猪肉中2-(三氟甲基)吩噻嗪的Ic-ELISA检测

doi:10.3969/j.issn.1000-1565.2023.06.010

摘要/Abstract

摘要： 为了快速检测猪肉中的镇静剂2-(三氟甲基)吩噻嗪(2-(trifluoromethyl)phenothiazine,TFPTZ),分别用偶氮苯甲酸法、混合酸酐法合成了半抗原、完全抗原,通过免疫新西兰大耳白兔获得了多克隆抗体,采用间接竞争酶联免疫方法(Ic-ELISA)对猪肉中TFPTZ进行了定量分析,实验条件:包被抗原浓度1.25 μg/mL,多克隆抗体稀释16 000倍,37 ℃包被3 h,封闭液为质量分数1%的牛血清白蛋白(BSA),TFPTZ稀释液为含体积分数10%的DMSO的PBS,竞争反应60 min.该方法相关系数R²为0.999 1,IC₅₀为1.500 2 μg/L,最低检测限IC₁₀为0.284 1 μg/L,线性(IC₂₀~IC₈₀)为0.430 7~5.225 2 μg/L,与5种TFPTZ结构类似物的交叉反应率小于1.2%,样品添加回收率为93.26%~109.13%,变异系数小于6%,实际样品检测结果与高效液相色谱法测定结果高度一致,表明建立的快速检测猪肉中TFPTZ的Ic-ELISA方法具有良好的准确度,适用于快速检测猪肉中TFPTZ的残留量.

关键词: 2-(三氟甲基)吩噻嗪, 抗原合成, 间接竞争酶联免疫方法(Ic-ELISA)

Abstract: In order to quickly analyze the sedative 2-(trifluoromethyl)phenothiazine(TFPTZ)in pork, haptens and complete antigens were synthesized using the azobenzoic acid method and the mixed anhydride method, respectively. Polyclonal antibodies were obtained by immunizing New Zealand white rabbits. Indirect competitive enzyme linked immunosorbent assay(Ic-ELISA)was used to quantitatively analyze TFPTZ in pork. The experimental conditions were as follows: the concentration of the coated antigen was 1.25 μg/mL, and the polyclonal antibody was diluted 16 000 times, Coated at 37 ℃ for 3 h, the blocking solution was 1% BSA of mass fraction, and the TFPTZ diluent is PBS of DMSO with a volume fraction of 10%. The competitive reaction lasted 60 minutes. The correlation coefficient R² of this method was 0.999 1, and the IC₅₀ was 1.500 2 μg/L, minimum detection limit IC₁₀ is 0.284 1 μg/L, linear range(IC₂₀~IC₈₀)0.430 7~5.225 2 μg/L, the cross reaction rate with five TFPTZ structural analogues is less than 1.2%, the sample addition recovery rate is 93.26%~109.13%, addition recovery rate is 93.26%~109.13%,- DOI:10.3969/j.issn.1000-1565.2023.06.010猪肉中2-(三氟甲基)吩噻嗪的Ic-ELISA检测刘广兴,韩晓利,李昳晴,王庭欣(河北大学质量技术监督学院,计量仪器与系统国家地方联合工程研究中心,河北省能源计量与安全检测技术重点实验室(筹),河北保定 071002)摘要:为了快速检测猪肉中的镇静剂2-(三氟甲基)吩噻嗪(2-(trifluoromethyl)phenothiazine,TFPTZ),分别用偶氮苯甲酸法、混合酸酐法合成了半抗原、完全抗原,通过免疫新西兰大耳白兔获得了多克隆抗体,采用间接竞争酶联免疫方法(Ic-ELISA)对猪肉中TFPTZ进行了定量分析,实验条件:包被抗原浓度1.25 μg/mL,多克隆抗体稀释16 000倍,37 ℃包被3 h,封闭液为质量分数1%的牛血清白蛋白(BSA),TFPTZ稀释液为含体积分数10%的DMSO的PBS,竞争反应60 min.该方法相关系数R²为0.999 1,IC₅₀为1.500 2 μg/L,最低检测限IC₁₀为0.284 1 μg/L,线性(IC₂₀~IC₈₀)为0.430 7~5.225 2 μg/L,与5种TFPTZ结构类似物的交叉反应率小于1.2%,样品添加回收率为93.26%~109.13%,变异系数小于6%,实际样品检测结果与高效液相色谱法测定结果高度一致,表明建立的快速检测猪肉中TFPTZ的Ic-ELISA方法具有良好的准确度,适用于快速检测猪肉中TFPTZ的残留量.关键词:2-(三氟甲基)吩噻嗪;抗原合成;间接竞争酶联免疫方法(Ic-ELISA)中图分类号:Q5-33 文献标志码:A 文章编号:1000-1565(2023)06-0638-08Detection of 2-(trifluoromethyl)phenothiazine in pork by Ic- ELISALIU Guangxing,HAN Xiaoli,LI Yiqing,WANG Tingxin(Hebei Key Laboratory of Energy Metering and Safety Testing Technology, National & Local Joint Engineering Research Center of Metrology Instrument and System, College of Quality and Technical Supervision, Hebei University, Baoding 071002,China)Abstract: In order to quickly analyze the sedative 2-(trifluoromethyl)phenothiazine(TFPTZ)in pork, haptens and complete antigens were synthesized using the azobenzoic acid method and the mixed anhydride method, respectively. Polyclonal antibodies were obtained by immunizing New Zealand white rabbits. Indirect competitive enzyme linked immunosorbent assay(Ic-ELISA)was used to quantitatively analyze TFPTZ in pork. The experimental conditions were as follows: the concentration of the coated antigen was 1.25 μg/mL, and the polyclonal antibody was diluted 16 000 times, Coated at 37 ℃ for 3 h, the blocking solution was 1% BSA of mass fraction, and the TFPTZ diluent is PBS of DMSO with a volume fraction of 10%. The competitive reaction lasted 60 minutes. The correlation coefficient R² of this method was 0.999 1, and the IC₅₀ was 1.500 2 μg/L, minimum detection limit IC₁₀ is 0.284 1 μg/L, linear range(IC₂₀~IC₈₀)0.430 7~5.225 2 μg/L, the cross reaction rate with five TFPTZ structural analogues is less than 1.2%, the sample addition recovery rate is 93.26%~109.13%, addition recovery rate is 93.26%~109.13%,- 收稿日期:2022-12-18 基金项目:河北省重点研发计划项目(18275502D) 第一作者:刘广兴(1995—),男,河北衡水人,河北大学在读硕士研究生,主要从事食品安全快速检测技术研究.E-mail:603658113@qq.com 通信作者:王庭欣(1965—),女,河北保定人,河北大学教授,主要从事食品安全快速检测技术研究.E-mail:tingxinwang@126.com第6期刘广兴等:猪肉中2-(三氟甲基)吩噻嗪的Ic-ELISA检测and the coefficient of variation is less than 6%. The actual sample detection results are highly consistent with the results determined by high-performance liquid chromatography, indicating that the established Ic-ELISA detection method of TFPTZ has good accuracy and is suitable for rapid detection of TFPTZ residues in pork.

Key words: 2-(trifluoromethyl)phenothiazine(TFPTZ), antigen synthesis, Indirect competitive enzyme linked immunosorbent assay(Ic-ELISA)

中图分类号:

Q5-33

刘广兴,韩晓利,李昳晴,王庭欣. 猪肉中2-(三氟甲基)吩噻嗪的Ic-ELISA检测[J]. 河北大学学报(自然科学版), 2023, 43(6): 638-645.

LIU Guangxing,HAN Xiaoli,LI Yiqing,WANG Tingxin. Detection of 2-(trifluoromethyl)phenothiazine in pork by Ic- ELISA[J]. Journal of Hebei University(Natural Science Edition), 2023, 43(6): 638-645.

参考文献

[1] 张秋云,杨洪生,谭秀慧,等.液相色谱-串联质谱测定水产品中15种苯二氮卓类镇静剂的药物残留[J].食品与机械,2022,38(11):60-67, 94. DOI: 10.13652/j.spjx.1003.5788.2022.80029.
[2] 中华人民共和国农业农村部,国家卫生健康委员会,国家市场监督管理总局.食品安全国家标准食品中兽药最大残留限量:GB 31650—2019[S].北京: 中国农业出版社,2019.
[3] 朱砾,翁毅仁,林治光,等.高效液相色谱法检测氯丙嗪血药浓度[J].上海精神医学,2004,16(4):206-208.
[4] CERKVENIK-FLAJS V. Determination of residues of azaperone in the kidneys by liquid chromatography with fluorescence dete-ction[J]. Anal Chimica Acta, 2007, 586(1/2): 374-382. DOI: 10.1016/j.aca.2006.11.010.
[5] 冯静,邹淼,陈曦,等.超高效液相色谱-串联质谱法检测牛肉中16种镇静剂类药物残留[J].食品安全质量检测学报,2019,10(10):3091-3096.
[6] 谭贵良,赵天珍,王文林,等.气相色谱-质谱法同时测定腊肠中7种镇静剂类药物残留[J].现代食品科技,2014,30(2):274-278. DOI: 10.13982/j.mfst.1673-9078.2014.02.009.
[7] MORENO L, LANUSSE C. Veterinary drug residues in meat-related edible tissues[M] //New Aspects of Meat Quality. Amsterda-m: Elsevier, 2017: 581-603. DOI: 10.1016/b978-0-08-100593-4.00024-2.
[8] FLEEKER J R, LOVETT L J. Enzyme immunoassay for screening sulfamethazine residues in swine blood[J]. J Assoc Off Anal Chem, 1985, 68(2): 172-174. DOI: 10.1093/jaoac/68.2.172.
[9] 郭尧君.蛋白质电泳实验技术[M].北京: 科学出版社,1999:123-156.
[10] MANDLI J, EL FATIMI I, SEDDAOUI N, et al. Enzyme immunoassay(ELISA/immunosensor)for a sensitive detection of pork adulteration in meat[J]. Food Chem, 2018, 255: 380-389. DOI: 10.1016/j.foodchem.2018.01.184.
[11] THONGRUSSAMEE T, KUZMINA N S, SHIM W B, et al. Monoclonal-based enzyme-linked immunosorbent assay for the det-ection of zearalenone in cereals[J]. Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2008, 25(8):997-1006. DOI: 10.1080/02652030701823159.
[12] 谭庶,杨金易,许吉华,等.鸡肉中金刚烷胺间接竞争ELISA检测方法的建立[J].食品科学,2020,41(10): 304-310. DOI: 10.7506/spkx1002-6630-20190123-293.
[13] 邢广旭,赵东,孙雪峰,等.酶联免疫法检测猪肉中的司帕沙星[J].现代食品科技,2022,38(2):266-271. DOI: 10.13982/j.mfst.1673-9078.2022.2.0527.
[14] PENG D P, WEI W, PAN Y H, et al. Preparation of a monoclonal antibody against amantadine and rimantadine and development of an indirect competitive enzyme-linked immunosorbent assay for detecting the same in chicken muscle and liver[J]. J Pharm Biomed Anal, 2017, 133:56-63. DOI: 10.1016/j.jpba.2016.11.009.
[15] 进出口动物源性食品中吩噻嗪类药物残留量检测方法液相色谱-质谱/质谱法: SN/T 1985—2007[S].
[16] 刘建静.氯丙嗪酶联免疫检测方法(ELISA)的建立[D].北京: 中国农业科学院,2009.
[17] 杨友辉,杨健,宋菲,等.抗EGFRvⅢ单链抗体间接ELISA方法的建立及条件优化[J].生物技术,2017,27(4): 342-348. DOI: 10.16519/j.cnki.1004-311x.2017.04.0056.
[18] 廖文彬.鸡肉中氟苯尼考残留的ELISA检测方法研究[D].广州: 华南农业大学,2020. DOI: 10.27152/d.cnki.ghanu.2020.000263.
[19] 殷萌琪.α-玉米赤霉醇类化合物和β-内酰胺类抗生素的广谱快速检测方法研究[D].无锡: 江南大学,2020. DOI: 10.27169/d.cnki.gwqgu.2020.001029.
[20] 盛恩泽.乙氧氟草醚单克隆抗体的制备及其免疫分析方法研究[D].南京: 南京农业大学,2018. DOI: 10.27244/d.cnki.gnjnu.2018.000046.
[21] 吴元元,金朵,付骋宇,等.拟除虫菊酯类农药多残留酶联免疫分析方法的条件优化[J].农药,2017,56(7): 510-514. DOI: 10.16820/j.cnki.1006-0413.2017.07.013.
[22] 张晓寒.环境中有机污染物的生物分析新方法的研究及应用[D].上海: 上海交通大学,2020. DOI: 10.27307/d.cnki.gsjtu.2020.000633. (