Reagents and chemicals

The following reagents were purchased from Sigma-Aldrich, St. Louis, MO, USA: clenbuterol (CL), sulfamethazine (SM₂), bovine serum albumin (BSA), ovalbumin (OVA), Freund's adjuvant complete (FCA), Freund's adjuvant Incomplete (FIA), Tween-20 and N', N-Dimethylformamide (DMF). Cy3-goat-anti-rabbit IgG, and HRP-goat-anti-rabbit IgG were purchased from Jackson ImmunoResearch, West Grove, PA, USA. 3,3'5,5'-Tetramethyl Benzidine dihydrochloride (TMB) was purchased from Amresco, Inc., Solon, OH, USA.

Hapten-carrier conjugation

The aromatic primary amine on CL and SM₂ were activated by diazotization and coupled to the phenol group of tyrosine residues on ovalbumin (OVA) as a carrier protein [11]. The detailed conjugation procedures are as follows: 10 mg of each hapten were first acidified (CL with 400 μl of 1 M HCl; SM₂ with 250 μl 0.5 M H₂SO₄). Then, 2% NaNO₂ was slowly added with stirring. After 1 h, 1 ml of 0.1 M carbonate buffer solution (pH 9.6) containing 35 mg OVA was added. The pH was maintained between 9 and 9.6 through the periodic addition of 0.5 M NaOH. The solution was allowed to stir overnight at 4°C. The conjugated product was dialyzed into PBS for 72 h, with PBS replacement at 8 h intervals. The synthetic products were combined with the same volume of sterile 80% glycerol and then frozen at -20°C. The formation of the artificial antigens was monitored by UV absorbance from 200 to 400 nm (TU-1810 V-spectrophotometer, Beijing Purkinje General Instruments, Beijing, China). Within this wavelength range, the haptens, OVA and the respective conjugates have distinct absorbance patterns and thus could be used to identify the formation of the conjugated product.

The efficiency of the diazotization was determined by the coupling ratios of the haptens to OVA. The conjugation ratios of CL to OVA and SM₂ to OVA are 16:1 and 3:1, respectively.

Antibody Production

One mg of each immunogen (CL-OVA and SM₂-OVA) were diluted with sterile saline and combined with an equal volume of complete Freund's adjuvant for the initial subcutaneous injection into New Zealand White Rabbits (SLAC Laboratory Animals, Shanghai, China). Second and third injections were mixed with incomplete Freund's adjuvant, and no adjuvant was used for the forth. The injections were 15 days apart, and two rabbits were immunized with each immunogen. Blood was collected before the start of immunizations as a negative control, and 10 days after each immunization for titer monitoring. 2 ml of blood was collected each time from the central ear artery and allowed to clot and retract at 37°C for 2 h and overnight at 4°C. The blood samples were then centrifuged at room temperature for 15 minutes at 3000 rpm and the sera were decanted into sterile tubes.

Antibody Titer Monitoring

Titers of the polyclonal antibodies against the two immunogens were determined by indirect ELISA. 96 well plates were coated with CL or SM₂ artificial antigens overnight at 4°C. The plates were washed 3 times with PBST (0.5%Tween-20 in PBS), and blocked with 150 μl of 0.1% (w/v) OVA per well at 37°C for 1.5 h. Antisera from each injection were diluted from 1:200 to 1:25,600 on a two-fold dilution series and 100 μl were applied to each well. Undiluted pre-immune sera were also assayed as the negative control. The plates were incubated at 37°C for 1 h, washed with PBST, and then incubated with HRP-anti-rabbit IgG (1:1000 dilution, 100 μl) at 37°C for 1 h. Finally, TMB (100 μl per well) was added and incubated at 37°C for 20 min, and 50 μl per well of 2 M H₂SO₄ was added to stop the enzymatic reaction. The plates were read with a ZS-2 microplate reader (Beijing Xin Feng Machine Electric Technical Instruments, Beijing, China). The final optical density reading was adjusted for background absorbance (OD = OD_{450 nm} - OD_{630 nm}).

We could observe an increase in the OD after each injection, indicating that the titers were rising. The pre-immune sera showed insignificant amount of absorbance, implicating that the antisera were specific. We determined that after four injections, a sufficient titer was achieved for both immunogens (OD reading of the sera of the fourth injection was at saturation at 1:200 dilution), and terminating bleeds were performed. The sera were collected as mentioned above and stored at -80°C.

Competitive indirect ELISA

Two 96-well plates were each coated with 5 μg per well of CL or SM₂ artificial antigen overnight at 4°C. The plates were washed 3 times with PBST, and blocked with 150 μl of 0.1% (w/v) OVA per well at 37°C for 1.5 h. A 1:12,800 dilution of the antisera were shown during tittering to achieve an OD in the working range, thus for both CL and SM₂, 50 μl of a 1:12,800 dilution of the corresponding antiserum were applied to each well, and 50 μl of the corresponding hapten standard solutions was added as the "competitor". The concentrations of the standard solutions of CL were 0, 1, 5, 10, 20, 50, 100, 1000, 5000, 12,500, and 25,000 ng/ml; for SM₂, the concentrations of the standard solutions were 0, 1, 10, 50, 100, 1000, 5000, 10,000 and 20,000 ng/ml. Each concentration was added to one row of twelve wells. The plates were incubated at 37°C for 1 h, and were processed the same way as in the indirect ELISA mentioned above.

Protein microarray immunodetection

Twenty nanogram of each artificial antigen were spotted onto a 7 × 7 array on P-L-L microarray slides (CapitalBio Corporation, Beijing, China) using an OmniGrid-100 Microarrayer (Genomic Solutions, Ann Arbor, MI, USA). Following a 2 h incubation in a humid chamber at 37°C, the slides were inverted and immersed into PBS (pH 7.5) containing 0.2% OVA (w/v). The slides were then turned right side up and immersed in a 2% BSA solution for 1 h at room temperature with gentle agitation. Next, the slides were washed twice (10 s each) at room temperature with PBST and twice with ddH₂O. The diluted antiserum (1:500 with PBS) and one of the corresponding standard hapten solutions (0, 5, 10, 20, 50, 100 and 1000 ng/ml) were mixed and added to an artificial antigen-spotted protein microarray slide. The slides were incubated with the antiserum-hapten mix for 1 h at room temperature and then washed three times with PBST and once with ddH₂O. Then, cy3-goat-anti-rabbit IgG (1:4000 diluted in PBST) was applied to the slides. After 1 h of incubation, the slides were rinsed with PBST and then washed as previously. The slides were dried by centrifugation and scanned using an Axon GenePix^® 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, USA) to detect the fluorescence signal.

IC₅₀determination

A dose-response curve was produced and was used to calculate the IC₅₀ by non-linear regression analysis using GraphPad Prism 5 software (GraphPad Software, Inc, La Jolla, CA, USA) For the ci-ELISA, the final optical density reading was adjusted for background absorbance (OD = OD_{450 nm} - OD_{630 nm}). The log of the hapten concentrations were plotted against the percentage of inhibition, which was calculated by the equation (OD_sample/OD_control) × 100%, where the control group (i.e., 0 ng/ml hapten) OD was considered to be the point of 100% activity.

The results of the protein microarray assays were reported as the average pixels of fluorescence at 532 nm of the 49 spots for each hapten concentration minus background pixels. The IC₅₀ of the protein microarray assays were determined by the same method of non-linear regression analysis as for the ci-ELISA.

Fortification of muscle homogenate with clenbuterol

The muscle tissue samples in control group were used in the fortification studies. Fortification was carried out by adding 25 μl of methanolic solutions containing clenbuterol concentrations of 30, 120 and 300 ng/ml to the tissue homogenate, resulting in fortification levels of 0.5, 2 and 5 ng/g, respectively [12, 13]. The fortified samples were used for calculation of recovery by ci-ELISA and protein microarrays.

Preparation of muscle samples from clenbuterol-fed chicken

Fifteen eight-week-old broilers (AA species, Zhengda Co., Beijing, China) were randomly divided into five groups and raised in brooders provided with fresh feed and water every day. The control group was given drug-free feed; the other four groups were given feed treated with 3 mg/kg CL (w/w) [12–14]. The broilers were fed for 14 consecutive days. One treated group was slaughtered after 0, 1 day, 7 days, and 14 days withdrawal periods, respectively. The muscle tissue samples were collected and frozen at -20°C until analysis. The muscle tissue samples were homogenized using a DI 25 basic Ultraturrax homogenizer (Ika-Werke, Staufen, Germany). Five gram of homogenate was mixed with 25 ml 50 mM HCl by shaking for 1.5 h. The homogenate was centrifuged, and the supernatant was collected in a tube containing 300 μl of 1 M NaOH and mixed for 15 min. 400 μl of 0.5 M KH₂PO₄ were then added and the mixture was stored at 4°C overnight. The next day the mixture was centrifuged at 2750 g for 15 min, and the supernatant was purified by RP-18 cartridges (Supelco, Bellefonte, PA, USA). Clenbuterol was eluted with methanol from the purified supernatant as described elsewhere [15]. The eluent was collected by vacuum and the solvent was evaporated under a nitrogen stream. Dried residue was redissolved in 400 μl of water, and 20 μl per well were analyzed by ci-ELISA and per slide by protein microarray analysis. The concentrations of the recovered CL were calculated against a standard curve made with 0, 1, 5, 10, 25, 50, 100, 250, 500 and 1000 ng/ml CL solutions for both methods. The total CL recovered were converted to and reported as per gram of tissue.