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.