Ultrasensitive Time-Resolved Fluoroimmunoassay for Saikosaponin a in Chaihu (Bupleuri Radix)

Abstract

The aim of this study is to establish a time-resolved fluoroimmunoassay (TRFIA) system for quantitative analysis of saikosaponin a (SSa) in the crude drug of Chaihu (Bupleuri Radix). A 96-well microplate coated with rabbit anti-mouse IgG was incubated with the methanol extracts of Chaihu samples and a mouse anti-SSa monoclonal antibody, and a Eu3+-labeled SSa-human serum albumin conjugate was used as the tracer. The established competitive TRFIA showed a good fourth order polynomial fitting from 0.01 to 10.0 μg/mL for standard SSa sample with a detection limit of 0.006 μg/mL. The intra- and inter-assay coefficients of variation of the assay were 7.3% and 8.9%, respectively, and the average SSa recovery was 119.2%. For samples of Chaihu extract, the results of this assay showed a good correlation with those by enzyme-linked immunosorbent assay established previously. This TRFIA system is ultrasensitive for detecting SSa with a wide detection range and a good stability and represents the first attempt of using TRFIA for quality evaluation of the crude drug of Chaihu.

Development of a Competitive Time‐Resolved Fluoroimmunoassay for Paclitaxel

“…TRFIA shares the same advantages as ELISA but has much better performance in terms of sensitivity, convenience and rapidness, according to our experience with the detection of saikosaponin a (Chao et al ., ). In the TRFIA system we established, a log‐logit model was adopted for curve fitting.…”

“…We further evaluated the accuracy and reliability of the TRFIA using recovery experiments. The samples spiked with 15, 20, and 50 μg of Tax yielded recovery rates of 108.6%, 116.4%, and 100.4%, respectively (mean 108.5%), which were slightly higher than that of icELISA; this might be attributed to the similar reason for a high %CV in TRFIA detection of saikosaponin a (Chao et al ., ). Nevertheless, the spiked recovery rates were all within the acceptable range (80–120%) (Peng et al ., ; Fang et al ., ), suggesting the accuracy of the TRFIA system.…”

“…In our previously established TRFIA and ELISA for detecting saikosaponin a, the polynomial fit model provided better fitting in calibration of the concentration and fluorescence intensity (Chao et al ., ). As to Tax in this study, the linear regression worked well as it did in almost all the previous reports.…”

“…Buffers used in TRFIA, i.e. the labelling buffer, elution buffer, blocking buffer, and washing buffer, were all the same as those used in TRFIA for saikosaponin a (Chao et al ., ). Buffers used in ELISA included phosphate buffered saline (PBS), PBS containing 5% skim milk (SPBS), PBS containing 0.05% Tween 20 (TPBS), substrate solution [0.1 M citrate buffer (pH 4.0) containing 0.003% hydrogen peroxide (H 2 O 2 ) and 0.3 mg/mL 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) diammonium salt (ABTS)].…”

“…Using lanthanide fluorescent chelates as fluorescence labels, TRFIA is commonly utilised in environmental contaminant monitoring, agricultural and food chemistry, laboratory testing, and forensic science (Zhang et al ., ; Sheng et al ., ; Zhang et al ., ; Xu et al ., ), but its use in crude drug analysis has not been described. We recently established a TRFIA system for the determination of saikosaponin a (Chao et al ., ), and compared with the conventional ELISA, TRFIA showed a 3–15 folds higher sensitivity with a reduced assay time by at least 1 h (Zhu et al ., ). In this study, we developed a TRFIA system using the anti‐Tax MAb 3A3 to provide another reliable, sensitive quantitative analytical method for Tax detection.…”