氧基硫代磷酸酯类有机磷农药奠定基础。
发明内容
本发明的一个目的在于提供一种针对二乙氧基硫代磷酸酯类有机磷农药的半抗原以及完全抗原,可有效地刺激被免疫动物产生灵敏度高、广谱性的抗体。
本发明的另一个目的在于提供上述二乙氧基硫代磷酸酯类有机磷农药半抗原以及完全抗原的制备方法,该方法简单易行。
本发明制备得到的二乙氧基硫代磷酸酯类有机磷农药半抗原、完全抗原可用于筛选广谱性识别二乙氧基硫代磷酸酯类有机磷农药的单克隆抗体,利用所制得抗体可建立二乙氧基硫代磷酸酯类有机磷农药的酶联免疫分析方法,应用于食品中可能残留的二乙氧基硫代磷酸酯类有机磷农药的快速检测。
本发明所提供的二乙氧基硫代磷酸酯类有机磷农药半抗原,其分子式为:
上述半抗原通过包括如下步骤的方法制备得到:
1)使得5-羟基-2-硝基苯甲酸甲酯与氯硫代磷酸二乙酯发生反应,得到中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯;
2)使得中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯在酸性条件下水解,得到5-((二乙氧基磷硫酰硫基)氧基)-2-硝基苯甲酸,即二乙氧基硫代磷酸酯类有机磷农药半抗原。
上述方法步骤1)中,所述反应可在无水碳酸钾存在下进行;
5-羟基-2-硝基苯甲酸甲酯与无水碳酸钾的摩尔比可为:1:2-3;
5-羟基-2-硝基苯甲酸甲酯与氯硫代磷酸二乙酯的摩尔比可为:1:1-1.5,具体可为1:1.2;
所述反应在加热回流条件下进行,所述反应的温度可为80℃,时间可为2~3小时;
所述反应在有机溶剂中进行,所述有机溶剂具体可为乙腈;
所述反应完成后,待反应体系冷却至室温,加入水淬灭反应,接着加入二氯甲烷萃取分层,收集有机层干燥浓缩后柱色谱分离得到中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯。
上述方法步骤2)中,所述水解在有机溶剂中进行,所述有机溶剂具体可为四氢呋喃;
所述水解在室温下进行,所述水解的时间可为4-12小时,具体可为12小时;
所述水解的具体操作为:将1份(摩尔份数)中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯溶于3-4份四氢呋喃,加入等体积的6mol/L盐酸,反应体系室温搅拌12小时,加入20份水淬灭反应,接着加入20份乙酸乙酯萃取分层,收集有机层干燥浓缩后柱色谱分离得到化合物:5-((二乙氧基磷硫酰硫基)氧基)-2-硝基苯甲酸,即二乙氧基硫代磷酸酯类有机磷农药半抗原。
本发明所提供的二乙氧基硫代磷酸酯类有机磷农药完全抗原,其结构式如下所示:
Pr表示偶联的载体蛋白。
上述二乙氧基硫代磷酸酯类有机磷农药完全抗原是由所述二乙氧基硫代磷酸酯类有机磷农药半抗原与载体蛋白偶联后得到的。
本发明还提供所述完全抗原的制备方法,即,通过碳二亚胺或混合酸酐法使所述二乙氧基硫代磷酸酯类有机磷农药半抗原与载体蛋白偶联制得完全抗原。
具体地,采用活化酯法将载体蛋白偶联于所述半抗原的羧基上。
所述制备完全抗原的具体操作如下:
1)将二乙氧基硫代磷酸酯类有机磷农药半抗原溶于二甲基甲酰胺中,加入二环己基碳二亚胺和N-羟基琥珀酰亚胺,室温搅拌得到A液;
2)将载体蛋白溶于碳酸盐缓冲液中,得到B液;
3)将上述A液滴加到B液中,搅拌,得到C液;
4)将C液置于透析袋,并于磷酸缓冲液中透析,收集透析袋中的溶液,即为二乙氧基硫代磷酸酯类有机磷农药完全抗原。
上述方法步骤1)中,二乙氧基硫代磷酸酯类有机磷农药半抗原、二甲基甲酰胺、二环己基碳二亚胺和N-羟基琥珀酰亚胺的用量比依次可为:20μmol∶1-2mL∶60-120μmol∶60-120μmol;具体可为:20μmol∶1mL∶60μmol∶60μmol;
所述室温搅拌的时间可为:18-24小时;
步骤2)中,所述载体蛋白具体可为牛血清蛋白BSA,制备完全免疫抗原;
所述载体蛋白为卵血清白蛋白OVA,制备完全包被抗原;
载体蛋白和碳酸盐缓冲液的用量比可为:0.4μmol∶3-6mL,具体可为:0.4μmol∶3mL;
二乙氧基硫代磷酸酯类有机磷农药半抗原与载体蛋白的摩尔比可为:20:1-40:1,具体可为20:1;
步骤3)中,所述搅拌在4-25℃(具体可为4℃)温度下搅拌10-12小时(具体可为10小时);
步骤4)中,所述透析过程中,每3小时换液一次,进行多次透析,具体可进行6次透析。
本发明还提供所述二乙氧基硫代磷酸酯类有机磷农药半抗原或所述二乙氧基硫代磷酸酯类有机磷农药完全抗原在制备二乙氧基硫代磷酸酯类有机磷农药的广谱性抗体中的应用。
本发明的再一目的是提供由上述二乙氧基硫代磷酸酯类有机磷农药完全抗原制备的广谱性抗体。
所述广谱性抗体包括多克隆抗体、单克隆抗体、重链抗体和重组抗体。
所述广谱性抗体的以下任一应用:
1)在检测二乙氧基硫代磷酸酯类有机磷农药中的应用;
2)在制备二乙氧基硫代磷酸酯类有机磷农药的ELISA检测用试剂盒中的应用;
3)在制备二乙氧基硫代磷酸酯类有机磷农药的胶体金检测试纸条中的应用。
本发明还提供由所述广谱性抗体制备的二乙氧基硫代磷酸酯类有机磷农药检测试剂或试剂盒。
本发明采用以上技术方案,以1份5-羟基-2-硝基苯甲酸甲酯溶解在3-4份乙腈中,加入2-3份无水碳酸钠和1.2份氯硫代磷酸二乙酯制得中间体:5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯。将中间体溶于3-4份四氢呋喃,加入等体积的6mol/L盐酸,制得化合物:5-((二乙氧基磷硫酰硫基)氧基)-2-硝基苯甲酸,即二乙氧基硫代磷酸酯类有机磷农药半抗原。将半抗原与载体蛋白偶联,制备得到完全抗原。本发明的制备方法简单易行,制备得到的二乙氧基硫代磷酸酯类有机磷农药半抗原、二乙氧基硫代磷酸酯类有机磷农药完全抗原,可有效地刺激被免疫动物产生灵敏度高、广谱性强的抗体,可用于免疫分析中,满足国内对二乙氧基硫代磷酸酯类有机磷农药残留的检测。
附图说明
图1为本发明制备得到的完全抗原PA-BSA(左)、PA-OVA(右)的紫外吸收图谱。
具体实施方式
下面通过具体实施例对本发明进行说明,但本发明并不局限于此。
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试剂、生物材料等,如无特殊说明,均可从商业途径得到。
实施例1
一种二乙氧基硫代磷酸酯类有机磷农药半抗原的制备方法,包括以下步骤:
1)将1份5-羟基-2-硝基苯甲酸甲酯溶解在3-4份乙腈中,加入2份无水碳酸钠和1.2份氯硫代磷酸二乙酯,反应体系80℃加热回流2小时,待反应体系冷却至室温,加入20份水淬灭反应,接着加入20份二氯甲烷萃取分层,收集有机层干燥浓缩后柱色谱分离得到中间体:5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯。
2)将1份5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯溶于3份四氢呋喃,加入等体积的6mol/L盐酸,反应体系室温搅拌12小时,加入20份水淬灭反应,接着加入20份乙酸乙酯萃取分层,收集有机层干燥浓缩后柱色谱分离得到化合物:5-((二乙氧基磷硫酰硫基)氧基)-2-硝基苯甲酸,即二乙氧基硫代磷酸酯类有机磷农药半抗原。
实施例2
以实施例1的二乙氧基硫代磷酸酯类有机磷农药半抗原制备二乙氧基硫代磷酸酯类有机磷农药完全抗原,包括以下步骤:
1)将二乙氧基硫代磷酸酯类有机磷农药半抗原溶于二甲基甲酰胺中,加入二环己基碳二亚胺和N-羟基琥珀酰亚胺,室温搅拌24小时得到A液;其中,二乙氧基硫代磷酸酯类有机磷农药半抗原、二甲基甲酰胺、二环己基碳二亚胺和N-羟基琥珀酰亚胺的用量比为20umol∶1mL∶60umol∶60umol;
2)将载体蛋白溶于碳酸盐缓冲液中,得到B液;其中,载体蛋白和碳酸盐缓冲液的用量比为0.4umol∶3mL;
3)将上述A液滴加到B液中并于4℃搅拌12小时,得到C液;
4)将C液置于透析袋,并于磷酸缓冲液中透析,每3小时换液一次,共透析6次,收集透析袋中的溶液,即为二乙氧基硫代磷酸酯类有机磷农药完全抗原。
所述载体蛋白为牛血清蛋白BSA,制备完全免疫抗原;
所述载体蛋白为卵血清蛋白OVA,制备完全包被抗原;
将二乙氧基硫代磷酸酯类有机磷农药半抗原、完全免疫抗原、完全包被抗原、牛血清载体蛋白、卵血清载体蛋白、用0.01mol PBS调整到1mg/mL后,在200-400nm波长下进行紫外吸收光谱扫描鉴定其结构,如图1:二乙氧基硫代磷酸酯类有机磷农药半抗原、载体蛋白(BSA、OVA)的最大吸收峰分别在304nm、292nm和245nm处,合成的免疫抗原、包被抗原的吸收曲线与相应的半抗原和载体蛋白相比均发生明显改变,所得化合物确为目标产物。
实施例3
二乙氧基硫代磷酸酯类有机磷农药完全抗原的免疫活性的鉴定。
用实施例2制备的二乙氧基硫代磷酸酯类有机磷农药完全抗原按表1方案免疫Balb/c小鼠,四免后第7天采眼眶静脉血清进行检测,判断所制备的二乙氧基硫代磷酸酯类有机磷农药抗原是否具有免疫活性。
表1免疫方案
Table1 Immunizing Procotol
1)将实施例2制备的二乙氧基硫代磷酸酯类有机磷农药完全抗原(1mg/mL)按500、1000、2000、4000、8000、16000、32000、64000倍稀释后加入酶标板,见表2,每孔100uL,37℃温育3h后取出,用PBST洗净;
2)向包被的酶标孔对照孔每孔加入50uL的样品稀释液(样品稀释液:500ml pH为7.5的PBS中加入0.1ml Tween-20,0.5g明胶),抑制孔加入50uL浓度为200ng/L的乙基对硫磷标样;取采集的小鼠血清,按表2稀释500、1000、2000、4000、8000、16000倍后加入酶标孔,每孔50uL,将酶标板置于37℃温育0.5h后取出,用PBST洗净;
3)向酶标孔中每孔加入1ug/mL的羊抗小鼠抗体(购买途径:北京中杉金桥生物技术有限公司及商品目录号:111-035-003),每孔100uL,随后将酶标板置于37℃温育0.5h后取出,用PBST洗净后向酶标孔中加入100uL的显色液,室温显色7min后,每孔加入50uL 50%浓硫酸终止反应,读取492nm吸光值;
4)实验结果见表2,可见随抗原、血清的稀释倍数加大,吸光值呈现逐渐下降的规律,小鼠血清效价定义为OD值为1.0时抗血清最大稀释倍数,抑制率=[(对照孔OD值-抑制孔OD值)/对照孔OD值]×100%,从表2可知,小鼠血清效价为4000,当包被抗原(1.0mg/ml)稀释倍数为1:500,血清稀释倍数为1:4000时,血清对乙基对硫磷的抑制效率最高,为71%。同时,选择该免疫小鼠进行融合,筛选所得的单克隆细胞株5H41H9分泌单克隆抗体对乙基对硫磷的抑制反应检测见表3:当包被抗原(1.0mg/ml)稀释倍数为1:2000,抗体稀释倍数为1:1000时,该单克隆抗体对500ng/mL乙基对硫磷抑制率达到90%,选取敌敌畏、杀扑磷、特丁硫磷、水胺硫磷、甲胺磷、马拉硫磷、蝇毒磷、喹硫磷、除线磷、甲拌磷、甲基对硫磷、倍硫磷、杀螟硫磷13种二乙氧基硫代磷酸酯类有机磷农药,考察以该单克隆抗体所建立的间接竞争酶联免疫检测方法(ic-ELISA)的广谱性见表4:单克隆抗体与蝇毒磷、甲拌磷、除线磷、甲基对硫磷、倍硫磷5种二乙氧基硫代磷酸酯类有机磷农药均存在不同程度的识别能力,表明以该单克隆抗体可建立特广谱性针对二乙氧基硫代磷酸酯类有机磷农药的ic-ELISA,同时,也说明该抗体对二乙氧基硫代磷酸酯类有机磷农药具有较好的广谱识别能力和抑制效果。
表2乙基对硫磷对免疫小鼠的血清抑制反应检测
Table 2The titer of the fusion mouse serum and its inhibitionreaction against Ethyl parathio
注:C表示酶标板中的对照孔,I表示酶标板中的抑制孔(200ng/mL乙基对硫磷)
表3抗原抗体最佳工作浓度筛选
Table 3Screening the optimal working concentration of coating andantibody
注:C表示酶标板中的对照孔,I表示酶标板中的抑制孔(500ng/mL乙基对硫磷)
表4单克隆抗体5H41H9对不同二乙氧基硫代磷酸脂类有机磷农药的IC50值
Table 4IC50 of monoclonal antibody 5H41H9 Against Different O,O-Diethyl Phosphorothioate Pesticides
Claims (10)
2.制备权利要求1中所述的二乙氧基硫代磷酸酯类有机磷农药半抗原的方法,包括如下步骤:
1)使得5-羟基-2-硝基苯甲酸甲酯与氯硫代磷酸二乙酯发生反应,得到中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯;
2)使得中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯在酸性条件下水解,得到5-((二乙氧基磷硫酰硫基)氧基)-2-硝基苯甲酸,即二乙氧基硫代磷酸酯类有机磷农药半抗原。
3.根据权利要求2所述的方法,其特征在于:
步骤1)中,所述反应在无水碳酸钾存在下进行;
5-羟基-2-硝基苯甲酸甲酯与无水碳酸钾的摩尔比为:1:2-3;
5-羟基-2-硝基苯甲酸甲酯与氯硫代磷酸二乙酯的摩尔比为:1:1-1.5;
所述反应在加热回流条件下进行,所述反应的温度为80℃,时间为2~3小时;
所述反应在有机溶剂中进行,所述有机溶剂具体可为乙腈;
所述反应完成后,待反应体系冷却至室温,加入水淬灭反应,接着加入二氯甲烷萃取分层,收集有机层干燥浓缩后柱色谱分离得到中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯。
4.根据权利要求2或3所述的方法,其特征在于:步骤2)中,所述水解在有机溶剂中进行,所述有机溶剂具体可为四氢呋喃;
所述水解在室温下进行,所述水解的时间为4-12小时;
所述水解的操作为:将1份摩尔份数中间体5-((二乙氧基磷硫基)氧基)-2-硝基苯甲酸甲酯溶于3-4份四氢呋喃,加入等体积的6mol/L盐酸,反应体系室温搅拌12小时,加入20份水淬灭反应,接着加入20份乙酸乙酯萃取分层,收集有机层干燥浓缩后柱色谱分离得到化合物:5-((二乙氧基磷硫酰硫基)氧基)-2-硝基苯甲酸,即二乙氧基硫代磷酸酯类有机磷农药半抗原。
6.制备权利要求5中二乙氧基硫代磷酸酯类有机磷农药完全抗原的方法,包括如下步骤:
1)将二乙氧基硫代磷酸酯类有机磷农药半抗原溶于二甲基甲酰胺中,加入二环己基碳二亚胺和N-羟基琥珀酰亚胺,室温搅拌得到A液;
2)将载体蛋白溶于碳酸盐缓冲液中,得到B液;
3)将上述A液滴加到B液中,搅拌,得到C液;
4)将C液置于透析袋,并于磷酸缓冲液中透析,收集透析袋中的溶液,即为二乙氧基硫代磷酸酯类有机磷农药完全抗原;
具体地,步骤1)中,二乙氧基硫代磷酸酯类有机磷农药半抗原、二甲基甲酰胺、二环己基碳二亚胺和N-羟基琥珀酰亚胺的用量比依次可为:20μmol∶1-2mL∶60-120μmol∶60-120μmol;所述室温搅拌的时间为:18-24小时;
步骤2)中,所述载体蛋白为牛血清蛋白BSA,制备完全免疫抗原;
所述载体蛋白为卵血清白蛋白OVA,制备完全包被抗原;
载体蛋白和碳酸盐缓冲液的用量比为:0.4μmol∶3-6mL;
二乙氧基硫代磷酸酯类有机磷农药半抗原与载体蛋白的摩尔比为:20:1-40:1,具体可为20:1;
步骤3)中,所述搅拌在4-25℃温度下搅拌10-12小时;
步骤4)中,所述透析过程中,每3小时换液一次,进行多次透析,具体可进行6次透析。
7.权利要求1所述的二乙氧基硫代磷酸酯类有机磷农药半抗原或权利要求5所述的二乙氧基硫代磷酸酯类有机磷农药完全抗原在制备二乙氧基硫代磷酸酯类有机磷农药的广谱性抗体中的应用。
8.由权利要求5所述的二乙氧基硫代磷酸酯类有机磷农药完全抗原制备的广谱性抗体。
9.权利要求8所述的广谱性抗体的以下任一应用:
1)在检测二乙氧基硫代磷酸酯类有机磷农药中的应用;
2)在制备二乙氧基硫代磷酸酯类有机磷农药的ELISA检测用试剂盒中的应用;
3)在制备二乙氧基硫代磷酸酯类有机磷农药的胶体金检测试纸条中的应用。
10.由权利要求8所述的广谱性抗体制备的二乙氧基硫代磷酸酯类有机磷农药检测试剂或试剂盒。
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CN115340986A (zh) * | 2022-09-22 | 2022-11-15 | 江南大学 | 一株分泌甲拌磷单克隆抗体的杂交瘤细胞株及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108593925A (zh) * | 2017-12-28 | 2018-09-28 | 华南农业大学 | 一种基于纳米抗体检测二乙氧基有机磷农药的酶联免疫试剂盒及其使用方法 |
KR20180128293A (ko) * | 2017-05-23 | 2018-12-03 | 주식회사 넥스바이오 | 유기인계 농약의 검출을 위한 단일클론 항체의 제조방법 |
CN111171073A (zh) * | 2020-01-07 | 2020-05-19 | 广西壮族自治区亚热带作物研究所(广西亚热带农产品加工研究所) | 一种对硫磷半抗原、完全抗原的制备方法与应用 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20180128293A (ko) * | 2017-05-23 | 2018-12-03 | 주식회사 넥스바이오 | 유기인계 농약의 검출을 위한 단일클론 항체의 제조방법 |
CN108593925A (zh) * | 2017-12-28 | 2018-09-28 | 华南农业大学 | 一种基于纳米抗体检测二乙氧基有机磷农药的酶联免疫试剂盒及其使用方法 |
CN111171073A (zh) * | 2020-01-07 | 2020-05-19 | 广西壮族自治区亚热带作物研究所(广西亚热带农产品加工研究所) | 一种对硫磷半抗原、完全抗原的制备方法与应用 |
Non-Patent Citations (4)
Title |
---|
PIAO, YUAN ZHE ET AL: "Development of ELISAs for the class-specific determination of organophosphorus pesticides", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
YOSHITAKE, AKIRA ET AL: "Synthesis of 14C-labeled O,O-dimethyl Synthesis of 14C-labeled O,O-dimethyl", 《RADIOISOTOPES》 * |
ZHAO, FENGCHUN ET AL: "Development and comparative study of chemosynthesized antigen and mimotope-based immunoassays for class-specific analysis of O,O-dimethyl organophosphorus pesticides", 《SCIENTIFIC REPORTS》 * |
谢桂勉等: "硫代磷酸二乙酯类农药半抗原设计及抗体识别特性", 《高等学校化学学报》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115340986A (zh) * | 2022-09-22 | 2022-11-15 | 江南大学 | 一株分泌甲拌磷单克隆抗体的杂交瘤细胞株及其应用 |
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