Baoping Qu

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.

Rapid, sensitive separation of the three main isoflavones in soybean using immunoaffinity chromatography

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.

Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium

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.

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.

Detection of total bile acids in biological samples using an indirect competitive ELISA based on four monoclonal antibodies

Total bile acids (TBAs) are sensitive indicators of hepatic injury, neonatal jaundice, and other metabolic diseases, and an enzyme cycling method has been used in clinical practice for the determination of TBA concentration for more than 20 years. However, this method has several limitations, particularly in terms of sensitivity and practicality. Here, we investigated a newly developed indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of 5 major components of TBA, using a combination of 4 different monoclonal antibodies (MAbs). We prepared 4 MAbs against 5 major bile acids, and determined their cross-reactivity. The coating antigen used in the new method represents a combination of 5 coating antigens (1 : 1 : 1 : 1 : 1 ratio). Inter-assay and intra-assay consistency, and the recovery rate were determined. The standard curve was determined to be linear between 0.2875 and 9.2 μmol L−1, while the IC50 value was 1.85 μmol L−1. Serum TBA concentrations determined using this method were consistent with values obtained by the enzyme cycling method, in both healthy mice and mice with cholestasis, and there is no significant difference between these methods (p > 0.05). We showed that icELISA can be used for the detection of bile acids in human saliva obtained from healthy volunteers, which is not possible with the enzyme cycling method. These results demonstrate that the new icELISA is valid and sensitive enough to determine TBA content in different types of biological samples, and it may be suitable for routine clinical practice.

Development of a sensitive and reliable enzyme-linked immunosorbent assay for detecting naringin in human saliva

In this work, a hybridoma secreting naringin monoclonal antibody (NAR mAb) was produced by fusing splenocytes from a mouse immunized against a NAR–bovine serum albumin conjugate with the hypoxanthine–aminopterin–thymidine sensitive mouse myeloma cell line (Sp2/0-Ag14). Then an indirect competitive enzyme-linked immunosorbent assay for NAR was developed and characterized using the mAb. In this assay, the effective measuring range was 3.91 ng mL−1 to 250 ng mL−1 of NAR. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, the concentration of NAR in traditional Chinese prescriptions and biological samples was determined using the novel method. The pharmacokinetic parameters of NAR determined in the saliva from healthy humans were also obtained. The method contributes to further research aimed at achieving a better understanding of the interactions of NAR with other drugs and at reducing the incidence of adverse reactions, by monitoring the saliva.