title: Protein unfolding allows use of commercial antibodies in an apolipoprotein M sandwich ELISA.
abstract: apoM is a member of the lipocalin superfamily and circulates in plasma attached to HDL particles. apoM plays a role in cholesterol metabolism and has recently been identified as transporter for the signaling lipid, sphingosine-1-phosphate (S1P), in plasma. S1P is implicated in several inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. The ability to accurately measure apoM is crucial for investigating its biological functions and possible clinical implications. However, reliable commercial methods have been lacking so far. Therefore, we have developed an assay that specifically recognizes human apoM in plasma using commercially available reagents. Commercial apoM antibodies were screened for compatibility in a sandwich ELISA-based assay. One optimal pair of antibodies was chosen, and sample preparation, buffers, and incubation times were optimized to generate a simple and reproducible method. Validation and comparison to a previously described ELISA for apoM confirmed that the assay displays a high degree of sensitivity, specificity, and precision. Our results show that commercially available antibodies can be used to accurately measure human plasma apoM. This method can be implemented in every laboratory and will help promote high quality research.
authors.affiliation: Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark; Department of Biomedical SciencesUniversity of Copenhagen, Copenhagen, Denmark.
authors.forename: Markus Høybye
authors.initials: MH
authors.lastname: Bosteen
authors.affiliation: Department of Clinical Chemistry, University of Lund, Malmö, Sweden.
authors.forename: Björn
authors.initials: B
authors.lastname: Dahlbäck
authors.affiliation: Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark; Department of Biomedical SciencesUniversity of Copenhagen, Copenhagen, Denmark. Electronic address: lars.bo.nielsen@regionh.dk.
authors.forename: Lars Bo
authors.initials: LB
authors.lastname: Nielsen
authors.affiliation: Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark; Department of Biomedical SciencesUniversity of Copenhagen, Copenhagen, Denmark. Electronic address: christina.christoffersen@regionh.dk.
authors.forename: Christina
authors.initials: C
authors.lastname: Christoffersen
mesh_terms: D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D001053:Apolipoproteins / Q000097:blood*; D000075243:Apolipoproteins M; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D054834:Lipocalins / Q000097:blood*; D058767:Protein Unfolding
chemical_list: C400523:APOM protein, human; D058846:Antibodies, Monoclonal, Murine-Derived; D001053:Apolipoproteins; D000075243:Apolipoproteins M; D054834:Lipocalins
keywords: antibodies; cholesterol; enzyme-linked immunosorbent assay; high density lipoprotein; lipoproteins; sphingosine phosphate