Development of an amplified luminescent proximity homogeneous assay for the detection of sulfonamides in animal‐derived products

Abstract In this study, we carried out an amplified luminescent proximity homogeneous assay (AlphaLISA) to detect sulfonamides (SAs) antibiotic residues in plasma, milk, pork, chicken, and fish. The SAs AlphaLISA method can detect 13 SAs with half‐inhibitory concentration (IC50) 2.11–29.77 ng/ml. The detection level of those SAs was 0.3–41.12 ng/ml in matrices, which satisfied the maximum residue limit (MRL) of the European Union, United States, and China. Our recoveries are in the range of 88% to 116.8% with a coefficient of variation less than 9.3% for different spiked food samples. We observed a good correlation between the AlphaLISA and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) with blood samples from injected rabbits. The established AlphaLISA method provided a no‐washing, rapid, high‐throughput screening tool for SAs in food quality control, which is suitable for small‐volume samples.

Several instrumental analysis techniques have been proposed for the determination of SAs residue (Premarathne et al., 2017;Wang et al., 2019;Xie et al., 2020;Yang et al., 2018;Zhao et al., 2018), including liquid chromatography (LC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and ultra-high-performance supercritical fluid chromatography (SFC). All these detection techniques have earned robust recognition in SAs and have many advantages, such as large dynamic linear range, low detection limits, and high productivity. However, most of these are complicated, time-consuming, and costly. Meanwhile, immunoassay methods are being developed rapidly in screening large numbers of sample because of its high throughput, short detection time, reduced sample consumption, and low overall cost Li et al., , 2020Liang et al., 2019). Enzyme-linked immunosorbent assay (ELISA) methods are one of the popular immunoassay methods for SA multiresidue screening (Adesiyun, 2020;Krall et al., 2018;Shelver et al., 2008).
However, ELISA usually requires many times washing which makes the whole detection time often last for 2-3 hr and cannot meet the need of rapid detection (Yu et al., 2015).
AlphaLISA technology is a homogenous light-induced chemiluminescence immunoassay in which donor beads (embedded phthalocyanine) can produce singlet oxygen ( 1 O 2 ) by photo-excitation (λ ex = 680 nm); the singlet oxygen then forms acceptor beads (embedded thioxene/EuIII-chelate mixtures) to generate fluorescence The AlphaLISA has many advantages over the ELISA; for example, its high sensitivity, relatively quick testing time, reduced hands-on workflow resulting from the ability to sequentially overlay the reagents, and it is easily adaptable to automation and high-throughput screening (Bielefeld-Sevigny, 2009;Li, Chen, et al., 2018;Wang et al., 2020). Therefore, AlphaLISA has been applied for the target detection, kinase assays, and protein-protein interactions (Armstrong et al., 2018;Lassabe et al., 2018;Zhao et al., 2019).
In the present study, we evaluated AlphaLISA system for SAs detection in plasma, milk, pork, chicken, and fish and compared its capability to conventional LC-MS/MS.

| Chemicals and instruments
The SAs holoantigen and SAs monoclonal antibody were produced by our laboratory. SA standard substance includes sulfameth-

F I G U R E 1
Principle of SAs detection based on AlphaLISA. (a) When there was no free SAs, the acceptor beads were in an emitting state ("bright") owing to the singlet oxygen ( 1 O 2 ) generated by donor beads upon photo-excitation at an effective assay distance of 200 nm; (b) in the presence of free SAs, the acceptor beads were in a nonemitting state ("dark") because the donor beads were out of the 200 nm range Goat anti-mouse IgG, Na cyanoborohydride powder (NaBH 3 CN), and carboxymethoxylamine were acquired from Sigma-Aldrich Co. Statistical analysis of the data was performed using RIDAWIN software from R-BioPharm AG (Darmstadt, Germany).

| Preparation of antigen and acceptor beads
The SAs holoantigen (10 mg/ml) was prepared in phosphate-buffered saline (PBS), to which 17.2 µL of 10 mmol/L Sulfo-NHS-LC-Biotin reagent solution was added according to the calculation provided in the reagent instructions. The reaction was then incubated at room temperature for 30 min. Excess nonreacted biotin and reaction by-products were removed by using a centrifugal filter (30 kDa). The final concentration of biotinylated SAs holoantigen was 1 mg/ml (15 μmol/L).
Next, 0.5 μL 5 mg/ml goat anti-mouse IgG acceptor beads was added into 500 μL AlphaLISA buffer; then 0.5 μL 10 mg/ml (63 μmol/L) SAs monoclonal antibody was added; and the mixture was incubated at 37°C for 1 hr with mild agitation in dark. The buffer and excess antibody were removed by centrifugation (4°C, 13,000 g, 60 min). The obtained preparations were store at 4°C.

| Characterization of acceptor beads
The dimensional characteristics of acceptor beads were conducted on TEM at an accelerating voltage of 300 keV. For this purpose, the naked acceptor beads and their conjugates with antibodies were dispersed in ultrapure water and applied onto a carbon-supported 300-mesh copper specimen grid to dry under a heat lamp prior to the detection.

| Procedure of AlphaLISA
In a typical experiment, 15 µl of samples, 10 µl of biotinylated SAs holoantigen, 10 µl of SAs monoclonal antibody, and 15 µl of anti-mouse IgG-coated acceptor beads were sequentially added into the microplate and incubated at 37°C in dark, and then, the streptavidin-coated donor beads were added to each well. After incubation for an additional 15 min at 37°C, the signal was read by AlphaLISA reader.

| Assay optimization
The concentrations of SAs holoantigen and SAs monoclonal antibody, and reaction time were examined to improve the sensitivity of the assay. To evaluate the effect of the antigen and antibody, different concentrations of the SAs holoantigen (0-25 nM) and the SAs monoclonal antibody (0-200 nM) were tested in a cross-over study designed using recommended concentrations of donor beads (17.91 ng/ml) and acceptor beads (5 ng/ml) without free SAs to obtain the best AlphaLISA signal. Then, fixing the concentration of the SAs holoantigen, changing the concentration of the SAs antibody along with that of the free SMT.
The effects of reaction time are also investigated by detecting the AlphaLISA signals of the SMT standard curve at different times from 20 to 70 min. The best SAs calibration curve was used to evaluate both the optimal concentration of antibody and total reaction time.

| Sensitivity and specificity of the SA AlphaLISA method
Under optimized conditions, the standard curves were generated by using SAs standard substance that was prepared by diluting appro- The concentration of each sulfonamide (C SA ) was obtained using the following equation: C SA = C SMT × CR, where C SMT is the concentration calculated from the calibration curve as SMT equivalents and CR is the cross-reactivity.

| Preparation of rabbits
The rabbits (2-3 kg) were injected intramuscularly with 10 mg/kg SMT in their hind legs every 24 hr for three consecutive days. Blood was sampled every 1 hr to obtain 10 blood samples from the rabbit ear-rim vein. In addition, a 10 ml aliquot of blood was collected prior to the SMT injection as a negative control. All of the blood samples were stored in tubes containing antithrombin, impurities were removed by centrifugation, and the samples were frozen at −30°C until analysis. The animal experiment was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) and was conducted in accordance with relevant guidelines and regulations.

| Preparation of samples
Milk, pork, chicken, fish, and plasma were used as animal-derived food sample for SAs residue analysis, and they were verified to be negative samples by LC-MS/MS. 4 ml or 4 g of samples was spiked with the SMT to three different levels (25, 50, and 100 ng/ml or ng/g), and each level of the sample was analyzed in triplicate.
For the AlphaLISA analysis, the sample was prepared according to the report (Dmitrienko et al., 2014). Basically, 4 g homogenized sample (pork, chicken, or fish) was extracted with 4 ml of ethyl acetate by vortex (10 min). After centrifugation (5 min, 2000 g, 25°C), 1 ml of supernatant was dried under a nitrogen stream (40°C) and then dissolved into 1 ml of PBS. 1 ml of n-hexane was added for defatting with centrifugation (5 min, 2000 g, 25°C); the lower aqueous phase was diluted 20-fold with PBS before analysis. As for liquid samples, 2.5 ml of skimmed milk or 1 ml of plasma was extracted with 5 ml of ethyl acetate by vortexed for 1 min. After centrifugation (2 min, 2000 g, 25°C), 2 ml of supernatant was dried, dissolved, defatted, and centrifuged as meat samples.
For the LC-MS/MS analysis, 1 ml sample of plasma (to which had been added 20 µL of phosphoric acid) and 3 ml of acetonitrile (precooled at 4°C for 1 hr) were vortexed for 5 min, followed by cooling at 4°C and centrifugation (10 min, 15,000 g, 25°C). The supernatant was dried under a nitrogen stream (40°C) and then reconstituted into 1 ml of acetonitrile-water (1:9) with 0.1% formic acid. Then, 1 ml of n-hexane was added for defatting, followed by centrifugation (5 min, 2000 g, 25°C); the lower aqueous phase was filtered through a 0.22µm filter prior to analysis.  effectively bound to the anti-mouse IgG-coated acceptor beads, which also confirmed the principle of SAs AlphaLISA methods (Figure 1).

| Optimization of assay conditions
Next, we used a two-step method for optimizing the concentration of antigen and antibody. First, the we obtained best AlphaLISA signal when the concentrations of SAs holoantigen and SAs monoclonal antibody were 12.5 and 200 nM, respectively, in a cross-over study (Table 1) Meanwhile, when the concentration of the SAs monoclonal antibody was below 1.5 nM, the AlphaLISA signals became weak and unstable, which was difficult to record. Therefore, 1.5 nM of the SAs monoclonal antibody was chosen as the optimal value. This two-step method was conducted stepwise, which differed from the strategy used in previous studies and resulted in more convincing optimization values.
Having obtained the optimal concentration of SAs holoantigen and SAs monoclonal antibody, we investigated effects of reaction time, because it takes time for the reaction between the target and antibody and we would like to find optimal time for practical applications. As shown in Figure 3b, the best IC 50 = 1.53 was achieved when the total reaction time of 30 min. Therefore, 30 min with reaction was selected as the optimal time and procedure for subsequent experiments.
Based on the results from the above optimization steps, we have come up with the following conditions; the concentrations of SAs holoantigen and SAs antibody were 12.5 nM and 1.5 nM; and the total reaction time was 30 min.

| Sensitivity and specificity of the SAs AlphaLISA method
Under the optimized conditions, the standard curves of STZ are described in Figure 4, and the LOD and IC 50 values for STZ were 0.015 ng/ml and 2.11 ng/ml, respectively. In this study, the broad specificity of the SAs AlphaLISA method was evaluated by the crossreactivities against 24 SAs. As shown in Table 2

| Validation of the AlphaLISA method
To assess the accuracy and precision of the SAs AlphaLISA method, 3 levels of SMT at 25, 50, and 100 ng/g were added to milk, pork, fish, chicken, and plasma, respectively. As shown in Table 3, the recovery values varied from 87.8% to 116.8%, with the CVs less than 9.3%, which confirmed the AlphaLISA method's effectiveness for SAs detection in different matrices. In addition, these results further support the feasibility of the developed SA AlphaLISA method for subsequent applications in monitoring animal-derived foodstuffs.

F I G U R E 3
In order to further evaluate the detection capability and the ac-

| CON CLUS IONS
In this study, we proposed a homogeneous immunoassay based on the AlphaLISA method to detect SAs antibiotic residues in plasma, milk, pork, chicken, and fish. The optimized SAs competi-

CO N FLI C T S O F I NTE R E S T
No conflict of interest declared.

E TH I C A L A PPROVA L
The animal testing involved in this study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) and was conducted in accordance with relevant guidelines and regulations.