Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10559763 | Talanta | 2011 | 9 Pages |
Abstract
Monoclonal antibodies against the hop-derived prenylated chalcone xanthohumol (X) and the prenylated flavonoids isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) were developed. Carboxylic acid haptens of X, IX and 8-PN were synthesized by linking a spacer to their C4â²-OH group followed by subsequent coupling to bovine serum albumin (BSA) to form conjugates that were employed as immunogens in BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the established hybridoma cell lines proved, in cross-reactivity studies, to possess highly specific binding capacities in an optimized competitive indirect ELISA. The immunoassays make use of immunogen-coated microtiterplates and a peroxidase-labeled anti-mouse IgG1 secondary antibody with ABTS as a chromogenic substrate. For X the IC50 value derived from the standard curve was 62.91 ng mLâ1, and for both IX and 8-PN 37.15 ng mLâ1. The assay was validated for the quantitative analysis of X, IX and 8-PN in urine and serum. A simple sample pretreatment procedure using a diethyl ether extraction was optimized and the recoveries and matrix effects were assessed. The validity of the established assay was tested and mean inter- and intra-assay variations in urine were 2.32% and 1.91%, respectively for X, 6.24% and 2.39%, respectively for IX and 7.18% and 0.74%, respectively for 8-PN. In serum, the mean inter- and intra-assay variations were 8.90% and 1.37%, respectively for X, 6.13% and 1.57%, respectively for IX and 6.13% and 2.43%, respectively for 8-PN. Furthermore, the method demonstrated excellent accuracy and significant correlation with measurements by an established and validated HPLC-MS method.
Keywords
EquolFPLCIC50TLCMWCOMSDENLNHSDCCTMBHmpATEMEDDMFo-phenylenediamine dihydrochloridepNPPpara-nitrophenylphosphateXanthohumolN-hydroxysuccinimidedicyclohexylcarbodiimide8-prenylnaringenin6-prenylnaringenin8-PNDMXFBSSDSDMEMABTSPBS17β-estradiol1H and 13C NMR3,3′,5,5′-tetramethylbenzidineBSADMSODulbecco's modified Eagle MediumenterodiolN,N,N′,N′-tetramethylethylenediamineopdbovine serum albuminUrineenterolactoneisoxanthohumolELISAEnzyme-linked immunosorbent assayDaidzeindimethylformamideDimethylsulfoxideSerumfetal bovine serumfast protein liquid chromatographyCoefficient of VariationPearson's correlation coefficientMass spectrometryGenisteinPhosphate-buffered salineMHzmegahertzMolecular Weight Cut OffMonoclonal antibodiesendpolyethylene glycolPEGGENthin-layer chromatographyGas chromatographyHATnegative controlquality controlCoumestrol
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Ciska Wyns, Lara Derycke, Bram Soenen, Selin Bolca, Dieter Deforce, Marc Bracke, Arne Heyerick,