Comprehensive defensin assay for saliva
Gardner, M., Rowland, M. D., Siu, A. Y., Bundy, J., Wagener, D., & Stephenson, J. (2009). Comprehensive defensin assay for saliva. Analytical Chemistry, 81(2), 557-566. DOI: 10.1021/ac801609r
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 ? (HNP1?3) and ? (HBD1?2) classes of defensins in saliva that are naturally observed in the concentration range of 1 ng/mL to 10 ?g/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 ?-defensin levels observed range from 1 to 10 ?g/mL and correlate well with known active concentrations against a wide variety of pathogens. The observed concentration range for ?-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.