Fluorescence Polarization Immunoassays for Potato Glycoalkaloids

Abstract

Fluorescence polarization immunoassays (FPIAs) were developed for potato glycoalkaloids (GAs) using a polyclonal antiserum and a monoclonal antibody (MAb). Fluorescently labeled solanidine (AMF-SOL) was synthesized by coupling 4'-(aminomethyl)fluorescein (4'-AMF) to the hemisuccinate derivative of solanidine using an active ester method. Both polyclonal antibody (PAb) and MAb could quantify the major potato GAs in the 20-100 nM range; however, the affinities of PAb and MAb differed slightly among the individual GAs. PAb displayed the greatest affinity for a-chaconine, while MAb was more sensitive to changes in -solanine levels. Affinity constants (ifaff) of PAb and MAb for AMF-SOL were estimated at 4.2 x 108 and 4.7 x 107 M-1, respectively. Potato samples containing low, medium, and high levels of GAs were successfully analyzed by the PAb FPIA. Equilibrium in the FPIA reaction was reached within 1-2 min, and standard curves were stable for at least 14 days.

Development of Practical HPLC Methods for the Separation and Determination of Eggplant Steroidal Glycoalkaloids and their Aglycones

“…[44] Several authors have explored novel methods for the determination of glycoalkaloids in recent years. Among these are the use of enzyme-linked immunosorbent assay (ELISA), which is useful only for the aglycone forms, [45] "fluorescence polarization immunoassay," [46] "solution phase immunoassay with capillary electrophoresis, [47] gas chromatography mass spectrometry as applied to the aglycone forms, [48] liquid chromatography mass spectrometry, [38,49] matrix-assisted laser-desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/MS), [50] "nonaqueous capillary electrophoresis with ultra violet (UV) detection (NACE-UV)" for the analysis of solasodine and solanidine aglycones, [51,52] NACE combined with ion-trap mass spectrometry, [52,53] and, lastly, derivatization of the glycoalkaloids followed by HPLC separation and chemiluminescence detection. [54] Each of these methods has its own particular merits; however, although glycoalkaloids show limited absorption of ultraviolet (UV) light, HPLC separation followed by UV detection can still be considered a viable method, due to the relative widespread availability of this instrumentation and the potential to detect not some, but all the glycoalkaloids of interest without a lengthy derivatization step.…”

Rapid Quantitation of Potato Glycoalkaloids by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…Immunoassays are also rapid and inexpensive to perform. Recently a fluorescence polarization immunoassay was developed, to improve on the variability inherently associated with solid phase immunoassays (Thomson and Sporns, 1995).…”

A Capillary Electrophoresis Laser-Induced Fluorescence Method for Analysis of Potato Glycoalkaloids Based on a Solution-Phase Immunoassay. 1. Separation and Quantification of Immunoassay Products

“…However, the detection of the reaction products in solution-phase immunoassays can be complicated and difficult. Thomson and Sporns (1995) described a solution-phase fluorescence polarization immunoassay for GAs in potatoes that relied on the increase in the polarization of fluorescence when the fluorescent antigen was bound to the antibody relative to the polarization of fluorescence of the free antigen in solution. Alternatively, the products of the antibody-antigen reaction can be analyzed by chromatographic or capillary electrophoretic techniques (Shahedo and Karnes, 1998).…”

“…Synthesis of Fluorescently Labeled Solanidine. Fluorescently labeled solanidine (AMF-SOL) was prepared according to the method of Thomson and Sporns (1995). AMF-SOL was isolated from the reaction mixture using normal-phase preparative thin-layer chromatography on silica gel Kieselgel 60F 254 20 × 20 cm, 1000 µm thick plates (E. Merck, Darmstadt, Germany) and a solvent system of ethyl acetate/methanol/aqueous ammonia (79:20:1, v/v/v).…”