A sensitive lateral flow immunoassay for the multiple residues of five adamantanes

ABSTRACT

Adamantanes (ADAs) are tricyclic decane saturated derivatives, which are used in the protection against the influenza virus. However, their abuse in animal husbandry has affected human health. To help prevent drug resistance and protect food safety, a rapid, portable, sensitive, and reliable method for onsite detection of ADAs was developed. Herein, we have produced a broad-specificity anti-ADAs monoclonal antibody (mAb), the subclass of which is IgG_2b, and its affinity constant is 7.25 × 10⁹ L/mol. From this, an antibody-based lateral flow immunoassay (LFIA) was developed for the rapid detection of five ADAs in food samples. When viewed with the naked eye, the LFIA had a visual detection limit of 0.1, 0.1, 1, 5, and 10 μg/kg and a cut-off value of 2.5, 2.5, 10, 100, and 100 μg/kg for somantadine, rimantadine, amantadine, 1-adamantanemethanol, 1-adamantyl methyl ketone respectively in chicken samples. The test results for the LFIA were very similar to that seen when using an enzyme linked immunosorbent assay (ELISA). Therefore, this paper-based LFIA provides a useful tool for rapid, onsite detection of ADAs in biological samples.

KEYWORDS:

• Adamantane
• gold nanoparticles
• monoclonal antibody
• lateral flow immunoassay

Introduction

Rapid, point-of-care (POC), and cost-effective detection methods are highly sought after by environment monitoring agencies, the health sector, and the food industry (Cate, Noblitt, Volckens, & Henry, 2015; Li, Tian, Al-Tamimi, & Shen, 2012; Mani, Kadimisetty, Malla, Joshi, & Rusling, 2013; Srisa-Art, Boehle, Geiss, & Henry, 2018; Wu et al., 2012). Among various of POC assays, paper-based testing devices have aroused great interest. Paper as a cheap, abundant, disposable, and easy to store material has become a desirable substrate for mass production of POC devices (Gong & Sinton, 2017; Nery & Kubota, 2013). In addition, the property of moving fluids by capillary action without the need for external pumping sources makes the advantages of paper in POC tests very attractive (Ge et al., 2012). As a result, sheet paper is the preferred platform for transforming conventional immunoassays for the diagnosis of disease, monitoring the environment, and detecting food toxins.

Amantadine (AMA) is an amino derivative of saturated tricyclic decane with confirmed antiviral effects (Eike et al., 2010). AMA has good efficacy for the prevention and early treatment of Asian influenza A virus and was the first Food and Drug Administration approved anti-influenza drug. Amantadine and its analogues including rimantadine (RMA), somantadine (SMA) have also been used to treat viral infection, Parkinson's disease, and Alzheimer's disease (Sharma, Lyons, & Pahwa, 2018; Singer, Papapetropoulos, Gonzalez, Roberts, & Lieberman, 2005). The favourable performance of AMA and its analogues for treating animal influenza made these drugs more popular in animal husbandry. As the use and dosage of these drugs increased, side effects appeared to gradually increase, especially drug resistance and residues in edible animal tissues. Therefore, it is essential to detect ADA residues in animal products to maintain human health and safety. The current analysis of ADAs mainly relies on instrumental assays (Farajzadeh, Nouri, & Alizadeh Nabil, 2013; Higashi, Uemori, & Fujii, 2005; Zhao et al., 2014). However, due to the use of expensive equipment, complex sample pre-treatment, length of time to assay, and the need for professional operators, these methods are unsuitable for rapid high throughput screening.

Here, a lateral flow immunoassay (LFIA) that used a paper sheet as the substrate for immobilized detection reagents for five different ADAs in biological samples was developed. Along with this LFIA, we have established a gold nanoparticle-based colorimetric signal readout system, in which the signals can be determined by eye or a portable scan reader. The test results of the LFIA when using real samples demonstrated that the LFIA is a simple, feasible, and efficient tool for onsite screening of these analytes.

Materials and methods

Chemicals and apparatus

Amantadine, RMA, SMA, 1-Adamantyl methyl ketone (AMK), 1-Adamantanemethanol (AMM), succinic anhydride, and ethyl 6-bromohexanoate were purchased from J&K Scientific Ltd. (Beijing, China). Bovine Serum Albumin (BSA), ovalbumin (OVA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), Freund's complete adjuvant, Freund's incomplete adjuvant, and gold chloride trihydrate were obtained from Sigma-Aldrich (St Louis, MO, USA). RPMI-1640 cell culture media, hypoxanthine-aminopterin-thymidine supplement, hypoxanthine-thymidine supplement, polyethylene glycol 1500, and fetal calf serum were purchased from Gibco BRL (Paisley, UK). All the reagents and solvents were of analytical grade or higher.

The sample pad (glass fibre membrane, CB-SB08), poly-vinyl chloride (PVC) sheets, and the absorption pad (SX18) were obtained from Goldbio Tech Co., Ltd (Shanghai, China). The nitrocellulose (NC) membrane (Pura-bind RP) was from Whatman-Xinhua Filter Paper Co., Ltd (Hangzhou, China).

The spectrograms for the haptens were recorded by an ultra-high-performance liquid chromatography series quadrupole time of flight mass spectrometer (Waters Technologies, American). The membrane dispenser was obtained from Xinqidian Gene-Technology Co., Ltd (Beijing, China), and a CM4000 Guillotine Cutting Module was purchased from Shanghai Kinbio Tech Co., Ltd (Shanghai, China). Other instruments used in this study were the multiskan MKS microplate reader (Thermo Lab systems Company, Beijing, China) and UV/Vis scanner (Bokin instruments, Tsushima, Japan).

Preparation of the haptens and antigens

Two different haptens, AMA-hapten and RMA-hapten, were synthesized in this study. The synthetic pathways employed were as follows:

AMA-hapten (6-(((3s, 5s, 7s)-adamantan-1-yl) amino) hexanoic acid): A mixture of AMA (2.00 g, 13.2 mmol), ethyl 6-bromohexanoate (2.93 g, 13.2 mmol) and NaOH (0.60 g, 15.0 mmol) were dissolved in 20 mL of DMF and the solution was stirred at 50°C for 15 h. After cooling to room temperature (RT), 200 mL of ultrapure water was added into the reaction mixture, and then the solution was extracted three times with dichloromethane. The organic layer was dried over anhydrous Na₂SO₄, and then concentrated under reduced pressure to produce a crude product, which was purified by column chromatography to generate the intermediate product ethyl 6-(((3s,5s,7s)-adamantan-1-yl) amino) hexanoate. This intermediate product was dissolved in 20 mL of methanol aqueous solution (v/v, MeOH: H2O = 5:1), and 0.58 g of LiOH was added to the solution. Then, the mixture was stirred at RT for 12 h, and the product was concentrated under reduce pressure in 20 mL of ultrapure water. The pH of this solution was adjusted to pH 7.0 with 1 M hydrochloric acid solution. The precipitate was then collected and dried to give a final product the AMA-hapten.

RMA-hapten (4-((1-((3r, 5r, 7r)-adamantan-1-yl)ethyl)amino)-4-oxobutanoic acid) was synthesized by the succinic anhydride method, which was the same as we reported in our previous work (Wang, Xie, et al., 2017; Wang, Zheng, et al., 2017). A mixture of RMA (1 g, 5.5 mmol) and succinic anhydride (0.66 g, 6.6 mmol) in 15 mL anhydrous pyridine was prepared and refluxed at 60°C for 12 h. Then, the mixture was dried using rotary evaporation, and the residue was dissolved in 15 mL of methanol aqueous solution (v/v, MeOH: H2O = 1:2). The solution was extracted three times with ethyl acetate. The organic layer was collected, and a precipitate was generated after adding 1 M hydrochloric acid solution. The precipitate was washed with ultrapure water and dried in a drying oven, and the RMA-hapten was obtained as a white solid.

Due to the presence of carboxyl groups in haptens, we chose the active ester method (Chen et al., 2017a; Liu et al., 2018; Zhao et al., 2017) to couple the small molecules to the carrier proteins. Briefly, a mixture of AMA-hapten (10.5 mg, 0.04 mmol) and NHS (13.7 mg, 0.12 mmol) was dissolved in 2 mL of DMF. The solution was stirred for 15 min and then 22.8 mg of EDC (0.12 mmol) was added into the solution. The mixture continued to react for another six hours at RT and was added dropwise to a carrier protein solution (30 mg of BSA dissolved in 10 mL of carbonate-bicarbonate buffer, pH 9.6). After stirring overnight, the reaction was dialyzed against 0.01M PBS (pH 7.4) for more than 2 days and the final antigen was stored at −20°C.

The other immunogen RMA-hapten-BSA and the coating antigens AMA-hapten-OVA and RMA-hapten-OVA were prepared in the same way.

Preparation of the mAb

Ten female BALB/c mice (6 weeks of age) were used and divided into two groups. One group underwent subcutaneous injection with the immunogen AMA-hapten-BSA, and the other group was treated with RMA-hapten-BSA. This process was similar to that used in our previous studies (Wang et al., 2016; Xu et al., 2014). After the third immunization, blood was extracted from the tail vein of each mouse, and the titre and specificity were determined by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) (Suryoprabowo, Liu, Peng, Kuang, & Xu, 2015). The mouse whose serum showed the highest titre and the best specificity was injected intraperitoneally with 25 μg of immunogen mixed with 100 μL of 0.9% saline solution to boost the immunization.

After three days, cell fusion between splenocytes from the immunized mouse and SP2/0 myeloma cells was carried out under sterile conditions (Xie et al., 2018; Zhao et al., 2017). The culture supernatants were collected and monitored by ELISA and ic-ELISA after 7 days. The selected hybridomas were subcloned according to the limiting dilution method. The target hybridomas were collected and expanded in culture. Then, the cell lines were injected intraperitoneally into three paraffin-primed BALB/c mice for producing ascites fluid. The mAb was purified from ascites fluid using the caprylic acid-ammonium sulfate precipitation method (Chunsheng et al., 2018; Xie et al., 2018; Xie, Chen, & Yang, 2012) and the mAb was stored at −20°C until needed for further experiments.

The subclass of the mAb was determined using a mouse mAb isotyping (IgA, IgM, IgG₁, IgG_2a, IgG_2b, IgG₃) kit (Cellway-lab, Luoyang, China). The affinity of the mAb against AMA and its analogues was determined according to previous studies (Beatty, Beatty, & Vlahos, 1987; Wang, Zheng, et al., 2017). In order to evaluate the performance of this mAb in the ic-ELISA, standard inhibition curves were established by plotting the optical density values at 450 nm obtained from each standard solution against the corresponding concentration of analyte. The half-maximal inhibitory concentration (IC₅₀) and the limit of detection (LOD) calculated from the standard inhibition curves were used to evaluate the sensitivity of the anti-ADAs mAb. Several ADAs, including SMA, RMA, AMM, and AMK, were selected for the cross-reactivity (CR) testing. The CR of the mAb was calculated using the following equation:$\mathrm{C}\mathrm{R}\left(\text{\%}\right)=\left(\mathrm{I}{\mathrm{C}}_{50}\mathrm{o}\mathrm{f}\mathrm{A}\mathrm{M}\mathrm{A}/\mathrm{I}{\mathrm{C}}_{50}\mathrm{o}\mathrm{f}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{a}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{o}\mathrm{g}\mathrm{u}\mathrm{e}\right)\times 100\text{\%}$

Preparation of detection reagents

In this study, the colloidal gold (CG)-labeled anti-ADAs mAb was chosen as the detection reagent for the LFIA.

The CG nanoparticles were produced using the sodium citrate reduction method according to previous studies (Liu et al., 2016; Song et al., 2019; Wang et al., 2018; Zhan et al., 2017). Briefly, 50 mL of 0.01% (w/v) HAuCl₄ solution in a Florence flask was boiled under constant stirring, and 2 mL of freshly prepared 1% (w/v) trisodium citrate was immediately added to the flask. The flask was constantly boiled until the colour of the solution changed to the colour of red wine. The solution was cooled to RT and stored at 4°C until use.

Colloidal gold-labeled antibody conjugates were prepared according to previous studies (Chen et al., 2017b; Liu et al., 2016; Peng et al., 2017). Firstly, the pH of the CG solution was adjusted to pH 8.8 with 0.1 mol/L K₂CO₃, and then 1 mg of mAb was slowly added into 100 mL of CG solution. The mixture was stirred for 30 min at RT. A total of 10 mL of 10% BSA aqueous solution was used to block any free CG particles which were added under constant stirring for another 30 min. The solution was centrifugation at 8,000 × g for 30 min to remove excess blocking agent and mAb. The precipitates were washed three times with resuspension buffer (0.02M borate buffer [pH8.4], 0.5% Tween, 0.6% BSA, and 5% sucrose). Finally, the CG-labeled mAb precipitate was resuspended in resuspension buffer and stored at 4°C.

Assembly of the test strip

As shown in Figure 1, the test strip consisted of the sample pad, absorption pad, nitrocellulose (NC) membrane, and polyvinyl chloride (PVC) backing card, and its assembly was performed according to previous methods (Kolosova, De Saeger, Sibanda, Verheijen, & Van Peteghem, 2007; Yang et al., 2018; Yu et al., 2015). The coating antigen and the goat anti-mouse IgG were both dispensed onto the NC membrane to form the test line (T line) and control line (C line), and then the membrane was dried at 37°C for 2 h in an air oven. The free sites on NC membrane were blocked with 0.01 M PBS containing 2% (w/v) BSA for 2 h at RT. The sample pad was treated by 0.01M PBS containing 1% (w/v) BSA, 0.2% (v/v) Tween 20, and 1% (w/v) and sucrose before assembly. After desiccation, the sample pad, NC membrane, and the absorbent pad were laminated onto the PVC backing card with a 2 mm overlap between each part. Finally, the prepared strip card was cut lengthways into 5 mm wide strips and the final product was stored under dry conditions at RM until use.

Figure 1. Schematic illustration of the LFIA for the detection of ADAs.

Sample pretreatment

Blank chicken samples, were obtained from a local market, and confirmed as negative for ADAs using LC-MS. The process of sample pre-treatment was described as follows: 3.0 ± 0.05 g of homogenized chicken samples were weighed and put into a centrifuge tube. One gram of anhydrous sodium sulfate and 6 mL of acetonitrile was added, and then the tube was vortexed for 5 min and centrifuged for 10 min at 4000 × g. Two millilitres of supernatant was added into a second tube and dried under nitrogen in a water bath at 60°C. The residues were dissolved with 1 mL of PBS containing 10% (v/v) methanol and stored at 4°C until needed.

Procedure and principle of the LFIA

For the sample assay, 10 μL of CG-labeled antibody solution, 40 μL of resuspension buffer and 100 μL of ADAs standard or sample extract solution were mixed and incubated for 5 min at RT. The sample pad of the test strip was dipped into the mixture and the solution migrated up the strip to the absorption pad by capillary forces, and the results were obtained in 10 min.

The LFIA principle is based on a competitive reaction, which was shown in Figure 1. If the sample contains ADAs, the CG-labeled mAb will firstly react with free ADAs in the sample extract and then the unreacted CG-labeled mAb will react with the coating antigens immobilized on the T line during migrating to the absorption pad. Therefore, the free small molecule will compete with the coating antigen immobilized on the T line for the limited amount of CG-labeled mAb. This implies that the colour of T line is related to the concentration of free ADAs in the extract solution. As the concentration of ADAs increases, the amount of unreacted CG-labeled mAb decreases, and the colour intensity of the T line decreases. The C line will always appear as a visible red line, indicating the validity of the test. In addition, because of the inverse relationship between the amount of ADAs in the sample and the colour intensity of the T line, the LFIA can be used for the quantitative detection of ADAs by using a hand held strip reader.

Results and discussions

Design and characterization of the haptens

The aim of this study was to produce a wide spectrum antibody, which can simultaneously recognize more than five ADA drugs. Therefore, the chemical structure of the hapten would need to include a structure common to all of the ADAs. In this study, two haptens were synthesized by using the succinic anhydride and the nucleophilic reaction methods, respectively, and their synthetic processes were shown in Figure 2.

Figure 2. The synthetic schemes of haptens for AMA. (a) The hapten (AMA-hapten) was synthesized by a nucleophilic substitution reaction. (b) The hapten (RMA-hapten) was synthesized by a succinic anhydride method.

As shown in Figure 3(a) and 3(b), the liquid chromatogram spectrum revealed that the major products were present at 7.05 min, and at the same retention time, the MS m/z 266 confirmed the presence of the AMA-hapten. The LC-MS analyses for the RMA-hapten were shown in Figure 3(c) and 3(d). The retention time of the RMA-hapten appeared at 9.85 min, and the MS chromatography revealed that the m/z 280 was consistent with the molecule weight of RMA-hapten, resulting in the successful synthesis of both the AMA and RMA-haptens.

Figure 3. LC–MS analysis of haptens of ADAs. (a) The liquid chromatogram spectrum of AMA-hapten; (b) the mass spectrum of AMA-hapten; (c) The liquid chromatogram spectrum of RMA-hapten; (d) the mass spectrum of RMA-hapten.

Screening of antisera and characterization of the mAb

The sera obtained from the two groups were assayed by ic-ELISA, and the results were shown in Table 1. The serum of each mouse was detected using homogeneous and heterogeneous coating antigens, and the results indicated that the best effect was obtained when applying the AMA-hapten-BSA as immunogen and the RMA-hapten-OVA as the coating antigen. As a result of this, mouse No. 4 was chosen for cell fusion.

Table 1. Titre and specficity of the immunized mice.^a

Immunogens/coating antigens		AMA-hapten-BSA/ RMA-hapten-OVA					RMA-hapten-BSA/AMA-hapten-OVA
Mouse No.		1	2	3	4	5	6	7	8	9	10
Titre	OD at 450 nm	2.141	1.623	2.402	2.293	2.373	2.451	2.728	2.512	2.627	2.935
Specificity^b	AMA 10 ng/mL	59%	56%	19%	57%	35%	<5%	<5%	<5%	<5%	<5%
	RMA 5 ng/mL	54%	57%	25%	60%	41%	<5%	<5%	<5%	<5%	<5%
	SMA 5 ng/mL	52%	60%	37%	65%	58%	<5%	<5%	<5%	<5%	<5%

^aThe data shown are at a coating antigen concentration of 0.3 µg/mL and an serum dilution of 1/3000.

^bThe specificity of each serum was primarily assessed by the [1-B/B₀]*100% values at three different standard solution, which were measured by ic-ELISA

A sensitive mAb against ADAs was purified from the ascites fluid by the caprylic acid-ammonium sulfate precipitation method. As shown in Figure 4(a), the subclass of the mAb was identified as IgG_2b by using a mouse mAb isotyping kit. A series of concentrations of mAb (1, 0.5, 0.25 and 0.125 μg/mL et al.) and coating antigen (1, 0.5, 0.25, and 0.125 μg/mL) were assembled in a checkerboard pattern to evaluate the affinity of the anti-ADAs mAb. The results were shown in Figure 4(b), and the affinity constant (K_aff) was calculated to be 7.25 × 10⁹ L/mol, and the CR of this mAb was shown in Table 2. The IC₅₀ values were 0.88, 0.43, 0.39, 7.81, and 7.42 ng/mL for AMA, RMA, SMA, AMK, and AMM, respectively. From the results it is clear that the IC₅₀ values of this antibody for AMA, RMA, and SMA are superior to that of AMK and AMM. One possible reason for this is that the amino group present in the structures of AMA, RMA, and SMA, and not present in AMK and AMM, and this may have a profound effect on the antibody production process.

Figure 4. Characterization of the mAb against ADAs. (a) Isotype determination of the anti-ADAs mAb. (b) Affinity testing results of the antibody.

Table 2. The characterization of anti-ADAs mAb.

Chemical compound	Structure	Standard curve and IC50 (ng/mL)	CR (%)
AMA			100
RMA			204
SMA			226
AMK			11
AMM			12

Optimization of the LFIA

In general, the recognition element is an indispensable part in any sensor device. In LFIA, the CG-labeled anti-ADAs mAb conjugates fulfil this role. A good CG-labeled antibody always results in a good performance when using LFIA. For this reason, the binding conditions for the antibody and CGs were optimized. The pH is one important factor influencing the synthesis of immune reagent conjugates. If the pH is equal to the isoelectric point of an antibody, the mAb is electrically neutral, and is therefore easier to absorb onto the surface of the CGs. Therefore, the pH value was optimized using different concentrations of K₂CO₃ to get close to the isoelectric point of the antibody. The results demonstrated that pH 8.8 represented an optimal value.

The quantity of mAb is another factor requiring optimization. In this study, a series of mAb contents (50, 100, 150, and 200 μg) were introduced to 10 mL of CG solution. The results revealed that when the concentration of antibody was 10 μg/mL, the consumption of antibody was minimal and the performance of the LFIA was optimal.

Previous studies (Li et al., 2011) have reported on a coating condition which included the coating buffer system and coating antigen. In our study, two coating antigens (RMA-hapten-BSA and RMA-hapten-OVA) and three coating buffers, including carbonate buffer (CB, pH 9.6), phosphate buffer (PB, pH 6.5) and PBS, were chosen to determine optimal combinations. The results revealed that the carrier protein BSA was better at avoiding matrix interference when compared with OVA, and the sensitivity of the LFIA for the five ADAs was better when using CB rather than PBS or PB. Therefore, the optimal combination was RMA-hapten-BSA and CB.

Finally, the effect of resuspension buffer with differing amounts of CG-labeled antibody were studied. The functions of the resuspension buffer are primarily to increase the flow rate of the sample solution, protect the immune reagents and improve the stability of the LFIA. In this study, a series of Tween 20 (v/v, 0.3%, 0.5%, and 1%), BSA (w/v, 0.2%, 0.4%, and 0.6%), and sucrose (w/v, 2%, 5%, and 8%) concentrations were optimized. The resuspension buffer containing 0.5% Tween 20, 0.6% BSA, and 5% sucrose was found to have the best effect. The amount of CG-labeled antibody will influence the sensitivity of the LFIA and the colour development of the T and C lines. High amounts of CG-labeled mAb will reduce the sensitivity, whereas low amounts will result in a weak T line. In this study, the best results occurred when the amount of CG-labeled antibody was 10 μg.

Analytical performance of the LFIA

Under optimal conditions, the detection of the LFIA for ADAs were evaluated by testing chicken samples spiked with ADAs. The results revealed that the LFIA could detect ADAs both qualitatively and quantitatively within 15 min.

For qualitative detection of ADAs, the results of the FLIA can be determined using the naked eye. As shown in Figure 5(a), the chicken samples spiked with the five different ADAs were tested using the LFIA. The visual limit of detection (vLOD) is defined as the minimum concentration of the target that can produce a detectable colour change on the T line when compared to the negative control, and the cut-off value is defined as the concentration of the target that can produce no colour on the T line. As shown in Figure 5(a), the vLOD of the LFIA were 0.1, 0.1, 1, 5, and 10 μg/kg for SMA, RMA, AMA, AMM, and AMK respectively, and the cut-off values were 2.5, 2.5, 10, 100, and 100 μg/kg for SMA, RMA, AMA, AMM, and AMK respectively in the chicken samples.

Figure 5. The test results of ADAs with LFIA. (a) Qualitative detection of ADAs in chicken samples (SMA: 0, 0.1, 0.25, 0.5, 1, 2.5 μg/kg; RMA: 0, 0.1, 0.25, 0.5, 1, 2.5 μg/kg; AMA: 0, 0.5, 1, 2, 5, 10 μg/mL; AMM: 0, 5, 10, 20, 50, 100 μg/kg; AMK: 0, 5, 10, 20, 50, 100 μg/kg); (b) Standard curves for detecting chicken samples using LFIA. The X-axis is shown as the logarithmic concentrations of ADAs; and T/T₀ defined as the ratio of the colour density of the spiked sample to that of the blank sample.

For quantitative detection of ADAs, a portable strip scan reader was used to test the colour intensity of the T line. The concentration of ADAs could be calculated from a standard curve established by plotting the T/T₀ values (the ratio of the colour density of the spiked sample to that of the blank sample) for each standard solution against the logarithmic concentration of the ADAs. As shown in Figure 5(b), five curves were constructed for quantitative detection of the ADAs. The LODs (the concentration of ADAs with a 10% inhibition of the colour density in the negative sample) of the LFIA were 0.06, 0.08, 0.5, 4.1, and 4.9 μg/kg for SMA, RMA, AMA, AMM, and AMK, respectively.

Recovery of ADAs in chicken samples

In order to evaluate the resistance of the LFIA to matrix interference, chicken samples spiked with different concentrations of ADAs (0, 0.2, and 0.5 μg/kg for SMA; 0, 0.2, and 0.5 μg/kg for RMA; 0, 2, and 5 μg/kg for AMA; 0, 25, and 50 μg/kg for AMM; 0, 25, and 50 μg/kg for AMK) were detected to determine recovery efficiency, and the results are shown in Table 3. The recovery ranged from 101.4∼110.5% for SMA, 101.9∼106.7% for RMA, 95.0∼110.3% for AMA, 89.4∼100.4% for AMM, and 91.5∼99.0% for AMK with the highest CV at 7.85%.

Table 3. Recovery and precision of LFIA for testing chicken samples spiked ADAs (n = 5).

ADAs	Spiked level (μg/kg)	Detection level (μg/kg) Mean ± SD	Recovery rate (%)	CV (%)
SMA	0	^aND	^bNC	NC
	0.2	0.22 ± 0.005	110.5 ± 2.3	2.10
	0.5	0.51 ± 0.02	101.4 ± 4.2	4.13
RMA	0	ND	NC	NC
	0.2	0.21 ± 0.006	106.7 ± 3.0	2.79
	0.5	0.51 ± 0.02	101.9 ± 4.8	4.70
AMA	0	ND	NC	NC
	2	2.21 ± 0.06	110.3 ± 3.2	2.94
	5	4.75 ± 0.37	95.0 ± 7.5	7.85
AMM	0	ND	NC	NC
	25	22.34 ± 0.65	89.4 ± 2.6	2.91
	50	50.18 ± 2.46	100.4 ± 4.9	4.89
AMK	0	ND	NC	NC
	25	24.75 ± 0.46	99.0 ± 1.8	1.87
	50	45.76 ± 0.79	91.5 ± 1.6	1.74

^aND, not detectable. ^bNC, not calculated.

Authenticity of LFIA

Ten chicken samples spiked with different concentration of ADAs were prepared and simultaneously analyzed by the LFIA and the ELISA kits. The comparison between the LFIA and ELISA is demonstrated in Table 4. The differences between the LFIA and the ELISA for testing chicken samples ranged between 0.3% and 16.4%, indicating that the results from the LFIA had a good correlation with the ELISA kit.

Table 4. Comparison of LFIA with ELISA kit for detecting ADAs residues in chicken samples (n = 3).

Sample No.		LFIA (μg/kg)	ELISA (μg/kg)	Difference between LFIA and ELISA (%)
SMA	1	0.17	0.16	7.9
	2	0.45	0.44	2.2
RMA	3	0.27	0.23	16.4
	4	0.54	0.57	5.9
AMA	5	1.69	1.76	3.9
	6	3.54	3.81	7.1
AMM	7	12.17	12.21	0.3
	8	21.29	20.72	2.7
AMK	9	19.03	21.43	11.2
	10	35	33.21	5.4

In summary, two haptens based on the common structure of ADAs were firstly designed, and then two immunogens based on the haptens were injected subcutaneously into mice. The mouse immunized with AMA-hapten-BSA was chosen to carry out cell fusion, and a sensitive mAb, having the ability to recognize five ADAs, was produced. Next, we developed a LFIA strip that could detect all five ADAs simultaneously within 15 min based on this broad-specificity mAb. The recovery experiments and the comparisons with ELISA indicated that the LFIA is capable to detecting ADAs in chicken samples. Therefore, the LFIA is a very convenient tool for the screening of large sample sizes at the point-of-source.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work is financially supported by National Key R&D Program (2018YFC1602901), the National Natural Science Foundation of China (21631005, 21874058, 51802125, 21771090 and 31771084), the Natural Science Foundation of Jiangsu Province and MOF (BK20180029, BK20180605, BE2016307).