Received Date: July 13, 2015; Accepted Date: July 24, 2015; Published Date: July 27, 2015
Citation: Qu B, Sai J, Qu H, Liu S, Cheng J, et al. (2015) Preparation of a Monoclonal Antibody Against Saikosaponin C and the Establishment of its Enzyme-Linked Immunosorbent Assay. Biochem Anal Biochem 4:196. doi:10.4172/2161-1009.1000196
Copyright: © 2015 Qu B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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A monoclonal antibody (MAb) 1E11D8 against saikosaponin c (SSc) was prepared by a cell fusion with splenocytes and hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line. The prepared anti-SSc MAb- 1E11D8 has a novel characteristic which shows high specific for saikosaponin c, saikosaponin d and saikosaponin a, three major oleanane-saponins in Radix Bupleuri. By using anti-SSc MAb-1E11D8, a realiable ELISA was developed for the detection of SSc. The system shows a full measuring range from 156.25 ng·mL-1 to 2500 ng·mL-1 in the case of SSc in indirect competitive ELISA (icELISA). The regression equation was y=-0.283 ln(C) +2.3301 with a correlation coefficient of 0.99. Precision and accuracy of the icELISA method were evaluated by the variations between replicates from well to well (intra-assay) and plate to plate (inter-assay). The values obtained for these parameters were within the normal range (<10%). The recovery rates ranged from 99.82 to 103.59%. The ELISA method was further validated to be of use for surveying SSc in traditional Chinese prescriptions.
Saikosaponins; Saikosaponin c; Monoclonal antibody; Immunoassays; Enzyme-linked immunosorbent assay
Immunoassays are preferable in the field of clinical analysis for viral proteins for years . Recently, various enzyme-linked immuosorbent assays (ELISA) for small molecules have been developed for both quantitative and qualitative analysis [2-5] due to the merit such as high throughput, simplicity, high reproducibility and sensativity. In our outgoing study, we have developed the preparation of MAbs against baicalin, puerarin, geniposide, hyodeoxycholic acid, Glycyrrhizic acid, paeoniflorin, ginsensoide Re, ginsenoside Rh1 and established their ELISA method [6-10]. Consequently, we applied the assays to study the pharmacokinetics and pharmacokinetic interactions between these bioactive compounds. The comparably low quantities of specimen required such as little as 5 μL of serum for determination in mice is really a merit for pharmacokinetic study. The ELISA method we developed is proved to be more suitable for quality control and pharmacokinetic study on traditional Chinese medicine compared with high-performance liquid chromatographic (HPLC) analyses since it can detect samples with little pretreatment.
Saikosaponin c (SSc) is one of the active ingredients isolated from Radix bupleuri, a traditional Chinese medicine which plays an important role in human disease prevention and treatment. Recent study has demonstrated that SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction . Until now, several HPLC analyses have been developed for the qualitative ananlysis of SSc . However, because SSc do not exhibit any absorption maximum in the ultraviolet (UV) region above 220 nm, it was usually analyzed below 210 nm and therefore it could not be easily detected by HPLC . The disadvantages of these methods continue to be high cost and limited availability. In view of these limitations, we sought to develop a more practical method for SSc based on our previous studies on the development of immunochemical techniques using monoclonal antibodies (MAbs) against small molecular weight compounds (Figure 1).
In this study, we developed a novel analysis method for determining SSc by using immunological technique. Monoclonal antibodies (MAbs) against SSc were prepared and used in enzyme-linked immunosorbent assay (ELISA) for analysis of SSc. This is the first report for determining SSc by ELISA system. Preparation of MAbs against SSc and its application to ELISA system are demonstrated in this paper.
Reagents and chemicals
The 96-well immunoplates were purchased from Corning Incorporated. Bovine serum albumin (BSA), ovalbumin (OVA), medium were obtained from Sigma-Aldrich Co., Ltd. Saikosaponin d, saikosapoin a, saikosapoin b1, saikosapoin b2, saikosaponin c and all other compounds were purchased from Chengdu Pufei De Biotech Co., Ltd and all had a purity of 98%. TMB was supplied by Sigma Co., Ltd. Peroxidase-labeled anti-mouse immunoglobulin G (IgG) was provided by Organon Teknika Cappel Products. Chinese medicines used in this paper were obtained from Beijing Tong Ren Tang Group Co., Ltd. (Beijing, China). All other chemical reagents of analytical grade were obtained from Sinopharm Chemical Reagents Beijing Co., Ltd.
An electronic balance (BS124-S) was procured from Sartorius AG. A spectrophotometric microtiter reader (for absorbance measurements) was obtained from Thermo Fisher Scientific (Multiskan MK3). Deionized water was prepared using an ultra-class ultraviolet (UV) plus water purification system (SG Co., Ltd.). Immunoreactions were carried out in an electro-heated standing-temperature cultivator (DRP-9082) purchased from Sumsung Experiment Instrument Co., Ltd.
The compositions of the buffers and solutions used in this study were as follows: phosphate-buffered saline (PBS)–NaCl (137 mmol·mL-1), Na2HPO4·12H2O (10 mmol·mL-1), KCl (2.68 mmol·mL-1), and KH2PO4 (1.47 mmol·mL-1), pH 7.4; carbonate-buffered saline–Na2CO3 (15 mmol·mL-1), NaHCO3 (35 mmol·mL-1), pH 9.6; washing buffer, PBS with 0.05% Tween-20 (PBS-T); blocking buffer, 1% gelatin in deionized water; TMB substrate solution, combination of Part A (10 mL; 24.3 mL of 0.1 mol·mL-1 citric acid, 25.7 mL of 0.2 mol·mL-1 Na2HPO4, and 50 mL of deionized water), Part B (0.5 mL; 2 mol·mL-1 of TMB in ethanol), and Part C (32 μL of 0.75% H2O2); stopping solution, 2 mol·mL-1 H2SO4.
Preparation of anti-SSc MAb
Saikosaponin c-bovine serum albumin conjugates (SSc–BSA) were synthesized using a periodate oxidation procedure based on a previously reported method with some modifications [14,15]. Briefly, SSc was dissolved in methanol at 10 mg·mL-1. Then, 1 mL of a freshly prepared odium periodate (0.1 M) was dropped into 1 mL of SSc solution. After stirring at 25°C for 1 h, 50 mM carbonate buffer solution (pH 9.6, 1 mL) containing BSA (10 mg) was added to the reaction mixture. pH was adjusted to 10 using 1 M Na2CO3 solution and the mixture was stirred at 25°C for 5 h. Then the mixture was dialyzed 6 times against CBS for 72 h. The SSc-OVA conjugate was synthesized using the same method as above.
BALB/c female mice (6 weeks of age) were immunized with the SSc–BSA conjugate. Joint Ethical Review Committee of the Beijing University of Chinese Medicine has approved our ethics application for animals. The first immunization (50 μg conjugate per injection) consisted of a subcutaneous multipoint injection in the back using a 1:1 emulsion of the conjugate to Freund’s complete adjuvant. The subsequent immunizations (50 μg conjugate per injection) were administered as a 1:1 emulsion in Freund’s incomplete adjuvant and performed at 2 week intervals. At the fourth and final immunization, a booster dose of 100 μg of conjugate dissolved in PBS without adjuvant was administered intraperitoneally. On the third day after the final immunization, splenocytes were isolated and fused with a hypoxanthine-aminopterinthymidine (HAT)-sensitive mouse myeloma cell line (Sp2/0-Ag14) by the polyethylene glycol (PEG) method. Hybridomas secreting MAbs that were reactive to SSc were cloned by the limited dilution method. Established hybridomas were cultured in hypoxanthine-thymidine (HT) medium.
Individual anti-SSc MAb was purified using a protein G FF column (0.46 × 11 cm; GE Healthcare, USA) . The ascites (50 mL) containing MAb was adjusted to pH 7 with 1 M Tris solution and loaded onto the column. The column was then washed with 20 mM phosphate buffer (pH 7.2) and the adsorbed antibody was eluted with 0.1 M of glycine buffer (pH 2.7). The eluted antibody was neutralized with 1 M Tris solution, dialyzed against 50× volumes of water for five changes at 4°C, and then lyophilized.
Determination of the MAb isotypes was carried out according to the instructions of the Mouse Monoclonal Antibody Isotype Kit. A sandwich ELISA method was applied to identify the subclasses of monoclonal antibodies against SSc.
The reactivity of anti-SSc MAb to SSc-OVA was determined by indirect ELISA (iELISA). SSc-OVA (100 μL/well) diluted in 50 mM carbonate buffer (pH 9.6) was added into the wells of a 96-well immunoplate and incubated for 1 h. This was then treated with 200 μL of blocking buffer (phosphate-buffered saline containing 5% skim milk, SPBS) for 2 h to eliminate non-specific adsorption. The plate was washed three times with washing buffer (phosphate-buffered saline containing 0.05% Tween 20, PBST) and filled with 100 μL of MAb solution at various dilutions in different rows. After 1 h, the plate was washed three times with PBST and incubated with 100 μL of a 10,000- fold diluted peroxidase-labeled goat anti-mouse IgG solution for 0.5 h, followed by another wash with PBST for three times. Then 100 μL of the substrate solution (0.1 mM citrate buffer containing 0.75% H2O2 and 2 mg/mL 3,3’,5,5’-tetramethylbenzi-dine (TMB)) was added to each well, and the plate was incubated for 15 min. The reaction was then terminated by adding 50 μL of 2 M sulfuric acid to each well. All reactions were carried out at 37°C. The absorbance was determined at 450 nm using a Biotek.
For indirect competitive ELISA (icELISA), SSc-OVA (1:10,000, 100 μL/well) was adsorbed in the wells of a 96-well immunoplate, which was then treated with 200 μL of 5% SPBS for 2 h to reduce non-specific adsorption. The plate was washed three times with PBST, and 50 μL of various concentrations of SSc solution were incubated with 50 μL of MAb solution for 1 h. The subsequent process was the same as that described for iELISA.
Validation of the icELISA Method
icELISA was used with SSc-OVA as the solid phased antigen and IgG solution. A range of SSc amounts was separately added to compete with the coated antigen. From this competition assay, a standard curve of inhibition for the measured range was created. Furthermore, the cross-reactivities (CRs, %) of SSc with structurally related compounds were determined according to Weiler and Zenk’s equation .
Several concentrations of SSc were measured with the developed icELISA method for six times in a day to determine the accuracy and variation of intra-assay (well to well), and for six consecutive days to determine that of inter-assay (plate to plate and day to day), which were all expresses by RSDs (%). Various concentrations of SSc were added for icELISA to calculate aassay recovery rate. For each level, six samples were analyzed, and the concentrations of SSc was determined by the icELISA.
An Agilent series 1260 HPLC instrument equipped with a quaternary pump, a diode-array detector, an autosampler and a column compartment was used for HPLC analyses. The sample was separated on a phenomenex luna 5u C18(2) 100A column (5 μm, 4.60×250 mm, phenomenex, USA), by isocratic elution with the mobile phase composed of water and CH3CN (43/57, V/V) at a flow rate of 1.0 mL·min-1, and analyzed by diode array detector with an excitation and emission wavelength of 203 nm. The chromatogram was recorded at 203 nm and the spectral data for all peaks were obtained in the range of 190–400 nm. The column temperature was kept constantly at 25°C.
All TCMs in a prescription were weighed in propotion, powdered and mixed together, and then 500 mg of the powder were extracted with MeOH (50 mL) under sonication five times, filtered using the Cosmonice filter W (pore size, 0.45-μm), and evaporated. The residues were redissolved in 5 mL of MeOH, and then the final sample solutions were diluted with deionized water properly when analyzed by icELISA.
Hyper-immunized BALB/c mice used to derive the cell clones described in this paper yielded splenocytes that were fused with a HAT-sensitive SP2/0 myeloma cell line according to the protocols established in this laboratory. A hybridroma-producing MAb reactive to SSc was obtained and named MAb-1E11D8. BALB/c male mice were intraperitoneally injected with 1E11D8 to induce ascites to obtain anti-SSc MAb. A sandwich ELISA method was employed to identify the subtype of the monoclonal antibody against SSc using the MAb isotyping kit. The data shows that the anti-SSc MAb-1E11D8 having lanmuda light chains was classified into the IgG1 subclass antibody group.
Sensitivity were analyzed by icELISA method. The 96-well immunoplate was coated with 1μg·mL-1 SSc-OVA and the optimal concentration of anti-SSc MAb was screened by iELISA. Various amounts of SSc were added to compete with the coated antigen from which the standard curve of inhibition was created. Figure 2 shows the competitive inhibition between the MAb and SSc-OVA produced by various concentrations of SSc by icELISA. Under this condition, a linear relationship between optical density and doses in the range of 156.25-2500 ng·mL-1 (R2=0.99) could be achieved with a half-maximal inhibitory concentration of 625 ng·mL-1.
Figure 2: Calibration curve of saikosaponin c. Various concentrations of saikosaponin c were incubated with monoclonal antibodies in wells pre-coated with saikosaponin c-OVA (1 μg·mL-1). After washing with PBS-T, the wells were again incubated with peroxidase-labeled anti-mouse immunoglobulin G and absorbance was measured at 450 nm.
Assay specificities were examined by icELISA with various related compounds, and the cross-reactivity rates (CRs) were calculated following Weiler and Zenk’s method . As shown in Table 1, anti- SSc MAb-1E11D8 reacted with SSa, and SSd at rates of 31.25% and 126.25%, respectively, but has no reactivity with other similar saponins and flavonoids. The higher reactivity to SSc, SSd, and SSa indicates that the SSc MAb can be used to determine SSc, SSa and SSd, which are major constituents of Radix bupleuri, thus it can be applied into quality control of Radix bupleuri.
|Salvianolic acid A|
The cross-reactivities were determined according to Weiler’s equation.
Table 1: Cross-reactivities (%) of MAb-1E11D8 against various compounds.
Table 2 shows the results of assay accuracy and variation. As expected, the intra-assay variations were lower than the inter-assay variations which may be due to variation of reagent preparation. The intra-assay RSDs were <3.0%, while the inter-assay RSDs were <8.9%, which indicate that this assay is very accurate and stable. As to assay recovery rate, SSc recoveries were 99.82–103.59% (mean, 101.71%) (Table 3).
|Concentration (ng·mL-1)||RSD (%)||Concentration(ng·mL-1)||RSD (%)|
|156.25||165.30 ± 12.38||7.49||147.75 ± 10.50||7.10|
|312.5||320.45 ± 19.56||6.10||325.08 ± 10.52||3.24|
|625||621.46 ± 3.54||4.99||601.01± 23.56||7.25|
|1250||1261.76 ± 9.75||0.77||1230.86 ± 19.14||9.09|
aAccuracy was evaluated by the determination of five concentrations of saikosaponin c. Six replicate wells of each were executed and indicated as mean ± SD. bAssay variation was described by RSD (%) of intra- and inter-assays. All measured values are mean ± SD for six replicate wells each.
Table 2: Assay accuracya and variationb
|Added amount (ng·mL-1)||Mean valuea (ng·mL-1)||Recoveryb (%)|
|200||204.06 ± 4.06||102.02|
|400||410.06 ± 5.12||102.51|
|600||601.63 ± 3.01||103.59|
|1200||1206.85 ± 6.03||100.60|
|2400||2395.66 ± 0.48||99.82|
aAll data are mean ± SD from triplicate wells of each sample. bThe zero level was used as control and the recovery rate was calculated as follows: recovery (%) = measured amount/added amount × 100%.
Table 3: Assay recovery rate.
In order to confirm the correlation SSc determination between ELISA and HPLC, correlation coefficient was calculated by fitting a straight line from analyses by the ELISA and HPLC methods. Table 4 shows the correlation of SSc concentration using HPLC and ELISA indicating good correlation. We decided the newly established ELISA can be used for the determination of SSc concentration without any pretreatment.
|content of SSc (μg·mL-1)|
|1||410.06 ± 5.12||1.25||383.09 ± 12.23||3.19|
|2||306 ± 4.90||1.60||315.08 ± 11.03||3.50|
|3||204.06 ± 4.06||1.99||189.55 ± 7.78||4.10|
Concentration of SSc determined by ELISA. All data were mean ± SD from six wells analysis of each sample.
Table 4: Contents of ssd determined by ELISA with anti-SSc MAb and HPLC
Since the ELISA assay was relatively quick, convenient, and economic compared with HPLC, we used it to determine the concentration of SSc in six prescriptions (Sini san, Xiao chai hu tang, Da cahi hu tang, Chai hu gui zhi tang, Bu zhong yi qi tang, and Huang lian jie du tang as shown in Table 5 with various Radix buplerui composition ratios without pretreatment. From these results, it is apparent that this ELISA method can be applied to determine SSc in medicine, which will be very useful and more convenient in product quality control and quantification in the future.
|Traditional Chinese prescriptions||Percentage of Bupleuri radix||Composition of Kampo medicine||Concentration of SSc (μg/g dwt.)|
|xiao-yao-san||1/55||Bupleuri radix 1g, Paeoniae radix alba 12g,Angelica sinensis 12g,Poria 12g Atractylodes 6g, Glycyrrhizae radix 6g||76.82 ± 17.94|
|Da-chai-hu-tang||1/7||Bupleuri radix 12g,Scutellariae radix 9g,Paeoniae radix alba 9g,Pinelliaerhizoma 9g, Aurantiifructusimmaturus 9g, Zingiberisrhizomarecens 15g, Jujubaefructus 15g, Rhei radix et rhizoma 6g.||83.54 ± 7.86|
|Si-ni-san||1/4||Bupleuri radix 6g, Paeoniae radix alba 6g, Aurantiifructusimmaturus 6g, Glycyrrhizae radix 6g||26.71 ± 5.01|
|Bu-zhong-yi-qi-tang||4/33||Astragali radix 16g, Ginseng radix 15g, Atractylodismacrocephalaerhizoma 10g, Glycyrrhizae radix 15g, Cimicifugaerhizoma 6g, Bupleuri radix 12g, Angelicaesinensis radix 10g, Citrireticulataepericarpium 6g, Zingiberisrhizomarecens 5g, Jujubaefructus 5g||34.20 ± 4.82|
|Huang-lian-jie-du-tang||0||Scutellariae radix 6g,Coptidis rhizome 9g, Phellodendrichinensiscoptex 6g, Gardeniaefructus 9g||NDb|
All data are mean ± SD from three wells analysis of each sample. NDb = not detectable.
Table 5: Total concentration of saikosaponin c (SSc) in various traditional Chinese medicines determined by enzyme-linked immunosorbent assaya.
This work was supported by the National Natural Science Foundation of China (81274043, 81373542, 81473338, 81430102) and the Classical Prescription Basic Research Team of the Beijing University of Chinese Medicine.The authors declare no conflicts of interest.