title: Development of an enzyme-linked immunosorbent assay for detection of CDCP1 shed from the cell surface and present in colorectal cancer serum specimens.
abstract: CUB domain containing protein 1 (CDCP1) is a transmembrane protein involved in progression of several cancers. When located on the plasma membrane, full-length 135kDa CDCP1 can undergo proteolysis mediated by serine proteases that cleave after two adjacent amino acids (arginine 368 and lysine 369). This releases from the cell surface two 65kDa fragments, collectively termed ShE-CDCP1, that differ by one carboxyl terminal residue. To evaluate the function of CDCP1 and its potential utility as a cancer biomarker, in this study we developed an enzyme-linked immunosorbent assay (ELISA) to reliably and easily measure the concentration of ShE-CDCP1 in biological samples. Using a reference standard we demonstrate that the developed ELISA has a working range of 0.68-26.5ng/ml, and the limit of detection is 0.25ng/ml. It displays high intra-assay (repeatability) and high inter-assay (reproducibility) precision with all coefficients of variation ≤7%. The ELISA also displays high accuracy detecting ShE-CDCP1 levels at ≥94.8% of actual concentration using quality control samples. We employed the ELISA to measure the concentration of ShE-CDCP1 in human serum samples with our results suggesting that levels are significantly higher in serum of colorectal cancer patients compared with serum from individuals with benign conditions (p<0.05). Our data also suggest that colorectal cancer patients with stage II-IV disease have at least 50% higher serum levels of ShE-CDCP1 compared with stage I cases (p<0.05). We conclude that the developed ELISA is a suitable method to quantify ShE-CDCP1 concentration in human serum.
authors.affiliation: Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: Yang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: Brittney S
authors.initials: BS
authors.lastname: Harrington
authors.affiliation: School of Medicine, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: Kevin C N
authors.initials: KCN
authors.lastname: Lau
authors.affiliation: Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: Lez J
authors.initials: LJ
authors.lastname: Burke
authors.affiliation: Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: Yaowu
authors.initials: Y
authors.lastname: He
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Qld 4059, Australia.
authors.forename: Mary
authors.initials: M
authors.lastname: Iconomou
authors.affiliation: Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: James S
authors.initials: JS
authors.lastname: Palmer
authors.affiliation: Colorectal Unit, Princess Alexandra Hospital, Woolloongabba Qld 4102, Australia.
authors.forename: Brian
authors.initials: B
authors.lastname: Meade
authors.affiliation: Wesley Hospital, Auchenflower, Qld 4066, Australia.
authors.forename: John W
authors.initials: JW
authors.lastname: Lumley
authors.affiliation: Mater Research Institute - The University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia. Electronic address: john.hooper@mater.uq.edu.au.
authors.forename: John D
authors.initials: JD
authors.lastname: Hooper
mesh_terms: D000368:Aged; D015703:Antigens, CD / Q000097:blood*; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor / Q000097:blood*; D015815:Cell Adhesion Molecules / Q000097:blood*; D002462:Cell Membrane / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000097:blood* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000097:blood*
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C432044:CDCP1 protein, human; D015815:Cell Adhesion Molecules; D009363:Neoplasm Proteins
keywords: CDCP1; Colorectal cancer; ELISA