Volume 33 Issue 9
Sep.  2020
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ZHOU Xing Hua, ZHANG Cai Qin, ZHANG Xun, SUN Cheng, LI Jie, XIAO Xiang, ZHAO Yan Sheng, OUYANG Qin, WANG Yun. Determination of Fipronil and Its Metabolites in Eggs by Indirect Competitive ELISA and Lateral-flow Immunochromatographic Strip[J]. Biomedical and Environmental Sciences, 2020, 33(9): 731-734. doi: 10.3967/bes2020.097
Citation: ZHOU Xing Hua, ZHANG Cai Qin, ZHANG Xun, SUN Cheng, LI Jie, XIAO Xiang, ZHAO Yan Sheng, OUYANG Qin, WANG Yun. Determination of Fipronil and Its Metabolites in Eggs by Indirect Competitive ELISA and Lateral-flow Immunochromatographic Strip[J]. Biomedical and Environmental Sciences, 2020, 33(9): 731-734. doi: 10.3967/bes2020.097

Determination of Fipronil and Its Metabolites in Eggs by Indirect Competitive ELISA and Lateral-flow Immunochromatographic Strip

doi: 10.3967/bes2020.097
Funds:  This study was supported by the National Key Research and Development Program of China [2017YFC1600801]
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  • Author Bio:

    ZHOU Xing Hua, female, born in 1982, Associate Professor, majoring in food safety and toxicology

  • Corresponding author: WANG Yun, Tel: 86-511-88797059, E-mail: wangy1974@ujs.edu.cn
  • &These authors contributed equally to this project.
  • Received Date: 2020-02-11
  • Accepted Date: 2020-06-02
  • &These authors contributed equally to this project.
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  • [1] Tingle CCD, Rother JA, Dewhurst CF, et al. Fipronil: environmental fate, ecotoxicology, and human health concerns. Rev Environ Contam Toxicol, 2003; 176, 1−66.
    [2] Durham E, Scharf M, Siegfried B, et al. Toxicity and neurophysiological effects of fipronil and its oxidative sulfone metabolite on European corn borer larvae (Lepidoptera: Crambidae). Pestic Biochem and Physiol, 2001; 71, 97−106. doi:  10.1006/pest.2001.2564
    [3] Zhang M, Bian K, Zhou T, et al. Determination of residual fipronil in chicken egg and muscle by LC–MS/MS. J Chromatogr B, 2016; 1014, 31−6. doi:  10.1016/j.jchromb.2016.01.041
    [4] Paramasivam M, Chandrasekaran S. Determination of fipronil and its major metabolites in vegetables, fruit and soil using QuEChERS and gas chromatography-mass spectrometry. Int J Environ Anal Chem, 2013; 93, 1203−11. doi:  10.1080/03067319.2012.708747
    [5] Tomasini D, Sampaio MRF, Cardoso LV, et al. Comparison of dispersive liquid–liquid microextraction and the modified QuEChERS method for the determination of fipronil in honey by high performance liquid chromatography with diode-array detection. Anal Methods, 2011; 3, 1893−900. doi:  10.1039/c1ay05221g
    [6] Li M, Sheng E, Cong L, et al. Development of immunoassays for detecting clothianidin residue in agricultural products. J Agric Food Chem, 2013; 61, 3619−23. doi:  10.1021/jf400055s
    [7] Xianjin L, Chunrong Y, Jian D, et al. Poly- and monoclonal antibody-based ELISAs for fipronil. J Agric Food Chem, 2007; 55, 226−30. doi:  10.1021/jf062045a
    [8] Natalia V, Ki Chang A, Bogdan B, et al. Development of an immunoassay for the detection of the phenylpyrazole insecticide fipronil. Environ Sci Technol, 2015; 49, 10038−47. doi:  10.1021/acs.est.5b01005
    [9] Kai Wang, Natalia V, Debin Wan, et al. Quantitative detection of fipronil and fipronil-sulfone in sera of black-tailed prairie dogs and rats after oral exposure to fipronil by camel single-domain antibody-based immunoassays. Anal Chem, 2019; 91, 1532−40. doi:  10.1021/acs.analchem.8b04653
    [10] Suquan S, Na L, Zhiyong Z, et al. Multiplex lateral flow immunoassay for mycotoxin determination. Anal Chem, 2014; 86, 4995−5001. doi:  10.1021/ac500540z
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Determination of Fipronil and Its Metabolites in Eggs by Indirect Competitive ELISA and Lateral-flow Immunochromatographic Strip

doi: 10.3967/bes2020.097
Funds:  This study was supported by the National Key Research and Development Program of China [2017YFC1600801]
  • Author Bio:

  • Corresponding author: WANG Yun, Tel: 86-511-88797059, E-mail: wangy1974@ujs.edu.cn
  • &These authors contributed equally to this project.
&These authors contributed equally to this project.
ZHOU Xing Hua, ZHANG Cai Qin, ZHANG Xun, SUN Cheng, LI Jie, XIAO Xiang, ZHAO Yan Sheng, OUYANG Qin, WANG Yun. Determination of Fipronil and Its Metabolites in Eggs by Indirect Competitive ELISA and Lateral-flow Immunochromatographic Strip[J]. Biomedical and Environmental Sciences, 2020, 33(9): 731-734. doi: 10.3967/bes2020.097
Citation: ZHOU Xing Hua, ZHANG Cai Qin, ZHANG Xun, SUN Cheng, LI Jie, XIAO Xiang, ZHAO Yan Sheng, OUYANG Qin, WANG Yun. Determination of Fipronil and Its Metabolites in Eggs by Indirect Competitive ELISA and Lateral-flow Immunochromatographic Strip[J]. Biomedical and Environmental Sciences, 2020, 33(9): 731-734. doi: 10.3967/bes2020.097
  • Fipronil is a phenylpyrrole insecticide that is widely used to control a variety of pests associated with crops in both agricultural and non-agricultural areas[1]. In the natural environment, fipronil can degrade to fipronil-desulfinyl, fipronil-sulfone, or fipronil-sulfide by photolysis, oxidation, or reduction, and these metabolites are more toxic to animal tissue than the parent molecule[2]. In China, the use of fipronil in agricultural areas is forbidden except as a corn or other coatedeed treatment agent[3]. In June 2017, the Netherlands Food and Consumer Product Safety Authority (NVWA) found a high concentration of fipronil in a sample batch of eggs, which would undoubtedly have a serious impact on the health of consumers. In short, a reliable and rapid method for monitoring fipronil and its metabolites in eggs must be developed.

    At present, detection methods for fipronil focus mainly on instrumental analytical techniques, including gas chromatography-mass spectrometry[4], liquid chromatography–tandem mass spectrometry [3], and high performance liquid chromatography[5]. These methods are highly sensitive and precise, but also generally time-consuming and require expensive equipment and the skills of a professional technician. As such, they are not suitable for the on-site detection of large numbers of samples. Immunoassays are widely used to detect pesticides in food due to their high throughput, sensitivity, speed, and low cost. However, the sensitivity of the antibody used in the enzyme-linked immunosorbent assay (ELISA) method is not high and only recognizes fipronil itself. The lateral-flow immunochromatographic assay (ICA) strip is used for the rapid on-site screening of large numbers of samples, but to the best of our knowledge, there is no lateral-flow ICA strip for fipronil and its metabolites.

    In this study, we obtained a highly sensitive and specific monoclonal antibody (mAb) against fipronil and its metabolites, and then established an indirect competitive (ic)-ELISA and lateral-flow ICA strip based on this mAb for the determination of fipronil and its metabolites in eggs.

    We synthesized a hapten by the hydrolysis of a nitrile group to a carboxylic group, and then coupled it with carrier proteins to produce a complete antigen by the activated ester method. BALB/c mice were immunized with the prepared immunogen and the mice sera were analyzed using the ic-ELISA method. We then selected the mouse that had exhibited good affinity and inhibition for cell fusion, from which we obtained hybridoma cells. These hybridoma cells were broadly cultured to prepare acties, from which we obtained a monoclonal antibody. We optimized the organic solvent, pH, and NaCl contents of the assay buffer used in the ic-ELISA method, and established standard curves under optimal conditions. The specificity of the mAb was then evaluated in a cross-reactivity experiment. To test the feasibility of the ic-ELISA method, we fortified the egg samples with different amounts of fipronil and its metabolites and performed analyses using the developed ic-ELISA.

    Gold nanoparticles (GNPs) were prepared using the sodium citrate reduction method and the resulting GNPs were assessed based on transmission electron microscopy (TEM) images and UV–vis spectroscopy. We prepared an antibody–GNPs conjugate solution as follows. After adjusting the pH of the GNPs solution (5 mL) to 8.5 with 0.2 mol/L K2CO3, we slowly added the prepared antibody (0.1 mg) to the GNPs solution while constantly stirring. After blocking the mixture with 0.5 mL 10% BSA (w/v), we then centrifuged it at 7,000 g for 30 min. Lastly, the antibody-GNPs conjugates was resuspended in PBS, which contained 2% (w/v) BSA, 1% (w/v) sucrose, and 0.02% (w/v) sodium azide. Fifty microliters of the antibody-GNPs conjugate were added to a 96-well microplate for freeze drying. We then sprayed coating antigen (1 mg/mL) and goat anti-mouse IgG (0.4 mg/mL) onto a nitrocellulose membrane as the test line (T line) and control line (C line), respectively, and cut the assembled strips into 2-mm widths.

    A lateral-flow ICA strip was developed based on a competitive immunoassay. We added 100 µL of sample extraction solution to a well containing the freeze-dried antibody-GNPs conjugate. This solution was mixed evenly with a pipettor and incubated for 5 min at room temperature, after which we inserted the strip into the well. By capillary action, the mixture migrated from the sample pad to the absorbent pad, was captured by the coating antigen on the T line and the goat anti-mouse IgG on the C line based on the antibody-antigen reaction. After 5 min, the detection results were visible to the naked eye.

    Fipronil cannot be used as an immunogen to induce an immune response because of its low molecular weight. It must be coupled with carrier proteins to become a complete antigen that has both immunogenicity and immunoreactivity. A hapten was synthesized by the hydrolysis of a nitrile group to a carboxylic group, as shown in Supplementary Figure S1 available in www.besjournal.com, with the molecular weight [(M-) = 454] of the target product consistent with the theoretical value [(M) = 455]. Then, the hapten could be covalently conjugated into proteins by activating the carboxylic groups. Hapten-BSA and hapten-OVA conjugates were used as an immunogen and coating antigen, respectively, and these two antigens were characterized by UV-vis spectroscopy. As shown in Supplementary Figure S2 available in www.besjournal.com, the shapes of the three curves are distinct, with obvious differences in the absorbance patterns of the conjugates as compared to those of the proteins and hapten. The hapten-BSA and hapten-OVA conjugates exhibited absorption peaks at 273 nm and 283 nm respectively, with the peaks of the two conjugates obviously differing from those of the hapten, BSA, and OVA. This confirmed that the hapten had been successfully conjugated with the carrier proteins.

    Figure S1.  The graph of hapten mass spectrometric identification.

    Figure S2.  The UV-vis absorption spectrum of (A) Hapten-BSA and (B) Hapten-OVA.

    The pH values of the antigen and antibody can affect their activities and even the number of available sites for the antigen–antibody reaction. As shown in Figure 1A, compared to other pH values, the Amax (A value of zero concentration at 450 nm) was not highest at pH 7.2, but had the lowest 50% inhibition concentration (IC50) value of 0.64 ng/mL and the highest Amax/IC50 ratio of 2.00. So overall, the pH of 7.2 was optimum. The NaCl content can affect the charge of the epitope and paratope groups and thereby limit the antigen-antibody reaction. As shown in Figure 1B, this optimum condition was reached at an NaCl content of 0.8%, which had the lowest IC50 value of 0.57 ng/mL and the highest Amax/IC50 ratio of 2.21. As we used acetonitrile to extract the target analytes from the egg samples, we first optimized the acetonitrile content in this study. Figure 1C shows that the 5% acetonitrile was the ideal condition, with the lowest IC50 value of 0.50 ng/mL and the highest Amax/IC50 ratio of 2.74. In short, a pH of 7.2, 0.8% NaCl content, and 5% acetonitrile content were chosen as the optimal conditions for performing the ic-ELISA for fipronil and its metabolites.

    Figure 1.  Optimization of ic-ELISA. (A) pH values. (B) NaCl content, (C) acetonitrile content, and (D) standard ic-ELISA curves for fipronil and its metabolites (n = 3).

    As shown in Figure 1D, from the standard curves plotted (absorbance at 450 nm) against the fipronil, fipronil-sulfide, fipronil-sulfone, and fipronil-desulfinyl concentrations and the respective equations y = 0.005 + 1.553 / [1 + (x/0.434) 1.410], y = 0.004 + 1.375 / [1 + (x/0.660)2.247], y = 0.010 + 1.424/[1 + (x/0.579)1.554], and y = −0.026 + 1.537/[1 + (x/0.376)0.914], the linear regression correlation coefficients (R2) were all 0.999. We defined the limit of detection (LOD) (IC20) of the ic-ELISA as the standard concentration yielding a 20% absorbance inhibition of the blank control[6]. The LODs (IC20) of the ic-ELISA for fipronil, fipronil-sulfide, fipronil-sulfone, and fipronil-desulfinyl were 0.16, 0.36, 0.24, and 0.08 ng/mL, respectively. Supplementary Table S1 available in www.besjournal.com shows that the IC50 values of fipronil, fipronil-sulfide, fipronil-sulfone and fipronil-desulfinyl were 0.43, 0.66, 0.58, and 0.38 ng/mL, respectively, which are better than the LOD of 5.99 ng/mL reported in another paper[7]. The cross-reactivity values of fipronil-sulfide, fipronil-sulfone, and fipronil-desulfinyl were 65.15%, 74.14%, and 113.16%, which are higher and more balanced than the results of 39.00%, 1.40%, and 25.00% reported elsewhere[8]. The sensitivity of the mAb against fipronil, fipronil-desulfinyl, fipronil-sulfone, and fipronil-sulfide improved by factors of 9, 76, 29, and 21, respectively, from those reported elsewhere[9]. Therefore, we can conclude that the antibody prepared in this study exhibited high sensitivity and good cross-reactivity with the metabolites of fipronil.

    AnalytesIC50 (ng/mL)CR (%)
    Fipronil0.43100.00
    Fipronil-sulfide0.6665.15
    Fipronil-sulfone0.5874.14
    Fipronil-desulfinyl0.38113.16

    Table S1.  The cross-reactivity values of related analytes by the ic-ELISA method

    The quality of the GNPs plays an important role in the development of latera-flow ICA strips, and determines the sensitivity and stability of the immunoassay. Figure S3A shows the UV-vis spectrum of the GNPs, which had a maximum absorption wavelength of 524 nm, indicating that the GNPs were successfully prepared. As shown in Figure S3B, the prepared GNPs were uniform in shape and size and were well dispersed with no areas of aggregation. The average diameter of the GNPs was (15.3 ± 0.86) nm.

    Figure S3.  The characterization of GNPs. (A) UV–vis spectrum and (B) TEM image.

    In the ic-ELISA, we spiked the egg samples with different amounts of fipronil and its metabolites, and determined their recovery rates and coefficients of variation (CV), the results of which are shown in Table 1. For the intra-assay, the recovery rates of fipronil, fipronil-sulfone, fipronil-sulfide, and fipronil-desulfinyl were 88.81%–105.55%, 83.66%–118.92%, 99.62%–107.33%, and 83.46%–107.24%, with CV values of 4.81%–10.34%, 0.46%–4.87%, 0.64%–7.23%, and 2.06%–9.61%, respectively. For the inter-assay, the recovery rates of these four drugs were 94.54%–107.12%, 97.80%–112.65%, 98.58%–104.35%, and 78.68%–96.32%, with CV values of 3.52%–10.43%, 8.35%–13.68%, 7.83%–13.32%, and 7.35%–16.10%, respectively. These high recovery rates and low CV values demonstrate the sensitivity and accuracy of the developed ic-ELISA for the quantitative detection of fipronil and its metabolites in egg samples.

    Spiked standard Spiked level (ng/g)Intra-assay (n = 3)Inter-assay (n = 3)
    Recovery (%)CV (%)Recovery (%)CV (%)
    Fipronil0.2105.55 ± 5.084.81103.10 ± 10.7510.43
    0.588.81 ± 9.1810.34107.12 ± 10.349.66
    1.092.80 ± 8.379.0294.54 ± 3.333.52
    Fipronil-sulfone0.3116.30 ± 0.530.46112.65 ± 11.8110.48
    0.683.66 ± 4.074.8797.80 ± 8.078.25
    1.2118.92 ± 3.242.51102.63 ± 14.0413.68
    Fipronil-sulfide0.4102.60 ± 5.345.20104.35 ± 9.378.98
    0.699.62 ± 0.640.64101.80 ± 13.5613.32
    1.0107.33 ± 7.767.2398.58 ± 7.727.83
    Fipronil-desulfinyl0.299.30 ± 2.052.0678.68 ± 12.6716.10
    0.583.46 ± 8.029.6187.47 ± 6.437.35
    1.0107.24 ± 3.052.8496.32 ± 9.359.71

    Table 1.  Recovery rates of fipronil and its metabolites in egg samples based on ic-ELISA

    In the lateral-flow ICA strip, the visual limit of detection (vLOD) is defined as the minimum concentration that shows a weaker T line color intensity that significantly differs from that of the negative control line[10]. As shown in Figure 2, for fipronil, fipronil-sulfide, and fipronil-sulfone, a lighter color on the T line at 10 ng/g was obviously visible for them all, whereas for fipronil-desulfinyl, a lighter color was observed on the T line at 5 ng/g. These results indicate that the developed lateral-flow ICA strip was suitable and accurate for the on-site detection of real samples for equipment-free analysis and that the strip analyses can be completed within 15 min. The results obtained by both of the developed methods were all below an LOD of 0.02 mg/kg in egg samples, as stipulated by the Codex Alimentarius Commission.

    Figure 2.  Sample analysis using the lateral-flow strip. (A) Fipronil: 0, 5, 10, and 20 ng/g; (B) fipronil-sulfone: 0, 5, 10, and 20 ng/g; (C) fipronil-sulfide: 0, 5, 10 ng/g, and 20 ng/g; and (D) fipronil-desulfinyl: 0, 2.5, 5, and 10 ng/g

    In this study, we developed a high-sensitivity ic-ELSIA and lateral-flow ICA strip based on an mAb for the detection of fipronil and its metabolites in egg samples. The 50% inhibition concentrations (IC50) of the ic-ELISA for fipronil, fipronil-sulfide, fipronil-sulfone, and fipronil-desulfinyl were 0.43, 0.66, 0.58, and 0.38 ng/mL, and their LOD values were 0.16, 0.36, 0.24, and 0.08 ng/mL, respectively. In our analysis of the egg samples, the recovery rates obtained in the ic-ELISA ranged from 83.46% to 118.96%, with an average CV less than 7.5%. The vLODs of fipronil, fipronil-sulfide, fipronil-sulfone, and fipronil-desulfinyl were 10, 10, 10, and 5 ng/g, respectively. Based on these results, we can conclude that both of the developed immunological methods are reliable in the determination of fipronil and its metabolites in egg samples at trace levels.

  • Conflicts of Interest All the authors declare that they have no conflicts of interest.

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