Development of an indirect competitive enzyme-linked immunosorbent assay for detecting flunixin and 5-hydroxyflunixin residues in bovine muscle and milk

ABSTRACT

The flunixin residues in foods of animal origin could pose hazards for human health. In this paper, a broad-selectivity polyclonal antibody was obtained using 5-hydroxyflunixin coupled with bovine serum albumin as the immunogen. The semi-inhibitory concentration (IC₅₀) of the polyclonal antibody was 1.43 ng/mL for flunixin and 0.29 ng/mL for 5-hydroxyflunixin, respectively, which were far below the maximum residue limits set by the United States, the European Union and China. The limits of detection were calculated to be 2.98 μg/kg for flunixin in bovine muscle and 0.78 μg/L for 5-hydroxyflunixin in milk. In spike and recovery tests, the average recovery ranged from 83% to 105%, with a satisfying coefficient of variance ranging from 5.8% to 11.3%. Furthermore, the ELISA method was validated by a well-established LC-MS/MS method. These results proved that the established method is suitable for flunixin and 5-hydroxyflunixin residue analysis.

KEYWORDS:

• Flunixin
• 5-hydroxyflunixin
• immunoassay
• bovine muscle
• milk

Introduction

Flunixin, (2-[[2-methyl-3-(trifluoromethyl)phenyl]amino]-3-pyridinecarboxylic acid, Figure 1), a nonsteroidal anti-inflammatory drug (NSAID), is widely used for the treatment of some diseases in animals, such as acute inflammation, mastitis, metritis, lack of lactation, musculoskeletal disorders, abdominal pain and sow endometritis because of its excellent analgesic and antipyretic effects (Anderson, Smith, Shanks, Davis, & Gustafsson, 1986; Cook, Meyer, Campbell, & Blikslager, 2009; Levionnois, Fosse, & Ranheim, 2018). It can inhibit cyclooxygenase synthesis and reduce inflammation and pain within 15 min of drug treatment (Huber, Arnholdt, Möstl, Gelfert, & Drillich, 2013; Yazar, Er, Uney, Altunok, & Elmas, 2007). The analgesic and antipyretic effects usually occur 1–2 h after treatment, and the maximum effect is maintained for between 2 and 16 h (Aké-López, Segura-Correa, & Quintal-Franco, 2005; Levionnois et al., 2018). Since October 2004, flunixin has been approved by the Food and Drug Administration for lactation and has been widely used in the United States, France, Switzerland, Germany and the United Kingdom (Shelver, Schneider, & Smith, 2016). In addition, flunixin has been approved by the Chinese Ministry of Agriculture for veterinary use in animal husbandry since 2007.

Figure 1. Chemical structures of flunixin and 5-hydroxyflunixin.

Flunixin is easily metabolised and eliminated in vivo, and approximately 90% of the original drug is excreted via urine and feces 24 h after drug treatment (Levionnois et al., 2018). The main metabolites of flunixin are 4-hydroxyflunixin, 5-hydroxyflunixin and 2-methylhydroxyl (Kissell et al., 2012; Lin et al., 2018). Among these compounds, 5-hydroxyflunixin (Figure 1) is identified as the marker residue in bovine milk, while flunixin is identified in bovine muscle (Jedziniak et al., 2013; Ngoh et al., 2003; Smith et al., 2015).

During the last two decades, many studies proved that flunixin and other NSAID residues in foods of animal origin could pose potential risks to human health such as gastrointestinal damage, hematopoietic and renal systemic toxicity, hepatotoxicity, and aseptic meningitis (Kissell, Baynes, Riviere, & Smith, 2013; Schulz, Anderson, Coetzee, White, & Miesner, 2011). To reduce these toxic effects, the European Union, the United States, Canada, Japan and China identified maximum residue limits (MRLs) or tolerances for flunixin and 5-hydroxyflunixin (Table 1) in the range of 6 μg/kg–60 μg/kg in animal tissues and milk (Zhu et al., 2013).

Table 1. The MRLs set by different countries for flunixin residues in foods of animal origin.

Analyte	US	EU	Canada	Japan	China
Flunixin in bovine muscle (μg/kg)	25	20	20	20	–
5-Hydroxyflunixin in milk (μg/L)	–	40	6	60	40

- : No MRLs were set for target analytes.

To prevent potential hazards to human health, various analytical techniques have been used to detect flunixin and other NSAID residues, i.e. high-performance liquid chromatography coupled with UV and mass spectrometry detectors (Asea, Patterson, Korsrud, Dowling, & Boison, 2001; Jedziniak, Szprengier-Juszkiewicz, & Olejnik, 2009; Liu et al., 2015; Peng et al., 2013; van Pamel & Daeseleire, 2015). Although these methods are very accurate and sensitive, they are costly and require complex sample pretreatment steps and trained personnel (Mukunzi, Suryoprabowo, Song, Liu, & Kuang, 2018; Qu et al., 2019; Wang et al., 2018; Zeng, Jiang, Liu, Song, & Kuang, 2018). Immunoassays are superior to chromatographic methods in screening tests for their simplicity, sensitivity, and high-throughput capabilities (Chen et al., 2017; Jiang et al., 2017; Kong et al., 2017; Wang, Luo, Chen, Huang, & Jiang, 2016). Recently, some immunoassays for detecting residues of flunixin and other NSAIDs have been reported (Lin et al., 2018). However, to the best of our knowledge, enzyme-linked immunosorbent assay (ELISA) methods for simultaneously detecting flunixin and 5-hydroxyflunixin residues have not been reported.

In this paper, we want to establish a rapid screening method for simultaneously detecting the residues of the parent drug and its hydroxyl metabolites (marker residues). In this study, flunixin was coupled with a carrier protein, and polyclonal antibodies were obtained after immunisation of rabbits with synthesised immunogens. Based on the produced antibody, we established an indirect competitive ELISA (icELISA) for the simultaneous detection of flunixin and 5-hydroxyflunixin residues. In addition, to establish a rapid and simple screening method, efforts were made to simplify time-consuming sample preparation. This established icELISA method was validated by the liquid chromatography tandem mass spectrometry (LC-MS/MS) method, and the results indicate that this icELISA method is suitable for the detection of flunixin and 5-hydroxyflunixin in bovine muscle and milk.

Material and methods

Chemicals

Flunixin (99%) was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). 5-hydroxyflunixin (98%) was purchased from Witega Laboratorien Berlin-Adlershof GmbH (Berlin, Germany). Bovine serum albumin (BSA), ovalbumin (OVA), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), Freund’s complete and incomplete adjuvant, horseradish peroxidase, and 3,3′,5,5′-tetramethyl-benzidine (TMB) substrate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated goat-anti-rabbit IgG was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). In addition, 96-well polystyrene microtiter plates were obtained from Costar Inc. (Milpitas, CA, USA).

Apparatus

A multimode microplate reader was obtained from BioTek Instruments Inc. (Winooski, VT, USA). A 30A ultrahigh-performance liquid chromatography system (Shimadzu, Kyoto, Japan) and an API 4000+ tandem mass spectrometer (AB SCIEX, USA) were used for final validation.

Antigen preparation

Because flunixin and 5-hydroxyflunixin are haptens, the immunogen was prepared by conjugating flunixin and 5-hydroxyflunixin with BSA through the carbodiimide method according to the methods in our previous studies (Jiang et al., 2015). Briefly, flunixin (11 mg) or 5-hydroxyflunixin (11 mg) was dissolved in 800 μL of N,N-dimethylformamide, and then NHS (8 mg) and EDC (13 mg) were added to the mixture. Afterwards, the reaction mixture was added to a BSA solution (10 mg dissolved in 4 mL of borate buffer) and reacted at room temperature for another 12 h. The hapten-protein conjugates were then dialysed against 0.01 M phosphate-buffered saline (PBS) for 3 days at 4°C. Similarly, the coating antigen was prepared using this method, and the only difference is that the coating antigen was conjugated with OVA.

Polyclonal antibody production

New Zealand white rabbits were immunised at a dosage of 1.0 mg/kg body weight and with the same volume of Freund’s complete adjuvant. Every two weeks, the rabbits were administered boost immunizations with the same dosage of immunogen emulsified in Freund’s incomplete adjuvant. The blood was collected by ear vein after each injection by subcutaneous multipoint injection, and then the antibody titre was determined by indirect ELISA. The antiserum was prepared by centrifugation of the whole blood, and the polyclonal antibody was purified from antiserum by chromatography.

Optimisation of the ELISA method

The icELISA approach can be described as follows: 96-well microtiter plates were coated with the coating antigen (100 μL/well) at a dilution of 1:2000 in PBS, and then the plates were incubated at 4°C overnight. Then, the plates were coated with 3% skim milk for 1 h at 25°C. After blocking, 50 μL of standard solutions and 50 μL of diluted polyclonal antibody solutions were added to each well, and the plates were incubated for 30 min. Then, diluted horseradish peroxidase-conjugated goat-anti-rabbit IgG was added to each well, and the plates were incubated for 30 min at 25°C. After another three washes, 100 μL of TMB substrate was pipetted, and the absorbance was measured after 50 μL of 2 M H₂SO₄ was added to stop the enzyme reaction.

Curve fitting, sensitivity and selectivity determination

Standards were run in triplicate wells, and average absorbance values of the standards were plotted against the analyte concentration on a logarithmic scale. Competition curves were fitted to a four parameter logistic equation from which IC₅₀ values were calculated (He et al., 2017; Jiang et al., 2013).$\frac{Y=\left(A\text{–}D\right)}{\left[1+{\left(X/C\right)}^B\right]+D}$Where A = the absorbance at the high asymptote of the curve, B = the slope at the inflection point of the sigmoid, C = the concentration of analyte resulting in 50% inhibition of tracer binding, D = the absorbance at the low asymptote of the curve and X = the calibration concentration.

To determine the selectivity, a cross-reactivity study was carried out under optimum conditions. The cross-reactivity of the antibody against flunixin, 5-hybroxyflunixin and other cross-reactants was calculated using the IC₅₀ values in the unit of pmol/mL according to the following equation (Jiang et al., 2016):$\mathrm{C}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{s}-\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}\left(\text{\%}\right)=\left(I{C}_{50}of5-\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{y}\mathrm{f}\mathrm{l}\mathrm{u}\mathrm{n}\mathrm{i}\mathrm{x}\mathrm{i}\mathrm{n}\right)/\left(I{C}_{50}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{g}\mathrm{s}\right)\times 100\text{\%}$

Real sample analysis

Bovine muscle and milk samples were purchased from local supermarkets and stored in a refrigerator (−20°C) until used. The flunixin and 5-hydroxyflunixin concentrations of each sample were quantified by LC-MS/MS before spike and recovery tests (General Administration of Quality Supervision, 2008). For sample extraction, the samples were thawed, and 1.0 ± 0.01 g of bovine muscle sample or 1.0 ± 0.01 mL of milk was combined with 4.0 mL of 0.5% hydrochloric acid (HCl) in acetonitrile. Samples were sonicated for 5 min and then centrifuged for 10 min at 5000 g. Then, 2 mL of the supernatant was collected and evaporated to dryness under a stream of nitrogen at 55°C. After reconstitution in 500 μL of assay buffer, the mixture was tested by the established icELISA.

Assay validation

A validation of the icELISA method was carried out according to the related content of the Commission Decision 2002/657/EC (European Commission, 2002). The limit of detection (LOD) was based on the average value of 20 blank samples plus three times the standard deviation (SD). The accuracy and precision of the method were represented by the recovery and coefficient of variation (CV), respectively. Concentrations of the tested compounds in naturally contaminated or spiked samples were confirmed by LC-MS/MS and then detected with the established ELISA for the final validation.

Results and discussion

Antigen synthesis and characterisation of the polyclonal antibody

The aim of this paper was to develop immunoassays for the rapid detection of flunixin and 5-hydroxyflunixin residues in foods of animal origin. The design of immunogens is critical for the success of rapid immunoassays. Flunixin and 5-hydroxyflunixin are small molecule haptens without immunogenicity (molecule weight <1000 Da), and they need to be coupled to carrier proteins to elicit a specific immune response in animals. The carboxyl, imido and hydroxyl groups in the molecular structure of flunixin and 5-hydroxyflunixin could be used for the synthesis of immunogens. The successful coupling of hapten to carrier protein was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry according to our previous studies, and the results showed qualitative differences between the carrier protein and the conjugate, which indicated the successful attachment of the hapten to the carrier protein.

The icELISA described in this paper was used to characterise the performance of the polyclonal antibody. In this study, rabbits immunised with flunixin-BSA and 5-hydroxyfluinxin-BSA produced antibodies. However, the antibody from rabbits immunised with 5-hydroxyfluinxin-BSA had better performance and also recognised flunixin. The synthesised coating antigens and the produced antibody were characterised by homologous and heterologous formats to select their best combinations, and the flunixin-OVA was selected as the coating antigen. When the dilution of antiserum was 1:2000 (after the third immunisation), the absorbance at 450 nm ranged between 1.5 and 2.0. Furthermore, the inhibition rate was > 50% when using 10 ng/mL flunixin as a standard. All of these characteristics indicated polyclonal antibodies would be suitable for detecting target analytes.

Optimisation of the icELISA

Suitable assay conditions are important for improving the sensitivity of icELISA. For this purpose, several key factors affecting the assay performance were studied as follows.

Optimisation of working concentrations of antibody and antigen: Competition experiments were carried out by checkerboard titration gradient dilution to determine the suitable concentrations of coating antigen and antibody. As shown in Table 2, the most suitable dilution ratio for coating antigen and antibody was 1:2000 and 1:2000 when a compromise was made between the A_max value (absorbance of assay buffer, ranging from 1.5 to 2.0) and the highest sensitivity (the absorbance at 10 ng/mL flunixin/absorbance of assay buffer).

Table 2. Optimisation of dilutions for coating antigen and antibody using checkerboard titration. The data shown in the table are absorbance values.

Antibody^a dilution	Flunixin concentration (ng/mL)	Coating antigen^b dilution
		1000	2 000	4000	8000
1 000	0	2.943	2.735	2.238	1.749
	10	1.611	1.437	1.313	0.907
2 000	0	2.387	1.872	1.382	1.108
	10	0.807	0.916	0.731	0.612
4 000	0	1.245	1.165	0.823	0.447
	10	0.674	0.714	0.411	0.215
8 000	0	0.965	0.759	0.571	0.438
	10	0.446	0.315	0.317	0.212

^aThe concentration of antibody was 2.4 mg/mL.

^bThe concentration of coating antigen was 3.6 mg/mL.

pH effect: Unsuitable pH conditions affect the binding between the antigen and antibody. To determine the optimum pH of the assay buffer, calibration curves were prepared in PBS buffers with different pH values between 5.0 and 9.0. Considering the A_max value and the sensitivity of the assay (IC₅₀), a slightly acidic pH (pH 6.5) is preferred for assay buffer (Figure 2).

Figure 2. Effects of pH (A), ionic strength (B) of assay buffer, and acetonitrile tolerance (C) on the assay performance of icELISA.

Ionic strength effect: Previous studies indicated that increasing the NaCl concentration improves the sensitivity by weakening the nonspecific binding of the antibody. To evaluate the effect of ionic strength, immunoassay performance was investigated in PBS with NaCl concentrations ranging from 0 to 0.5 M. As shown in Figure 2, a NaCl concentration of 0.05 M was optimal when compromising between the A_max value and sensitivity. (IC₅₀ exhibited a minimum value, and at the same time, the A_max/IC₅₀ showed the maximum value).

Organic solvent effect: In this paper, acetonitrile was used to extract the target analytes from food matrices, and thus, the acetonitrile residues in the sample extract may affect the assay performance. Calibration curves were performed with PBS buffer containing different proportions of acetonitrile. As shown in Figure 2, the assay sensitivity and absorbance decreased with increasing acetonitrile concentration. After the assay buffer with different acetonitrile amounts was tested, 0% acetonitrile matched the sample extract calibration curve and was used in the established ELISA.

Curve fitting and sensitivity and selectivity determination

In this study, an icELISA method was established, and the standard solutions were 5-hydroxyflunixin diluted in PBS at concentrations of 0.03–24.3 ng/mL. Whether the established method could be applied practically depends on its repeatability. Herein, repeatability was investigated by five tests at every analyte concentration. The results showed that the CVs of absorbance at six different concentrations levels were in the range of 3.5%-6.8%, which indicated a good repeatability of icELISA.

To assess sensitivity and selectivity, IC₅₀ and cross-reactivity (CR%) values were determined by the established icELISA. A concentration series of standard solutions was prepared and measured in the optimised assay conditions. The standard calibration curves are shown in Figure 3, and the IC₅₀ value was calculated to be 1.43 ng/mL for flunixin and 0.29 ng/mL for 5-hydroxyflunixin. The quantification linear range was as broad as 0.13–68.7 ng/mL for flunixin, whereas the linear working range was determined to range from 0.076 ng/mL to 16.3 ng/mL for 5-hydroxyflunixin. The IC₅₀ values obtained in this work were much lower than those in some previous reports, and the results indicated that the icELISA fabricated in this work displayed better performance.

Figure 3. Calibration curves of icELISA for flunixin and 5-hydroxyflunixin residue analysis. The data are average values of triplicate samples (average ± SD).

The selectivity of the icELISA was assessed by the cross-reactivity against the analogs compared with flunixin, including 5-hydroxyflunixin, and other related NSAID analytes under the same experimental conditions. Table 3 gives the IC₅₀ values of other structurally related analytes and their cross-reactivity values. When the cross-reactivity with 5-hydroxyflunixin was set as 100%, the cross-reactivity with flunixin was calculated to be 20.3%. However, there was a small amount of cross-reactivity (< 0.1%) with the other related NSAID compounds because they are not structurally similar to the target haptens. The results suggested that these analogs should not interfere with flunixin and 5-hydroxyflunixin detection.

Table 3. IC₅₀ and cross-reactivity values using 5-hydroxyflunixin as a standard.

Analyte	Chemical Structure	IC50 (ng/mL)	Cross-Reactivity (%)
5-Hydroxyflunixin		0.29	100
Flunixin		1.43	20.3
Aspirin		>200	<0.1
Paracetamol		>200	<0.1
Ibuprofen		>200	<0.1
Celecoxib		>200	<0.1
Naproxen		>200	<0.1
Nabumetone		>200	<0.1
Diclofenac acid		>200	<0.1
Indometacin		>200	<0.1
Nimesulide		>200	<0.1

Real sample analysis

In published analytical methods, flunixin and 5-hydroxyflunixin residues have mostly been determined by the use of LC-MS/MS. In the sample pretreatment procedures, acid or enzyme hydrolysis is a critical part for the release of tissue-bound residues into solution because of the high (99%) protein binding of flunixin (Horii, Ikenaga, Shimoda, & Kokue, 2004). In previous studies (Lin et al., 2018), the samples were directly diluted or extracted for the analysis of flunixin residues in foods of animal origin. This method may be used for satisfactorily detecting artificially spiked samples; however, high protein binding increases the potential risk of false negative rates. In this paper, different approaches in the extraction step were applied. First, the samples were extracted and then diluted by assay buffer for analysis. As shown in Figure 4A and B, the calibration curves built directly from bovine muscle and milk extracts did not match the calibration curves built in assay buffer. Considering the high protein binding, the spiked bovine muscle and milk samples were acidified with HCl for the precipitation of proteins, and the supernatant was extracted with acetonitrile in the presence of sodium chloride to simultaneously extract flunixin and its main metabolite 5-hydroxyflunixin from bovine muscle and milk. The extraction efficiency was investigated using the incurred sample containing flunixin and 5-hydroxyflunixin that had been taken 12 h after injection. For this purpose, four samples were taken, and three of them were acidified with 200 µL of 1 N, 3 N, and 6 N HCl. The samples were left to stand for 12 h and then analyzed according to the procedure. The results from acidified samples were compared to the results from a blank sample extract spiked with the target analytes, whose value was regarded as 100%. As presented in Figure 4C, the recovery values meet the requirements of spike and recovery tests when the samples were acidified using 3N HCl in the sample extract procedures.

Figure 4. Evaluation of matrix effects of bovine muscle (A) and milk (B) on the calibration curves. Extract efficiency (C) from acidified incurred samples were calculated as the percentage of results obtained with the assay buffer.

Assay validation

In this paper, LOD, accuracy, repeatability and reproducibility were investigated for assay validation. Based on the average values determined from blank samples plus three times the standard deviations, the LODs were calculated to be 2.98 μg/kg for flunixin in bovine muscle and 0.78 μg/L for 5-hydroxyflunixin in milk samples, which were lower than the MRLs set by the European Commission, United States and China. A spike and recovery test was carried out to assess the accuracy, repeatability and within-laboratory reproducibility. The blank samples were spiked at three concentration levels (5, 10, and 20 μg/kg for flunixin and 2, 5, and 10 μg/L for 5-hydroxyflunixin). After the sample pretreatments, the extract was tested by the established icELISA. As shown in Table 4, the average recoveries were in the range of 83%–94% for flunixin and 81%–105% for 5-hydroxyflunixin, which were within an acceptable range. Repeatability and within-laboratory reproducibility, both expressed as the CVs, were 5.8%–10.2% for flunixin and 6.8%–11.3% for 5-hydroxyflunixin, respectively. The results suggested that the established method was accurate for use in real sample analysis.

Table 4. Average recovery and CV values for flunixin and 5-hydroxyflunixin detection in bovine muscle and milk (n = 3).

Analyte	Food matrix	Spiked concentration	Intra-assay		Inter-assay
			Recovery (%)	CVs (%)	Recovery (%)	CVs (%)
Flunixin	Bovine muscle	5^a	83	8.9	88	9.3
		10^a	87	5.8	94	10.2
		20^a	92	7.6	91	9.8
5-Hydroxyflunixin	Milk	2^b	81	8.5	85	11.3
		5^b	86	7.4	105	8.3
		10^b	97	6.8	92	9.6

^aThe unit of the spiked concentration is μg/kg.

^bThe unit of the spiked concentration is μg/L.

To further evaluate the accuracy and precision of the established ELISA, a well-established LC-MS/MS method was used to test the same spiked concentrations of flunixin. As shown in Figure 5, the naturally contaminated and artificially spiked samples were investigated by icELISA and LC-MS/MS. The results of icELISA showed few significant difference from those of LC-MS/MS, which indicates that icELISA could be applied for target analyte analysis with good effectiveness and applicability.

Figure 5. Correlation between icELISA and LC-MS/MS in detecting flunixin residues in bovine muscle (A) and 5-hydroxyflunixin residues in milk (B). The data are average values of triplicate samples (average ± SD).

Flunixin has been widely used for disease treatment in animal husbandry because of its excellent analgesic and antipyretic effects. The flunixin and 5-hydroxyflunixin residues in foods of animal origin could pose hazards for human health. In this paper, we developed an icELISA for the rapid screening of flunixin residue in bovine muscle and 5-hydroxyflunixin residue in milk. The produced antibody in this study could simultaneously recognise flunixin (IC₅₀: 1.43 ng/mL) and 5-hydroxyflunixin (IC₅₀: 0.29 ng/mL), and the calculated LODs of the developed icELISA were 2.98 μg/kg for flunixin in bovine muscle and 0.78 μg/L for 5-hydroxyflunixin in milk. In 2018, Lin et al successfully produced a sensitive anti-flunixin meglumine monoclonal antibody 2H4, and the produced antibody had an IC₅₀ of 0.29 ng/mL, a LOD (IC₁₀) of 0.1 ng/mL, and a linear range of 0.08664 ng/mL – 0.97226 ng/mL. Based on the developed antibody and icELISA, Lin et al. developed an effective and portable immunochromatographic strip assay with a cutoff value of 0.29 ng/mL flunixin meglumine in milk (Lin et al., 2018). Compared with previous research (Lin et al., 2018), we used 5-hydroxyflunixin to synthesise immunogens, and the produced antibody showed a satisfying cross-reactivity (20.3%) with is parent drug flunixin. Based on the produced antibody, we established an icELISA for the simultaneous screening of flunixin residue in bovine muscle and 5-hydroxyflunixin residue in milk before confirmation by a mass spectrometry method.

Conclusions

In summary, an icELISA was established to detect flunixin residue in bovine muscle and 5-hydroxyflunixin residue in milk before confirmation by a mass spectrometry method. Compared with traditional methods, several merits of the proposed assay were highlighted. First, the proposed polyclonal antibody could be used to detect flunixin and its marker residues. In addition, the sample pretreatment was more suitable than traditional methods for analyzing a large number of samples within 120 min. In real-sample tests, the results from the icELISA agreed well with those from the LC-MS/MS method. From a practical point of view, this icELISA method can be carried out in any routine laboratory without the requirement of special facilities.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work is financially supported by National Natural Science Foundation of China [31602103], Science and Technology Planning Project of Guangdong Province, China [2017B020207009 and 2016A020210055], Shenzhen Basic Research Project [JCYJ20170817095240632 and JCYJ20160307114724751], Sanming Project of Medicine in Shenzhen [SZSM201611068], and the Scholarship offered by China Scholarship Council [CSC201808440085].