Development of IC-ELISA and immunochromatographic strip assay for the detection of flunixin meglumine in milk

ABSTRACT Flunixin meglumine (FM) is a novel nonsteroidal anti-inflammatory drug for animals, which has antipyretic, analgesic, and anti-inflammatory effects. The drug, which was originally used to relieve inflammation in horses, musculoskeletal disorders, and pain, has been approved for use in endotoxemia, infectious diseases in swine, etc. A sensitive anti-FM monoclonal antibody 2H4 was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic strip assay for the detection of FM in milk. The complete antigen and coating antigen were conjugated with bovine serum albumin and ovalbumin, respectively. The monoclonal antibody 2H4, with a half inhibition concentration of 0.29 ng/mL, had a limit of detection of 0.432 ng/mL and a linear range of detection of 0.08664–0.97226 ng/mL. A sensitive and convenient immunochromatographic strip assay was developed with an FM cutoff value of 0.29 ng/mL. The developed methods were suitable for the detection of FM in milk.


Introduction
Flunixin meglumine (FM) is a novel nonsteroidal anti-inflammatory drug that was approved for veterinary uses by the Ministry of Agriculture in China in June 2007 (Choi et al., 2016;Coetzee, 2011;Johnson & Myers, 2015;Miciletta, Cuniberti, Barbero, & Re, 2014;Stafford & Mellor, 2011). In October 2004, FM was approved for use in lactating dairy cows by the FDA and has been widely used in the United States, France, Switzerland, Germany, and Britain. FM impacts prostaglandin synthesis by inhibiting cyclooxygenase (COX) and reduces inflammation and pain within 15 min of administration. Compared with FM, antipyrine, butazodine, and antondin pyrazolone are more frequently used in animals. Its long-term use causes aplastic anemia, neutropenia, thrombocytopenia, proteinuria, interstitial nephritis, and liver damage. However, in the treatment of claudication and swelling of joints, FM is four times more effective than phenylbutazone on a weight basis.
FM is easily metabolized and eliminated in vivo. The main FM metabolites are 4-hydroxy flunixin, 5-hydroxy flunixin, and 2-methylhydroxy. After 24 h of administration, approximately 90% is excreted via urine and feces.

Chemicals and instruments
The instruments used in this study consisted of a UV/VIS scanner (Bokin Instruments, Tsushima, Japan), a Multiskan MKS microplate reader (Thermo LabSystems Inc., Beijing, China), a vortex machine (Shanghai Huxi Analysis Instrument Factory Co. Ltd, Shanghai, China), a membrane dispenser (Xinqidian Gene-Technology Co. Ltd, Beijing, China), and a water bath (Shanghai Instrument Group Co. Ltd., Supply & Sales Co., Shanghai, China).

Antigen preparation
Complete antigen (FM-EDC-BSA) was prepared by conjugating hapten with carrier protein using the carbodiimide (EDC) method and identified by UV/VIS spectroscopy (Gu, Liu, Song, Kuang, & Xu, 2016;Kong, Liu, Song, Kuang, & Xu, 2016;Sun, Liu, Song, Kuang, & Xu, 2016). FM hapten (2.2 mg) was dissolved in 400 μL of DMF, and 1.6 mg of NHS and 2.6 mg of EDC were successively added to the mixture. The mixture was stirred at room temperature (RT) for 8 h and added to 10 mg of BSA in 4 mL of BB. The mixture was stirred at RT for 18 h, dialyzed against 0.01 M PBS for 3 d with six changes of dialysate, and stored at −20°C.
FM was conjugated to OVA (FM-EDC-OVA) by the EDC method and was used as coating antigen. FM hapten (1.6 mg) was dissolved in 300 μL of DMF, and 1.2 mg of NHS and 1.9 mg of EDC were successively added to the mixture. The mixture was stirred at RT for 8 h and added to 5 mg OVA in 2 mL of BB. The mixture was stirred overnight at RT, dialyzed against 0.01 M PBS for 3 d, and stored at −20°C. All conjugates were identified by UV/VIS spectroscopy.

FM monoclonal antibody preparation
Female BALB/c mice (6-8 weeks of age) were immunized with complete antigen (FM-EDC-BSA) in FCA (80 μg per mice) by subcutaneous injection for the first immunization. Booster immunizations were performed 3 weeks after the first immunization. Each mouse was injected with FIA mixed with isometric complete antigen (40 μg per mice). Tail blood sampling was carried out after the third immunization; serum was analyzed by IC-ELISA (Suryoprabowo, Liu, Peng, Kuang, & Xu, 2014). The mouse with the highest inhibitory rate and titer was intraperitoneally injected with complete antigen (20 μg). After 3 weeks, spleen cells from the immunized mouse were fused with Sp2/0 myeloma cells to produce hybridoma cells (Peng, Liu, Kuang, Cui, & Xu, 2017), which were screened by IC-ELISA. The limited dilution method was performed to subclone positive clones. After three subcloning procedures, we obtained a hybridoma cell line capable of producing specific monoclonal antibodies (mAbs). The selected hybridoma cell line was cultured and intraperitoneally injected into mice for ascite production (Wang et al., 2017). Antibodies were purified by octanoic acid-ammonium sulfate precipitation (Li et al., 2014;Yan, Liu, Xu, Kuang, & Xu, 2015). After dialysis against PBS at 4°C for 3 d, antibodies (2 mg/mL) were stored at −20°C.

Development of IC-ELISA
We measured FM mAb titer by IC-ELISA and determined the optimal working concentration of coating antigen and FM mAb by square matrix titrimetry. FM-EDC-OVA (1 µg/ mL, coating antigen) was diluted three times with CB by gradient dilution; 100 µL was transferred to 96-well microtiter ELISA plates. The plates were dried at 37°C for 2 h and washed three times with PBST. Blocking reagent (200 µL per well) was added, and the plate was incubated at 37°C for 2 h. The blocked plates were washed three times with washing buffer, dried, and stored at 4°C.
FM standard was diluted with 0.01 M PBS buffer solution containing potassium salt, and FM antibody was diluted with antibody dilution (PBS containing 0.1% gelatin). FM standard (50 µL) and antibody (50 µL) were successively added into each well, and the plates were incubated at 37°C for 30 min. After washing the wells three times, HRPlabeled goat anti-mouse IgG was diluted 3000-fold with antibody dilution and added into each well. The plates were incubated at 37°C for 30 min. Following four washes, substrate buffer mixed with solutions A and B at 5:1 (v/v) ratio was added into each well and incubated at 37°C for 15 min. Following the addition of stop solution, absorbance was measured at 450 nm in a microplate reader. A standard curve of mAb 2H4 against FM concentration was drawn. The 50% inhibitory concentration (IC 50 ) of FM mAb, which was the half maximal inhibitory concentration.

Labeling FM mAb with colloidal gold particles
Colloidal gold-labeled FM mAb was prepared as previously reported with slight modifications (Feng et al., 2015). First, the pH of colloidal gold solution was adjusted to ∼8.8 with 0.1 M K 2 CO 3 . Second, 1 mL of gold nanoparticle and 4 µL purified FM mAb 2H4 were continuously stirred at RT for 50 min. BSA (10% w/v, 40 µL) was added, and the mixture was incubated at RT for 2 h. Following centrifugation at 8000 rpm for 20 min for three times, the resulting pellet was washed with gold-labeled resuspension (20 mmol tris containing 0.1% polyethylene glycol (PEG), 0.1% tween, 5% sucrose, 5% trehalose, 0.2% BSA, and 5% polyvinyl pyrrolidone (PVP), pH 8.2). Colloidal gold-labeled FM mAb was resuspended in PVP and stored at 4°C.

Preparation of the immunochromatographic strip
For the preparation of the immunochromatographic strip, polyvinyl chloride (PVC) backing card, sample pad, absorption pad, and nitrocellulose (NC) membrane were assembled in layers. The NC membrane contained the control line and test line. Using a membrane dispenser, coating antigen (FM-EDC-BSA) was applied to form the test line and goat anti-mouse antibody was applied to form the control line (1 µL/cm). The strip was dried at 37°C and stored in a desiccator.

Principle of the immunochromatographic strip assay
Standard FM (150 µL) was mixed with 50 µL colloidal gold-labeled FM mAb in a microtiter well. Following incubation at RT for 5 min, the mixture was added onto the sample pad, where it migrated to the absorption pad by capillary action. The results were visualized by the naked eye within 5 min. In FM-negative samples, colloidal gold-labeled FM mAb migrates and is captured by FM-EDC-BSA, leading to a deep color. In FM-positive samples, colloidal gold-labeled FM mAb is combined with FM in the sample; therefore, less colloidal gold-labeled FM mAb is able to combine with FM-EDC-BSA on the test line, leading to a light color on the test line.
The principle of the immunochromatographic strip assay is based on the competition between FM present in the sample and the coating antigen present on the test line for colloidal gold-labeled FM mAb. FM-EDC-BSA was fixed on the test line of the NC membrane. In FM-positive samples, FM competes with the coating antigen fixed on the test line and conjugated with colloidal gold-labeled FM mAb. With increasing FM concentration in the sample, the color intensity of the test line becomes weaker until it finally disappears. Accordingly, there is an inverse proportional relationship between the color intensity of the test line and the concentration of FM in the sample.

Analysis of milk samples
Milk samples were purchased from local supermarkets. The samples were spiked with FM and measured by IC-ELISA.

Identification of complete antigen and coating antigen
The carboxyl groups on FM are crucial for the conjugation reaction to carrier proteins (Figure 1). BSA and OVA conjugated to FM by EDC were used as complete antigen and coating antigen, respectively.
The complete antigen and coating antigen were identified by UV/VIS spectroscopy ( Figure 2). The characteristic absorption peaks of FM were obtained at 325-350 nm and 285 nm. BSA and OVA had an absorption peak at 278 nm, and FM-EDC-BSA and FM-EDC-OVA had an absorption peak at 325-350 nm, which was higher than that of FM within the same range. The primary peak of the conjugates was between the absorption peaks of BSA/OVA and FM. These results revealed that FM was successfully conjugated to carrier proteins.

Characteristic of mAb 2H4
FM-EDC-BSA was used as immunogen in mice, and FM-EDC-OVA was used as coating antigen in IC-ELISA. Following cell fusion, ascites were purified by octanoic acid-ammonium sulfate precipitation, and the antibodies were identified by IC-ELISA. MAbs 1H5, 2C10, 2F10, and 2H4 were obtained. MAb 2H4 was selected as the most sensitive antibody among all mAbs. A standard curve generated between mAb 2H4 and FM concentration (y = 0.0857 + 1.324/[1+ (x/0.290) 1.145 ]) had a linear regression correlation coefficient (R 2 ) of 0.99928.  IC 50 of mAb 2H4 was 0.29 ng/mL, and limit of detection (LOD) (IC 10 ) was 0.0432 ng/mL with a linear range (IC 20 to IC 80 ) (Li et al., 2017) of 0.08664-0.97226 ng/mL (Figure 3).   shows that pad 1 and pad 2 were added 0 and 5 ng/mL FM standard, respectively. The optimum conditions consisted of 1 mL of gold nanoparticle with 4 µL K 2 CO 3 , 8 µg/mL antibody, and 0.5 mg/mL coating 1. We selected FM-EDC-BSA as the coating antigen for subsequent experiments.
Different FM standard concentrations (0, 0.1, 0.25, 0.5, 1, 2.5, and 5 ng/mL) were added to whole milk. Spiked whole milk (100 μL) was added to the sample pad (Figure 7). The color of the test line was weak at 0.1 ng/mL FM and disappeared at 5 ng/mL FM. LOD was 0.1 ng/mL with a cutoff value of 5 ng/mL.

Conclusions
In this study, we developed a highly sensitive mAb 2H4 for the determination of FM, with an IC 50 of 0.29 ng/mL, an LOD (IC 10 ) of 0.1 ng/mL, and a linear range of 0.08664-0.97226 ng/mL. Furthermore, through the optimization of coating antigen and resuspensions, an effective and portable immunochromatographic strip assay was developed with a cutoff value of 0.29 ng/mL FM in milk. The immunochromatographic strip assay is a simple, fast, portable, and sensitive method for the detection of FM in milk.

Disclosure statement
No potential conflict of interest was reported by the authors.

Notes on contributors
Lu Lin got her bachelor from Dalian Ocean University, Dalian, China in 2015 and then she began to study in Jiangnan University (Wuxi, China) for her Master Degree in food science. Her research interests are immunoassay applications in food.
Wei Jiang got her bachelor from Yangzhou University, Yangzhou, China in 2015 and then she began to study in Jiangnan University (Wuxi, China) for her Master Degree in food science. Her research interest includes immunoassay development for food safety.
Liguang Xu earned his Ph.D. in food science in 2012 from Jiangnan University, Wuxi, China and then became a faculty in college of food science and technology of Jiangnan University. His research is focused on the synthesis and controllable assembly of nanoparticles, especially noble metal nanoparticles.
Liqiang Liu got his Ph.D. in food science in 2014 from Jiangnan University, Wuxi, China and then became a faculty in college of food science and technology of Jiangnan University. His research interests are immunochromatographic strip design and application.
Shanshan Song got her Master degree in food science in 2012 from Jiangnan University, Wuxi, China and then became a research assistant in college of Food science and technology of Jiangnan University. Her research interests are monoclonal antibody development.
Hua Kuang got her Ph.D. from China Agricultural University in 2009 and then began to work as a faculty in college of food science and technology of Jiangnan University. She is currently a full professor in food safety. Her research interests are biosensor development.