Development of an immunochromatographic strip assay for ractopamine detection using an ultrasensitive monoclonal antibody

ABSTRACT

A sensitive gold nanoparticle immunochromatographic strip assay and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) were developed for the detection of ractopamine (RCT), a beta-adrenergic agonist. The immunogen and coating antigen were synthesized by the carbodiimide method and conjugated with keyhole limpet hemocyanin and ovalbumin, respectively. The highly sensitive and specific monoclonal antibody was prepared for RCT, with a 50% inhibition concentration of 0.05 ng/ml and had no cross-reactivity with other beta-adrenergic agonists. An ultrasensitive and rapid immunochromatographic strip assay was developed with an RCT cutoff value of 2 ng/ml. Both developed methods can be used for RCT detection in swine urine.

KEYWORDS:

• Ractopamine
• monoclonal antibody
• immunochromatographic strip assay
• ELISA
• swine urine

Introduction

Ractopamine (RCT) is a beta-adrenergic agonist that is added to cattle feed to reduce fat deposition and increase muscle production (Zhang, Gong, Zhang, Xi, Li, Chen, et al., 2014). It has been reported that RCT increases feed efficiency, reduces costs, and improves cattle marketing (Wang et al., 2015). However, RCT residues have negative effects on human health, leading to metabolic disturbances, arrhythmias, and muscle tremors (Hu, Liu, Li, & Li, 2010). Consequently, RCT has been officially banned in the European Union, Japan, and China (Dong, 2012). Therefore, it is important to develop a rapid and sensitive RCT detection method.

The current RCT detection methods include electrochemical and fluorescence detection (Liu, Chen, et al., 2011; Liu, Lin, et al., 2011; Rajkumar, Li, & Chen, 2013; Wang, Yong, He, Du, Ma, Dan, et al., 2013), gas chromatography (Fang et al., 2011), liquid chromatography-mass spectrometry (Amelin, Korolev, & Tret'yakov, 2015; Dong, Xi, Xia, Ding, Li, Zhang, et al., 2011; Liu, Yan, & Zhang, 2014; Liu, Ning, Qu, Peng, Dong, Gao, et al., 2012; Wei et al., 2014), ultra-performance liquid chromatography-tandem mass spectrometry (Jing-Kai, Teh-Ia, Lie-Chwen, & Tung-Hu, 2014), high-performance liquid chromatography (Li, Zhang, Deng, Yang, Liu, Xiao, et al., 2010), capillary electrophoresis (Anurukvorakun, Buchberger, Himmelsbach, Klampel, & Suntornsuk, 2010), and immunoassays (Cao et al., 2013; Mei, Xue, Chao, Bo, Wen, Heng Yi, et al., 2014; Sairi & Arrigan, 2015). Even though these methods are very sensitive, they require complex and time-consuming pretreatment steps and the instruments are costly.

Immunoassays, which have been widely used for RCT detection, have a high-throughput and are specific, user-friendly, and quick. Immunoassays include enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FLA), time-resolved fluoroimmunoassay, and immunomagnetic-proximity ligation assay (Gao et al., 2014; Jiang, Zhu, & Liu, 2014; Jing, Xintian, Jingli, Donghui, & Zhenhua, 2014; Radu, Aciu, Constantin, Leauta, Dermengiu, Hostiuc, et al., 2012; Yue, Wu, Li, & Xu, 2009). In this study, we produced an ultrasensitive and specific monoclonal antibody (mAb) based on a hapten containing an RCT fragment. Additionally, we developed both an immunochromatographic strip assay and ELISA for RCT detection.

Materials and methods

Chemicals

Ractopamine hydrochloride, trimethylamine, sodium triacetoxyborohydride [NaBH(OAc)₃], octopamine, ethyl levulinate, ovalbumin (OVA), keyhole limpet hemocyanin (KLH; MW: 5,000,000 Da), 2-methylpropyl carbonochloridate, N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), incomplete Freund's adjuvant (FIA), complete Freund's adjuvant (FCA), enzyme immunoassay-grade horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin, 3,3′,5,5′-tetramethylbenzidine (TMB), Tween-20, HRP and gelatin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture reagents (e.g. polyethylene glycol solution, HAT supplement, HT supplement, and 1640 cell culture) were obtained from Life Technologies Corporation (Shanghai, China). The materials of creating test strips were purchased from JieYi Biotech. Co., Ltd. (Shanghai, China), including glass fiber membrane for the sample pad, NC membrane used as immobilizing the coating antigen, H5076 filter paper used for the absorbent pad and Ahlstrom 8964 for the conjugated pad.

Solutions

Coating buffer (CB; 0.05 M carbonate bicarbonate, pH 9.6), blocking buffer (0.2% gelatin in coating buffer), washing buffer (PBST: 0.05% Tween-20 (v/v) in 0.01 M PBS), and stop solution (2 M sulfuric acid) were prepared. The substrate buffer consisted of solutions A (citric acid, H₂O₂, and Na₂HPO₄) and B (0.06% v/v TMB in glycol) at 5:1 (v/v) ratio. Swine urine was obtained from a local farm (Wuxi, China).

Antigen preparation

The RCT hapten was synthesized by the reductive amination reaction with octopamine (Liu, Kuang, Peng, Wang, & Xu, 2013) (Figure 1). The immunogen and coating antigens were RCT hapten-EDC-KLH and RCT hapten-EDC-OVA, respectively. RCT was linked to the proteins by an active ester method (Suryoprabowo, Liu, Peng, Kuang, & Xu, 2014). Firstly, 10 mg RCT hapten was dissolved in 800 μl of dimethylformamide (DMF) and 1 ml of 0.1 M 2-[N-morpholino] ethanesulfonic acid, pH 4.7. Subsequently, 22.6 mg of EDC and 13.6 mg of NHS were added. The solution was stirred overnight at room temperature and in the dark (solution 1). KLH (11 mg) and OVA (14.8 mg) were mixed with 2 ml of 0.1 M CB, pH 9.6 (solution 2 and solution 3, respectively). Solution 1 was divided in half and added to solutions 2 and 3. The two solutions were stirred overnight at room temperature and dialyzed against 0.01 M PBS for 3 days to remove unbound RCT. Finally, UV and polyacrylamide gel electrophoreses were used to confirm the presence of antigens, which were subsequently stored at −20°C.

Figure 1. Synthesis of the hapten of RCT.

RCT mAb preparation

Female BALB/c mice (6–8 weeks of age) were subcutaneously immunized with immunogen RCT hapten-KLH mixed with FCA at 100 μg per mouse. FIA was incorporated to the subsequent boost injection (50 µg/mouse), which was performed on day 28 following the first immunization and repeated every 4 weeks thereafter (Li, Ji, Chen, Liu, Xu, Peng, et al., 2008; Xing & Kuang, 2013). Following the third injection, mice blood sera were analyzed by indirect competitive (ic)-ELISA. The mouse with the highest inhibition and titer was intraperitoneally injected immunogen RCT hapten-KLH with 25 µg. After 21 days, the mouse splenocytes were fused with Sp 2/0 myeloma cells for hybridoma cell production. Hybridoma cells were selected by ELISA and subcloned three times. The selected hybridoma cell culture was injected into mice for the production of ascites (Deng et al., 2012). Antibodies were purified by caprylic acid and ammonium sulfate precipitation, dialyzed against PBS for 3 days, and stored at −20°C.

ELISA

We used the checkerboard method to optimize the concentration of antibody and antigen. In this experiment, 100 μl coating antigen (RCT-OVA) was added to each well in microtiter plates and diluted with coating solution. The coated plates were incubated either at 37°C for 2 h or at 4°C for 12 h and washed 3 times with PBST. Blocking buffer (200 μl/well) was added to the plates, which were subsequently incubated at 37°C for 2 h. The blocked plates were washed twice with washing buffer. RCT standard (50 μl), which was diluted with swine urine, and antibody (50 μl) were added into each well, and the plates were incubated at 37°C for 30 min. Following three washes, HRP (100 μl), previously diluted with antibody dilution buffer (PBST with gelatin) at 1:3000, was added and incubated at 37°C for 30 min. Finally, TMB substrate solution freshly prepared was added (100 μl/well). The plates were washed four times and incubated at 37°C for 15 min. Following the addition of stop solution (50 μl/well), absorbance was measured at 450 nm in a microplate reader (purchased from BioTek) (Guan, Guo, Liu, & Kong, 2015). Standard mAb curves were generated by plotting B/B₀ against log[RCT]. Antibody sensitivity was determined by 50% inhibition concentration (IC₅₀).

The ELISA conditions were optimized to improve its sensitivity. The effects of pH (5.0, 6.0, 7.4, 8.6, 9.6), methanol concentrations (0%, 10%, 20%, and 30%, v/v), and NaCl concentrations (0%, 0.4%, 0.8%, 1.6%, and 3.2%) were evaluated.

Cross-reactivity

Clenbuterol, salbutamol, phenylethanolamine A, arformoterol, and formoterol were used to assess cross-reactivity (CR) with RCT by ic-ELISA. CR was calculated by the following formula, CR (%) = (IC₅₀ of RCT/IC₅₀ of the tested analogues)×100%.

Preparation of colloidal gold-labeled mAb

The preparation of colloidal gold-labeled mAb has been previously described (Sun et al., 2012). Purified mAb was slowly mixed with gold solution (20 ml), resulting in solution A. Solution A (pH 8.2) was mixed with 0.1 M K₂CO₃ at room temperature for 2 h. Bovine serum albumin (BSA, 10%, 100 ml) was added, thereby resulting in solution B. Solution B was stirred for 2 h and centrifuged three times at 8000 rpm for approximately 20 min each time. Gold-labeled mAb was re-suspended in borate solution and stored at 4°C (Guo, Liu, et al., 2015).

Immunochromatographic strip preparation

The immunochromatographic strip consists of four parts: a conjugate pad, a nitrocellulose (NC) membrane, a sample pad, and an absorbent pad. The control and test line were loaded onto the goat anti-mouse IgG, and the coating antigen was added to the NC membrane using a membrane dispenser. There are two lines (control and test) with a 0.5 cm separation in-between. The NC membrane was dried at 37°C for 30 min and attached to the back of the plate. Therefore, the NC membrane, a conjugate pad, and sample pad were loaded to the back of the plate. The absorbent pad was attached to the upper section of the plate. Finally, the assembled plate was cut into 3-mm wide strips (Guo, Song, et al., 2015).

Immunochromatographic strip assay

Swine urine sample (∼80 μl) was added to the sample pad and, by capillarity, it migrated to the absorption pad. Gold-labeled mAb became soluble and migrated to the coating antigen of the NC membrane. In RCT-negative swine urine, the gold-labeled mAb was captured by the coating antigen resulting in a visible red test line. A control line may also be generated because of redundant mAb captured by goat anti-mouse IgG. In RCT-positive swine urine, the free RCT would compete with the immobilized RCT for the gold-labeled mAb; however, mAb would not complex with the immobilized RCT, leading to the disappearance of the test line. At high RCT concentrations in the sample, the test line would have a faint color.

Analysis of spiked swine urine samples

RCT-negative swine urine was confirmed by a commercial immunochromatographic strip assay (Beijing wei de wei kang Biotech Co, Ltd). Swine urine was purified by centrifugation (8000 rpm, 10 min), spiked with different RCT concentrations, and analyzed by ic-ELISA.

Results and discussion

Conjugated antigen preparation

KLH was selected as the carrier protein for immunization, while OVA was selected as the coating antigen. The production of immunogen and coating antigen was based on the EDC method. In the EDC method, the proportion of organic phase and water phase is crucial, because EDC activity in water is higher than in organic solutions. In this method, DMF was approximately 30% (v/v). Additionally, the pH value should be approximately 4.7 in the active system, and kept between 9 and 10 when the active solution was added into the protein solution.

The immunogen RCT hapten-KLH was confirmed by UV–VIS spectroscopy (Figure 2(a)), and the coating antigen was confirmed by polyacrylamide gel electrophoresis (Figure 2(b)). The main UV peak of the RCT hapten was 27 nm, and KLH carrier protein were at 280, 350 nm, respectively. And the main UV peak of the RCT hapten-KLH was between 270 and 280 nm, it had the offset. The conjugated antigen also had the carrier protein KLH UV peak 350 nm. The findings revealed that the production of immunogen and coating antigen was successful. Peak 1 corresponded to OVA, the carrier protein, while peaks 2, 3, and 4 corresponded to conjugated proteins (the coupling ratio in RCT-EDC-OVA was 60, 80, and 100 M _(RCT)/M _(OVA), respectively). The molecular weight of OVA is 45,000 Daltons. If the RCT hapten was successfully conjugated with the carrier protein KLH, the molecular weight of RCT hapten-KLH was higher than OVA, and it resulted that the stripe of RCT hapten-KLH was behind of the OVA. The findings proved that RCT hapten was successfully linked with carrier protein KLH.

Figure 2. (a) UV–VIS spectra of RCT immunogen (RCT-EDC-KLH); (b) polyacrylamide gel electrophoresis image of coating antigens (1: OVA; 2: RCT:OVA = 60; 3: RCT:OVA = 80; 4: RCT:OVA = 100 M_(RCT) : M_(OVA)).

Preparation of mAb

MAb was obtained following ascite purification and confirmed by ic-ELISA. The cell lines 1B4, 2B11, 3E7, 4B3, 5B4, and 6D1 had similar IC₅₀ values (0.05 ng/ml) in negative swine urine. The cell line 1B4 was selected for the immunochromatographic strip assay.

Optimization of ic-ELISA

RCT-EDC-OVA was selected as the coating antigen at a concentration of 0.1 µg/ml, with an antibody concentration of 0.06 µg/ml. The optimization experiments were based on the monoclonal antibody 1B4.

The effect of pH (5.0–9.6) was evaluated. As shown in Figure 3(a), the Absorbance value_max (Abs_max)/IC₅₀ value at pH 7.4 was the highest among the tested pH values. Furthermore, IC₅₀ (0.05 ng/ml) was the lowest and Abs_max was the highest at pH 7.4. Therefore, pH 7.4 was selected for subsequent experiments.

Figure 3. (a) The influence of pH; (b) The influence of ionic strength; (c) The influence of methanol content; (d) Standard inhibition curve for RCT by the optimized conditions.

The effect of ionic strength was evaluated using four different NaCl concentrations (0.4–3.2%) in PB (Figure 3(b)). Abs_max was similar between 0.4% and 0.8% NaCl; however, IC₅₀ was lower at 0.8% than at 0.4% NaCl. At > 0.8% NaCl, Abs_max decreased. Therefore, 0.8% NaCl was selected for subsequent experiments.

Finally, the effect of different methanol concentrations (0–30%, v/v) was evaluated. With increasing methanol concentration, Abs_max decreased and IC₅₀ increased (Figure 3(c)). IC₅₀ was lower at 0% methanol than at other methanol concentrations. Therefore, 0% methanol was selected for subsequent experiments.

Therefore, the optimal conditions were pH 7.4, 0.8% NaCl in PB buffer, and 0% methanol concentration. At these conditions, IC₅₀ was 0.05 ng/ml and IC₂₀ (20% inhibition concentration) was 0.0286 ng/ml (Figure 3(d)).

Cross-reactivity

The specificity of the synthesized mAb was assessed by determining CR with other beta-adrenergic agonists such as salbutamol, clenbuterol, phenylethanolamine A, arformoterol, and formoterol, The developed RCT antibody had high specificity (Table 1). The CR values with the beta-adrenergic agonists were < 0.1%.

Table 1. CR of related compounds

Chemicals	Structure	IC50 (ng/mL)	CR (%)
RCT		0.05	100
SAL		>100	<0.1
CL		>100	<0.1
Phenylethanolamine		>100	<0.1
Arformoterol		>100	<0.1
Formoterol		>100	<0.1

RCT recovery by ic-ELISA

Swine urine was spiked with different RCT concentrations (0.025–0.1 ng/ml) and analyzed by ELISA. RCT recovery was 110–120% (Table 2), and the effect of the urine matrix led to the recovery was very high. The intra-day coefficient variation (CV) was 0.34–3.87%, and the inter-day CV was 0.14–1.46%.

Table 2. Recoveries of RCT in swine urine samples.

Samples	Spiked level (ng/ml)	Mean (ng/ml) ± SD	Recovery (%)	CV (%) (n = 3)
				Intra-assay	Inter-assay
Urine	0.025	1.37 ± .004	120	0.34	0.23
	0.05	1.07 ± .006	120	0.88	0.14
	0.1	0.53 ± .015	110	3.87	1.46

RCT detection by immunochromatographic strip assay

The immunochromatographic strip was assayed by the dry method. Swine urine does not require complex pretreatment steps. The results revealed that swine urine was RCT-negative based on commercial immunochromatographic strip assays. In this study, 3 ng/ml RCT was added to swine urine. Firstly, we used the wet method to assess the optimal antibody and coating antigen concentrations. Using coating antigen (RCT-BSA-60) and different antibody concentrations, we optimized the assay. The results revealed that the optimum conditions were 1 ml of gold nanoparticle with 4 μl K₂CO₃, 8 μg/ml antibody, and 1 mg/ml coating antigen. However, these conditions were not suitable for subsequent experiments. Therefore, we selected RCT-EDC-OVA 60 as the coating antigen at a concentration of 0.5 ng/ml (Figure 4). At 1 ml of gold nanoparticle with 6 μl K₂CO₃, the RCT cell line 1B4 was selected for subsequent experiments.

Figure 4. The sensitivity of the immunochromatographic assay in urine for RCT different antibodies (0.3 ng/ml from left to right).

Different RCT standard concentrations (0–3 ng/ml) were added to swine urine. An aliquot (80 µl) of the spiked urine sample was added to the sample pad of the strips. The results were visible in 5 min, and as showed in Figure 6 and Table 3, the strip performed well when the concentrations (0, 1, 2, and 3 ng/ml) were analyzed, the RCT strip cutoff value was 2 ng/ml.

Table 3. Accuracy and reproductivity evaluation of the RCT strip.

RCT concentration (ng/ml)	0	1	2	3
Control line	+++	+++	+++	+++
Test line	+++	+	−	−
Results	Negative	Weakly positive	Positive	Positive

Notes: +++: Highly red color. +: Weakly red color. −: No test line is observed.

There are several RCT-detecting immunochromatographic strips commercially available (Figure 5) with RCT cutoff values of 3 ng/ml (Figure 6(a), the company A; Figure 6(b), the company B; Figure 6(c), the company C; Figure 6(d), the company D) and 5 ng/ml (Figure 6(e), the company E; Figure 6(f), the company F). In general, the optimum RCT-detecting strips have an RCT cutoff value of 3 ng/ml. In this study, the RCT cutoff value was 2 ng/ml in swine urine. Therefore, our developed immunochromatographic strip assay is practical for the detection of RCT residues in urine samples.

Figure 5. Comparison with other market products. (a) The company A for RCT, it cuts off at 3 ng/ml; (b) The company B for RCT, it cuts off at 3 ng/ml; (c) The company C for RCT, it cuts off at 3 ng/ml; (d) The company D for RCT, it cuts off at 3 ng/ml; (e) The company E for RCT, it cuts off at 5 ng/ml.; (f) The company F for RCT, it cuts off at 5 ng/ml.

Figure 6. By optimized the gold nanoparticle, coating and labeled antibody, the cut off value of immunochromatographic strip in urine was at 2 ng/ml for RCT antibody 1B4.

Conclusions

In this study, we developed an ultrasensitive mAb for RCT detection, with an IC₅₀ of 0.05 ng/ml. CR was very low, indicative that the developed mAb was highly specific. The developed mAb can be applied for RCT detection in swine urine samples. By optimizing the coating antigen and gold nanoparticle, a rapid and portable immunochromatographic strip assay was also successfully developed with an RCT cutoff value of 2 ng/ml in swine urine.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes on contributors

Huaiying Gu obtained her bachelor’s degree from Southwest University for Nationalities, Chengdu, China in 2013 and then her master’s degree in food science from Jiangnan University (Wuxi, China). Her research interest includes immunoassay development for veterinary drugs.

Liqiang Liu earned 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 include immunochromatographic strip design and application.

Shanshan Song received her Master’s 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 interest includes monoclonal antibody development.

Hua Kuang obtained 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 nanoscience and food safety. Her research interests are biosensor development.

Chuanlai Xu is a full professor of Food science and technology of Jiangnan University. He earned his Ph.D. in 2002. His research interests are fast detection technology and food safety evaluation.

Additional information

Funding

This work is financially supported by the Key Programs from MOST [2012BAK08B01], and grants from Natural Science Foundation of Jiangsu Province, MOF and MOE [BE2013613, BE2013611, BK20140003, 201310128, 201310135].