Hapten Synthesis and the Development of an Ultrasensitive Indirect Competitive ELISA for the Determination of Diethylstilbestrol in Food Samples

An ultrasensitive indirect competitive enzyme-linked immunosorbent assay (ic ELISA) using monoclonal antibodies (mAbs) was developed for the specific detection of diethylstilbestrol (DES) residues. To establish an ELISA based on mAbs, hapten diethylstilbestrol mono-carboxypropyl-ether (DES-MCPE) was chemically synthetized and then conjugated to bovine serum albumin (BSA) for immunization in mice. This ic ELISA was further optimized for DES determination. The sensitivity of the ic ELISA was found to be 0.49 μg/kg and the limit of detection was 0.075 μg/kg. DES residues in salmon meat and pork were tested with the recovery range from 74.0 to 85.2% and the coefficient of variation (CV) was less than 10%. Parallel analysis of DES samples from salmon meat showed comparable results from the ic ELISA with high-performance liquid chromatography. The ic ELISA provides a useful screening method for the quantitative detection of DES residues in animal-derived food.

Hapten synthesis. A derivative of DES, called DES-MCPE, was chemically synthetized using ethyl 4-bromobutyrate as follows. Fifty-four milligrams of DES was dissolved in 2 mL of anhydrous dimethyl sulfoxide (DMSO), then 27.5 mg of potassium carbonate was added, and the mixture was stirred at room temperature for 1.5 h in the dark. Seventeen milligrams of ethyl 4-bromobutyrate was added to the mixture and stirred at room temperature for 8 h in the dark. The reaction product was transferred to 10 mL of pre-cooled dilute hydrochloric acid solution (pH 2), and 5 mL of ethyl acetate was added with double distilled water for extraction. The extracts were rinsed with double distilled water and placed into a vacuum concentrator (jouan-rc1010z, France) to extract the organic solvent. The extracted product was dissolved in 2 mL of methanol solution, and then 2 mol/L sodium hydroxide solution was added. Once the product was fully dissolved, an appropriate amount of hydrochloric acid solution was added dropwise to maintain the pH value of the solution between 2 to 4. The above steps were repeated, and the product was extracted by ethyl acetate, rinsed with double distilled water, and vacuum dried to obtain the DES-MCPE.
Hapten-carrier protein conjugation. The hapten of DES-MCPE was conjugated to BSA or OVA by the EDC/NHS method 23 . Forty-two milligrams of DES-MCPE was dissolved in 2 mL of dimethyl sulfoxide, and 25 mg of EDC, 0.6 mL of anhydrous dimethylformamide, and 15 mg of NHS were added to the mixture. The mixture was stirred at room temperature for 4 h in the dark and centrifuged at 2900 g for 5 min to collect the supernatant. Sixty milligrams of BSA was fully dissolved in a mixture of 1 mL of anhydrous dimethylformamide and 2 mL phosphate buffer saline (PBS) (PH 8). In ice bath conditions, the supernatant was added dropwise to the BSA solution and stirred at 4 °C for 8 h. The reaction product was dialysed against PBS (PH 7.2) for 8 h.
Preparation of monoclonal antibodies. For the DES immunogen, subcutaneous injections were given to three female BALB/c mice (at the animal experimental centre of Zhengzhou University, Zhengzhou, China). For the first immunization, 0.2 mL of Freund's complete adjuvant was emulsified with the conjugate (60 μg of immunogen in 0.2 mL of PBS) (1:1, v/v) for injection into a mouse weighing 15~22 g. At intervals of 21 days, Freund's incomplete adjuvant was used for fortified subcutaneous immunization. Ten days after the fourth immunization, 10 μL of blood sample from each mouse was obtained, and the collected antiserum was evaluated by indirect competition ELISA (ic ELISA). Sp2/0 myeloma cells were fused with the splenocytes of the selected mouse using PEG-1500 72 h after intraperitoneal injection of 100 μg of immunogen in 150 μL of PBS buffer. Ten days later, www.nature.com/scientificreports www.nature.com/scientificreports/ hybridomas were selected by ic ELISA, and lowest value of IC 50 were considered positive, subcloning was performed by the limited dilution method. The ascitic fluids of positive hybridomas were produced in mice with liquid paraffin in the peritoneal cavity.
Development of the ic ELISA. An ic ELISA was developed according to the description of Sun et al. 24 .
Serum samples were screened for polyclonal or monoclonal antibodies specific to DES using an ic ELISA. Antigen for detection (2 μg/mL OVA conjugate in carbonate buffer, 50 μL well −1 ) was coated in 96-well ELISA plates at 37 °C for 2 h. Then, each well was washed four times with phosphate buffered solution [PBST, PBS + 0.05% Tween-20 (V:V)] and blocked with 220 μL/well of 5% pig serum in PBST at 37 °C for 60 min, and the plates were washed four times with PBST and dried at room temperature. Fifty microlitres of diluted DES in PBS was added to each well, and 50 μL of diluted antibody solution was added. After 20 min in a constant temperature incubator at 37 °C, the plate was washed four times with PBST, and goat anti-mouse IgG labelled horseradish peroxidase (GAMIgG-HRP) was diluted 1:1000 and added at 50 μL/well. 3, 3' , 5, 5'-Tetramethylbenzidine (TMB) colour liquid was added to each well and incubated until the colour development of each well was ideal. Finally, 50 μL of 2 mol/L sulfuric acid was added to terminate the reactive liquid and the absorbance values of the optical density at a wavelength of 450 nm (OD 450nm ) were mensurated using a microplate reader 550 (Bio-Rad, Richmond, CA, USA).

Optimization of the ic ELISA.
To improve the sensitivity of the ic ELISA, we optimized different parameters, including the antibody-working concentration, coating concentration, coating time, blocking solution, diluted concentration of GAMIgG-HRP, and time of TMB colorization. Concentrations of the working antibody and coating antigen were optimized by checkerboard titration using DES-MCPE-OVA as the coating antigen 25 . Briefly, the DES-MCPE-OVA was coated on the ELISA plate at concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 μg/ mL and the mAbs towards DES were diluted with PBS at dilutions of 1:4.0 × 10 3 , 1:8.0 × 10 3 , 1:1.6 × 10 4 , 1:3.2 × 10 4 , 1:6.4 × 10 4 , 1:1.28 × 10 5 , 1:1.28 × 10 5 , and 1:5.12 × 10 5 . The OD 450nm value was obtained by microplate reader. A well was selected when its OD 450 nm value was approximately 1.0, and the difference of its OD 450nm value was significant relative to its adjacent wells. The concentrations of DES-MCPE-OVA and anti-DES mAbs corresponding to this well were considered optimal. For other parameters, the immunoassay performance was evaluated using the Amax (maximal absorbance)/IC 50 ratio. ELISA sensitivity is positively correlated with the Amax/IC 50 ratio 26 . Sample preparation. DES-negative salmon meat and pork were purchased from a local supermarket, and both were analysed by LC-MS. Eight-grams of salmon meat was cut into small pieces, homogenized using a high-speed homogenizer, and 16 mL of methanol was added. The mixture was shaken on a vortex oscillator for 25 min and centrifuged for 9 min at 3300 g. The supernatant was filtered and concentrated to 8 ml. The negative pork was also processed using the same operation. were tested in sextuplicate by ic ELISA and HPLC. One sample t test was used to analyse the measured data for the difference between ic ELISA and HPLC methods.

Results and Discussion
Design of haptens and characterization of DES conjugates. For developing an immunoassay to detect DES, it is very important to synthesize effective haptens to produce an anti-DES antibody. A derivative of DES, called DES-MCPE (Fig. 1), was chemically synthesized using ethyl 4-bromobutyrate, which can be conjugated to BSA using the EDC/NHS method (Fig. 2). Coupling ratios of DES-MCPE to BSA and OVA were 12.8:1 and 11.5:1, respectively.
In this work, the antigen was synthesized 6 times to be successful, and the antiserum of mice was not sensitive against DES when the hapten had not been purified, so synthesizing the antigen is meticulous work. To allow efficient conjugation, a spacer arm including 4-6 carbons has been reported to be optimal 28 . A spacer group   www.nature.com/scientificreports www.nature.com/scientificreports/ obtain sensitive, specific antibodies for developing an immunoassay based on DES-MCPE-BSA to detect DES. We also tried to synthesize another antigen, DES-hemisuccinate (DES-HS), which was chemically modified via the succinic anhydride method of Hongsibsong et al. 29 and then conjugated to BSA by the mixed anhydride method of Wainer et al.; 30 the coupling ratios of DES-HS to BSA and OVA were 9.4:1 and 7.6:1, respectively. However, DES-HS-BSA elicited antibodies which showed higher potency in indirect ELISA (>1:128000), but poor reactivity with DES in ic ELISA.
Development and optimization of ic ELISA. An ic ELISA was established using the mAbs 4C7, based on the competitive binding of free DES in the sample and coated DES-MCPE-OVA. The optimal conditions for the ic ELISA were determined by chessboard ELISA as follows: the diluted concentration of the mAb was 1:6.4 × 10 4 , and the coating concentration of DES-MCPE-OVA was 0.5 µg/mL. For other parameters, the highest Amax / IC 50 ratio was selected; for example, the ELISA plate was coated with DES-MCPE-OVA at 37 °C for 120 min (Fig. 3A), blocked for 60 min with 5% porcine serum at room temperature (Fig. 3B), the diluted concentration of GAMIgG-HRP was 1:1 × 10 3 (Fig. 3C), and TMB was used for colour development at room temperature for 10 min (Fig. 3B).
Sensitivity, specificity and reproducibility of the ic ELISA. According to the optimized reaction conditions, reference solutions of DES at concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μg/kg were analysed using the ic ELISA. Assays were performed in triplicate, and typical results are shown in Fig. 4. The immunoassay displayed an IC 50 of 0.49 μg/kg, and the limit of detection was 0.075 μg/kg for DES determination.
DES and structural analogues, including hexestrol, dienestrol, progesterone, oestradiol, bisphenol A and estriol were added to salmon meat samples and tested. The ic ELISA gave 100%, 7.66% and 3.83% CR for DES, hexestrol and dienestrol, respectively, and produced no CR with other compounds (<0.01%) including progesterone, oestradiol, bisphenol A and estriol (Table 1).
To determine the reproducibility of the ic ELISA, salmon meat and pork samples containing 2.0, 10.0, and 50.0 μg/kg DES were tested using ic ELISA. The recoveries ranged from 74.0 to 89.6% for reproducibility. The CVs of the ic ELISA were all less than 10.0% (Table 2).
Comparison between ic ELISA and HPLC. The performance of the ic ELISA was validated by HPLC at three levels with authentic samples. Concentrations of DES in the salmon meat at 8.0, 16.0, and 32.0 μg/kg were determined using an ic ELISA and HPLC. Statistical analysis using one sample t-test did not show a significant difference between ic ELISA and HPLC (Table 3).

Conclusion
An ic ELISA was established using a high-affinity, specific mAb, 4C7, against DES for the detection of DES residues. Upon optimization, the IC 50 was found to be 0.49 μg/kg, and the limit of detection was 0.075 μg/kg in the reference solutions. Recovery from salmon meat and pork samples was tested and found to range from 74.0 to 85.2%. The major advantages of the ic ELISA are that it is relatively more cost-effective and requires a shorter time than chromatographic instrument analysis, and it is easy to operate by professionals. Therefore, the ic ELISA has the potential to be used as a rapid screening tool for detecting DES residues in animal-derived food samples.
Ethical statement. All BALB/c mice in this experiment were approved by the animal ethics committee of Zhoukou Normal University (approval No. ZKNU-1-2018022701-1002) and were used in accordance with all applicable institutional and governmental regulations concerning the ethical use of animals. This article does not contain any studies with human participants performed by any of the authors.