Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications

Journal of Chromatography B, 953–954 (2014) 120–125

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Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications Huihua Qu b , Guiliang Zhang a , Yifei Li a , Hui Sun a , Ye Sun a , Yan Zhao a,∗ , Qingguo Wang a a b

School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China Center of Scientific Experiment, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China

a r t i c l e

i n f o

Article history: Received 27 December 2013 Accepted 29 January 2014 Available online 15 February 2014 Keywords: Puerarin Monoclonal antibody ELISA Immunoaffinity column chromatography

a b s t r a c t An enzyme-linked immunosorbent assay (ELISA) was developed, and its application in immunoaffinity column chromatography was studied using a monoclonal antibody (MAb) against puerarin. Splenocytes isolated from a female BALB/c mouse immunised with a puerarin–bovine serum albumin (BSA) conjugate were fused with SP2/0 myeloma cells. The hybridoma cell line secreting MAb against puerarin (AA9) was acquired by screening and limiting dilution. The antibody generated was highly specific for puerarin with <0.01% cross-reactivity with over 50 structurally related chemicals, except for baicalein (51.8%). Using AA9, we developed an immunoassay for puerarin with a linear detection range of 10 ng/ml to 1 ␮g/ml. This assay system was further validated using intra- and inter-assays and recovery experiments. In addition, puerarin levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. Finally, we developed and validated protocols for knocking puerarin out of its parent medicine completely. In conclusion, we successfully developed a reliable ELISA and an immunoaffinity column for puerarin detection and knockout, which are useful tools for exploring the role of puerarin in formulated Chinese medicines. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Radix puerariae is the dried root of Pueraria lobata (Willd.) Ohwi, which is a semi-woody, perennial, and leguminous vine native to Southeast Asia. This root has been used for more than 2000 years as herbal medicine for the treatment of fevers, acute dysentery, diarrhea, diabetes, cardiovascular and cerebrovascular diseases [1]. To explore the underlying mechanism of its biological action, studies of its traditional applications, phytochemistry, pharmacological activities, toxicology, quality control, and potential interactions with conventional drugs have been reported [2]. There are over seventy phytochemicals that have been identified in radix puerariae, of which puerarin (PU), which is an isoflavonoid, is believed to be the major constituent responsible for the pharmacological effects of this herbal medicine. Indeed, data from animal and in vitro studies suggest that PU has beneficial effects for the Parkinson’s

Abbreviations: PU, puerarin; PLL, polylysine; BSA, bovine serum albumin; MAb, monoclonal antibody; EXT, extract; KO, knockout; HPLC, high performance liquid chromatography; TLC, thin layer chromatography; BBS, blocking buffer. ∗ Corresponding authors at: Beijing University of Chinese Medicine, School of Basic Medical Sciences, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China. Tel.: +86 1 6428 6705; fax: +86 1 6428 6821. E-mail addresses: [email protected] (Y. Zhao), [email protected] (Q. Wang). 1570-0232/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2014.01.051

disease (PD) [3], diabetes [4], angina pectoris [5,6], alcohol-caused liver injury [7], colon cancer [8], breast cancer [9], hepatic fibrosis [10], lead-induced hepatotoxicity and hyperlipidaemia [11], and ischemic stroke [12,13]. For PU-containing medicine, the PU injection has significant effects on angina pectoris due to coronary heart disease and it has been found to be safe with no obvious side effects [14]. In addition, this medicine effectively improves the cardiac arrhythmia and prognosis of chronic heart failure patients [15] and enhances the susceptibility of leukaemia cells to cytotoxic drugs from resistance to response [16]. Because PU has such important effects, it is vital to measure the concentration of PU from various preparations. Therefore, a rapid and sensitive method for monitoring the PU concentration in Chinese medicines or Chinese herbal compounds and pharmacological research is needed. The purification and knockout of PU from medicine is also an important issue. Commercial isolation of PU typically includes several isolation steps, such as crystallisation, column chromatography, and liquid partitioning. However, these methods are far from satisfactory for analytical purposes such as high sensitivity, reproducibility, large amounts of extraction solvents that are required, and the time-consuming nature of the methods. Therefore, it is imperative to establish a desirable approach. Various approaches for the separation or quantification of PU in radix puerariae have been reported, such as high-performance

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liquid chromatography (HPLC) with ultraviolet detection [14], HPLC or ultra-performance liquid chromatography (UPLC) with electrochemical detection (ECD) or diode array detection (DAD) combined with mass spectrometry (MS) [15–17], thin layer chromatography (TLC), and ultraviolet(UV) visible spectrophotometry [18,19]. To establish the fingerprints of radix puerariae, HPLC-diode array detection-flow injection-chemiluminescence coupled with HPLCelectrospray ionisation-MS methods have also been reported to separate and identify PU and other chemicals [20,21]. These methods suffer from various limitations, such as a high cost, component degradation, lengthy time, low recovery rates, or complicated pretreatment, especially for in vivo metabolism research. Therefore, it is necessary to establish a new and simple method for PU analysis. An immunoassay with a polyclonal antibody for the determination of PU has been recently established [22,23]. However, an immunoassay with monoclonal antibodies (MAbs) for PU determination does not currently exist. In addition, we developed a hybridoma to produce the anti-PU MAb(AA9), which can then be produced in relatively large quantities as needed, without the need to raise new antibody from animals. Therefore, in the current study, we generated a hybridoma to produce the MAb against PU and developed an ELISA to measure PU in various samples and an immunoaffinity column to knock PU out from its original medicinal plants. 2. Experimental 2.1. Materials and methods PU was purchased from the National Institute for Food and Drug Control (NIFDC, China, purity of 98%). The PU injection was purchased from Xiehe Pharmaceutical Co., Ltd. (Beijing, China), and various Chinese medicines were obtained from Beijing Tong Ren Tang Group Co., Ltd. (Beijing, China), Sanjiu Medical & Pharmaceutical Co., Ltd. (Beijing, China), Cachet Pharmaceutical Co. Ltd. (Beijing, China), AnguoshiTongli Herbal Medicine Co., Ltd. (Anguo, China), and Guangzhou Pharmaceutical Holdings Ltd. (Guangzhou, China). Sodium periodate was obtained from Sinopharm Chemical Reagents (Beijing, China). Bovine serum albumin (BSA), polylysine (PLL) and Freund’s complete and incomplete reagents were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). All of the other chemicals and reagents were of analytical grade and purchased from Sinopharm. The CNBr-activated sepharose 4B and protein G FF columns were obtained from GE Healthcare Co. (USA).

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plate and then expanded using a 15:6:1 solvent mixture of chloroform, methanol and water followed by detection with a sulphuric acid–ethanol solution. With the UV approach, the characteristic spectra of PU, BSA, PU–BSA, PLL, and PU–PLL were simultaneously analysed. Conjugation of the hapten to BSA or PLL was also confirmed by comparison of their spectra. 2.4. Animal treatment Female BALB/C mice (6 weeks old) were purchased from Vital River Laboratories (Beijing, China). The mice were fed a standard rodent diet (Keaoxieli Animal Feed Co. Ltd., Beijing, China) ad libitum and housed in an environmentally controlled (23 ± 2 ◦ C; 12 h light/dark cycle) animal facility. All experimental protocols were approved by the Committee on Ethics of Animal Experiments at Beijing University of Chinese Medicine, China. 2.5. Immunisation The immunisations were performed at 2 week intervals. The mice were intraperitoneally (i.p.) injected with an initiation shot of 50 ␮g of the PU–BSA conjugate in PBS emulsified with an equal volume of Freund’s complete adjuvant in the initial immunisation. The second and third immunisations, which contained 50 ␮g of the PU–BSA conjugate in Freund’s incomplete adjuvant, were injected subcutaneously at 2 and 4 weeks after the initial injection. The fourth immunisation involved injection with a solution of PU–BSA(100 ␮g) in PBS without adjuvant. In one day, mice blood was obtained from the tail vein, and sera were tested for their titer by indirect ELISAs using PU–PLL as a solid-phase antigen. 2.6. Cell fusion and preparation of anti-PU MAb Three days after the final immunisation, splenocytes were isolated and fused with a hypoxanthine–aminopterin–thymidine (HAT)-sensitive mouse myeloma cell line, SP2/0 (Sciencell Research Laboratory; Carlsbad, CA, USA), according to the polyethylene glycol (PEG) method [26,27]. Briefly, 1 ml of PEG was added dropwise to the cell pellet after centrifugation of the blended splenocytes and myeloma cells (at a ratio of 5:1) and incubated for 1 min at 37 ◦ C. Then, the HAT medium (Sigma-Aldrich) was added. The hybridoma was transferred to 96-well plates for cell culture. The cells producing MAb reactive to PU as identified by an indirect ELISA were cloned by the limiting dilution method [28,29]. The established hybridoma was cultured in HT medium.

2.2. Synthesis of PU–BSA conjugate 2.7. Purification of the MAb The conjugates were synthesized using a periodate oxidation procedure according to a previously reported protocol with some modifications [24,25]. Briefly, PU was dissolved in methanol at 10 mg/ml. Then, 1 ml of a freshly prepared sodium periodate solution (0.1 M) was added dropwise into 1 ml of the PU solution. The mixture was stirred at 25 ◦ C for 1 h followed by the addition of 1 ml of glycol. After stirring for 12 h, 8 mg of BSA was added, and the final pH was adjusted to 9.0 using a 0.05 M carbonate buffer (pH 9.6). After 6 h, the mixture was dialysed six times against phosphatebuffered saline (PBS). The dialysate of the PU–BSA conjugate was stored at 4 ◦ C for detection and immunisation. The PU–PLL conjugate was synthesized using the same method as the PU–BSA conjugate synthesis method described above. 2.3. Determination of the PU–BSA conjugate by TLC and UV The PU–BSA conjugates were determined using TLC and UV, as previously reported [18,19]. For the TLC analysis, 5 ␮l of PU, BSA, or PU–BSA was loaded on the start line of the chromatography

Ascites obtained from the abdominal cavity irritated with an established hybridoma of BALB/c mice was purified using a protein G FF column (GE Healthcare) [30]. After adjusting the solution to a pH of 7 with a 1 M Tris solution (pH 9), the ascites containing IgG were filtered through a 0.22 ␮m membrane and loaded on top of the column. After binding, the column was washed with PBS (pH 7.2), and the bound IgG was eluted with a 0.1 M glycine buffer (pH 2.7). The eluted fraction was neutralised with a 1 M Tris solution (pH 9) and dialysed against water (50× volume) for five cycles at 4 ◦ C followed by lyophilisation. 2.8. Indirect ELISA using PU–PLL The reactivity of MAb to PU–PLL was determined by an indirect ELISA. PU–PLL (1 ␮g/ml, 100 ␮l/well) dissolved in 50 mM carbonate buffer (pH 9.6) was added to the wells of a 96-well Maxisorp immunoplate and incubated for 1 h. The plate was washed three times with washing buffer (PBS containing 0.05% Tween 20, PBST)

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and then was treated with 200 ␮l of the blocking buffer (PBS containing 5% skim milk, BBS) for 1 h to eliminate nonspecific absorption. The plate was washed three times with PBST again and filled with 100 ␮l of the MAb solution at various dilutions. After 1 h, the plate was washed three times with PBST and incubated with 100 ␮l of a 10,000-fold diluted peroxidase-labelled goat anti-mouse IgG solution for 1 h. After washing the plate three times with PBST, 100 ␮l of the substrate solution [0.1 M citrate buffer (pH 4) containing 0.003% H2 O2 and 0.1 mg/ml of 3,3 ,5,5 -tetramethylbenzidine (TMB)] was added to each well and incubated for 15 min. The reaction was terminated by adding 2 M sulphuric acid. All of the reactions were performed at 37 ◦ C, except for the substrate step. Absorbance at 405 nm was recorded using a Biotek ELx 800 microplate reader.

2.9. Indirect competitive ELISA (icELISA) The reactivity of MAb to PU was determined by an icELISA. The procedures are mostly the same as described above, but in this part, we incubated 50 ␮l of the PU solution at various dilutions and 50 ␮l of the MAb solution for 1 h instead of 100 ␮l of the MAb solution at various dilutions.

2.10. Quantitative analysis of PU by HPLC HPLC analysis was performed according to a previously reported protocol with modifications [31]. The HPLC system used in this study was an Agilent 1260 Infinity with an Agilent ZORBAX SB-C18 column (5 ␮m, 0.46 × 150 mm, Agilent, USA) maintained at room temperature. The components were separated by gradient elution using water (solvent A) and methanol (solvent B) at a constant flow rate of 1.0 ml/min. The gradient profile was as follows: 0–10 min, from 0% to 23% solvent B; 30–50 min, from 23% to 35% solvent B; 50–60 min, from 35% to 70% solvent B. 10 ␮l of each sample was injected and monitored at 250 nm.

2.11. Development of icELISA using anti-PU MAb(AA9) The calibration curve and the standard curve for PU inhibition were established by an icELISA which was described above. The sensitivity of the assay is the concentration at which 50% inhibition was reached. The linear range of the calibration curve represents the test range of the assay. The cross-reactivities (CRs, %) of PU and structurally related compounds were calculated according to Weiler’s equation [32]. The recovery rate was determined according to a previously reported protocol [33]. Various amounts of PU were added to a standard control extract of radix puerariae in which the amount of PU had been determined. Therefore, the content of PU in all of the samples was assayed by an icELISA, and the recovery rate was obtained.

2.12. Sample preparation To determine the concentration of PU, powdered radix puerariae (50 g) was extracted with boiling water for 30 min and filtered through gauze, and then, the water extract was diluted with 30% ethanol. The pooled extract was centrifuged at 4000 rpm for 5 min, and the supernatant was collected and filtered through a 0.45 ␮m polyethersulfone (PES) membrane prior to the assay. An icELISA was performed as the same samples were simultaneously injected into the HPLC system. The analysis was repeated six times.

OH OH CH2OH

HO HO

O

A

O C

HO B

OH

O

A

O C

B

HO OH O

Fig. 1. Chemical structures of puerarin and baicalein.

2.13. Determination of PU in the biological samples Golden hamsters from Vital River Laboratory were maintained on a 12 h light–dark cycle with light from 7 a.m. to 7 p.m. in a temperature (22 ◦ C ± 2 ◦ C) and humidity (50% ± 10%) controlled room. They were treated after a 1-week acclimation period, at which time their body weights were close to 200 g. All of the procedures were approved by the Committee on Ethics of Animal Experiments, Beijing University of Chinese Medicine, China. The PU diluted in normal saline was administered to golden hamsters by i.p. injection at a dose of 20 mg/kg. For the dynamic study, six golden hamsters were sacrificed at each selected time point (i.e., 5, 15, 30, 45, 60, 120, 270, 420, and 720 min) using pentobarbital. After the hippocampal tissues were isolated, they were processed by the addition of PBS, sonicated 5 times, and centrifuged at 10,000 × g for 30 min at 4 ◦ C. The supernatant was stored at −40 ◦ C prior to the ELISA assay. 2.14. Immunoaffinity chromatography The protective additive in the CNBr-activated Sepharose 4B (Pharmacia GE) was washed away by 1 mM HCl (pH 4.0, 20 ml/g). The purified anti-PU MAb(AA9) was coupled to a slurry of CNBr-activated Sepharose 4B in coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCl, pH 8.3) and used to prepare the immunoaffinity column. The excess anti-PU MAb(AA9) was washed away by five medium volumes of coupling solution followed by an alternative pH buffer, which contains 0.1 M Tris–HCl buffer (pH 8.0 containing 0.5 M NaCl) and 0.1 M acetic acid/sodium acetate buffer (pH 4.0 containing 0.5 M NaCl). The medium was washed with five volumes of an alternative pH buffer for three cycles. After use, the immunoaffinity column was washed and equilibrated with 0.01 M PBS buffer, and stored at 4 ◦ C until the next use. The entire procedure was performed at room temperature except for the incubation. All of the procedures mentioned above were carried out according to the GE Healthcare Instructions. 2.15. Knockout of PU by the immunoaffinity column The water extract (EXT) of radix puerariae was filtered through a 0.45 ␮m PES membrane, and the concentration of PU was determined by HPLC. An appropriate amount of EXT was loaded on top of the medium followed by washing with 0.01 M PBS. The filtrate fractions were collected as the knockout extract (KO-EXT) and for determination of PU. After the PU signal peak disappeared, elution of PU was initiated with 0.1 M glycine–HCl buffer (containing 0.5 M NaCl, pH 2.7). The fractions were collected as puerarin eluent (PU). All of the procedures were performed at room temperature. 3. Results and discussion 3.1. Synthesis of PU–BSA and PU–PLL conjugates PU is poorly immunogenic because it is a hapten with a low molecular weight (Fig. 1). In general, PU was conjugated to the

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Fig. 2. Synthetic pathway of the puerarin-BSA conjugate.

carrier BSA using a periodate sodium synthetic method for the production of an antibody against the hapten. The glucose moiety of PU was oxidised by NaIO4 and conjugated to a free amine of BSA by the formation of a Schiff base to yield the PU–BSA conjugate (Fig. 2). The PU–PLL conjugate, which is a coating antigen for ELISA, was synthesized in the same manner. In the TLC experiments, under the same condition, the PU–BSA and PU–PLL conjugates stayed at the starting line like the protein. However, these conjugates stained the same way as PU after 10% sulphuric acid–ethanol processing. This phenomenon indicates that PU–BSA and PU–PLL exhibit characteristics of both the carrier protein and PU, and this result confirmed that PU–BSA and PU–PLL were successfully conjugated. The UV spectra of PU–BSA, PU, and BSA were recorded, and the results indicated that the absorption wavelength of PU and BSA were 254 nm and 281 nm, respectively. The UV absorption characteristics of PU and BSA are significantly different, while the UV absorption of PU–BSA and PU–PLL conjugates exhibited the characteristics of both the PU and carrier protein. Based on these results, we concluded that PU–BSA and PU–PLL were successfully conjugated (Table 1). 3.2. Generation of anti-PU MAb(AA9) Six-week old female BALB/c mice were immunised with the PU–BSA conjugate, and the desired hybridoma secreting MAb against PU was cloned by the limiting dilution method after screening by an icELISA. Ascites was purified by a protein G FF column. Finally, the anti-PU MAb, designated AA9, was obtained for further experiments. Competitive inhibition between MAb and PU–PLL by various concentrations of PU was investigated using a PU calibration curve in the icELISA. Under these conditions, a linear relationship between the optical density and doses ranging from 10 ng/ml to 1 ␮g/ml (R2 = 0.997) was achieved with a detection limit of 181.3 ng/ml (Fig. 3). 3.3. Cross-reactivities of anti-PU MAb(AA9) and assay To analyse specificity, the cross-reactivities of this MAb AA9 compared to compounds structurally related to PU and other natural products were evaluated. Table 2 shows the cross-reactivity of MAb AA9 examined by the indirect competitive ELISA and the half maximal inhibitory concentrations (IC50 ) of PU and other chemicals, which were determined according to the method reported by Weiler et al. [30]. MAb AA9 exhibited no cross-reactivity with any Table 1 The UV visible spectra of PU, BSA, and PU–BSA. Compound

Concentration (␮g/ml)

A254 nm a

A281 nm a

PU BSA PU–BSA

25 1000

1.6698 0.2804 1.4962

0.8790 0.6128 1.0590

a The absorbance of PU, BSA, and PU–BSA at 254 nm and 281 nm, which is the maximum absorption wavelength of PU and BSA.

Fig. 3. Standard curve of inhibition by PU using MAb AA9 in an indirect competitive ELISA.

of the other flavonoids, except for a 58.1% reactivity to baicalein. The reason that MAb AA9 cross-reacted with baicalein is unclear and requires further study. 3.4. Accuracy and variation The accuracy and variation of the assay were evaluated by relative standard deviations (RSDs) of intra- (well to well) and inter(plate to plate and day to day) assays performed with the icELISA. The RSDs were <3.5% for the intra-assay results and <7.5% for the Table 2 Cross-reactivities of the MAb AA9 against various compounds. Compound

Cross-reactivity (%)a

Puerarin Baicalein Baicalin Rutin Hesperidin Amygdalin Hyperin Paeoniflorin Geniposide Naringin Gastrodin Saikosaponin a Saikosaponin d Ginsenoside Rb1 Ginsenoside Re Notoginsenoside R1 Rhein Ferulic acid Cholic acid Salvianolic acid Glycyrrhetic acid Glycyrrhizic acid Berberine

100 58.1 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

a The cross-reactivities were determined according to Weiler’s equation in Ref. [32].

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Table 3 The accuracy and variation of the assay. PU (ng/ml) 20 40 80 160 320

Concentrationa (ng/ml) 20.3 ± 0.5 40.1 ± 2.3 81.4 ± 0.7 161.6 ± 4.8 322.8 ± 8.1

Table 5 Content of PU in various Chinese patent medicines determined by ELISA and HPLCa . CVb (%) Intra-assay

Inter-assay

2.5 3.4 0.8 2.9 2.5

5.8 5.5 5.5 5.5 7.4

a The accuracy was evaluated by determination of five concentrations of PU. Three replicate wells of each were executed and indicated as mean ± SD. b The variation of the assay was described by the CV (%) of the intra- and interassay results. All measured values are presented as mean ± SD for three replicate wells of each concentration in three plates and in three consecutive days. The variations from well to well and plate to plate are indicated as the CV of the intra- and inter-assay, respectively.

Table 4 The recovery rate of the assay. Added amount (␮g)

Mean valuea (␮g)

0 10 40 160 640

100.3 ± 0.5 110.6 ± 2.3 136.1 ± 2.7 268.6 ± 9.3 767.7 ± 8.1

Recoveryb (%) 105.5 90.3 105.4 104.3 Average 101.4

All data are presented as mean ± SD from triplicate wells of each sample. The zero level was used as a control, and the recovery rate was calculated as follows: recovery (%) = (measured amount − control)/added amount × 100%. a

b

inter-assay results. These results indicated that the assay was accurate and stable (Table 3). 3.5. Recovery rate The recovery experiment was performed to evaluate the reliability of the assay. The recovery rate of each sample of puerariae radix containing 1 mg of PU and various amounts of precisely added PU were calculated by the icELISA. As shown in Table 4, the average recovery rate was 101.4% ± 7% (mean ± SD, n = 3) for 10 to 640 ␮g of spiked PU. Based on its accuracy and consistency, this assay was sufficiently reliable for the determination of PU in various samples. 3.6. Correlation between HPLC and icELISA analysis of PU in radix puerariae and a Chinese herbal compound using MAb AA9 After establishing the PU quantification ELISA method, we measured the concentrations of PU in radix puerariae and Chinese medicines with icELISA and compared the results with those determined by HPLC. The correlation co-efficiency between the two methods was linear, as shown in Fig. 4. As shown in Table 5, the

Fig. 4. Correlation between the PU content determined by ELISA and HPLC.

Chinese patent medicine

Content (w/w, mg/g) ELISA

HPLC

Radix puerariae SANJIU Ganmaoqingre granule Ganmaozhike granule Jingfukang tablet Jingtong tablet Xinkeshu tablet Yufengningxin tablet Ganmaoqingre granule

10.1 ± 0.2 1.12 ± 0.04 0.21 ± 0.02 0.43 ± 0.07 0.94 ± 0.02 1.19 ± 0.03 0.61 ± 0.05 0.15 ± 0.03

10.05 ± 0.11 1.14 ± 0.03 0.24 ± 0.02 0.45 ± 0.05 0.96 ± 0.04 1.21 ± 0.04 0.58 ± 0.03 0.14 ± 0.02

a

All data are presented as mean ± SD from triplicate well analysis of each sample.

concentrations of PU in several Chinese medicines, which was measured using our developed icELISA, were highly consistent with those obtained by HPLC from the same samples (R2 = 1). Therefore, we are confident that this icELISA method can be applied to determine PU levels in herbal and biological samples. As the HPLC methods suffer from various limitations, such as a high cost, component degradation, lengthy time, low recovery rates, or complicated pre-treatment, especially for in vivo metabolism research, this icELISA method would be useful and more convenient for product quality control and content determination as well as pharmacokinetic and target studies in the future. 3.7. Measurements of PU concentrations in the hippocampus by icELISA using MAb AA9 Using our developed ELISA method, we determined the concentrations of PU in the hippocampus of golden hamsters during a 24 h period following PU administration. The concentrations of PU in the hippocampus increased rapidly 30 min after PU administration and reached a maximum level (1.61 ␮g/ml) at 60 min. Then, the PU level decreased rapidly from 120 min to 290 min, and the second peak (0.62 ␮g/ml) increased and decreased much more slowly from 300 min to 720 min (Fig. 5). 3.8. Knockout of PU by an immunoaffinity column using anti-PU MAb AA9 PU possesses numerous pharmacological activities, which can be eliminated by knockout of PU from its parent material. In the present study, the results indicated that highly pure PU and the KO-EXT could be obtained by immunoaffinity column chromatography. This method is a novel and important tool for exploring the biological role of PU in medicines containing this compound.

Fig. 5. Dynamic change of the PU concentration in the hippocampus as determined by the immunoassay. The PU concentration in the hippocampus reached the peak around 60 min, and a second peak formed at approximately 420 min. The data are shown as the mean ± SD of four replicates.

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Acknowledgements The authors wish to thank Elsevier WebShop for providing language help and proof reading the article, Huiming Ren for assistance with the immunoaffinity chromatography experiments, and XingKai Jiang for assistance with hapten synthesis for the conjugates. This research was supported by the National Natural Science Foundation of China (81373542, 81274043), Beijing University of Chinese Medicine (The Team of Applied Basic Research of Classical Prescription), Major drug discovery national subject (2010ZX09502-002). Fig. 6. Elution profile of the radix puerariae water extract using the immunoaffinity column and monitored by ELISA. From 0 to 40 ml, the knockout fraction, there is little PU detected, and from 40 to 110 ml, the elute fraction, a rapid peak of PU appeared around 60 ml. This profile of PU content analyzed by the immunoaffinity column was demonstrated to be a good success.

The water extract of radix puerariae was loaded on top of the immunoaffinity column and washed with the washing solvent. A small peak (fractions 0–40 ml) containing overcharged PU and a large peak (fractions 40–110 ml) were determined by ELISA (Fig. 6). The collected fractions from 0 to 40 ml was KO-EXT, while the fractions from 40 to 110 ml contained pure PU. The capacity of the immunoaffinity column for PU (22 ␮g/ml) could give a sufficient yield for further in vitro and in vivo pharmacological analyses. As indicated in this experiment, the antibody was stable when exposed to the washing, eluent, and regenerate solvents after repeated use of more than 50 times under the same conditions. More importantly, the PU could be completely eliminated by the immunoaffinity column conjugated with anti-PU MAb(AA9). Using this method, the exact pharmacological activity of PU in radix puerariae and its potential interaction with other components could be studied. 4. Conclusions To the best of our knowledge, we developed the first MAb against PU and a subsequent ELISA method, as well as a method for purifying and knocking out PU, which provides a simpler, more efficient, and sensitive approach for determining the PU content in drug materials and biological samples. This reagent and assay method also provides useful tools for exploring the pharmacokinetics and targets of PU. Authors’ contributions The manuscript was written via contributions from all of the authors. All of the authors have approved the final version of the manuscript.

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