Oxidation method with sodium iodide was used to synthesize immunogenic antigen (POL Ⅲ-BSA) and coating antigen (POL Ⅲ-OVA) of polyphylin Ⅲ.UV spectroscopy showed that polyphylin Ⅲ was successfully conjugated with BSA and OVA.After immunized by polyphylin Ⅲ,the mice can produce anti-polyphylin Ⅲ antibodies specifically.The ELISA test results showed the anti-polyphylin Ⅲ antibodies with titer up to 1:16 000 and the linear range was from 1 ng· L-1 ～100 μg · L-1 and measured the cross reaction of polyphylin Ⅲ with polyphylin Ⅰ,polyphylin Ⅱ and polyphylinⅦ.The research successfully prepared the conjugate antigen polyphylin Ⅲ and its antiserum to produce anti polyphylin Ⅲ monoclonal antibody and to establish a fast assay for polyphylin Ⅲ content by and ELISA.

We investigated a newly developed indirect competitive enzyme-linked immunosorbent assay for the determination of 5 major components of TBA, which works efficiently in different types of biological samples, and may be suitable for routine clinical practice.

目的 制备特异性敲除甘草酸的免疫亲和色谱柱.方法 抗甘草酸单克隆抗体与CNBr活化的Sepharose 4BTM凝胶偶联制备甘草酸特异性敲除免疫亲和色谱柱,利用HPLC法测定敲除液与洗脱液中甘草酸质量浓度,考察甘草酸免疫亲和色谱柱的最大载样量、敲出率、精密度、准确度及稳定性等参数.结果 甘草酸免疫亲和色谱柱的最大载样量为1.326 11 mg;日内及日间精密度总RSD分别为0.65％,相对误差(RE)为0.37％;免疫亲和色谱柱稳定性实验显示32 d内甘草酸敲除率的RSD为1.37％,稳定性良好.结论 制备的甘草酸特异性敲除免疫亲和色谱柱能快速、高效、稳定地敲除甘草酸.

OBJECTIVE:To analyze the transdermal profile of pseudoephedrine and amygdalin in the Traditional Chinese Medicine majiepingchuan in rat skin and to reveal their interaction.METHODS:A Franz diffusion cell was used in vitro to evaluate the transdermal parameters of cumulative transdermal flux (Q(tot)), cumulative transmission (T(tot)), and mean penetration rate (Kp) of pseudoephedrine and amygdalin in majiepingchuan. Linear regression analyses of Q(tot) over time of pseudoephedrine vs amygdalin and their ratios was adopted for correlation evaluation.RESULTS:At 1, 2, 4, 6, and 8 h, the Q(tot), T(tot) and Kp of pseudoephedrine showed a good correlation with that of amygdalin.CONCLUSION:There was a small difference in the ratios of Q(tot), T(tot) and Kp between pseudoephedrine and amygdalin, and a correlation between them.

In this study, a rapid (within 10 min) quantitative lateral-flow immunoassay using a quantum dots (QDs)-antibody probe was developed for the analysis of puerarin (PUE) in water and biological samples. The competitive immunoassay was based on anti-PUE monoclonal antibody conjugated with QDs (detection reagent). Secondary antibody was immobilized on one end of a nitrocellulose membrane (control line) and PUE-bovine serum albumin conjugate was immobilized on the other end (test line). In the quantitative experiment, the detection results were scanned using a membrane strip reader and a detection curve (regression equation: y=−0.11 ln(x)+0.979, R2=0.9816) representing the averages of the scanned data was obtained. This curve was linear from 1 to 10 μg/mL. The IC50 value was 75.58 ng/mL and the qualitative detection limit of PUE was 5.8 ng/mL. The recovery of PUE added to phosphate-buffered saline and biological samples was in the range of 97.38–116.56%. To our knowledge, this is the first report of the quantitative detection of a natural product by QDs-based immunochromatography, which represents a powerful tool for rapidly screening PUE in plant materials and other biological samples.

Daidzin, genistin, and glycitein are major isoflavone compounds in soybean that are indispensable nutrients in traditional Chinese foods. Generally, strategies for detecting and separating soy isoflavones have been based on HPLC and chromatographic techniques, which are tedious and time-consuming procedures. In the present study, we developed an ELISA-based approach for daidzin detection using a broad-specificity monoclonal antibody (clone number: AA9) with an effective detection range of 10-10 000 ng/mL. Subsequently, we prepared an immunoaffinity column by coupling the monoclonal antibody AA9 to CNBr-activated Sepharose 4B. Our results demonstrate that the immunoaffinity column can efficiently and specifically extract daidzin, glycitein, and genistin from numerous structurally similar soy isoflavones in leguminous plants, thereby providing a new method for the extraction of target components from similar compounds in natural products.

Objective To determine the contents of baicalin in Gegen Qinlian Tablet produced by seven manufacturers, a Chinese patent drug used widely in acute gastroenteritis, virus diarrhea, and bacillary dysentery, by using indirect competitive enzyme-linked immunosorbent assay ( ic-ELISA ) . Methods The ic-ELISA method was established by using anti-baicalin monoclonal antibody ( anti-BA MAb) , in which the linear relation, accuracy and the recovery rate were measured. Based on the ic-ELISA method, the contents of baicalin in Gegen Qinlian Tablet from different manufacturers were deter-mined. Results The linear range of the ic-ELISA method established was from 16. 157 to 1 280 mg/L, the relative standard deviation (RSD) was≤5. 1% in the intra-assay, and≤9. 0% in the inter-assay, with the average recovery rate of 107 . 1% ( RSD=4 . 9%) . The baicalin contents in Gegen Qinlian Tab-let from seven manufacturers ranged from 4. 45 to 13. 41 mg per tablet. Conclusion The ic-ELISA method for the baicalin determination was convenient, accurate, and repeatable, which could be applied to the quality control of traditional Chinese herbal compound containing baicalin.

Vernonia anthelmintica is one of the widely used increased pigment medicine. It has various effects of modulating immune system,enhancing the activity of tyrosinase,prompting the release of melanin,supplementing trace elements and so on. It is commonly used in the treatment of vitiligo,liver diseases and pneumosilicosis clinically. This paper describes the research advances of treating Vitiligo with Vernonia anthelmintica from the aspect of chemical contents and pharmacological mechanism study to clinical application. This paper also provides reference for the mechanism of using Vernonia anthelmintica for vitiligo and the new drug investigation.

In this work, a novel monoclonal antibody specific for naringin was prepared and characterized. Subsequently, an indirect competitive enzyme-linked immunosorbent assay for naringin was developed, with an effective range from 4.8 to 156 ng/mL naringin. Next, an immunoaffinity column was obtained by coupling anti-naringin monoclonal antibodies to CNBr-activated Sepharose 4B and a rapid immunoaffinity chromatography assay for naringin was developed. The immunoaffinity column was used to separate naringin from Citrus aurantium. The results showed that 1 g of the dry Sepharose 4B can couple 10 mg of immunoglobulin G. And the immunoaffinity column can efficiently and specifically capture approximately 250 μg of naringin without cross reacting with its structurally similar compounds. Moreover, our results indicate that the application of immunoaffinity chromatography can simplify the pretreatment and the isolation process greatly compared to conventional methods, providing a potential method for extracting the target component from structurally similar compounds in natural products.

This work developed a novel immunochemical approach for the quality control of saikosaponin d using an enzyme-linked immunosorbent assay. Splenocytes from mice immunized with the saikosaponin d-bovine serum albumin conjugate were fused with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line, and a hybridoma secreting monoclonal antibody against saikosaponin d was successfully obtained. The prepared anti-saikosaponin d monoclonal antibody 1E7F3 has a novel characteristic, showing weak reactivity with compounds that are structurally related to saikosaponin d. Using monoclonal antibody 1E7F3, a specific and reliable enzyme-linked immunosorbent assay was developed to detect saikosaponin d. The system shows a full measurement range from 156.25 to 5000.00 ng × mL(-1). Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10.00%. The recovery rates ranged from 92.36% to 101.00%, meeting the requirements for biological samples. There was a good correlation between the enzyme-linked immunosorbent assay and high-performance liquid chromatography analyses of saikosaponin d, and the saikosaponin d levels in formulated Chinese medicines were successfully determined. Furthermore, immunoaffinity column chromatography was established using this anti-saikosaponin d monoclonal antibody, and the elution profile of saikosaponin d was detected by a Bio-Rad QuadTec UV/Vis detector at 203 nm. The results demonstrate that we generated a reliable and more efficient assay system for measuring saikosaponin d and provide a potential approach for purifying and separating saikosaponin d.

OBJECTIVE:To determine the effects of different formulations of Banxia Xiexin Decoction ( , BXD) on the pharmacokinetics of baicalin (BAL) in mice.METHODS:Pungent, bitter, and sweet components of BXD (totaling 7 Chinese herbs) were formulated into the following groups: K (bitter herbs), XK (pungent and bitter herbs), KG (bitter and sweet herbs), and BXD (all 7 herbs) groups. These different formulations were administered intragastrically in mice, and blood was collected via the tail vein for continuous monitoring. BAL, which is a main active constituent in Scutellaria baicalensis Georgi., was detected in this study. Indirect competitive enzyme-linked immunosorbent assays (icELISAs) based on anti-BAL-monoclonal antibodies were employed to determine BAL concentrations in each group.RESULTS:The concentrations of BAL in blood samples from mice in the K and XK groups were lower than those in other groups. In all groups, BAL concentrations peaked at around 1-1.5 h and again at 5-7 h. There were no significant differences in the timing of peak BAL concentrations between groups. However, the peak concentrations and area under curve (AUC)0-36 h in the KG and BXD groups were almost 3 times of those in the K and XK groups.CONCLUSIONS:Differing compatibilities of BXD caused dissimilar pharmacokinetics of BAL. Moreover, we demonstrated a method for the continuous detection of blood concentrations of Chinese medicines in mice, and icELISA may be a feasible technique for the study of pharmcokinetic mechanisms of Chinese medicine.

In the present study, a novel monoclonal antibody (MAb) specific for icariin (ICA) was prepared and characterized. A hybridoma‑secreting MAb against icariin was produced by fusing splenocytes immunized with an ICA‑bovine serum albumin conjugate with a hypoxanthine‑aminopterin‑thymidine‑sensitive mouse myeloma SP2/0 cell line. The antibody showed high specificity for ICA with almost no cross‑reactivity against the majority of structurally‑related chemicals. Subsequently, an indirect competitive enzyme‑linked immunosorbent assay (ELISA) for ICA was established and characterized. In this assay, an effective measuring range of 10‑1,000 ng/ml of ICA (R2=0.9828) was detected. Intra‑ and inter‑assay repeatability and precision were achieved with a relative standard deviation (RSD) of <10%. A mean recovery of 95‑115% was obtained, with an RSD of <10%. In addition, the levels of ICA in traditional Chinese herbal prescriptions were determined, and correlation between the ELISA and high‑performance liquid chromatography analyses of total ICA was obtained. These results demonstrated that a reliable ELISA method had been successfully developed to determine ICA in traditional Chinese herbs and may contribute to further clinical investigations.

Background: Icariin (ICN) is the main bioactive natural flavonoid in plants of the Epimedium genus and is valued in traditional Chinese medicine owing to its beneficial effects, including enhancement of cardiovascular function and antitumorigenic activity. Current methods for detecting ICN employ expensive instruments that require a high level of skill for operation. As such, a more simple and rapid assay is desired. Methods: A colloidal gold-conjugated anti-ICN monoclonal antibody was prepared and an Immuno-chromatographic Strip (ICS) test was developed for detecting ICN in plant preparations. Results: The prepared anti-ICN MAbs show weak reactivity with compounds that are structurally related to ICN. Using anti-ICN MAbs, a specific and reliable ELISA was developed to detect ICN. The system shows a full measurement range from 10 ng/mL to 1280 ng/ml with regression equation is y = -0.158ln(x) + 1.3336 (R-2 = 0.9924). Moreover, the usefulness of the ICS assay using anti-ICN mAb as a qualitative method was confirmed for the analysis of ICN in different samples by comparing to indirect competitive enzyme-linked immunosorbent assay. This ICS assay based on the competitive immunoassay system showed high specificity for ICN and had a detection limit of 500 ng/ml. Conclusion: Our results indicate that the ICS assay can be a powerful tool for rapid screening of plant materials used in traditional Chinese medicine owing to its ease of manipulation and low cost. In addition, it can potentially be used for the detection of ICN in other types of biological sample.

Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA. (C) 2015 Elsevier B.V. All rights reserved.

The icELISA for NAR using anti-NAR mAb will be of great use in determining NAR in foods, medicines and human saliva; which provides a potential tool for clinical safe medications.

Objective: To establish a quick enzyme-linked immunosorbent assay(ELISA) method for the determination of baicalin(BA).Methods: Indirect competitive ELISA was developed by using anti-BA monoclonal antibody(anti-BA MAb),and then it was applied to BA measurement in the traditional Chinese medicine injection Jingzhi Qingkailing.Results: The standard curve of established ELISA was linear between 2.67 and 1029.00 ng.mL-1.The average recovery was 100.9%,and the relative standard deviation(RSD) of measurements was 3% in the intra-assay,and 4% in the inter-assay.The test of samples could be finished in 4hs by using this ELISA method.The analysis result of ELISA method was consistent with that of HPLC test in BA determination of Jingzhi Qingkailing injection.Conclusion: Quick ELISA method for the BA determination is well established and can be applied to the quality control of traditional Chinese herbal medicine / compound preparation which contains baicalin.