Tharita Kitisripanya

Anti-miroestrol polyclonal antibodies: A comparison of immunogen preparations used to obtain desired antibody properties

Immunogen quality is one important factor that contributes to desirable antibody characteristics. Highly specific antibodies against miroestrol can be used to develop a quality control immunoassay for Pueraria candollei products. In this study, we investigated how various immunogen preparations affect antibody properties. The results show that immunogen prepared using the Mannich reaction provides antibodies with higher specificity and sensitivity against miroestrol than immunogen prepared with the periodate reaction. The results suggest the Mannich reaction maintains the original structure of miroestrol and generates useful antibodies for developing immunoassays.

Development of a Rapid Immunochromatographic Strip Test for the Detection of Mulberroside A

INTRODUCTION Mulberroside A (MuA) is the major active anti-tyrosinase compound in the root bark extract of Morus alba L. (Moraceae). Typically, MuA is widely employed as an active ingredient in whitening cosmetics. A rapid and simple assay system utilizing a small quantity of test sample is essential for the detection of MuA in large number of samples. An immunoassay using highly specific MuA polyclonal antibodies may be useful for the determination of small quantities of MuA in test samples. OBJECTIVE To establish a rapid qualitative MuA test, an immunochromatographic strip test was developed using anti-MuA polyclonal antibodies (anti-MuA PAb). METHODOLOGY The qualitative assay was based on a competitive immunoassay where the detection reagent consisted of anti-MuA PAb colored with colloidal gold particles. The capture reagent was a MuA-ovalbumin (MuA-OVA) conjugate immobilized on the test strip membrane. RESULTS A sample containing MuA and the detection reagent were incubated together with immobilized capture reagent on a nitrocellulose membrane. When MuA was present, it competed with the immobilized conjugates on the strip membrane to bind a limited amount of colored antibodies; thus, a positive sample showed no color on the capture spot zone. The detection limit for the strip test was 2 µg/mL. The developed immunochromatographic strip test was utilized to determine MuA in plants, medical preparations and cosmetic samples. CONCLUSION This immunochromatographic strip test is advantageous as a rapid, simple and sensitive screening method for the detection of MuA in plant extracts, cosmetic samples and pharmaceutical products.

Development of an indirect competitive immunochromatographic strip test for rapid detection and determination of anticancer drug, harringtonine

Harringtonine (HT) is a natural compound, which is mainly produced by the genus Cephalotaxus, and has been clinically utilized in China for the treatment of acute leukemia and lymphoma. However, the amounts of HT in the Cephalotaxus species are very small; therefore, plant tissue cultures have been focused upon to enhance HT production. Qualitative/quantitative methods for HT detection are required to screen superior cell lines. We developed a one-step indirect competitive immunochromatographic assay (ICA) using colloidal gold nanoparticles conjugated with highly specific monoclonal antibodies against HT (MAb 1D2) for simple, rapid, and sensitive detection of HT in plant samples. This ICA can be completed in 15min after dipping the strip into analytes with a limit of detection of ∼313ng/mL. In developed ICA, fiber pad which is usually used for conventional ICA, was not used to shorten the time for preparing chromatographic strip, resulting in a decrease in the volume of valuable analytes (20μL). Considering simplicity, rapidity, and sensitivity of the developed ICA, this study could be applied to a fieldwork study for finding new natural resources containing HT.

Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an identical marker, in White Kwao Krua using a monoclonal antibody

&NA; Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen‐rich plant widely used among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have been added as an active ingredient for skin rejuvenation and breast enlargement effects in various functional foods. However, most of the products on the market containing WKK have not been sufficiently standardized with respect to the active compound or identical marker. To control the quality of these plant materials, an enzyme‐linked immunosorbent assay (ELISA) using anti‐isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and was thus selected to develop the ELISA. Based on the validation analysis and the tested performance of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant. HighlightsAntibodies against isomiroestrol were first generated in this study.To determine isomiroestrol, an identical marker of WKK, ELISA was established using anti‐isomiroestrol 12C1‐mAb.The developed ELISA can be applied for quantitative analysis of isomiroestrol in WKK samples.

A pilot pharmacokinetic study of miroestrol and deoxymiroestrol on rabbit sera using polyclonal antibody-based icELISA analysis

Miroestrol (ME) and deoxymiroestrol (DME) are the most potent phytoestrogens and bioactive markers in Pueraria candollei var. mirifica tuberous roots. To understand their pharmacokinetic profiles, a pharmacokinetic study of ME and DME, at 0.43 and 0.21 mg per kg body weight, respectively, in three rabbits was performed after orally administering a single dose of P. candollei var. mirifica enriched fraction extract. Two established polyclonal antibody‐based indirect competitive enzyme‐linked immunosorbent assays were validated to determine ME and DME in rabbit sera. In rabbits, the area under the 0‐ to 48‐hr concentration‐time curve of ME and DME were 854.92 and 1,692.84 ng·h/ml, respectively. The maximum concentration of ME was measured 1 hr after administration as 69.62 ± 8.28 ng/ml, and the maximum concentration of DME was measured at 3 hr as 81.8 ± 5.43 ng/ml. These results provide an initial approach for designing and studying the relationship between the ME and DME levels and their therapeutic effects based on their pharmacokinetic profiles.

Development of a colloidal gold nanoparticle-based immunochromatographic strip for the one-step detection of miroestrol and puerarin

Pueraria candollei or White Kwao Krua (Leguminosae) is an indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation, particularly in elder women. Among the bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold-based detection system for the simultaneous detection of miroestrol and puerarin in a one-step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limits of miroestrol and puerarin were 0.15 and 4.5 μg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme-linked immunosorbent assay and presented a good correlation between these two methods. This is the first report on the development of an ICS that can detect miroestrol and puerarin in one step. The developed ICS provides a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.

An Enzyme‐linked Immunosorbent Assay for Genistein 7‐O‐[α‐rhamnopyranosyl‐(1→6)]‐β‐glucopyranoside Determination in Derris scandens using a Polyclonal Antibody

INTRODUCTION Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-β-glucopyranoside (GTG) is a major bioactive compound in Derris scandens. It is responsible for anti-inflammatory activity by inhibition of cyclooxygenase and lipoxygenase. There are many commercial products of D. scandens available in Thailand. OBJECTIVE To develop an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of GTG in plant material and derived products using a polyclonal antibody. METHODS An immunogen was synthesised by conjugating GTG with a carrier protein. The polyclonal antibody against GTG (GTG-PAb) was produced in New Zealand white rabbits. The ELISA method was validated for specificity, sensitivity, accuracy, precision and correlation with HPLC. RESULTS The polyclonal antibody was specific to GTG and genistin within the range of compounds tested. The GTG ELISA was applied in the range 0.04-10.00 μg/mL with a limit of detection of 0.03 μg/mL. The recovery of GTG in spiked Derris scandens extracts ranged from 100.7 to 107.0%, with a coefficient of variation less than 7.0%. The intra- and inter-assay variations were less than 5.0%. The ELISA showed a good correlation with HPLC-UV analysis for GTG determination in samples, with a coefficient of determination (r2 ) of 0.9880. CONCLUSION An ELISA was established for GTG determination in Derris scandens. The GTG-PAb can react with GTG and genistin, but genistin has not been found in the plant. Therefore, the ELISA can be used for high throughput quality control of GTG content in D. scandens and its products. Copyright