title: Fluorochrome-linked immunoassay for functional analysis of the mannose binding lectin complement pathway to the level of C3 cleavage.
abstract: The humoral response to invading pathogens is mediated by a repertoire of innate immune molecules and receptors able to recognize pathogen-associated molecular patterns. Mannose binding lectin (MBL) and ficolins are initiation molecules of the lectin complement pathway (LCP) that bridge innate and adaptive immunity. Activation of the MBL-dependent lectin pathway, to the level of C3 cleavage, requires functional MASP-2, C2, C4 and C3, all of which have been identified with genetic polymorphisms that can affect protein concentration and function. Current assays for MBL and MASP-2 lack the ability to assess activation of all components to the level of C3 cleavage in a single assay platform. We developed a novel, low volume, fluorochrome linked immunoassay (FLISA) that quantitatively assesses the functional status of MBL, MASP-2 and C3 convertase in a single well. The assay can be used with plasma or serum. Multiple freeze/thaw cycles of serum do not significantly alter the assay, making it ideal for high throughput of large sample databases with minimal volume use. The FLISA can be used potentially to identify specific human disease correlations between these components and clinical outcomes in already established databases.
authors.affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, United States.
authors.forename: Mary C
authors.initials: MC
authors.lastname: Walsh
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Shaffer
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Guikema
authors.forename: Simon C
authors.initials: SC
authors.lastname: Body
authors.forename: Stanton K
authors.initials: SK
authors.lastname: Shernan
authors.forename: Amanda A
authors.initials: AA
authors.lastname: Fox
authors.forename: Charles D
authors.initials: CD
authors.lastname: Collard
authors.forename: Michael
authors.initials: M
authors.lastname: Fung
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Taylor
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Stahl
mesh_terms: D000595:Amino Acid Sequence; D001769:Blood / Q000276:immunology; D050577:Complement C3-C5 Convertases / Q000032:analysis*; D037582:Complement Pathway, Mannose-Binding Lectin / Q000276:immunology*; D005456:Fluorescent Dyes / Q000032:analysis*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D037241:Mannose-Binding Lectins / Q000097:blood*; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000032:analysis*; D008969:Molecular Sequence Data
chemical_list: D005456:Fluorescent Dyes; D037241:Mannose-Binding Lectins; D050577:Complement C3-C5 Convertases; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases