Comprehensive defensin assay for saliva.

Analytical chemistry (2008-12-17)
Michael S Gardner, Megan D Rowland, Amy Y Siu, Jonathan L Bundy, Diane K Wagener, James L Stephenson
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Visiprep Manifold Collection Rack (included with standard and DL models), for use with 24-port model, includes base, 2 support rods, center plate, plate for 10 mm test tubes, 8 retaining clips
Supelco
Visiprep Manifold Collection Rack (included with standard and DL models), for use with 12-port model, includes base, 3 support rods, center plate, plate for 10 mm test tubes, 12 retaining clips
Supelco
Visiprep Manifold Plate (included with standard and DL models unless otherwise noted), for use with 12-port model, 20 mL scintillation vials, not included with standard and DL models
Supelco
Visiprep Manifold Plate (included with standard and DL models unless otherwise noted), for use with 12-port model (standard and DL), 16 mm test tubes
Supelco
Visiprep Manifold Plate (included with standard and DL models unless otherwise noted), for use with 12-port model, 2 mL autosampler vials
Supelco
Visiprep Manifold Plate (included with standard and DL models unless otherwise noted), for use with 24-port model, 16 mm test tubes
Supelco
Visiprep Manifold Plate (included with standard and DL models unless otherwise noted), for use with 24-port model, 2 mL autosampler vials

Social Media

LinkedIn icon
Twitter icon
Facebook Icon
Instagram Icon

MilliporeSigma

Research. Development. Production.

We are a leading supplier to the global Life Science industry with solutions and services for research, biotechnology development and production, and pharmaceutical drug therapy development and production.

© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Reproduction of any materials from the site is strictly forbidden without permission.