Development of an Enzyme-Linked Immunosorbent Assay for Determination Of Miroestrol Using an Anti-miroestrol Monoclonal Antibody

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Chemicals and reagents
• Preparation of miroestrol conjugates
• Generation of anti-miroestrol monoclonal antibody
• Indirect ELISA
• Indirect competitive ELISA using anti-miroestrol monoclonal antibody
• Recovery of miroestrol from Pueraria candollei extract
• Preparation of immunoaffinity gel for immunoaffinity chromatography
• Miroestrol knockout extract
• HPLC analysis
• Plant materials and extraction
• References

Abstract

Miroestrol is a chromene with potent estrogenic activity present in Pueraria candollei, commonly known as White Kwao Krua. Although this compound is only present in low amounts in the plant, it plays an important role in the estrogenic action of P. candollei products. As a tool for further studies about the efficacy and safety of P. candollei as a phytoestrogenic supplement, we generated a novel monoclonal antibody against miroestrol. This anti-miroestrol monoclonal antibody was used to develop an immunoassay for the determination of miroestrol content, which can be used for quality control purposes of P. candollei. The developed ELISA against miroestrol has a calibration range of 10–780 ng/mL miroestrol, a limit of detection of 3.5 ng/mL, and a limit of quantitation of 12.2 ng/mL. According to the validation analysis, the established ELISA is precise, accurate, specific, and sensitive for miroestrol detection in plants. Furthermore, the anti-miroestrol monoclonal antibody was used to prepare an immunoaffinity column for the isolation of miroestrol from the tuberous root of P. candollei. The column provides a simple procedure for miroestrol isolation, with a capacity of 3.91 µg of miroestrol per 1 mL of immunogel.

Introduction

The tuberous roots of Pueraria candollei Wall ex Benth var. mirifica (Airy Shaw & Suvat.) Niyomdham and Pueraria candollei Wall ex Benth var. candollei (Fabaceae), commonly called “White Kwao Krua”, have been used in Thai traditional medicine to increase longevity, stimulate appetite, and for rejuvenation [1], [2]. Several biological activities of P. candollei have been reported, such as estrogenic [3], [4], [5], antioxidant [6], and osteoporosis prevention effects [7], [8]. As a recognized source phytoestrogens, P. candollei has been used to alleviate menopausal symptoms such as hot flashes, night sweating, and frustration [9], [10], [11]. In the global market, various products derived from the P. candollei tuberous root are sold as health supplements and for cosmetic purposes. Due to the variation in the phytochemical composition of P. candollei from different sources [12], standardization and quality control of P. candollei products is necessary to ensure their efficacy and absence of toxicity.

Previously, our group developed an ELISA for total isoflavonoid analysis in P.candollei using polyclonal antibodies [13]. In addition to isoflavonoids, which are major compounds in P. candollei, miroestrol and deoxymiroestrol ([Fig. 1]) are characteristic minor constituents that have been reported to possess the highest estrogenic activity among known phytoestrogens [14]. Miroestrol was shown to occur as an air oxidation product of deoxymiroestrol during the harvesting and processing of the plant material [15]. Considering that miroestrol shows higher stability and might be more relevant than deoxymiroestrol for the estrogenic activity of processed P. candollei samples, this compound can represent an appropriate reference standard to control the quality of P. candollei products, such as dietary supplements and cosmetics.

However, miroestrol is present in low amounts in P. candollei (0.0012–0.0080 % dry weight) [16], [17]. The signals of other compounds may interfere with miroestrol in HPLC analysis [18] and multiple steps are needed to isolate miroestrol. Thus, our research group developed an immunoassay using polyclonal antibodies for miroestrol analysis in P. candollei samples in an ELISA [19], further modified as an enhanced chemiluminescence (ECL) ELISA [20]. The developed immunoassay showed high specificity and sensitivity against miroestrol, and sample preparation was relatively simple. Furthermore, we have investigated immunogen preparation to obtain an antibody against miroestrol with the desired characteristics, including improved specificity and antibody titer [21]. In the current study, a monoclonal antibody (mAb) against miroestrol was now generated to provide a sustainable source of antibody for immunoassay development.

Immunoaffinity column chromatography is a type of liquid chromatography in which a stationary phase contains an antibody or antibody-related substance that specifically binds to an analyte [22]. The isolation of active compounds in herbal plants is usually time-consuming and requires large volumes of organic solvents. Efforts to develop alternative approaches have included the use of immunoaffinity columns using mAb against bioactive compounds. Using this approach, various phytochemicals, such as forskolin [23], solasodine glycosides [24], ginsenosides [25], and puerarin [26], were successfully isolated from crude extracts of plant materials.

In the current study, anti-miroestrol mAb was not only applied for the development of an ELISA but also used to demonstrate the effectiveness of immunoaffinity column chromatography for isolation of miroestrol from P. candollei samples.

Results and Discussion

Miroestrol analysis methods using HPLC and ELISA were previously reported by our group [18], [19], [20], [21]. The high sensitivity and high specificity of immunoassays made them suitable for samples containing multiple ingredients. Initially, polyclonal antibodies (pAbs) derived from an immunogen prepared by a periodate reaction were used for development of ELISA and ECL-ELISA that showed high specificity and sensitivity against miroestrol [19], [20]. However, there was some inconsistency in the specificity of pAbs between different immunizations. Later, an immunogen prepared by the Mannich reaction, which can better preserve the structure of miroestrol, was used for rabbit immunization. The results showed that the pAbs derived from the immunogen prepared by this method exhibited higher specificity and higher sensitivity against miroestrol [21]. Hence, the immunogen used for the generation of mAb in this study was prepared by the Mannich reaction. The MALDI-TOF mass spectra of cBSA (MW 67 013.26) and miroestrol-cBSA (MW 69 535.22), which was used for the immunization, is shown in [Fig. 2]. The hapten number in the synthetic immunogen was seven molecules of miroestrol per molecule of cBSA.

After cloning by limiting dilution, the hybridomas were screened for reactivity against miroestrol by indirect ELISA. The monoclonal hybridoma designated 1H1 was selected and scaled up. The cell culture supernatant was collected for purification of anti-miroestrol mAb. The characteristics of the anti-miroestrol mAb were evaluated by indirect ELISA. [Fig. 3] shows the reactivity of the anti-miroestrol mAb (A) against miroestrol and the competitive inhibition of miroestrol against the mAb (B). The linearity range of the mAb against miroestrol is between 10–780 ng/mL with a limit of detection (LOD) of 3.5 ng/mL and limit of quantitation (LOQ) of 12.2 ng/mL. The percentage of cross-reactivity was determined as a measure of the specificity of the mAb against miroestrol compared with other chromenes and isoflavonoids found in P. candollei tubers and some further chemical compounds. The anti-miroestrol mAb 1H1 exhibited cross-reactivity less than 0.5 % with the chromenes, deoxymiroestrol and isomiroestrol, and ≤ 0.04 % with the other tested compounds ([Table 1]). As to the chromenes, the binding of mAb 1H1 could involve the OH group at C-14 since the chemical structures of deoxymiroestrol and isomiroestrol differ from that of miroestrol at this position ([Fig. 1]). In previous studies, pAbs obtained by immunization with immunogens prepared by the periodate reaction for the determination of isoflavonoids in P. candollei, such as anti-puerarin and anti-daidzin pAbs, showed high cross-reactivity with other isoflavonoid derivatives [13], [27]. In addition, an anti-miroestrol pAb derived from immunogen prepared by the periodate reaction also showed cross-reactivity against deoxymiroestrol (6.7 %) and isomiroestrol (1.0 %) [19]. The developed anti-miroestrol mAb 1H1 shows higher specificity than anti-miroestrol pAbs in previous studies [19]. Our results confirm that the specificity of the antibody depends on the conjugation reaction.

The developed mAb shows high specificity against miroestrol, reduces animal use, and can be homogenously produced on a large scale. Therefore, the anti-miroestrol mAb was used to develop an ELISA for miroestrol analysis and to prepare an immunoaffinity column for the isolation of miroestrol from P. candollei samples. To validate the developed ELISA, we investigated its precision and accuracy. Intra- and inter-plate assays were performed to evaluate the percentage relative standard deviation (RSD) ([Table 2]). The RSDs of intra- and inter-plate assays were in the ranges of 0.92 to 3.82 % and 3.23 to 5.10 %, respectively. The recovery percentage of miroestrol from a spiked (2.5–20 µg/mL) P. candollei extract was within the acceptable range (90 to 110 %) [28] ([Table 3]). Based on its accuracy and consistency, the developed ELISA is sufficiently reliable for determination of miroestrol in P. candollei samples.

To investigate the validity of the developed ELISA, HPLC was used to determine the miroestrol content in various P. candollei samples compared to ELISA ([Table 4]). The miroestrol content in each P. candollei sample, as determined by ELISA and HPLC, exhibited a high correlation coefficient (R² = 0.9955).

In a further step, the monoclonal antibody was used to prepare an anti-miroestrol immunogel. The carbohydrate moiety of the anti-miroestrol mAb was coupled with the hydrazide functional group of Affi-gel, as illustrated in [Fig. 4]. The percentage of coupling efficiency was 65.17 % of mAb. Five milliliters of anti-miroestrol immunogel was prepared. The elution profile that resulted from the application of a standard of miroestrol to the immunoaffinity column is shown in [Fig. 5]. The amount of eluted miroestrol was 3.84 µg per 1 mL of immunogel. Then, a P. candollei extract containing the same amount of miroestrol was applied and eluted, which afforded a miroestrol amount of 3.91 µg per 1 mL of immunogel. Thus, other compounds in the P. candollei did not appear to affect the capacity of the immunogel. The immunoaffinity column using the anti-miroestrol mAb enabled simple and efficient isolation of miroestrol from P. candollei, and reduced the amount of organic solvent used. To investigate the isolation performance of the immunoaffinity column, the miroestrol content in the eluted fraction was compared with standard miroestrol and miroestrol obtained from a P. candollei sample by HPLC, as shown in [Fig. 6]. The HPLC chromatogram showed that miroestrol was specifically bound and isolated by the immunoaffinity column using the anti-miroestrol mAb.

In this study, we first generated a monoclonal antibody with high specificity against miroestrol. The anti-miroestrol mAb was used as material to develop an ELISA and an immunoaffinity column. The developed ELISA provides high reliability and effectiveness to control the quality of P. candollei products. In addition, this ELISA might also be applied to determine the miroestrol content of biological fluids in clinical studies. Finally, miroestrol isolated by the immunoaffinity column and the knockout extract could be used to investigate the role of miroestrol in the pharmacological action of P. candollei.

Materials and Methods

Chemicals and reagents

Miroestrol (> 98 % purity) was isolated from P. candollei roots and confirmed by NMR comparison with an authentic standard provided by Dr. Chaiyo Chaichantipyuth, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand. Enriched RPMI-1640 Dulbeccoʼs-Ham F12 (eRDF) and RD-1 supplement were obtained from Kyokuto Pharmaceutical Industrial. Bovine serum albumin (BSA), ovalbumin (OVA), PEG solution 50 % (w/v), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were obtained from Sigma-Aldrich. FBS, HAT (50×), and HT (100×) supplements were purchased from Gibco. Peroxidase-conjugated goat affinity purified antibody to mouse IgG (Fc) was obtained from MP Biomedicals for use as a secondary antibody. Affi-Gel Hz 10×Coupling Buffer Concentrate and Affi-Gel Hz Hydrazide Gel were purchased from Bio-Rad. All other reagents purchased were of analytical grade.

Preparation of miroestrol conjugates

Carrier proteins used for conjugation with miroestrol were prepared in a cationized form as described by Muckerheide et al. [29]. Then, miroestrol conjugate was prepared as described previously [21]. Briefly, 2.5 mg of miroestrol was dissolved in 600 µL absolute ethanol and 400 µL 37 % formaldehyde. The mixture was dropped into cationized bovine serum albumin (cBSA) solution [5 mg cBSA in 2 mL MES (2-(N-morpholino)-ethane sulfonic acid) buffer (pH 4.7; 0.1 M) and NaCl (0.9 M)]. The reaction was performed at 37 °C under stirring for 10 h. The reaction product was dialyzed against distilled water and then lyophilized. The conjugated product was analyzed by MALDI-TOF mass spectrometry (matrix assisted laser desorption/ionization-time of flight mass spectrometry). To prepare the coating reagent for ELISA, cationized ovalbumin (cOVA) was used instead of cBSA for conjugation with miroestrol.

Generation of anti-miroestrol monoclonal antibody

Five-week-old male BALB/c mice (20 g) were purchased from the National Laboratory Animal Center, Mahidol University, Thailand. Animal handling and the treatment protocol were approved by the Animal Ethic Committee of Khon Kaen University (Record No. AEKKU 18/2557; Date of Approval: 19–06–2014). For the first immunization, BALB/c mice were injected intraperitoneally with 0.2 mL of miroestrol-cBSA conjugate at a concentration of 0.5 mg/mL in PBS emulsified with an equal volume of complete Freundʼs adjuvant. Two weeks later, a half dose of conjugate was injected as a second immunization using incomplete Freundʼs adjuvant. The next boost was after a 10- to 14-day interval without adjuvant. Serum was collected from each mouseʼs tail vein at 5-day intervals after each boost and checked for antibody titer using indirect ELISA. A mouse with a high titer of antibody against miroestrol was selected and sacrificed by cervical dislocation. The spleen was removed and fused with myeloma (sp2/0-Ag14) by the PEG method [30]. The ratio of splenocytes to myeloma was 10 : 1. Fused cells were plated into a 96-well cell culture plate in eRDF with a 15 % FBS supplement. One day after fusion, HAT supplement was added into the fused cells for selection of hybridoma. Two weeks later, the cell culture supernatants in each well were screened for antibody-secreted hybridoma and inhibition against miroestrol by indirect ELISA. A hybridoma that secreted an antibody against miroestrol was used for cloning by limiting dilution [31]. Afterward, the monoclonal hybridoma was selected and scaled up. The hybridoma culture supernatant was collected and the anti-miroestrol mAb was purified using a protein A Sepharose 4 FF affinity column (Amersham Biosciences). The purified anti-miroestrol mAb was used for development of the immunoassays in this study.

Indirect ELISA

The antibody titer of the immunized mouseʼs sera and the reactivity of the anti-miroestrol mAb were determined by indirect ELISA using miroestrol-cOVA as the coating reagent. The procedure was performed as previously reported, with some modifications [21]. One hundred microliters of miroestrol-cOVA 5 µg/mL in carbonate (sodium) buffer (pH 9.6; 50 mM) was placed in each well of a 96-well plate and then the plate was incubated for 1 h. The coating reagent was washed with 0.05 % Tween 20 in phosphate buffered saline (pH 7.0; 0.01 M) (T-PBS). Each well was filled with 300 µL of 1 % gelatin in PBS and incubated for 1 h. After the gelatin was removed, 50 µL of 20 % ethanol and 50 µL of diluted mouse sera (or anti-miroestrol mAb) in T-PBS, at varied concentrations, were added and incubated for 1 h. The diluted sera (or anti-miroestrol mAb) were discarded, and peroxidase-conjugated goat anti-mouse IgG antibody was added to each well. After incubation for 1 h, the plate was washed and 100 µL substrate solution [citrate buffer (pH 4.0; 100 mM) containing 0.003 % H₂O₂ and 0.3 mg/mL ABTS] were added. After incubation at 37 °C for 20 min, the absorbance was measured at 405 nm by a microplate reader. The antibody titer was expressed as the dilution factor that provides an absorbance value of 1 at 405 nm. To determine the reactivity of anti-miroestrol mAb, data were plotted for absorbance at 405 nm over a concentration of mAb on a logarithmic scale to determine the optimal concentration for further analysis.

Indirect competitive ELISA using anti-miroestrol monoclonal antibody

Indirect competitive ELISA was developed using anti-miroestrol mAb and applied for analysis of miroestrol content in the P. candollei samples. In the procedure, all steps were performed as indirect noncompetitive ELISAs, except after the gelatin removal step. Each well received 50 µL 20 % ethanol as a blank or various concentrations of miroestrol in 20 % ethanol and 50 µL anti-miroestrol mAb solution (0.32 µg/mL). The data were plotted as absorbance at 405 nm over the concentration of miroestrol on a logarithm scale in which absorbance was represented by A/A0 (A and A0 refer to the absorbance of the sample and blank, respectively). The LOD of the mAb was calculated as the concentration of miroestrol that provided 10 % inhibition against the anti-miroestrol mAb. The LOQ of mAb was defined as the lowest concentration of miroestrol used in the calibration curve. The percentage of cross-reactivity was calculated from the half maximal inhibitory concentration (IC₅₀) of miroestrol and other compounds against the reactivity of the anti-miroestrol mAb as shown in Equation 1.

Recovery of miroestrol from Pueraria candollei extract

Indirect competitive ELISA using the anti-miroestrol mAb was used for the determination of miroestrol recovery from spiked P. candollei extracts. P. candollei extract was accurately spiked with miroestrol at final concentrations of 2.5, 5, 10, and 20 µg/mL in triplicate. The amount of miroestrol in the unspiked P. candollei extract was 1.45 µg/mL. The percentage of miroestrol recovery was calculated with Equation 2.

Preparation of immunoaffinity gel for immunoaffinity chromatography

An immunoaffinity column against miroestrol was prepared according to the Affi-Gel Hz manualʼs instructions and a previous report [24]. Purified anti-miroestrol mAb (5 mg) was dialyzed in Affi-Gel Hz coupling buffer (pH 5.5) overnight at 4 °C and oxidized with NaIO₄ by gentle stirring in a closed container protected from light at room temperature for 1 h. Immediately after oxidation, glycerol was added to the reaction at a final concentration of 20 mM, mixed for 10 min, and then dialyzed against coupling buffer (pH 5.5) at 4 °C. The oxidized and desalted mAb was coupled to 5 mL of Affi-Gel Hz hydrazide gel in coupling buffer and stirred at room temperature for 24 h. After the coupling reaction was complete, the immunoaffinity gel slurry was poured into a plastic column. For coupling efficiency determination, the column eluent was collected to measure unbound proteins using Bradfordʼs reagent. After that, the column was washed with one volume of PBS. Then, the gel was equilibrated with PBS containing 0.02 % sodium azide and stored at 4 °C until use.

Miroestrol knockout extract

The immunoaffinity column was washed with at least five bed volumes of PBS before use at room temperature. Standard miroestrol was used to determine the capacity of the immunoaffinity column against miroestrol. Miroestrol (80 µg) in PBS was loaded into the immunoaffinity column and incubated at 4 °C for 2 h. Then, the column was washed with three bed volumes of PBS and eluted with two bed volumes of 1 M guanidine HCl with 20 % ethanol in PBS as the fraction containing miroestrol. The column was regenerated with PBS containing 0.02 % sodium azide and stored at 4 °C until later use. The miroestrol concentration of each collected fraction was determined by ELISA.

Dried P. candollei tuberous roots were macerated with chloroform, ethylacetate, and ethanol, in that order. The ethanolic extract was dissolved in PBS and filtered with a 0.45-µm filter. The miroestrol content of the filtrate was determined by ELISA. Then, a volume of filtrate containing 80 µg of miroestrol was applied to the immunoaffinity column to remove miroestrol.

HPLC analysis

The HPLC system was used according to previously reported procedures [32], with some modifications. HPLC analysis was performed on an Agilent 1100 series (Agilent Corp.) equipped with a degasser (JP40715322), pump (DE40926753), UV/Vis detector (DE40521586), and autosampler (DE33225330). A reverse-phase column (LiChroCart, 125 mm × 4 mm, 5 µm; Merck) was used. Briefly, the column oven was set at 30 °C, and the detection wavelength was set at 280 nm, with the reference wavelength set at 360 nm. Twenty microliters of each sample solution was injected. The mobile phase consisted of 1.5 % acetic acid in water (A) and acetonitrile (B). The flow rate was set at l mL/min, and a linear gradient was applied: 15 to 20 % solvent B from 0 to 15 min, 20 to 40 % solvent B from 15 min to 40 min, and 40 to 100 % solvent B from 40 min to 45 min, and finally 100 % solvent B for 10 min.

Plant materials and extraction

Tuberous roots of P. candollei var. mirifica were obtained from Nakhon Ratchasima province and Khon Kaen province in May 2014. The tuberous roots of P. candollei var. candollei were obtained from Ubon Ratchathani province and Surat Thani province in January 2015. Plant samples were identified by Dr. Thaweesak Juengwatanatrakul, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, Thailand. Voucher specimens (NI-PSKKU 070–073) were deposited at the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand. The tuberous roots were dried at 50 °C and ground to powder. Each sample (200 mg) was extracted in a microtube with 500 µL ethanol under sonication (35 kHz) for 20 min at room temperature. The extract was centrifuged at 5000 rpm for 10 min and the supernatant was collected. Extraction was repeated three more times. The combined supernatants were evaporated until they were completely dried and then redissolved in 1 mL of 20 % ethanol. The extracts were stored at − 20 °C until analysis.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the Thailand Research Fund Organization (Grant numbers PHD/0050/2556), and the Graduate Research Fund of Khon Kaen University (Grant number 57212105). The authors thank Dr. Chaiyo Chaichantipyuth, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand, for the gift of an authentic standard miroestrol.

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Correspondence

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