Quantification of human β-defensin-2 and -3 in body fluids : Application for studies of innate immunity

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 53; no. 4; pp. 757 - 765
• Main Authors: GHOSH, Santosh K, GERKEN, Thomas A, SCHNEIDER, Keith M, ZHIMIN FENG, MCCORMICK, Thomas S, WEINBERG, Aaron
• Format: Journal Article
• Language: English
• Published: Washington, DC American Association for Clinical Chemistry 01.04.2007
• Subjects: Adolescent; Adult; Aged; Analytical, structural and metabolic biochemistry; beta-Defensins - analysis; Biological and medical sciences; Blister; Body Fluids - chemistry; Bronchoalveolar Lavage Fluid - chemistry; Cervix Uteri; Child; Child, Preschool; Enzyme-Linked Immunosorbent Assay; Female; Fundamental and applied biological sciences. Psychology; Humans; Immunity, Innate; Infant; Investigative techniques, diagnostic techniques (general aspects); Male; Medical sciences; Middle Aged; Mucins - analysis; Saliva - chemistry; Vagina; Human; Body fluid; Clinical biology; Natural immunity; Biochemistry; Molecular biology; Defensin; Quantitative analysis
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2006.081430

Human beta-defensins (hBDs) are epithelial cell-derived antimicrobial and immunoregulatory cationic peptides. Our objective was to establish an analytical tool to quantify inducible hBD-2 and -3 in body fluids. We developed sandwich ELISAs using commercially available capture and detection antibodies and determined optimal assay conditions (with 250 mmol/L CaCl(2)) to overcome masking by endogenous components of body fluids. We used recombinant hBD as calibrators and for recovery testing. hBD-2 and -3 detection limits were approximately 75 ng/L and approximately 3 microg/L, respectively. Mean (SD range) values in saliva samples from healthy donors (n = 60) were 9.5 (1.2-21) microg/L for hBD-2 and 326 (50-931) microg/L for hBD-3. We did not detect hBD-3 in suction blister fluid (BF; n = 10) or bronchoalveolar lavage (BAL; n = 5) from healthy participants. We detected low hBD-2 peptide concentrations in BF and BAL, 0.16 (0.03-0.32) and 0.04 (0-0.049) microg/g total protein, respectively. We observed no correlation of hBD-2 in BF and saliva or BAL and saliva from the same person. In vaginal swabs from healthy women (n = 2), mean hBD-2 and -3 concentrations were 3.42 and 103 microg/g total protein, respectively. Cervicovaginal lavage from the same women contained mean concentrations of 1.46 and 55.5 microg/g total protein. These ELISA assays can measure inducible hBD peptide concentrations in body fluids by overcoming masking effects of anionic molecules. This approach may therefore be applicable for quantifying these peptides in health and disease.