title: Comprehensive defensin assay for saliva.
abstract: Defensins are highly basic cationic peptides that are important components of the innate and adaptive immune response pathways. In addition, these peptides are involved in CD8+ T cell response to HIV-1, increased pulmonary infection risk among cystic fibrosis patients, upregulated levels of HNP-5 for patients with ulcerative colitis and Crohn's disease, and monitoring HNP-3 levels as a tumor classification scheme for cutaneous T cell lymphomas, and have promise in the pharmaceutical field as a new class of antibiotics. Here we present a parallel assay for the alpha (HNP1-3) and beta (HBD1-2) classes of defensins in saliva that are naturally observed in the concentration range of 1 ng/mL to 10 microg/mL. The method utilizes solid phase extraction of saliva samples combined with liquid chromatography-tandem mass spectrometry to identify and quantitate defensin targets. The approach involves limited sample manipulation and is easily amenable to automation. The saliva samples analyzed are derived from a large cohort study focused on examining the role of polymorphisms in genes of innate and adaptive immunity in modulating the response to vaccination for two gastrointestinal tract infections: typhoid and cholera. The alpha-defensin levels observed range from 1 to 10 microg/mL and correlate well with known active concentrations against a wide variety of pathogens. The observed concentration range for beta-defensins was between the detection limit and 33 ng/mL and had a sensitivity level that was comparable to immunoassay-based detection. This method is easily adapted for use in a clinical immunology setting and can be modified for other biological matrixes. This assay will facilitate examination of the production, secretion, and regulation of defensin peptides in a direct fashion to coordinate levels of these compounds with gender, age, response to vaccination, gene copy number, and oral health.
authors.affiliation: Biomarker and Systems Biology Research Program, Research Triangle Institute, 3040 Cornwallis Road, Research Triangle Park, North Carolina 27709-2194, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Gardner
authors.forename: Megan D
authors.initials: MD
authors.lastname: Rowland
authors.forename: Amy Y
authors.initials: AY
authors.lastname: Siu
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Bundy
authors.forename: Diane K
authors.initials: DK
authors.lastname: Wagener
authors.forename: James L
authors.initials: JL
authors.lastname: Stephenson
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D015331:Cohort Studies; D023082:Defensins / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D007111:Immunity, Cellular; D008969:Molecular Sequence Data; D012463:Saliva / Q000737:chemistry*; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000379:methods*; D023084:alpha-Defensins / Q000032:analysis; D023083:beta-Defensins / Q000032:analysis
chemical_list: D023082:Defensins; D023084:alpha-Defensins; D023083:beta-Defensins