Highly sensitive immunochromatographic assay for simultaneous determination of azaperone and azaperol in pork

Graphical abstract


Introduction
Azaperone (AZN) is a neuroleptic drug belonging to butyrophenones, which is commonly used as sedative for farm animals, especially pigs (Benson & Thurmon, 1979;Taghizadeh, Mohammadnia, Ghalkhani, & Sohouli, 2021). The behavioral inhibition of AZN is mainly mediated by blocking dopamine receptors located in the brain (Osama & Blakely, 2019;Schwarz et al., 2018). The inhibitory effect reduces the ability of animal response towards external signals, which makes them relatively indifferent towards the surrounding environment. AZN is used for treatment of various indications of animals (anesthesia, sedation, stress and anti-aggressiveness) to prevent premature death and loss of meat quality during breeding or transportation (Pohlin et al., 2020;Vitali et al., 2020). However, AZN is usually illegally added for improving fodder conversion rate by reducing animal activity, which can promote weight gain. The accumulation of AZN in edible animal tissues is caused by the improper use of drug dosage or non-observance of withdrawal period (Oliveira et al., 2017), which thus cause damage to the health of consumers through the food chain to result in heart rate reduction, skin vasodilation, cardiovascular effects and so on (Lin, Wu, Cui, Song, Kuang, & Xu, 2020). It is worth noting that azaperol (AZL), the main metabolite of AZN, can be used as the marker residue to screen AZN (Rauws, Olling, Freudenthal, & Ham, 1976). Moreover, AZL also possesses pharmacological activity. Therefore, so the maximum residual limit (MRL) is regulated to be the sum of AZN and AZL (Stolker & Brinkman, 2005). The European Commission stipulates the MRL of AZN and AZL at 100 µg/kg in muscle, skin and fat, kidney and liver of pigs, respectively. In China, the MRL is set to be 60 µg/kg in muscle, skin and fat by the Ministry of Agriculture and Rural Affairs.
Notably, the most important components are molecular recognition element and the signal transduction element in ICA (Liu, Zhang, Chang, Zhang, Brennan, & Li, 2015;Majdinasab, Zareian, Zhang, & Li, 2019). At present, only one literature has reported the detection of AZN by ICA method (Lin et al., 2020). However, the reported monoclonal antibody (mAb), a molecular recognition element, was unable to detect AZN and AZL simultaneously due to its weak cross-reactivity (CR) with AZL. As a signal transduction element, gold nanoparticle (AuNP) is commonly applied in the ICA, which has stable performance and low price. Meanwhile, some other types of materials have been attempted as signal element, such as fluorescent nanoparticles, magnetic nanoparticles, chemiluminescent materials, and latex beads (Yang, Xu, & Zhou, 2019;Zvereva, Hendrickson, Zherdev, & Dzantiev, 2020). These materials may improve the sensitivity. However, the complicated preparation procedure increases the workload and cost. The common mode of ICA depends on the traditional direct label technology, namely directed combination between specific antibodies and AuNP. There is also an indirect labeling technique which combines the anti-species antibodies with AuNP. Compared with the direct ICA, the free specific antibodies of the indirect ICA have more active sites to bind with the target analytes in the sample solutions than the antibodies immobilized on the surface of AuNP because of its faster Brownian motion and higher biological activity (Byzova, Urusov, Zherdev, & Dzantiev, 2018;Petrakova, Urusov, Gubaydullina, Bartosh, Zherdev, & Dzantiev, 2017). Therefore, the indirect format of ICA shows a better sensitivity than the direct format during the detection process.
With the above considerations, we designed a novel hapten according to the structures of AZN and AZL to produce the mAb with high affinity which could recognize them at the same time. Subsequently, the indirect format of ICA with high specificity and sensitivity was established. This analysis method realized the rapid, sensitive and on-site detection of AZN and AZL simultaneously, which exhibited good application prospect in actual samples.
Murine myeloma SP2/0 cells were supplied by the British National Animal Health. Six weeks old female BALB/c mice were provided by the Laboratory Animal Center of Zhengzhou University (Zhengzhou, China). The animal experiments were all approved by the Animal Ethics Committee and supervised under the guidelines of the Key Laboratory of Animal Immunization, Henan Academy of Agricultural Sciences. At the end of the experiment, the slaughter method of animals was carbon dioxide inhalation.
The Microplate Reader 550 was supplied by Bio-Rad (Richmond, USA). NanoDrop One was provided by Thermo Fisher (Madison, USA). XYZ Biostrip Dispenser, TSR3000 membrane strip reader and CM4000 Cutter were acquired from Bio-Dot (Richmond, USA).

Preparation of hapten and antigen
In this work, AZL, the derivative of AZN, was employed to synthesized AZL-SA which was used as the hapten by the succinic anhydride method. The scheme for the synthesis of AZL-SA was presented in Fig. 1 (a). The processes were briefly described as follows. 6 mg of AZN was dissolved in 1.5 mL anhydrous pyridine solution. Subsequently, 2.8 mg of SA and 0.2 mg of DMAP were added. The mixture solution was stirred and refluxed at 60℃ for 24 h. After the reaction, the solution was dried by rotary evaporation. Then the sample was redissolved in 600 µL of N, N-Dimethylformamide. Finally, the obtained hapten AZL-SA was characterized by LC-MS/MS analysis.
AZL-SA-KLH and AZL-SA-OVA conjugates, used as immunogen and coating antigen, respectively, were prepared using EDC/NHS method by combination of AZL-SA with KLH and OVA (Chen et al., 2020). The coupling reaction was conducted at room temperature. 4.0 mg of NHS and 6.6 mg of EDC were appended into the AZL-SA solution and reacted for 30 min. In addition, 5 mg of KLH and 4 mg of OVA were dissolved in 1 mL of 0.05 M carbonate-buffer saline (CBS, pH 9.6), respectively. 0.25 mL of the activation was slowly added into the KLH solution and incubated for 4 h with continuous stirring to obtain the immunogen. As for the coating antigen, the remaining activation was added into the OVA solution under the same conditions. After the incubation, the mixture was dialyzed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 3 d. The supernatant was collected after centrifuging at 5000g for 2 min and frozen at − 20 ℃ for later use. The AZL-SA-KLH and AZL-SA-OVA conjugates were characterized by ultraviolet/visible (UV/Vis) spectroscopy.

Production of monoclonal antibody against AZN
According to the reported protocol (Sun et al., 2014), four female BALB/c mice were immunized. The immunogen was AZL-SA-KLH, and the dose of each immunization was 20 µg with a volume of 200 µL. The mice were injected for four times at an interval of 21 d. The first immunogen was emulsified with the same volume of FCA, while FIA was instead of FCA in the following three immunizations. The indirect competitive ELISA (ic-ELISA) was used to measure the titer and inhibition rate of the mouse serum against AZN at 10 days after the fourth injection. The mouse with the highest titer and inhibition rate to AZN was chosen for the next experiment. The chosen mouse was given intraperitoneal injection with 40 µg AZL-SA-KLH conjugate. After 72 h, the spleen cells were fused with SP2/0 cells by PEG 1500. The process of cell fusion was carried out as previously reported. After that, the fused cells were dispersed on Ninety-six-well culture plates and cultured in the incubator at 37 • C with 5 % carbon dioxide. After 8 days, screening target hybridomas was performed to identify the cell supernatant by ELISA. Subsequently, the selected cell lines were subcloned using finite dilution method to obtain the stable antibody-producing clones. The stable cell line was amplified and intraperitoneally injected into the mouse. After seven days, the ascites was collected and purified by octanoic acid-ammonium sulfate method.

Characterization of mAb against AZN
The titer, specificity, sensitivity, affinity and subtype were used to evaluate the characterization of the mAb against AZN. The subtype determination of the mAb was performed with a MMIK. The titer and affinity were determined by ELISA. ELISA was performed using a standard method that has been reported (Na et al., 2020b). The affinity constant (K a ) calculated by Beatty method was identified to evaluate the affinity of the mAb, and the calculation formula was as follows: Where, n was the multiple of two different concentrations of coating antigen, [Ab t ] and [Ab] were the concentration of mAb at 50 % absorbance of the maximum value.
To evaluate the specificity and sensitivity, ic-ELISA was performed by adding a series of standard compounds before addition of antibodies. The 50 % inhibitory concentration (IC 50 ) was used to determine the sensitivity, which was defined as the concentration of the standard compounds which inhibited the binding of coating antigen to the antibodies by 50 %. The specificity was assessed by the CRs of AZN and a series of related analytes including AZL, XYL, CAR, CHL, DRO and HAL. The CR was calculated using the following formula:

Preparation of AuNP-labeled anti-AZN mAb and RAGI
Firstly, the preparation of AuNP was carried out by the classical reduction method (Sun et al., 2016). The steps were as follows: 2.0 mL of 1 % (w/v) chloroauric acid was drawn into 198 mL of deionized water and heated to boil with a vigorous stirring. Then 3.2 mL of 1 % sodium citrate was added into chloroauric acid solution with stirring. It was observed that the color of the mixture changed from gray to red. After boiling and stirring continuously for an additional 5 min, the solution was stopped heating and naturally cooled to room temperature. Finally, the AuNP solution was stored at 4 ℃.
The antibody and AuNP were combined by the hydrophobic and electrostatic interactions. The AuNP-labeled mAb was obtained according to the established methodology from literature (Na et al., 2020a). Firstly, the pH of AuNP solution was adjusted to 8.2 by addition of 0.2 M potassium carbonate. For confirming the optimal ratio between antibody and AuNP, 10 µL of the antibody was successively twofold diluted by ultrapure water in microwell. 125 µL of AuNP solution was dropped into each well for incubating 10 min at room temperature, and then 125 µL of 10 % sodium hydroxide liquor was added to terminate the reaction. The optimal ratio between antibody and AuNP was considered as the minimum ration that did not cause the color changes of the mixture. Subsequently, 60 µL of mAb (2.43 mg/mL) was allocated into 20 mL of AuNP liquor and incubated for 2 h at room temperature. After incubation, 1 mL of 20 % BSA was spiked for incubation at 0.5 h to block the residual binding sites on AuNP surface. Next, the resulting deposit was collected with a centrifugation at 12,000g for 0.5 h. Finally, the AuNP-labeled mAb was dissolved in 10 mL of resuspension buffer (20 mM, pH 9.0) including 0.03 % sodium azide, 3 % sucrose and 1 % BSA, which was stored at 4℃ for further use. The steps of preparing the AuNP-labeled RAGI were the same as above except that 160 µL of GAMI (0.25 mg/mL) was added into 20 mL of the AuNP solution.

Assembly of the ICA
The ICA device was composed of an adhesive backing board, an absorbent pad (20 cm × 1.6 cm), a nitrocellulose membrane (20 cm × 2.5 cm), a conjugate pad (20 cm × 0.8 cm) and a sample pad (20 cm × 1.6 cm). The absorption pad pulled fluid by capillary force to allow the analyte across the assembly pads. On the nitrocellulose membrane, there was a test (T) line and a control (C) line. The two lines with a 0.45 cm distance were formed by the XYZ Biostrip Dispense at a spraying rate of 0.9 µL/cm, and then dried for 4 h at 42 ℃. The sample pad was treated by 0.1 mol/L PBS containing 0.3 % Tween 20, 5 % sucrose, 0.05 % sodium azide and 1 % BSA, and then positioned at 42 ℃ for 4 h. The conjugate pad was used for receiving recognition labels, which ensured the stability within the warranty period. The recognition labels were sprayed onto the conjugate pad at a rate of 5 µL/cm and laid at 37℃ for 1 h. The absorbent pad, nitrocellulose membrane, conjugate pad and sample pad were superimposed on backing board in turn and cut into strips (2.8 mm width) by a CM4000 Cutter.

Principle of the ICA
According to the principle of competitive immunoassay, two formats (direct and indirect mode) of the ICA were designed. In the direct format, AuNP-labeled anti-AZN mAb was immobilized on the conjugate pad. The T and C line were AZL-SA-OVA and goat anti-mouse IgG, respectively. Generally, the ICA was inserted into the treated sample solution which was migrated successively cross the conjugate pad and membrane towards the absorption pad by capillary force. The results of the sample solution by ICA were presented in Fig. 2(a). In the case of the sample without AZN, the AuNP-labeled anti-AZN mAb was trapped by the coating antigen on the T line to emerge a red line. When AZN presented, AuNP-labeled anti-AZN mAb reacted with the free AZN, which led to less or no combination between it and AZL-SA-OVA. The more target content led to the less AuNP-labeled anti-AZN mAb which would be captured and the weaker color of T line. Regardless of presence or absence of the target, free or coupled AuNP-labeled anti-AZN mAb was always arrested by goat anti-mouse IgG on C line to form a red color, which indicated the effectiveness of the ICA.
In the indirect format, the difference from the direct format was that C line on the nitrocellulose membrane was IgG from mouse and the conjugate pad was the AuNP-labeled GAMI. Before inserting the ICA into the sample solution well, a certain amount of mAb was added and incubated at room temperature for 5 min. The test result was similar to the direct mode, as shown in Fig. 2(b). In the absence of analytes, the mAb would bind to the AuNP-labeled GAMI and was captured by AZL-SA-OVA on T line to generate a red band. In the presence of analytes, the mAb was firstly reacted with the analytes in the sample solution and made fewer mAb binding to the AuNP-labeled GAMI. Therefore, the conjugate compound of mAb and AuNP-labeled GAMI was less intercepted by AZL-SA-OVA to result in a lighter or colorless T line.

Pretreatment of sample
The pork samples in this study were derived from Henan Academy of Agricultural Sciences, which was previously purchased in the market and determined to be negative by LC-MS. Initially, 5 g of pork was ground into homogenate and transferred into a centrifuge tube (50 mL). Then 20 mL of acetonitrile was added and vibrated for 2 min, followed by ultrasonic treatment at room temperature for 5 min. The supernatant and precipitate were separated by centrifugation at 5000g for 5 min. After collecting the supernatant, 10 mL of acetonitrile was again added into the precipitate for repeated extraction. Then the two extracts were mixed together and dried under nitrogen at 40 ℃. Finally, the residue was redissolved in 5 mL PBS including 5 % acetonitrile for further use.

Analysis of the ICA performance
To analyze the ICA performance, a series of concentrations of AZN standard solution were spiked into the treated samples. For the direct ICA, seven concentrations of AZN were selected as 80, 40, 20, 10, 5, 2.5 and 0 ng/g. The concentrations of AZN were 8, 4, 2, 1, 0.5, 0.25 and 0 ng/g in the indirect format. The sensitivity of the ICA in the semiquantitative test was considered to be the visual detection limit (cutoff value). It was interpreted as the minimum concentration of AZN when T line color disappeared. In addition, the G/D × area of the relative optical density (ROD) values of T line were defined as the quantitative detection and measured using a strip reader. Based on the values decreased when the target concentration increase, a standard curve was obtained. The IC 50 and the detection limit (LOD, defined as the target concentration causing 10 % inhibition) were calculated by the standard curve and considered as the sensitivity of the ICA for the quantitative detection. To verify the specificity of the ICA, six related compounds were chosen for the cross reaction.
The recovery experiment was performed by adding three concentrations (0.5, 1, 2 ng/g) of AZN or AZL to the pork samples. Whereafter, T line was detected by a strip reader and the target residues were calculated based on the standard curve. To evaluate the accuracy of the ICA, each sample was determined in triplicate to confirm the coefficient of variation (CV, %). The intra-assay reproducibility was obtained from the same batch and the inter-assay reproducibility was acquired from three different batches.

Hapten and antigen identification
A small compound <5000 Da cannot directly induce the production of specific antibodies due to lack of T cell epitopes, which is named as hapten without immunogenicity (Wang, Beier, & Shen, 2017). Therefore, small molecules must be attached to carrier proteins in order to generating an immune response. It is well known that the design of hapten and the preparation of antigen are the keys to obtain mAb with high quality. In previous study, Lin et al. prepared the hapten by introducing a linker arm containing four carbon atoms and an active carboxyl group to the benzene ring of AZL, which is then linked with KLH to synthesize the immunogen (Lin et al., 2020). However, the CR value to AZL of the mAb obtained by the hapten was <2.5 % influenced by the mode and location of hapten binging to carrier protein. It indicated that the product could not be used to recognize both AZN and its metabolite AZL.
In this study, we need to consider that the produced mAb could be used to recognize both AZN and its metabolite AZL. Considering that the only difference was a ketone group between AZN and AZL, the hapten was derived by the succinic anhydride method. Then, the hapten AZL-SA was obtained, which could bind to the carrier protein more easily by retaining the common structures to the maximum extent. Simultaneous, the introduction of carboxyl group increased the connecting arm so that the specific structure of hapten could show more prominently on the surface of complete antigen. The design of the hapten facilitated the production of highly specific mAb for AZN and AZL. The synthesized derivatives were identified by LC-MS/MS. Fig. 1(b) showed that the m/z [M + H] + of the main product was 430.2136 for the positive ion analysis. Hence the molecular weight of the compound was 429. It indicated that AZL-SA (C 23 H 28 FN 3 O 4 ) was successfully synthesized.
The conjugates were initially identified by scanning UV/Vis spectroscopy as shown in Fig. 1(c) and (d). The protein KLH had absorption peaks at 280 nm and 350 nm. Notably, AZL had a strong absorption peak at 238 nm and a weak absorption peak at 300 nm. The synthesized conjugate AZL-SA-KLH not only exhibited a strong absorption peak at 231 nm due to the shift of AZL, but also had the characteristic absorption peak of KLH at 280 nm. The weak absorption of the conjugate at 300-400 nm also reflected the weak absorption peak of AZL at 300 nm and the weak absorption peak of KLH at 350 nm. Meantime, the conjugates AZL-SA-OVA and AZL-SA-KLH had similar waveforms. These characteristics indicated that AZN was effectively coupled with KLH/ OVA. The formed conjugates could be used as artificial antigens for subsequent experiments.

Characterization of mAb
A monoclonal cell line 35D1D2 simultaneously identifying AZN and AZL, was produced by cell fusion and hybridoma screening. The titer of mAb represents the absolute number of mAb involved in the antigen-antibody reaction system. The strength of affinity reflects the magnitude of the binding ability between the antigen and the antigen. The K a value is equilibrium dissociation constant between the antibody and its antigen. According to noncompetitive ELISA analysis, the titer of the mAb was 1:1.024 × 10 6 and the affinity was 1.28 × 10 8 L/mol. Basted on the ic-ELISA, the sensitivity of the mAb was determined. The concentration of coating antigen and mAb affects the performance of ELISA. In this study, the optimal concentrations of coating antigen and mAb were 0.23 and 0.38 µg/mL, respectively, through chessboard method. As shown in Fig. 3(b), the linear equation of mAb against AZN and AZL was y = − 0.32947x + 0.3508 (R 2 = 0.9916) and y = − 0.35707x + 0.31694 (R 2 = 0.9919), respectively. It was worth noting that the IC 50 of AZN and AZL was 0.35 and 0.31 ng/mL which is lower than that reported (Lin et al., 2020). It indicated that the sensitivity of mAb for AZN was higher than that before. Especially, it can precisely recognize AZL at the same time.
In Fig. 3(a), it revealed that the subclass of mAb from 35D1D2 was IgG2b, and its light chain was Kappa. In addition, the specificity of mAb was estimated based on the CRs of several related analytes by ic-ELISA. The specificity of mAb directly confirms whether the mAb can be applied to complex analysis of real samples. When the CR of mAb is <10 %, it can be considered to show no cross-reactivity (Lin et al., 2020). The results were shown in Table 1, the CRs of the mAb with XYL, CAR, CHL, DRO and HAL were all <0.035, indicating the excellent specificity to AZN and AZL. In general, the mAb secreted by 35D1D2 was a suitable antibody for the ICA with satisfactory affinity, specificity and sensitivity.

Sensitivity of the direct ICA and the indirect ICA
The direct and indirect ICA strips were prepared to detect AZN for comparison. In general, the main parameters affecting the direct ICA are the concentrations of AuNP-labeled mAb and captured antigen. In the direct format, the amounts of AuNP-labeled mAb and AZL-SA-OVA were optimized to achieve the minimum LOD of AZN. After adjusting the color of T line, we found that the jetting rate and the concentration of conjugated AZL-SA-OVA were 6 µL/cm and 0.24 mg/mL, respectively. In the indirect format, the amount of AuNP-labeled GAMI, AZL-SA-OVA and mAb (24.34 µg/mL) was firstly optimized as follows: 6 µL/cm, 0.18 mg/mL, and 3 µL, respectively. The sensitivity of the direct and indirect ICA was shown in Fig. S1. Intuitively, the cutoff value of the direct ICA was 80 ng/g, and indirect ICA was 8 ng/g. It was obviously indicated that the sensitivity was improved by indirect ICA. It may be due to that the free mAb had higher biological activity and longer reaction time between mAb and targets. Compared with the reported ICA, the established indirect ICA is not only 1.25 times more sensitive for the detection of AZN, but also can simultaneously detect its metabolite AZL (Lin et al., 2020). The ICA method allows the detection of total AZN and its metabolite AZL, which can meet the requirements of the European Commission or other countries.

Analysis of the indirect ICA in pork
The whole experiment process of the indirect format was performed within 15 min including two consecutive stages. In the first stage, the mAb was put into the sample extract to react for 5 min. In the second stage, the ICA was inserted into the extract liquid for testing. The visual image of the ICA was shown in Fig. 4. A series of concentrations of AZN (8, 4, 2, 1, 0.5, 0.25, 0 ng/g) and AZL (8, 4, 2, 1, 0.5, 0.25, 0 ng/g) were added to the sample extract for detecting by indirect format. When 8 ng/ g AZN or AZL were contained in the extracts, the ICA was colorless. Thus, the cutoff values of AZN and AZL for the pork samples were both judged to be 8 ng/g. For the quantitative test, T line was read by a strip reader. Thus, the standard curve was generated by taking the logarithm of target analyte concentration as X axis and the color intensity ratio of T line between spiked sample and non-spiked sample as Y axis. Hence, the equation of standard inhibition curve for AZN and AZL was y = − 0.55105x + 0.42786 (R 2 = 0.9972) and y = − 0.57488x + 0.3725 (R 2 = 0.98755), respectively. Based on the standard equation, the IC 50 of AZL was 0.60 ng/g which was slightly less than that of AZN (0.74 ng/g). Meanwhile, the LOD values of them were also acquired, 0.14 ng/g for AZN and 0.12 ng/g for AZL. The working range was defined as the concentration of target analytes that inhibited the corresponding signal by 20 % to 80 % to be 0.21-2.59 ng/g for AZN and 0.18-2.00 ng/g for AZL. The ICA realizes the synchronous detection of AZN and its  metabolite AZL in pork samples, meeting the detection limits recommended by the European Commission or other countries. The method is a rapid immunoassay, which can be used for semi-quantitative or quantitative determination of AZN and AZL in large quantity of samples.

Recovery validation
The recovery test was conducted to validate the precision and accuracy of the ICA. AZN and AZL with the concentrations of 0.5, 1, 2 ng/g were spiked into the pork samples, respectively. According to the extraction method in 2.8, the samples were treated for testing with three times repeated. The same batch test strips were used for intra-assay and three different batches were used for inter-assay. Table 2 showed that the recovery rates for AZN and AZL in intra-assay were in the range from 88.12 % to 91.40 %, and the CVs were from 6.13 % to 8.69 %. The recovery rates for AZN and AZL in inter-assay were in the range from 87.61 % to 91.12 %, and the CVs were from 7.16 % to 10.52 %. As is well-known, the recovery rates within 25 % and CVs within 15 % are acceptable (Na et al., 2020a). Consequently, the established ICA was effective and reliable for the determination of AZN and AZL in pork.

Conclusions
This study developed an indirect ICA for simultaneously rapid and sensitive detection of AZN and its metabolite AZL. The establishment of this strategy included two key designs: one was to synthesize highly similar hapten AZL-SA through preserving the common structure of AZN and AZL. Subsequently, a satisfactory mAb was produced, which could recognize both of them. The other was to use the indirect format to improve its sensitivity, which allowed mAb to retain high biological activity for combining with the target. The detection process was completed within 15 min, the visual detection limit of AZN and AZL were both 8 ng/g, the IC 50 and LOD values for AZN were 0.74 and 0.14 ng/g, and for AZL were 0.60 and 0.12 ng/g, respectively. The indirect ICA was simple, low-cost and sensitive, and suitable for rapid on-site detection of AZN and AZL in pork. This strategy opens up a facile but powerful avenue of rapid on-site detection platforms for other small molecular targets in food samples.

Statement
All animal experiments were approved by the Animal Ethics Committee and conducted under the guidelines of the Key Laboratory of