title: Production of monoclonal antibodies against hop-derived (Humulus lupulus L.) prenylflavonoids and the development of immunoassays.
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 C4'-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 IgG(1) secondary antibody with ABTS as a chromogenic substrate. For X the IC(50) value derived from the standard curve was 62.91 ng mL(-1), and for both IX and 8-PN 37.15 ng mL(-1). 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.
authors.affiliation: Laboratory of Pharmacognosy and Phytochemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000, Ghent, Belgium.
authors.forename: Ciska
authors.initials: C
authors.lastname: Wyns
authors.forename: Lara
authors.initials: L
authors.lastname: Derycke
authors.forename: Bram
authors.initials: B
authors.lastname: Soenen
authors.forename: Selin
authors.initials: S
authors.lastname: Bolca
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
authors.forename: Marc
authors.initials: M
authors.lastname: Bracke
authors.forename: Arne
authors.initials: A
authors.lastname: Heyerick
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D003429:Cross Reactions; D005419:Flavonoids / Q000032:analysis / Q000276:immunology*; D027582:Humulus / Q000737:chemistry*; D006825:Hybridomas / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D005419:Flavonoids