Quantification of human beta-defensin-2 and -3 in body fluids: application for studies of innate immunity.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Ghosh
Santosh K
SK
Department of Biological Sciences, Case School of Dental Medicine, Cleveland, OH 44106-4905, USA.
Gerken
Thomas A
TA
Schneider
Keith M
KM
Feng
Zhimin
Z
McCormick
Thomas S
TS
Weinberg
Aaron
A
D000293:Adolescent; D000328:Adult; D000368:Aged; D001768:Blister; D001826:Body Fluids / Q000737:chemistry*; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D002584:Cervix Uteri; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007113:Immunity, Innate; D007223:Infant; D008297:Male; D008875:Middle Aged; D009077:Mucins / Q000032:analysis; D012463:Saliva / Q000737:chemistry; D014621:Vagina; D023083:beta-Defensins / Q000032:analysis*
C422950:DEFB103A protein, human; C106641:DEFB4A protein, human; D009077:Mucins; D023083:beta-Defensins