A sensitive and specific indirect competitive enzyme­linked immunosorbent assay for the detection of icariin.

In the present study, a novel monoclonal antibody (MAb) specific for icariin (ICA) was prepared and characterized. A hybridoma­secreting MAb against icariin was produced by fusing splenocytes immunized with an ICA­bovine serum albumin conjugate with a hypoxanthine­aminopterin­thymidine­sensitive mouse myeloma SP2/0 cell line. The antibody showed high specificity for ICA with almost no cross­reactivity against the majority of structurally­related chemicals. Subsequently, an indirect competitive enzyme­linked immunosorbent assay (ELISA) for ICA was established and characterized. In this assay, an effective measuring range of 10­1,000 ng/ml of ICA (R2=0.9828) was detected. Intra­ and inter­assay repeatability and precision were achieved with a relative standard deviation (RSD) of <10%. A mean recovery of 95­115% 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 high­performance 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.

Correlation between the transdermal characteristics of pseudoephedrine and amygdalin in majiepingchuan in vitro.

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.

Mechanism of baicalin compatibility in chinese medicine formula Banxia Xiexin Decoction () by pharmacokinetics and indirect competitive enzyme-linked immunosorbent assays in mice.

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.

Rapid lateral-flow immunoassay for the quantum dot-based detection of puerarin.

In this study, a rapid (within 10min) 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=-0.11ln(x)+0.979, R(2)=0.9816) representing the averages of the scanned data was obtained. This curve was linear from 1 to 10µg/mL. The IC50 value was 75.58ng/mL and the qualitative detection limit of PUE was 5.8ng/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.

Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody.

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.

Development of a Fluorescence-Linked Immunosorbent Assay for Baicalin.

Previously, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for baicalin (BAL) and used this assay to investigate the pharmacokinetics of BAL in mice. In this study, an anti-BAL monoclonal antibody (MAb) was purified by the caprylic acid method and then labelled with fluorescein isothiocyanate (FITC). Subsequently, an indirect competitive fluorescence-linked immunosorbent assay (icFLISA) was developed to detect baicalin (BAL) using FITC-labelled anti-BAL MAbs. Characterization of the assay demonstrated an effective BAL measurement range of 6.4 ng/mL to 500 µg/mL (R(2) = 0.997). The relative standard deviations (RSDs) for both intra-assay and inter-assay repeatability and precision were below 10 %. This icFLISA for BAL is simple, rapid and sensitive, with a 390-fold larger linear range and a 2-fold lower limit of detection (LOD) compared with the previously developed icELISA. We observed a strong correlation between the results determined by the icFLISA and icELISA methods. Overall, this study provides a useful method for detecting BAL in medicines, enabling in vivo visualization research.