An Ultrasensitive Immunoassay Strip for Simultaneously Detecting Cyproheptadinehydrochloride and Six Phenothiazinesin Feedstuffs Based on a Monoclonal Antibody

Background: An antihistamine cyproheptadine (CPH) and phenothiazines (PZs)sedative hypnoticshave a similar tricyclic structure, these drugs are often illegally added to food animal feedstuffs due to their price advantage and signicant effect in promoting growth and improving meat quality. However, the abuse of these drugs may lead totheir residues in animal products, thereby causing harmful effects such as allergies and dermatological reactions on human health.To supervise the use of prohibited drugs and ensure food safety, it is necessary to establish a simple and effective screening method to detect CPH and PZs.In this research, an articial antigen against cyproheptadine(CPH) was successfully synthesized by bromoacetic acid substitution method. An ultrasensitive and broad-specic monoclonal antibody (mAb) able to recognize CPH and six phenothiazines (PZs) was produced. Based on the gold-labeled mAb, an immunochromatographic strip (ICS) was established. Results:The 50% inhibition concentration (IC 50 ) of the produced mAb against CPH was identied as 0.036 ng mL -1 by ELISA, and the cross-reactivities for six PZs were from 6.33% to 63.16%. The visual detection limits(cut-off values) of the developed ICS ranged from 5 to 100ngg -1 in feedstuffs. Take a reading by strip reader, the IC 50 was from 0.570 to 7.750ng g -1 . In addition,a recovery experiment was carried out to verify the reliability of the ICS. The intra-assay recoveries were from 79.83% to 103.38% with the highest coecient of variation (CV) of 11.31%. The inter-assay recoveries were from 79.00% to 96.63% with the highest CV of 12.66%. Conclusions: We have successfully produced a broad-spectrum monoclonal antibody and established an ICS for simultaneouslydetecting CPH and six PZs drugs.In brief, the proposed ICS was considered suitable for qualitatively and quantitatively monitoring CPH and PZs in feedstuffs.


Introduction
Cyproheptadine (CPH), an antihistamine drug, is extensively employed to treat allergic disorders caused by histamine overproduction in humans [1,2]. In addition, CPH can stimulate the appetite by inhibiting 5hydroxytryptamine activity in the hypothalamus, leading to increases in growth rate and body weight [3,4]. CPH has a similar effect on animals as clenbuterol, thus it has been utilized as a feed additive. Currently, in consideration of the allergic and other potential hazards for consumers posed by CPH residues in animal edible tissues [5], CPH has been banned as a veterinary drug for food-producing animals according to EU Directive 2001/82 [6]. In the announcement No. 176 and 1519 of China's Ministry of Agriculture, CPH was prohibited to use in feedstuffs and drinking water [7]. However, many investigations have shown illegal use or overuse of CPH by farmers to promote growth of animals due to its price advantage and effectiveness [6,8]. It follows that an available test tool is urgently needed to monitor the illegal use of CPH.
Phenothiazines (PZs) are a class of sedative and hypnotic drugs with a common structure of thioaniline rings. PZs included promethazine (PTZ), chlorpromazine (CPZ), perphenazine (PPZ), uphenazine (FPZ), acepromazine (APZ) and thioridazine (TDZ) were normally used in the treatment of human psychiatric diseases. In animal husbandry, PZs are used as feed additives and are conducive to weight gain by limiting animal movement and reducing the consumption of nutrients. Meanwhile, PZs are applied as anti-stress drugs in the transportation of food producing animals [9]. However, the use of PZs in these ways can leave residues in the edible tissues of food-derived animals and thus endanger human health. It was found that CPZ is a potential risk for engendering adverse reactions, such as dermatological reactions, leukocytosis, obstructive and orthostatic hypotension, leukopenia and jaundice [10]. Although the toxicity of other phenothiazines remains unclear, they may have the same pathogenicity as chlorpromazine because of the common structure. Considering the potential harm of PZs to consumers, the European Union banned the use of CPZ in food animals in any form [11]. According to the announcement NO. 176 and NO. 235 of the Ministry of Agriculture of China, PZs have also been banned as feed additives and must be undetectable in edible tissues of animals [12].
Up to now, a few analytical techniques have been developed for the detection of CPH, such as capillary electrophoresis-electrochemiluminescence assay [1], high performance liquid chromatography (HPLC) [6,13], liquid chromatography-tandem mass spectrometry (LC-MS/MS) [8,14,15], gas chromatographymass spectrometry (GC-MS) [2] and so on. There are also many instrumental analytical methods for the detection of PZs in biological matrixes, including HPLC [16,17], GC-MS [18][19][20], LC-MS/MS [13] and ultrahigh-performance liquid chromatography-Q-Trap tandem mass spectrometry (UPLC-MS/MS) [21]. These instrumental detection methods show the advantages of good stability and high sensitivity [22]. However, above techniques and instruments require skilled technical support, and are expensive and timeconsuming [23,24]. Furthermore, they are not practical for on-site detection or rapid screening of large numbers of samples [25,26]. A simple, rapid, reliable and low-cost method is imperatively needed to monitor illegal use and deal with high-throughput screening of CPH or PZs.
Immunochemical methods, especially colloidal gold immunochromatographic strips (ICS), are receiving increasing attention due to their convenient and practicality [27,28]. At present, many test strips have been reported on the detection of various veterinary drugs and feed additives, including aminoglycoside antibiotic [29]. sulfonamides antibacterial agents [30], vancomycin [31], paromomycin [32], polymyxin B [33] and sildena l [34]. To the best of our knowledge, none immunochromatographic assays have been established to detect CPH or PZs. In this study, an arti cial immunogen CPH-BSA was designed and prepared, then a mAb with high a nity and sensitivity was obtained. Based on the antibody, one ICS was developed to simultaneously screen for CPH and PZs in feedstuffs.

Materials And Methods
Chemicals, reagents and apparatus

Preparation of immunogen and coating antigen
Here, the hydroxyl group-containing COH was introduced into the carboxyl group by substitution with bromoacetic acid, and conjugated with BSA or OVA using EDC/NHS method, the route was shown in Fig.  1(d). Firstly, 0.1 mmol of COH was dissolved in 1 mL DMF, and 1 mmol of K 2 CO 3 and 0.15mmol of bromoacetic acid were added carefully. This substitution reaction was performed at room temperature under stir for 4 h. The reaction solution was then centrifuged at 5000 × g for 10 min, the supernatant was carefully collected and adjusted to pH 5.0 with HCl. EDC (0.15 mmol) and NHS (0.15 mmol) were added into the supernatant and react for 15 min at room temperature. Finally, half the volume of the solution was added dropwise to 1.5 mL of 0.1 mmol L -1 BSA solution and another half to 1.5 mL of 0.1 mmol L -1 OVA solution, then incubated for 4 h at room temperature. The mixtures were dialyzed in PBS for 3 d at 4℃.

Preparation of anti-CPH mAb
The production of anti-CPH mAb was performed as described previously [35]. Four female BALB/c mice were subcutaneously injected with 200 µL of a 1:1 (v/v) mixture of PBS and FCA containing 50 µg immunogen. At 21-day interval, the mice received another three further immunizations with the same dosage of immunogen but using FIA instead of FCA for the emulsi cation of the antigen respectively. Sera were collected on the tenth day after the fourth immunization and tested for anti-CPH activity by enzyme-linked immunosorbent assay (ELISA). The mouse with highest sensitivity and titer of anti-CPH antibodies was selected as the spleen donor. The selected mouse was immunized intraperitoneally with 50 µg of CPH-BSA in PBS, then three days later the spleen cells were used for fusion with myeloma cells using PEG1500 [36]. The supernatant of the hybridoma cells was tested by ELISA. The hybridoma cells that secreted high anti-CPH activity antibodies were subcloned by limiting dilution. To obtain large numbers of monoclonal antibodies against CPH, the BALB/c mice received an intraperitoneal injection of the selected hybridoma (0.5-1.0 × 10 6 cells for each mouse). The ascites uid was collected after ten says and puri ed by caprylic acid and ammonium sulfate method (CA-SA).

Competitive ELISA
The development of the indirect competitive ELISA (ic-ELISA) was performed according to conventional protocols [37]. The optimum coating antigen and mAb concentrations for competitive ELISA were determined by the bi-dimensional titration assay. The indirect competitive ELISA (ic-ELISA) process was as follows. First, a series of standard solutions were added to the reaction plate with 100 µL per well, and the mAb solution was added expect blank wells. The plate was incubated at 37℃ for 15 min and then washed with PBST (PBS containing 0.05% Tween 20) four times. Second, 100 µL of HRP-anti-IgG was added per well and the plate was incubated at 37℃ for 30 min. After washing step, 100 µL of the TMB dilution buffer was added per well, then the plate was incubated at 37℃ for 15 min. Finally, the reaction was terminated with 50 µL of 2 mol L -1 H 2 SO 4 per well, and the optical density (OD) values at 450 nm were read by a microplate reader. Furthermore, the standard curve was constructed from the ratio of Preparation of colloidal gold-mAb probe and ICS Colloidal gold was prepared as previously described with average particle diameter of approximately 24 nm [38]. The nanoparticles were conjugated with mAb as reported with slight modi cation [39]. Concisely, the pH of the colloidal gold solution (10 mL) was adjusted to 8.2 with 0.2 M K 2 CO 3 , then 1 mL of mAb solution (150 µg mL -1 ) was added dropwise under gently stirring. After reaction at room temperature for 30 min, 2 mL of 10 % BSA was added to block excess gold nanoparticles, and reacted for a further 15 min at room temperature. The mixed solution was centrifuged at 12000 × g for 30 min under 4℃. The supernatant was cautiously removed and 2 mL of the gold pellets were resuspended in borate buffer (pH 9.0). The conjugate solution was stored at 4℃ for use.
The assembly of ICS was shown in Fig. 2. The strip consisted of a semirigid polyethylene sheet (backing card), a nitrocellulose (NC) membrane, a sample pad, a conjugate pad and an absorbent pad. [38] The NC membrane (20 mm × 30 mm) was spotted with one test line (T line) applied with CPH-BSA and one control line (C line) with goat anti-mouse IgG, with 0.5 cm between the two lines. The conjugate pad was treated with colloidal gold-mAb solution. The sample pad was saturated with TB solution (0.01M PBS containing 1%BSA, 1% sucrose and 0.05% Tween 20). The assembled card was cut into 3 mm wide strips, then sealed and stored under dry condition.

Test procedure and principle
The competitive immunoassay theory was applied to this ICS, shown in Fig. 2. Brie y, 100 µL of sample solution was dripped onto the sample pad, which owed all the way through the NC membrane to the absorbent pad by capillary action. In the absence of an analyte in the sample, gold-labelled mAb gradually released as the solution owed could bind to the capture antigen to form antibody-antigen complexes and are intercepted on the T line. This line appeared bright red. Conversely, when enough dose of analyte existed in the sample, the analyte would block the gold-labelled antibodies and avoided their coupling with the capture antigen, the T line was colorless. The lower dose of analyte in a sample, the more obvious T line. The C line was always visible red regardless whether the sample contained analyte or not. Otherwise, the strip should be discarded and a new strip used.

Results And Discussion
Characterization of immunogen CPH is not immunogenic due to its small molecular weight. To produce highly sensitive antibodies, CPH must be e ciently conjugated with the carrier protein. BSA is the most commonly used carrier protein because it is cost-effective. The structures of CPH and COH were shown in Fig. 1(a) and Fig. 1(b), CPH has no active group for coupling and COH has a free hydroxyl group, therefore, COH was selected for structure modi cation. Bromoacetic acid substitution is a feasible method with high reaction e ciency and mild reaction process. In our study, a carboxyl group was introduced to COH by substituting the hydroxyl group, then carboxylated COH was connected to the free amino group of BSA by EDC/NHS. Afterwards, the conjugated reactant was qualitatively analyzed by UV spectrophotometry. As shown in Fig. 1(c), the maximum absorption peaks of carrier protein BSA and CPH were at 280 nm and 285 nm, respectively. However, the immunogen CPH-BSA had a shifted peak at between 280 and 285 nm. Accordingly, the synthesis of the immunogen was considered successful.

Characterization of the mAb
After a series of screening, a target cell line (10D3-C3) was obtained. The mAb of 10D3-C3 was produced and puri ed by the conventional CA-SA. The properties of the mAb were determined by ELISA. The a nity constant (Ka) of 10D3-C3 was estimated as 7.353 × 10 9 L mol -1 . The titer of the mAb was as high as 1:1.102 × 10 6 and the mAb belonged to IgG2a sub-class. As shown in Table 1, the IC 50 value was calculated from the inhibition standard curve of CPH or PZs by ic-ELISA, ranged from 0.036 to 0.569 ng g -1 . Additionally, there was little cross-reaction between CPH and other analogues including diclazuril (DIC), benzimidazole (BMD) and diclofenac sodium (DFS). The cross-reaction (CR) rates were less than 1×10 -6 , indicated that 10D3-C3 possessed satisfactory speci city. The ultra-sensitive mAb was the vital basis for developing ICS.
Optimization and analytical characteristics of the ICS According to previous reports [34], the parameters affecting the performances of ICS are mainly pH, the concentrations of gold-labeled mAb and capture antigen. The color depth of T line is the evaluation standard for parameter optimization. After a series of color comparisons, the optimal pH range was from 7 to 8.5, thus the readily available PBS was chosen as the sample re-suspension. The optimal concentrations of capture antigen and mAb were determined to 0.15 mg mL -1 and 12.5 µg mL -1 .
To assess the sensitivity of the ICS, a series of analyte standard were diluted with processed sample solution and tested by the strips. As shown in Fig. 4, with increasing concentration of analyte standard, there was a change in the color of T line from dark to light as observed by the naked eye. Moreover, the cut-off value was obtained at the lowest concentration of analyte, where the T line was colorless. As shown in Fig. 4 and Table 2, the cut-off values of the ICS for CPH and PZs were from 5 to 100 ng g -1 . The G/D × A (area) of the relative optical density (ROD) and G/peak values of T lines were measured by Bio-Dot TSR3000 Membrane Strip Reader. The sigmoidal dose-response curves were generated by concentrations of analytes and the G/D × A (area) of the ROD. The linear equation (LE) for CPH was y = -5377x + 0.8019 (R 2 = 0.9916), the APZ's LE was y = -5738x + 0.8781 (R 2 = 0.9935), the FPZ's LE was y = -5101x + 0.8412 (R 2 = 0.9979), the PPZ's LE was y = -5485x + 0.8154 (R 2 = 0.9976), the PTZ's LE was y = -5521x + 0.8391 (R 2 = 0.9958), the CPZ's LE was y = -5304x + 0.7944 (R 2 = 0.9960) and the TDZ's LE was y = -4904x + 0.7977 (R 2 = 0.9965). The IC 50 values were computed from the standard curves, ranged from 0.570 to 7.750 ng g -1 , the LOD values ranged from 0.103 to 1.354 ng g -1 . The above results showed that the established ICS was highly sensitive and su cient for monitoring CPH and six PZs.

Recovery of CPH in feedstuff samples
The accuracy of the ICS was evaluated by a recovery experiment. Feedstuffs containing CPH (0.4, 0.8 and 1 ng g -1 ), PTZ (3, 5 and 15 ng g -1 ) or TDZ (1, 3 and 5 ng g -1 ) were extracted and separately detected by the ICS. The measured G/D × A (area) of the ROD values were inserted into the standard curve equations and the sample concentration values were calculated. The results were shown in Table 3. To determine intraassay reproducibility, each sample was tested in triplicate with the same batch of strips, the recoveries ranged from 79.83% to 103.38% and the highest coe cient of variation (CV) were 11.31%. To determine inter-assay reproducibility, three different batches of strips were applied to test the samples in triplicate.
Recoveries were from 79.00% to 96.63% and the highest CV was 12.66%. In general, recoveries between 75% and 125% and the CV below 15% were considered acceptable. Thus, the test strip exhibited satisfactory accuracy.

Conclusions
In this study, we successfully designed the arti cial hapten and prepared an immunogen CPH-BSA.
Subsequently, an ultrasensitive and broad-speci c mAb recognizing both CPH and PZs was prepared. Further, an ICS device for simultaneous detection of CPH and PZs in feedstuffs was established. The overall analysis time was within 10 min by the naked eye and the cut-off values were from 5 to 100 ng g -1 . In summary, the ICS was an ideal, practical, cost-effective tool for preliminarily screening CPH and PZs chemical hazards in feedstuffs, also provided the potential to detect CPH and PZs in food matrixes.   Table 2. Sensitivity of the ICS Table 3. Recoveries and inter-assay and intra-assay precision of the immunoassay strips for CPH spiked in feedstuff Analyte Concent ration (ng g -1 ) Intra-assay Inter-assay Chemical structures of CPH (a) and COH (b); the UV spectroscopy of CPH, BSA and CPH-BSA (c);the preparation of immunogen CPH-BSA (d).

Figure 1
Chemical structures of CPH (a) and COH (b); the UV spectroscopy of CPH, BSA and CPH-BSA (c);the preparation of immunogen CPH-BSA (d).

Figure 2
Schematic illustration of ICS for detecting analyte.

Figure 2
Schematic illustration of ICS for detecting analyte.