Wenchao Shan

Development of ELISA for detection of Rh1 and Rg2 and potential method of immunoaffinity chromatography for separation of epimers.

In this work, hybridomas producing anti-ginsenoside-Rh1 monoclonal antibodies (MAbs) were generated. These MAbs were subsequently used to create indirect competitive enzyme-linked immunosorbent assays (icELISAs). A linear correlation was obtained for G-Rh1 concentrations in the range from 26 to 512ng/mL. The regression equation was y=1.979-0.201Log2(X) with a regression coefficient of 0.9898. 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 recovery rates ranged from 93.16% to 108.43%. Testing with the icELISA demonstrated that the MAbs were specific for 20(S)-Rh1 and 20(S)-Rg2 with no cross-reactivity against 20(R)-Rh1 and 20(R)-Rg2. The immunoaffinity chromatography column (IAC) was constructed by covalently coupling monoclonal antibody (MAb) against G-Rh1 to CNBr-activated Sepharose 4B. When 20(R)-type-Rg2 passed through the IAC column, it was adsorbed, but the amount adsorbed was lower than that when 20(S)-type-Rg2 ran through the column. The differences in adsorption between the 20(S) and 20(R) type ginsenosides bring a new approach or method to separate 20(S)-Rg2 and 20(R)-Rg2 by IAC. Our results indicate that the icELISA is a sensitive and efficient approach for the identification of epimers, and the application of IAC using MAbs against small molecules provides a totally new thought and potential method for resolving epimers.

Development of an enzyme‐linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti‐glycyrrhizic acid monoclonal antibody

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti-glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line (Sp2/0-Ag14). Subsequently, an indirect, competitive enzyme-linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12-2500 ng/mL. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine.

A Sensitive and Specific Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Paeoniflorin and Its Application in Pharmacokinetic Interactions between Paeoniflorin and Glycyrrhizinic Acid.

This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay based on monoclonal antibodies against paeoniflorin to study the effects of different doses of glycyrrhizinic acid on the pharmacokinetics of paeoniflorin in mice. An anti-paeoniflorin monoclonal antibody was produced from a hybridoma created through a fusion of splenocytes immunized with paeoniflorin-bovine serum albumin and conjugated with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line SP2/0. The resultant antibody was used to develop and validate a rapid, specific and sensitive, indirect competitive enzyme-linked immunosorbent assay for the measurement of paeoniflorin (linear range 4.8-312.5 ng · mL(-1)). The intraday and interday precision values of the indirect competitive ELISA method were well within the recommended range (≤ 10 %), and the recovery rate was, on average, 101.13 %. Pharmacokinetic parameters obtained from mouse blood samples at various intervals following the oral administration of paeoniflorin and glycyrrhizic acid at three doses (1 : 0.3, 1 : 1, 1 : 3, respectively) demonstrated that the highest dose of glycyrrhizic acid inhibits the absorption of paeoniflorin.

Enzyme-Linked Immunosorbent Assay for Hyodeoxycholic Acid in Pharmaceutical Products Using a Monoclonal Antibody

Herein is reported an immunochemical approach to determine hyodeoxycholic acid using hybridoma-secreting monoclonal antibodies. The hyodeoxycholic acid-specific antibody was produced by fusing splenocytes immunized with a hyodeoxycholic acid-bovine serum albumin conjugate with a hypoxanthine–aminopterin–thymidine-sensitive mouse myeloma cell line (SP2/0). The antibody was highly specific for hyodeoxycholic acid, with less than 0.05 percent cross-reactivity to over fifty structurally related compounds. The antihyodeoxycholic acid monoclonal antibody was then used to develop a rapid, specific, and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of hyodeoxycholic acid in pharmaceutical compounds. The linear dynamic range was from 0.48 to 62.5 nanograms per milliliter with an IC50 value of 8 nanograms per milliliter. The icELISA results correlated well with a conventional high-performance liquid chromatography method for the determination of hyodeoxycholic acid (R2 = 0.9982). This study shows that the icELISA method was successfully applied to the quantification of hyodeoxycholic acid in pharmaceutical products.

Development of an enzyme-linked immunosorbent assay for chenodeoxycholic acid using an anti-chenodeoxycholic acid monoclonal antibody

CDCA is one of the major bile acids in humans and has important clinical significance and therapeutic applications. In this work, a new monoclonal antibody (MAb) specific for chenodeoxycholic acid (CDCA) was prepared and characterized. A hybridoma secreting an anti-CDCA MAb was produced by fusing splenocytes from a mouse immunized against a CDCA–BSA conjugate with the hypoxanthine–aminopterin–thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0-Ag14. The antibody was highly specific for CDCA, exhibited only weak reactions with CA and DCA and did not react with other structurally related chemicals. Subsequently, a MAb-based enzyme-linked immunosorbent assay (ELISA) against CDCA was developed and characterized. In this assay, we detected an effective CDCA measurement range of 32 ng mL−1 to 1024 ng mL−1 (R2 = 0.9962). Both intra-assay and inter-assay repeatability and precision were achieved with relative standard deviations (RSD) below 10%. Additionally, the CDCA levels in medicines and samples were determined with high sensitivity and efficiency. This study provides a useful method for detecting CDCA in medicines and will contribute to the further clinical research.

Development of immunoaffinity chromatography to specifically knockout baicalin from Gegenqinlian Decoction

Specific knockout technology provides a powerful tool to confirm the role of target compounds in a plant or its derived prescriptions, and this principle is the same as that with knockout genes. In this study, we generated an immunoaffinity column conjugated with an anti-baicalin monoclonal antibody and then loaded Gegenqinlian Decoction extracts, followed by washing with deionized water and an elution solvent. The results of the high-performance liquid chromatography fingerprints and high-performance liquid chromatography with mass spectrometry showed that the immunoaffinity column was able to specifically knockout baicalin, oroxylin A-7-O-glucuronide, wogonoside, wogonin, and baicalein from Gegenqinlian Decoction. A reliable one-step method to specifically knockout baicalin was established with an immunoaffinity column. Gegenqinlian Decoction and its knocked-out fraction induced the expression of superoxide dismutase and were compared in human umbilical vein endothelial cells cultured with a high glucose concentration; the results showed that the Gegenqinlian Decoction and its knocked-out fraction showed no significant difference, which indicated that the baicalin, oroxylin A-7-O-glucuronide, wogonoside, wogonin, and baicalein that were knocked out by the immunoaffinity column might not be key compounds for the induction of Gegenqinlian Decoction superoxide dismutase secretion.

Preparation of a Monoclonal Antibody Against Saikosaponin C and the Establishment of its Enzyme-Linked Immunosorbent Assay

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.

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.