Journal of Hebei University(Natural Science Edition) >

2023 , Vol. 43 >Issue 6: 638 - 645

DOI: https://doi.org/10.3969/j.issn.1000-1565.2023.06.010

Detection of 2-(trifluoromethyl)phenothiazine in pork by Ic- ELISA

  • LIU Guangxing ,
  • HAN Xiaoli ,
  • LI Yiqing ,
  • WANG Tingxin
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  • 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

Received date: 2022-12-18

  Online published: 2023-12-15

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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)

Cite this article

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 . DOI: 10.3969/j.issn.1000-1565.2023.06.010

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