Detection of Human β-Defensins by Western Blot and ELISA

Human beta-defensins (hBDs) are epithelial cell derived antimicrobial and immunomodulatory peptides. Detection of these proteins in body fluids has not been well established. Objective: To conduct western blot and ELISA protocols for efficient detection of hBD2 and hBD3. Methods: Western blot analysis involved using recombinant hBD2 (rhBD2) or rhBD3 (Quinones-Mateu et al, AIDS, 2003) (100ng-200pg) and detecting using various polyclonal antibodies, respectively. For ELISA, rhBD2 peptide (80ng/ml-1pg/ml) was measured using an antibody sandwich technique. HBD2 antibody (Peprotech, Rocky Hill, NJ) was coated on Nunc Maxisorp ELISA plates (Fisher, Pittsburg, PA). After blocking with BSA, rhBD2 was added, followed by addition of the biotin conjugated antibody. POD conjugated streptavidin, followed by ABTS (Roche Diagnostics, Alameda, CA), were used to measure signal at 415 nm with a multichannel microplate reader. Results: Western analysis showed vendor dependent antibody detectability. The anti-hBD2 antibody from Santa Cruz (Santa Cruz, CA) detected down to 1.0 ng; Peprotech, 6 ng; Cell Sciences (Canton, MA), 10 ng; anti-hBD2 from H. Komatsuzawa (Hiroshima University, Japan) via T. Kawai (Forsyth Institute, Boston,MA), 100 ng; Alpha Diagnostic (San Antonio, TX), no detection at 100 ng. The anti-hBD3 antibody from Orbigen (San Diego, CA) detected down to 3.0 ng, as did the product from Novus Biologics (Littleton, CO). ELISA for hBD2 showed a linear detection range from 2 ng to 7.86 pg. Neither canine nor orcine salivary mucins interfered with detection. The ELISA detected presumptive hBD2 in human saliva which, when spiked with rhBD2 did not mask its detection. Conclusion: Sensitivity of detection of rhBD2 by Western blot is anti-hBD2 antibody vendor dependent. Our ELISA is able to detect pg amounts of rhBD2. Future studies will determine if this can be used to reliably assess hBD levels in body fluids. Supported by NIH/NIDCR RO1 DE015510.