Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Ultrasensitive Time-Resolved Fluoroimmunoassay for Saikosaponin a in Chaihu (Bupleuri Radix)

  • Zhi Chao , (ZC); (YW)

    Affiliation School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China

  • Qian Cui,

    Affiliation School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China

  • Enwei Tian,

    Affiliation School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China

  • Weiping Zeng,

    Affiliation School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China

  • Xuan Cai,

    Affiliation School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China

  • Xiaolei Li,

    Affiliation School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China

  • Hiroyuki Tanaka,

    Affiliation Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

  • Yukihiro Shoyama,

    Affiliation Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo 859–3298, Japan

  • Yingsong Wu (ZC); (YW)

    Affiliation School of Biotechnology, Southern Medical University, Guangzhou 510515, China

Ultrasensitive Time-Resolved Fluoroimmunoassay for Saikosaponin a in Chaihu (Bupleuri Radix)

  • Zhi Chao, 
  • Qian Cui, 
  • Enwei Tian, 
  • Weiping Zeng, 
  • Xuan Cai, 
  • Xiaolei Li, 
  • Hiroyuki Tanaka, 
  • Yukihiro Shoyama, 
  • Yingsong Wu


The aim of this study is to establish a time-resolved fluoroimmunoassay (TRFIA) system for quantitative analysis of saikosaponin a (SSa) in the crude drug of Chaihu (Bupleuri Radix). A 96-well microplate coated with rabbit anti-mouse IgG was incubated with the methanol extracts of Chaihu samples and a mouse anti-SSa monoclonal antibody, and a Eu3+-labeled SSa-human serum albumin conjugate was used as the tracer. The established competitive TRFIA showed a good fourth order polynomial fitting from 0.01 to 10.0 μg/mL for standard SSa sample with a detection limit of 0.006 μg/mL. The intra- and inter-assay coefficients of variation of the assay were 7.3% and 8.9%, respectively, and the average SSa recovery was 119.2%. For samples of Chaihu extract, the results of this assay showed a good correlation with those by enzyme-linked immunosorbent assay established previously. This TRFIA system is ultrasensitive for detecting SSa with a wide detection range and a good stability and represents the first attempt of using TRFIA for quality evaluation of the crude drug of Chaihu.


Chaihu (Bupleuri Radix), a common traditional Chinese medicinal herb derived from the dried roots of Bupleurum chinense DC. or B. scorzonerifolium Willd., has been used for medical purposes in China for more than 2000 years. According to ancient Chinese medical literatures, Chaihu is capable of regulating the exterior and interior metabolisms, dispersing evil heat from the superficies, soothing the liver, and promoting yang and qi (representing “life energy” or “life force” in Traditional Chinese Medicine theories) [1, 2]. Among the complex constituents, saikosaponins, which have a typical oleanan-type skeleton as the aglycon, have been identified by modern techniques as the major biological active constituents in Chihu. Of these saikosaponins, saikosaponin a (SSa) (Fig 1), a major saponin, has been shown to possess versatile bioactivities to suppress inflammation [3] and oxidation, protect liver function [4], induce tumor cell apoptosis, inhibit carcinogenesis [59], and induce cell differentiation [10]; research evidence also demonstrated its activities in immunomodulation [11], promoting corticosterone secretion [12], and lowering plasma cholesterol [13]. Because of the uneven quality of Chaihu in the market [2], quantification of SSa is critical to ensure the effectiveness of the crude drug [1], thus an accurate, sensitive, and convenient method for determination of SSa in Chaihu is essential.

Several techniques have been developed for analyzing SSa in Chaihu and various Chaihu products, including thin-layer chromatograph scanning (TLCS) [14], high-performance liquid chromatography (HPLC) [1519], HPLC coupled with evaporative light scattering detector (ELSD) [20], HPLC coupled with mass spectrometry [21], ultraperformance liquid chromatography (UPLC) [22], UPLC coupled with mass spectrometry [23, 24], and capillary electrochromatography [25]. Chromatography-based analytical techniques are the most frequently used modality for quantitative and/or qualitative analysis of SSa. But because SSa has a rather short maximum absorption wavelength (205 nm), interference easily occurs in SSa detection using ultraviolet or diode array detector (DAD) to lower the detection sensitivity. In addition, this strategy also requires sophisticated equipment (eg, a mass spectrometer), complicated sample pretreatment, and the use of toxic organic solvents in the mobile phase.

Immunological approaches provide valuable alternatives for SSa analysis. The first attempt of immunologically based SSa detection was made by Jung et al., who used anti-SSa polyclonal antibody for quantitative analysis of SSa [26]. But as polyclonal antibodies recognize non-specific antigenic determinants and also cross-react with the non-target proteins, the specificity of the assay was considerably lowered. In a more recent study, an enzyme-linked immunosorbent assay (ELISA) system was established using an anti-SSa monoclonal antibody (MAb) [27]. Due to the high specificity of the monoclonal antibody, this assay was successfully applied in quality inspection of commercial Chaihu crude drugs [28].

Different from the approaches described above, time-resolved fluoroimmunoassay (TRFIA) represent an ultrasensitive technique using lanthanide elements and their chelates as the tracer with unique fluorescence properties. This technique is commonly used in laboratory medicine, agriculture, food industry, environmental health study, and forensic science [2932]. But currently few studies have been reported to describe its application in crude drug analysis. In this study, we aimed to establish a TRFIA-based system for SSa detection for providing an accurate and more sensitive, less time-consuming method for quality control of Chaihu.

Materials and Methods


Standard SSa sample was purchased from National Institutes for Food and Drug Control, China. Bovine serum albumin (BSA) and human serum albumin (HSA) was obtained from Sigma-Aldrich (St. Louis, USA). Rabbit anti-mouse IgG was purchased from Rockland Immunochemicals (Limerick, USA). A previously prepared murine monoclonal antibody 1G6 against SSa was used [27], which was a highly specific IgG with a light chain of κ-type and showed low cross-reactions with saikosaponin c, d and b2 (at rates of 2.65%, 3.76%, and <0.25%, respectively). The crude drug samples of Chaihu were purchased from the local pharmacies. Based on the macroscopic characteristics and the description in Chinese Pharmacopoeia, the samples were identified by the author (ZC) as the roots of B. chinense. Samples No.4 and No.5 were of poor quality because of the presence of the stem remnants to suggest apparent adulteration. The other chemicals used were routine reagents of the analytical grade.

Synthesis of Antigen-Carrier Protein Conjugate

SSa-HSA conjugate was synthesized using a model based on the periodate oxidation method. Briefly, the standard sample of SSa (3.2 mg) was dissolved in 0.5 mL of methanol (MeOH), in which a sodium periodate (10.0 mg)/H2O (1.0 mL) solution was added dropwise. The mixture was stirred at room temperature for 1 h to allow the oxidative reaction, followed by the addition of HSA (5.0 mg) in 50 mM carbonate buffer (1.0 mL; pH 9.6) and further stirring at room temperature for 12 h. The reaction mixture was dialyzed against H2O at 4°C for 2 days and then lyophilized to obtain SSa-HSA conjugate.

Eu3+ Labeling of Antigen-Carrier Protein Conjugate

The synthesized SSa-HSA conjugate was dissolved in the labeling buffer containing 50 mM Na2CO3-NaHCO3 (pH 8.5) and 155 mM NaCl and gently mixed with Eu3+-DTTA (isothiocyannatobenzyl diethylenetriaminetetraacetic acid) dissolved in the same buffer. The mixture was vortexed gently at room temperature for 16–20 h before being loaded onto a sepharose CL-6B column (Pharmacia Company, Chalfont St Giles, UK) for purification. The column was eluted with the elution buffer (containing 50mM Tris-HCl, pH7.8, 0.9% NaCl and 0.05% NaN3), and the fractions from the first peak with the highest Eu3+ count were pooled. BSA solution was then added at a final concentration of 0.2% into the pooled fractions. Finally, the Eu3+-labeled SSa-HSA conjugate was aliquoted and stored at 4°C until use.

Competitive TRFIA

Fig 2 shows the schematic diagram of the TRFIA system for SSa detection. The 96-well immunoplate (Nunc, Roskilde, Denmark) was coated with 100 μL of rabbit anti-mouse IgG (4 μg/mL) dissolved in 50 mM carbonate buffer (pH 9.6) at 4°C overnight, and then blocked with 300 μL of the blocking buffer (50 mM Tris-HCl, 0.9% NaCl, 0.1% BSA and 0.05% NaN3, pH 7.8) to reduce the nonspecific reactions. In each well, 25 μL of standard SSa samples at different concentrations or test samples dissolved in 10% MeOH solution were added and incubated for 1 h with 100 μL of anti-SSa MAb solution (1:4000) and 100 μL of Eu3+-labeled SSa-HSA conjugate (1:800). After three washings with the washing buffer (50 mM Tris-HCl, 0.9% NaCl, 0.1% BSA, 0.05% Tween 20 and 0.05% NaN3, pH 7.8), 200 μL of the enhancement solution (100 mM acetate-phthalate buffer, pH 3.2, containing 0.1% Triton X-100, 15 μM β-naphthoyltrifluoroacetate, and 50 μM tri-n-octylphosphine oxide) was added to each well and incubated for 5 min. The fluorescence intensity was measured on a Victor3 1420 Multilabel Counter (PerkinElmer, Waltham, USA) equipped with filters for Eu3+ (613 nm). The excitation wavelength was 340 nm and the emission wavelength was 613 nm with a delay time of 0.40 ms, window time of 0.40 ms, and cycling time of 1.0 ms. Curve fitting and calculation of the concentrations in the samples were performed using Multicalc software (PerkinElmer, Waltham, USA), where a spline algorithm on logarithmically transformed data was employed.

Fig 2. Schematic diagram of the TRFIA system for SSa detection.

Rabbit anti-mouse IgG (“Y” shape in grey) was coated onto the plate; SSa (yellow oval dot), anti-SSa MAb (“Y” shape in green), and Eu3+ (red dot) -labeled SSa-HSA (purple oval dot) conjugate were mixed and incubated; after washing off of the extra reactants, the enhancement solution (blue drip) was added and the fluorescence intensity was measured.

Preparation of Test Samples and Standard Solutions

All the commercial Chaihu samples were prepared into dried powder using a steel grinder. The dried Chaihu powder (20 mg) was extracted with 0.5 mL of MeOH containing 5% NH4OH with sonication for 10 min, and the extract was centrifuged at 9000 g for 5 min to collect the supernatant. For each sample, the extraction was repeated six times. The pooled supernatants from six rounds of extraction were evaporated with N2 gas, and the residue was dissolved in 5.0 mL MeOH and diluted at 1:10 with H2O to obtain the test sample solution.

The standard SSa sample was precisely weighed and dissolved in MeOH to prepare a 5.0 mg/mL stock solution. Gradient concentrations of the stock solution were prepared by serial dilutions with 10% MeOH (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.10, 0.20, 0.40, 0.80, 2.0, 5.0, 10.0 and 20.0 μg/mL) as the SSa standards. The test sample and standard SSa solutions were used for subsequent TRFIA and ELISA analysis.

Calibration Curve

Serially diluted SSa standard solutions at 14 different concentrations (0.001 to 20.0 μg/mL) were used to establish the calibration curve. The detection limit was determined by calculating the minimum detectable amount of standard SSa that could be significantly distinguished from zero (mean binding at zero dose at two times of the SD) [33, 34].

Intra- and Inter-assay Variation

The intra-assay variation was tested by determining the percent coefficient of variations (%CVs) for 3 SSa samples of varying concentrations in 10 duplicate wells across the microtiter plate. The inter-assay variation was determined by evaluating the SSa samples in triplicate plates on 3 consecutive days using the same reagent lots, and the %CVs were calculated.

Recovery Experiments

Various amounts of the standard SSa sample (25, 50, and 100 μg) were spiked into dried Chaihu crude drug powder (20 mg), and the total SSa was extracted for detection following the procedures described previously. For each level, three replicates were analyzed.

Correlation between TRFIA and ELISA Analysis

ELISA of the samples following the previously reported protocols was conducted to validate the results of the established TRFIA [27, 28]. In brief, a 96-well immunoplate was coated with 100 μL of 1 μg/mL SSa-HSA and blocked for 1 h with 300 μL of phosphate buffered saline (PBS) containing 5% skim milk. Fifty microliters of various concentrations of the standard SSa sample or the test samples dissolved in 10% MeOH solution were incubated with 50 μL of anti-SSa MAb solution (1:1000) for 1 h. The plate was washed 3 times with PBS containing 0.05% Tween 20, and then 100 μL of 1: 1000 diluted peroxidase-labeled anti-mouse IgG was added and incubated for 1 h. Finally, 100 μL of the substrate solution [0.1 M citrate buffer (pH 4.0) containing 0.003% H2O2 and 0.3 mg/mL 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)] was added to each well and incubated for 10 min. The absorbance was measured by a microplate reader at 405 nm. The interpolated concentrations of the samples obtained by each method were then compared by correlation analysis.

Data Analysis

The data were analyzed using the Statistical Package for Social Sciences (SPSS, version 19.0). For correlation studies, Spearman correlation coefficient was employed and the level of significance was set to alpha = 0.01. Paired signed rank test was used to determine the statistical significance between the results by TRFIA and ELISA (alpha = 0.05).


Assay Sensitivity

The dilution of Eu3+-labeled SSa-HSA conjugate that emitted 75% of the full fluorescence intensity (saturate solid phase fluorescence) was chosen as the working titer. A dose-response curve was obtained by averaging 10 individual curves normalized by the reporting fluorescence values (given as %B/B0, where B was the mean fluorescence intensity for each standard and B0 the mean fluorescence intensity for a zero SSa concentration). The calibration curve (Fig 3, S1 File) derived by processing the data of SSa concentration and the fluorescence intensity with log-logit function derived from fourth order polynomial fitting (logit Y = ln [Y/(1-Y)], Y = B/B0) [34] showed a detection range of 0.01–10.0 μg/mL for SSa detection. The detection limit calculated from 3 curves, each prepared in duplicate, was 0.006 μg/mL. Compared with the conventional ELISA, which had a detection range of SSa from 0.026 to 1.5 μg/mL [27] (0.16–2.5 μg/ml in another case [28]), the TRFIA system showed a sensitivity about 3–15 times greater and a much wider detection range.

Fig 3. Standard curve of SSa content detected by TRFIA.

The main body represents the dose-response curve between SSa concentrations and the reporting fluorescence values (B/B0), whereas the inserted smaller figure demonstrates the calibration curve acquired after log-logit treatment, showing a good fourth order polynomial fit. Each point represents the mean±SD of 10 assays in duplicate.

Assay Variation

The intra-assay (well-to-well) and inter-assay (plate-to-plate) variations were determined to assess the reproducibility and precision of this assay. The intra- and inter-assay maximum relative standard deviations (RSDs) were 7.9% and 11%, respectively (Table 1).

Recovery of SSa by Competitive TRFIA in Spiked Samples

Chaihu crude drug sample No.2 was randomly selected for the recovery experiment. Various amounts of standard SSa sample (25, 50, 100 μg) were added to 20.0 mg dried crude drug powder (containing 116.9 μg SSa after calculation), and the total amount of SSa was measured by competitive TRFIA. The recovery of spiked SSa was calculated as follows:

The recovery rates were between 108.9% and 126.9% with reasonable RSD values (9.8% − 14%), as shown in Table 2. The average recovery rate was 119.2%.

SSa Contents in Chaihu Crude Drug Samples Determined by TRFIA

Table 3 shows the results of quantitative analysis of SSa contents in the crude drug samples of Chaihu purchased from the local pharmacies. In the 10 commercial Chaihu samples, the measured SSa contents in the methanol extract ranged from 0.27 to 8.77 μg/mg, and 2 samples showed SSa contents lower than the minimum requirement documented in the Chinese Pharmacopeia.

Correlation between TRFIA and ELISA for Detecting SSa Contents in Chaihu Crude Drug Samples

For crude drug samples, SSa contents determined by TRFIA were similar to those by ELISA (Table 3, S1 File). The Spearman correlation coefficient (R) between the results by TRFIA and ELISA was 0.903 (P<0.01, Fig 4, S1 File). Paired signed rank test showed no significant difference between the SSa contents measured by the two methods (P = 0.153).

Fig 4. Correlation of SSa contents in Chaihu crude drug samples determined by TEFIA and ELISA.

SSa contents in Chaihu crude drug samples determined by TRFIA (Y-axis) were well correlated with those determined by ELISA (X-axis), with a Spearman correlation coefficient (R) of 0.903 (P < 0.01).


Chromatography-based approaches represent the current mainstream for saikosaponin analysis. As the maximum absorbance wavelength of SSa is 205 nm (near the end of the ultraviolet spectrum), ultraviolet- or DAD-based detection of SSa has a low sensitivity and requires a high purity of the mobile phase, complicated sample pretreatment, and sophisticated equipment such as an evaporative light-scattering detector and even a mass spectrometer. The immunological approaches are free of such limitations of chromatographical methods and allows more convenient assay of biomedical samples. In fact, the previously established ELISA has been shown to allow simultaneous analysis of SSa for a large number of samples [27, 28].

The TRFIA system using anti-SSa MAb we established showed a wide detection range (0.01–10.0 μg/mL) and a low detection limit (0.006 μg/mL), and was about 3–15 times more sensitive than ELISA. This assay was also less time-consuming than ELISA (by at least 1 hour) because of the omission of a secondary antibody. The validation experiments confirmed the sensitivity, reproducibility, and accuracy of the TRFIA system. The maximum intra- and inter-assay variations of this system (7.9% and 11%, respectively) were both <15%, suggesting its sufficient precision and good repeatability for SSa detection [35]. The recovery rates (between 108.9% and 126.9%, 119.2% on average) met the acceptance criteria (80%– 120%), and thus confirmed the accuracy of this assay system [36, 37].

Nevertheless, we noted a higher level of imprecision (higher %CV) of this new assay at higher SSa concentration levels compared with ELISA. Because of the greater sensitivity of the established TRFIA, even small variations of SSa content in the test samples would be greatly amplified, which may lead to a high %CV in detection. We adopted a simplified calibration curve with 5 standards to allow a high-throughput detection of SSa in samples; this, however, may give rise to the limitation of the assay precision and lead to a larger CV. Moreover, the skills in sample manipulation such as pipetting also affect the variation margin, especially for high SSa content samples.

We tested SSa contents in 10 commercial Chaihu samples using the established TRFIA system. The results were similar to those reported previously [28], which confirmed the validity and reliability of the TRFIA system. Samples No. 4 and No. 5 were found to have low SSa contents (below the minimal limit prescribed in Chinese Pharmacopoeia) because of serious adulteration with the aerial parts of the plant that contained no saikosaponins. This result is consistent with our macroscopic inspection of the crude drug before TRFIA determination.

Correlation between TRFIA and ELISA for detecting SSa contents in Chaihu crude drug samples was investigated. Commonly, linear regression is adopted in calibrating the concentration and absorbance in ELISA, but in this case, the data collected for ELISA showed a non-linear curve. We found fifth order polynomial fit model could provide a better fitting.

The correlation coefficient between the results of TRFIA and ELISA was 0.903 (P<0.01), demonstrating a high correlation between them and hence the good reliability of TRFIA. Paired signed rank test also confirmed the comparability between the results by the two methods (P>0.05). These results suggest the value of this TRFIA system for SSa detection in Chaihu crude drugs without any complicated pretreatments.

Although TRFIA has been used for at least 30 years, no reports have been available to describe its use in quantitative analysis of the bioactive constituents in traditional and folk Chinese crude drugs. We for the first time established a TRFIA system for SSa detection, which is the most sensitive method for such purposes so far. Given its high sensitivity, convenience and rapidness in high-throughput analysis, we believe that this technique has great potential in analysis of various Chaihu products and in detection of trace amount of SSa in different samples.

Previously, we developed several MAbs against bioactive constituents like ginsenosides [38], glycyrrizin in licorice [39], paclitaxel in yew trees [40], sennosides in rhubarb [41], and baicalin in Scutellaria [42]. These MAbs potentially allow the establishment of corresponding TRFIA systems for quality control and inspection of traditional Chinese drugs by detecting their bioactive components.

Supporting Information

S1 File. Raw data for curve fitting and SSa quantification.


Author Contributions

Conceived and designed the experiments: ZC YW. Performed the experiments: QC WZ XC XL. Analyzed the data: ZC QC ET. Contributed reagents/materials/analysis tools: HT YS. Wrote the paper: ZC QC YS YW.


  1. 1. National Pharmacopoeia Committee. Pharmacopoeia of the People’s Republic of China. Beijing: Chemical Industry Press; 2010.
  2. 2. Chao Z, Zeng WP, Liao J, Liu L, Liang ZB, Li XL. DNA Barcoding Chinese Medicinal Bupleurum. Phytomedicine 2014; 21: 1767–1773. pmid:25444445
  3. 3. Lu CN, Yuan ZG, Zhang XL, Yan R, Zhao YQ, Liao M, et al. Saikosaponin a and its epimer saikosaponin d exhibit anti-inflammatory activity by suppressing activation of NF-κB signaling pathway. Int Immunopharmacol. 2012; 14: 121–126. pmid:22728095
  4. 4. Wu SJ, Lin LY, Chu CC, Tsai YH, Chao JC. Curcumin or saikosaponin a improves hepatic antioxidant capacity and protects against CCl4-induced liver injury in rats. J Med Food 2008; 11: 224–229. pmid:18598162
  5. 5. Motoo Y, Sawabu N. Antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines. Caner Lett. 1994; 86: 91–95.
  6. 6. Wu WS. ERK signaling pathway is involved in p15INK4b/p16INK4a expression and HepG2 growth inhibition triggered by TPA and saikosaponin a. Oncogene 2003; 22: 955–963. pmid:12592382
  7. 7. Chen JC, Chang NW, Chung JG, Chen KC. Saikosaponin a induces apoptotic mechanism in human breast MDA-MB-231 and MCF-7 cancer cells. Am J Chin Med. 2003; 31: 363–377. pmid:12943168
  8. 8. Sun Y, Cai TT, Zhou XB, Xu Q. Saikosaponin a inhibits the proliferation and activation of T cells through cell cycle arrest and induction of apoptosis. Int Immunopharmacol. 2009; 9: 978–983. pmid:19375524
  9. 9. Kim BM, Hong SH. Sequential caspase-2 and caspase-8 activation is essential for saikosaponin a-induced apoptosis of human colon carcinoma cell lines. Apoptosis 2011; 16: 184–197. pmid:21107704
  10. 10. Tsai YJ, Chen IL, Hong LY, Wu RT. Induction of differention in rat C6 glioma cells with Saikosaponins. Phytother Res. 2002; 16: 117–121. pmid:11933111
  11. 11. Yen MH, Lin CC. The immunomdulatory effect of saikosaponin derivatives and the root extract of Bupleurum kaoi in mice. Phytother Res. 1995; 9: 351–358.
  12. 12. Nose M, Amagaya S, Ogihara Y. Corticosterone secretion-inducing activity of saikosaponin metabolites formed in the alimentary tract. Chem Pharm Bull. 1989; 37: 2736–2740. pmid:2611932
  13. 13. Hattori T, Ito M, Suzuki Y. Studies on antinephritic effects of plant components in rats (1). Effects of saikosaponins original-type anti-GBM nephritis in rats and its mechanisms. Nihon Yakurigaku Zasshi 1991; 97: 13–21. pmid:2045012
  14. 14. Yen MH, Lin CC, Chuang CH, Liu SY. Evaluation of root quality of Bupleurum species by TLC scanner and the liver protective effects of “xiao-chai-hu-tang” prepared using three different Bupleurum species. J Ethnopharmacol. 1991; 34: 155–165. pmid:1795519
  15. 15. Park IS, Kang EM, Kim N. High-performance liquid chromatographic analysis of saponin compounds in Bupleurum falcatum. J Chromatogr Sci. 2000; 38: 229–233. pmid:10890745
  16. 16. Li XQ, Gao QT, Chen XH, Bi KS. High performance liquid chromatographic assay of saikosaponins from Radix Bupleuri in China. Biol Pharm Bull. 2005; 28: 1736–1742. pmid:16141550
  17. 17. Zhou Q, Hao JP, Gao KQ, Guan Q. Analysis on saikosaponin of sixteen Bupleurum chinense from Shanxi. Nat Prod Res Dev. 2012; 24: 644–647.
  18. 18. Wei H, Liu Q, Wei WG, Cui CL, Zhang Q. Determination of saikosaponin a, d in different extracts of Bupleurum longicaule Wall. ex DC. var. giraldii Wolff by HPLC. Chin Trad Patent Med. 2013; 35: 342–345.
  19. 19. Zhang GS, Zhang XY, Feng CH, Li DX, Rao XY, Hu PY, et al. Simultaneous determination of saikosaponins a, b2, c, d, f in Radix Bupleuri by HPLC-UV. Chin J Inform Trad Chin Med. 2014; 21: 74–77.
  20. 20. Zhang HN, Zhao YY, Liang H, Huang LQ, Zhang QY, Zhang ZX. Simultaneous HPLC-ELSD determination of saikosaponins a, c, d, f in Radix Bupleuri. Chin J Pharm Anal. 2007; 27: 1150–1153.
  21. 21. Bao YW, Li C, Shen HW, Nan FJ. Determination of saikosaponin derivatives in Radix Bupleuri and in pharmaceuticals of the Chinese multiherb remedy xiaochaihu-tang using liquid chromatographic tandem mass spectrometry. Anal Chem. 2004; 76: 4208–4216. pmid:15253665
  22. 22. Tang FL, Fu X, Zhu ZZ, Cai GM, Yuan B, Zhang ZY. UPLC determination of saikosaponin a, d in roots of Bupleurum smithii Wolff var. parvifolium Shan et Y. Li. Chin J Pharm Anal. 2011; 31: 1654–1657.
  23. 23. Guo Z, Peng B, Li ZY, Pan RL. Study on transformation rule of saikosaponin a and saikosaponin d under different extraction conditions by UPLC-QTof-MS. Nat Prod Res Dev. 2014; 26: 716–720.
  24. 24. Chen Y, Wang JY, Yuan L, Zhou L, Jia XB, Tan XB. Interaction of the main components from the traditional Chinese drug pair Chaihu-Shaoyao based on rat intestinal absorption. Molecules 2011; 16: 9600–9610. pmid:22095024
  25. 25. Lin XL, Xue L, Zhang HY, Zhu CF. Determination of saikosaponins a, c, and d in Bupleurum chinese DC from different areas by capillary zone electrophoresis. Anal Bioanal Chem. 2005; 382: 1610–1615. pmid:16007438
  26. 26. Jung DW, Shibuya M, Ebizuka Y, Yoshimatsu K, Shimomura K, Sung CK. ELISA for the determination of saikosaponin a, an active component of Bupleuri Radix. Chem Pharm Bull. 1998; 46: 1140–1143. pmid:9692221
  27. 27. Zhu SH, Shimokawa S, Tanaka H, Shoyama Y. Development of an assay system for saikosaponin a using anti-saikosaponin a monoclonal antibodies. Biol Pharm Bull. 2004; 27: 66–71. pmid:14709901
  28. 28. Ma LL, Tan MM, Chao Z. Quality inspection of commercial Bupluri Radix samples in Guangzhou: an ELISA for determining saikosaponin a content using anti-saikosaponin a monoclonal antibody. J South Med Univ. 2011; 31: 1006–1008.
  29. 29. Yu T, Gao SX, Yin AH, Tang YP, Wu YS, Li LL, et al. A sensitive time-resolved fluoroimmunoassay for determination of median levels of pregnancy-associated plasma protein a in pregnant women in China. J Immunoassay Immunochem. 2013; 34: 365–75. pmid:23859787
  30. 30. Secundo F, Bacigalupo MA, Scalerab C, Quici S. Rapid time-resolved fluoroimmunoassay for diethylstilbestrol in cow milk samples with a highly luminescent Tb3+ chelate. J Food Compos Anal. 2012; 25: 221–225
  31. 31. Sheng YJ, Eremin S, Mi TJ, Zhang SX, Shen JZ, Wang ZH. The development of a fluorescence polarization immunoassay for aflatoxin detection. Biomed Environ Sci. 2014; 27: 126–129. pmid:24625404
  32. 32. Liu ZJ, Yan X, Hua XD, Wang MH. Time-resolved fluoroimmunoassay for quantitative determination of thiacloprid in agricultural samples. Anal Methods 2013; 5: 3572–3576.
  33. 33. Liang QN, Zhou JW, Liu TC, Lin GF, Dong ZN, Chen ZH, Chen JJ, Wu YS. Development of a time-resolved fluorescence immunoassay for herpes simplex virus type 1 and type 2 IgG antibodies. Luminescence 2014; 30: 649–654. pmid:25377426
  34. 34. Xu WW. Evaluation index for in vitro diagnostic products(IVD) during R&D. J Mol Diagn Ther. 2010; 2: 140–144.
  35. 35. Crowther JR. The ELISA Guidebook. Totowa: Humana Press Inc.; 2001.
  36. 36. Peng K, Xu KY, Liu LN, Hendricks R, Delarosa R, et al. Critical role of bioanalytical strategies in investigation of clinical PK observations, a Phase I case study. mAbs 2014; 6: 1500–1508. pmid:25484037
  37. 37. Fang JH, Zhang MZ, Li Y, Chen ZL. Development of ELISA method for rapid detection of neomycin residue in animal tissue. J Chin Univ Metrol. 2009; 20: 211–213.
  38. 38. Sritularak B, Morinaga O, Yuan CS, Shoyama Y, Tanaka H. Quantitative analysis of ginsenosides Rb1, Rg1, and Re in American ginseng berry and flower samples by ELISA using monoclonal antibodies. J Nat Med. 2009; 63: 360–363. pmid:19377908
  39. 39. Fujii S, Tuvshintogtokh I, Mandakh B, Munkhjargal B, Uto T, Morinaga O, et al. Screening of Glycyrrhiza uralensis Fisch. ex DC. containing high concentrations of glycyrrhizin by Eastern blotting and enzyme-linked immunosorbent assay using anti-glycyrrhizin monoclonal antibody for selective breeding of licorice. J Nat Med. 2014; 68: 717–722. pmid:24845807
  40. 40. Chao Z, Tan MM, Paudel MK, Sakamoto S, Ma LL, Sasaki-Tabata K, et al. Development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) using highly specific monoclonal antibody against paclitaxel. J Nat Med. 2013; 67: 512–518. pmid:23007175
  41. 41. Morinaga O, Uto T, Sakamoto S, Tanaka H, Shoyama Y. Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies. Fitoterapia 2009; 80: 28–31. pmid:18926890
  42. 42. Paudel MK, Putalun W, Sritularak B, Morinaga O, Shoyama Y, Tanaka H, et al. Development of a combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin. Anal Chim Acta 2011; 701: 189–193. pmid:21801887