title: Development of a monoclonal antibody-based ELISA for the hedgehog inhibitors cyclopamine and KAAD-cyclopamine.
abstract: Cyclopamine (1) was isolated from the plant Veratrum californicum Durand (Liliacea) and identified as the teratogen responsible for severe craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in central Idaho. More recently, cyclopamine (1) was found to inhibit the hedgehog (Hh) signaling pathway which plays a critical role in embryonic development and is implicated in several types of cancer. Thus, cyclopamine (1) and cyclopamine derivatives have been targeted as potential pharmaceutical treatments for certain cancers and other diseases associated with the Hh signaling pathway. A monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay was developed to detect and measure cyclopamine (1) and cyclopamine derivatives in biological samples. The limits of detection of the assay for cyclopamine (1), 3-keto-N-aminoethyl aminocaproyl digyrocinnamoyl-cyclopamine (8), and N-(4-l-rhamnopyranosyl-1H-1,2,3-triazol-1-yl)-methylcyclopamine (11) were 2.9 pg, 0.41 pg and 2.6 pg, respectively. This assay was also found to be useful for the detection and measurement of cyclopamine (1) in sera from mice that had been dosed with cyclopamine (1). The simple ELISA method described herein demonstrates the potential of using these techniques for the rapid screening of biological samples for the presence and levels of cyclopamine (1) and other cyclopamine derivatives that are Hh inhibitors with anticancer potential.
authors.affiliation: Poisonous Plant Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1150 E. 1400 N., Logan, UT 84341, USA. stephen.lee@ars.usda.gov
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Lee
authors.forename: Kip E
authors.initials: KE
authors.lastname: Panter
authors.forename: Dale R
authors.initials: DR
authors.lastname: Gardner
authors.forename: Benedict T
authors.initials: BT
authors.lastname: Green
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Welch
authors.forename: Jianjun
authors.initials: J
authors.lastname: Zhang
authors.forename: Cheng-Wei Tom
authors.initials: CW
authors.lastname: Chang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000972:Antineoplastic Agents, Phytogenic / Q000097:blood; D002934:Cinnamates / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors; D057230:Limit of Detection; D051379:Mice; D008810:Mice, Inbred C57BL; D012756:Sheep; D015398:Signal Transduction / Q000187:drug effects; D014704:Veratrum Alkaloids / Q000097:blood*
chemical_list: C542465:3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine; D000911:Antibodies, Monoclonal; D000972:Antineoplastic Agents, Phytogenic; D002934:Cinnamates; D053823:Hedgehog Proteins; D014704:Veratrum Alkaloids; C000541:cyclopamine