A lateral flow immunochromatographic strip test for rapid detection of hexoestrol in fish samples

A lateral flow immunochromatographic strip test was developed for rapid and sensitive on-site detection of hexoestrol (HES) residues in fish samples with colloidal gold labelling of the anti-HES monoclonal antibody. The strip is composed of a sample pad, a conjugate reagent pad, an absorbent pad and a test membrane containing a control line and a test line. The sensitivity (half inhibitory concentration, IC50) of the strip in the detection of fish extract samples was confirmed to be 1.86 µg kg−1, and the limit of detection value was 0.62 µg kg−1. For intra-assay and inter-assay reproducibility, recoveries of HES-spiked samples ranged from 86.3% to 92.3% and 85.8% to 93.4%, coefficients of variation were 2.91–4.64% and 4.24–5.17%, respectively. High-performance liquid chromatography was employed to confirm the performance of the strip. The strip test takes less than 10 min, and thus provides a repaid method for on-site detection of HES residues.


Introduction
Hexoestrol (HES, 4,4'-(1,2-diethyl-1,2-ethanediyl) bisphenol), also known as hexoestrolum, is a kind of synthetic non-steroidal oestrogen, which can maintain the female character of the organism, promote the growth and development of reproductive organs, and adjust a positive and negative feedback on the hypothalamus and pituitary gland [1,2]. It has been mainly & 2018 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. used in clinics for treatment of amenorrhoea, uterine hypoplasia, dysfunctional uterine bleeding, menopausal syndrome, senile vaginitis and prostatic cancer [3][4][5][6]. Though the use of HES in animals is banned in many countries including China, the USA and many European countries, unfortunately, HES has been illegally used in animal production as a growth-promoting additive for increasing feed conversion rate on account of the assimilation of protein and for its cost-efficiency [7]. Subsequently, the accumulation of b-agonist residues with long half-life in the meat of HES-treated animals could result in genital anomalies in human beings, and make women contract cancer through the food chain [8,9]. Histological lesions of liver and kidney, precocious puberty and boy feminization characterize the clinical profile of exposure to HES in humans [10]. Therefore, developing new methods for monitoring HES is necessary and important.
Traditional methods for the determination of HES include high-performance liquid chromatography (HPLC), gas chromatography -mass spectrometry (GC-MS) and high performance liquid chromatography -mass spectrometry (HPLC-MS) [11 -14]. However, these methods require extensive sample preparation, expensive instruments and professional operation. Alternatively, enzyme-linked immunosorbent assay (ELISA) has been successfully developed for screening HES in urine or tissues [15,16], but, ELISA also needs incubation and washing steps and is mainly confined to laboratories. Since the development of the lateral flow immunochromatographic strip test (LFIST) for on-site detection of the concentrations of biotech crops in samples of ground grain [17], it has become a more popular method for the qualitative or semi-quantitative detection of various analytes [18][19][20][21][22] for its advantages of a one-step process (only addition of specimen), rapidity (less than 5 min) and costeffectiveness [23,24].
In this paper, we describe the development of an LFIST for the rapid detection of HES residues in fish using an anti-HES monoclonal antibody (mAb). The LFIST employs a competitive format in which HES in the sample competes with HES-bovine serum albumin (BSa) on the test line for binding to the limited amount of colloidal gold-labelled anti-HES mAb, and is suitable for on-spot screening of large fish samples.

Preparation of artificial antigens
Structure modification of HES was performed in accordance with the instruction described elsewhere [25] with minor modification, namely, under the protection of nitrogen stream, 550 mg of HES and 1.9 g of K 2 CO 3 -Al 2 O 3 carrier reagent were dissolved in 20 ml of anhydrous acetone and 0.2 ml 4-bromobutyric acid ethyl ester, and the solution was heated in the dark for 12 h. The solid was removed by filtering. The filtrate was concentrated by rotary evaporation to about 5 ml in volume. The product was separated by thin-layer chromatography (TLC) using a chloroform-methanol mixture rsos.royalsocietypublishing.org R. Soc. open sci. 5: 180504 (95 : 5, v/v). The silica gel plate of the product was extracted with methanol and the solvent of the film was evaporated to achieve hexoestrol-mono-ether-butyrate-ethyl (HES-MEBE). The HES-MEBE was saponified and hydrolysed; the product was HES-mono-caroxyl-propyl-ethyl (HÈ S-MCPE).
An active ester method was used to prepare immunogen (HES-BSA) and coating antigen (HES-OVA) [26]. Briefly, 35.0 mg of HES-MCPE, 12 mg of NHS and 20 mg of EDC were dissolved in 1 ml of DMF for 4 h at room temperature (RT). Sixty-six milligrams of BSA dissolved in 2.0 ml of phosphate buffered saline (PBS) were added to the above solution and stirred for 12 h at 48C. The reaction product was purified by dialysis against 1 l of PBS for nine changes to remove the uncoupled free hapten and stored at 2208C. The conjugation ratio was characterized by UV.

Production of monoclonal antibody against hexoestrol 2.3.1. Immunization of mice
Six seven-week-old BALB/c female mice were injected subcutaneously in back with immunogen (HES-BSA). The first immunization dose was 80 mg HES-BSA emulsified with an equal dosage of FCA. Five weeks later, four immunizations were given at three week intervals with the same amount of HES-BSA emulsified with the same dosage of FIA. Antisera were collected at two weeks after the five immunizations and were screened for anti-HES activity by competitive inhibition ELISA (ci-ELISA). One mouse that produced the highest anti-HES activity was used for fusion. The mouse received a final intraperitoneal injection of 80 mg of the conjugation in PBS. After 3 days, the spleen of the super-immune mouse was excised for cell fusion.

Cell fusion and hybridoma screening
Hybridomas secreting anti-HES antibodies were generated according to a previous study [27]. First, PEG1500 was used to carry out fusion of NS0 cells and the spleen cell from the intraperitoneal injection of the mouse. The fused cells, mouse peritoneal macrophages and the selective HAT medium were then allotted into ELISA plates, respectively, and were incubated together. Nine days later, supernatants of hybridoma colonies were gleaned and detected by indirect ELISA antibody binding to HES. The positive wells of hybridoma cells were subcloned by limiting dilutions. Anti-HES mAb were produced using the mouse ascites method. The Ig subtypes of the mAb were determined by a commercialized mouse antibody isotype kit [28]. The affinity (K a ) of mAb was determined by indirect ELISA [29].

Indirect enzyme-linked immunosorbent assay and competitive inhibition enzyme-linked immunosorbent assay
Indirect ELISA was performed as follows: 96-well ELISA plates were coated with HES-OVA (detecting antigen) in 0.05 mol l 21 carbonate -bicarbonate buffer (pH 9.6) and incubated for 2 h at 378C. Then the wells were blocked with 5% skimmed milk at 378C for 1 h. Twofold serially diluted serum samples or cell culture supernatant was added and incubated at 378C for 30 min. GaMIgG-HRP was added and incubated for another 30 min at 378C. The optical densities (ODs) were measured at 450 nm with a microplate reader after colour development with TMB (3,3 0 , 5,5;-tetramethylbenzidine) chromogen solution and termination with 2 mol l 21 H 2 SO 4 . The ci-ELISA was carried out as follows: ELISA plates were coated as described above, and serially diluted free HES in PBS was then added together with the antibody and incubated for 30 min at 378C. The following procedures were identical to those employed for indirect ELISA. During the steps, TBST (TBS containing 0.05% Tween 20 (v/v), pH 7.4) was used as a wash buffer to remove unbound antigens or antibodies.

Production of colloidal gold-labelled anti-hexoestrol monoclonal antibody
Colloidal gold nanoparticles (24 nm diameters) were made by reduction in gold chloride with 1% sodium citrate. The 100 ml colloidal gold was adjusted to pH 8. One hundred and twenty microlitres of optimal concentration of mAb solution were mixed with 12 ml of colloidal gold solution at RT, 20 min later, then 1 ml of 10% BSA solution in 0.02 mol l 21 Na 2 B 4 O 7 . 10H 2 O solution was added to the mixture and stirred at RT for another 12 min. The labelled mAb was centrifuged at 15 000g at 48C for 30 min. The supernatant was discarded. The precipitate was washed two times with 0.02 mol l 21 sodium borate ( pH 9.0) containing 0.1% NaN 3 and1% BSA. The colloidal gold-labelled mAb were stored in the 0.02 mol l 21 sodium borate buffer at 48C.
2.5. Assembly of the strip 2.5.1. Immobilization of capture reagents HES-BSA and goat anti-mouse IgG were sprayed in the NC membrane in the form of dots at 1 ml cm 21 as the test and control lines (T and C lines), respectively. The T and C lines were both set at 5 mm separation from the centre of the NC membrane, and were dried for 20 min at 378C. The final stage of the NC membrane was stored in a dry, closed environment.

Manufacture of conjugate pad, sample pad and absorbent pad
One millilitre of colloidal gold-labelled mAb to HES was added in 2 ml of sodium borate buffer to prepare the conjugate solution. A conjugate pad was produced by dipping 7 Â 300 mm glass fibre (Millipore) in the conjugate solution. After drying for 50 min at 378C, the pad was stored at 48C.
The mentioned materials, absorption pad (40 Â 300 mm) and support plate (75 Â 300 mm) were installed according to the procedure described by Yang et al. [29].

Basic test principle and process
The LFIST for detecting HES in fish samples is based on a competitive format. The anti-HES mAb labelled with colloidal gold was used as a probe. HES-BSA conjugates and goat anti-mouse IgG were dispensed onto the nitrocellulose membrane as the T line and C line, respectively. During detection applied to the strip, free HES in the sample would compete with HES-BSA on the test line for binding to mAb-gold conjugate. The colour of the test line is in a reverse relationship with the concentration of HES in the sample. For qualitative and semi-quantitative detection, the colour of the test line can be evaluated directly by the naked eye. For quantitative assay, the OD of a test line can be measured with a test strip reader, and according to the regression equation from the standard curve, the level of the HES residue can be calculated [30 -33].

Sample pretreatment for lateral flow immunochromatographic strip test
The fish samples from the market were identified to be negative by HPLC-MS. Six grams of negative fish meat (carassius, contains no HES) were homogenized, mixed with 12 ml of methanol, vortexed vigorously for 1 min, shocked for 10 min and centrifuged at 4000g for 5 min. The supernatant (1200 ml) was evaporated at 608C under a gentle flow of nitrogen. The residue obtained was resuspended in 600 ml of the mixed solution PBS and methanol (4 : 1, V/V) as negative samples [34,35].  The specificity of the test strip was identified by cross-reactivity (CR), adding other similar competitors to the negative fish samples, including diethylstilbestrol, dienestrol, bisphenol A, estradiol, estriol and provera at a concentration of 1 mg ml 21 . The CR was calculated by the equation: CR (%) ¼ ((IC 50 of HES)/IC 50 of the other competitors) Â 100%.
To estimate the accuracy, fish samples containing 4, 12 and 24 mg kg 21 of HES were tested by one batch of the test strips for replication analysis (n ¼ 6). For inter-assay precision, three batches of the test strips were used to detect the given samples in triplicate. Accuracy and precision were expressed as coefficients of variation (CV).

Comparison of lateral flow immunochromatographic strip test and high-performance liquid chromatography
Fish samples, containing five different HES concentrations (2.4, 9.6, 16.2, 36.3 and 28.8 mg kg 21 ), were detected by the strip and HPLC. HPLC was performed in accordance with the following conditions: the column was Diamonsil C18 (250 Â 4.6 mm, 5 mm), the mobile phases were a 20 mmol l 21 phosphoric acid and acetonitrile aqueous solution (60 : 40, V/V), the flow velocity was 0.5 ml min 21 , the column temperature was held at 308C, and the injection volume of the sample solution was 20 ml [36]. The test values by two methods were compared to the given concentrations by a one-sample t-test, and the test values of the two methods were also compared by an independent-sample t-test.
The differences between the test values and the relative given concentrations were statistically significant at p , 0.05, the same as the differences between the relative test values of the two methods.

Production and properties of monoclonal antibody
After 9 days, growing hybridomas in almost every microtiter plate well could be observed. Via an indirect ELISA, OD 450nm of the supernatants of each well was screened. The larger value cells (OD 450nm . 2.0) were tested by a ci-ELISA to obtain the least IC 50 of the supernatants. The recultured hybridomas 1C4, 3D6, 3E4, 4B9 were injected intraperitoneally into the BALB/c mouse to collect ascites fluid containing anti-HES mAb. The titre of HES-mAb was 1 : 1.28 Â 10 5 , 1 : 5.12 Â 10 5 , 1 : 2.56 Â 10 5 and 1 : 2.56 Â 10 5 , respectively; the affinity constant (K a ) was 9.0 Â 10 9 , 4.5 Â 10 10 , 3.2 Â 10 10 and 2.7 Â 10 10 l mol 21 , respectively; the IC 50 value was 1.903, 1.635, 1.768 and 1.792 ng ml 21 ; and the subclass of all anti-HES mAb was IgG1. The mAb 3D6, having both the highest titre and the lowest IC 50 , was chosen to be labelled by colloidal gold.

Optimization of the strip
To obtain the best performance of the strip, several conditions were optimized as follows: the pH value of the colloidal gold was 8.2, the concentration of anti-HES mAb labelled with colloidal gold was 5.7 mg ml 21 , the colloidal gold-labelled HES mAb was sprayed onto the conjugate pad at thickness of 15 ml cm 21 . In addition, HES-BSA and goat anti-mouse IgG were coated on NC membrane at a rate of 0.8 and 0.8 mg ml 21 , respectively. In a previous study, anti-HES polyclonal antibody was prepared and applied in a one-step membrane-based competitive colloidal gold-based immunoassay in lateral flow format for the determination of HES in swine urine and liver [37]. However, the sensitivity of the test strip was far higher than 30 mg kg 21 by unaided visual assessment, which is much higher than ours, suggesting the strip developed in this study is more sensitive.   (table 2). Therefore, the test strip for HES was highly specific and showed negligible CR with provera, estradiol, bisphenol A and estriol.

Reproducibility of hexoestrol in fish samples
To determine the reproducibility of the strip test, fish samples containing 4.0, 12.0 and 24.0 mg kg 21 of HES were tested using three different batches of strips. As shown in table 3, for intra-assay and interassay reproducibility, recoveries were from 86.3 to 92.3% and from 85.8 to 93.4%, respectively. The intra-and inter-assay CV of the LFIST were both less than 6.0% (table 3). These data indicated that the results given by the strip test were highly reproducible.

Advantages and development trends of immunoassay test strips
In China, test strips are widely used in experiments in the field of food safety inspection. Instrumental analysis cannot deal with so many samples. The strip can really promote the progress of food safety inspection technology, achieving universal inspection. Unfortunately, large-scale instrument inspections cannot meet universal inspection requirements in terms of time, place and economy. Multi-target, high-throughput and digitization are the future development trend of the test strip. There are two technical approaches to achieving multi-target and high-throughput detection. Firstly, the antibodies against a common epitope of a certain class of targets are produced and a single test line is used to achieve the joint detection of multiple similar targets. Secondly, the chips of the test strips are designed. Multi-detection lines or a matrix are used to achieve the joint inspection of multiple similar or different targets. Digitization is the realization of quantitative detection. The development of portable card readers compensates for the arbitrariness of human eye observation and the non-portability of current card reading devices, and realizes digitized detection under certain conditions [30,38].

Conclusion
The use of a high-affinity mAb against HES enables us to establish a colloidal gold-based LFIST for detecting HES in fish meats. The major advantages of the one-step strip test are rapidity (less than 10 min), integration (all needed reagents were integrated in the strip), sensitivity, specific and costeffectiveness. Therefore, the strip test holds great potential as an on-site screening tool for monitoring HES residues in fish meats.
Ethics. All mice in the experiment were approved by the animal Ethics committee of Zhoukou Normal University, China (no. ZKNU-1-2016090501-1001) and in accordance with all applicable institutional and governmental regulations concerning the ethical use of animals.