Received on October 9, 2006
Accepted on January 16, 2007

Clinical Immunology

Quantification of -Defensin-2 and -3 in Body Fluids: Application for Studies of Innate Immunity

¹ Department of Biological Sciences, Case School of Dental Medicine
² Department of Pediatrics and Biochemistry, Case Western Reserve University, Cleveland, OH
³ Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH
⁴ Department of Biological Sciences, Case School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106-4905.

^* To whom correspondence should be addressed. E-mail: aaron.weinberg{at}case.edu.

Background: Human -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.

Methods: We developed sandwich ELISAs using commercially available capture and detection antibodies and determined optimal assay conditions (with 250 mmol/L CaCl₂) to overcome masking by endogenous components of body fluids. We used recombinant hBD as calibrators and for recovery testing.

Results: hBD-2 and -3 detection limits were 75 ng/L and 3 µg/L, respectively. Mean (SD range) values in saliva samples from healthy donors (n = 60) were 9.5 (1.2-21) µg/L for hBD-2 and 326 (50-931) µg/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) µg/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 µg/g total protein, respectively. Cervicovaginal lavage from the same women contained mean concentrations of 1.46 and 55.5 µg/g total protein.

Conclusion: 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.