Tetramethylbenzidine substrate

Cytokines and nitrite (an indirect indicator of NO production) were measured in cell culture supernatants from BMM and PuM un-treated or treated with either IFN-γ or AEC_sup at 4, 24 and 48 h after the end of infection with BCG. For ELISA determinations, the commercially, TNF, IL-6, CXCL10/Interferon gamma-induced protein 10 kDa (IP-10), IL-1β (R&D Systems) and IL-12, (Mabtech) were used to determine the cytokine levels in the culture supernatants according to the manufacturer's recommendations. The enzyme-substrate reaction was developed using p-nitrophenyl phosphate (Sigma) for IL-12 and tetramethylbenzidine substrate (R&D Systems) for the rest of determinations. Depending on the substrate used, the optical density was measured in a multiscan ELISA reader (Anthos Labtech Instruments, Salzburg, Austria) at 405 or 450 nm. NO production was determined by measuring nitrite concentration using the Griess reaction according to the manufacturers protocol (Sigma).

Determination of anti-P. gingivalis and anti-Escherichia coli LPS-specific antibodies by enzyme-linked immunosorbent assay (ELISA). To optimise our evaluation anti-P. gingivalis antibody assessed by ELISA., we performed two standardised ways for coating: Whole extract or lipopolysaccharide (LPS) components. LPS from P. gingivalis (InvivoGen, Toulouse, France) was coated on 96-well plates (Nunc, Dominique Dutscher, Brumath, France) at 10 µg/mL (diluted in carbonate buffer, pH 9.6) and incubated overnight at 4 °C. We used LPS from Escherichia coli (E. coli, InvivoGen, Toulouse, France) as control with the same dilution. Wells were washed three times with phosphate buffered saline (PBS)-Tween (0.005%). Plasma were diluted to 1:600 in PBS containing 1% of bovine serum albumin (BSA) and incubated (in duplicate) for 2 h at room temperature. Plates were washed as described above and incubated with peroxidase-conjugated goat anti-human IgG H + L (Jackson ImmunoResearch, West Grove, PA, USA) (diluted 1:50 000 in PBS) for 2 h at room temperature. After a final wash, detection was made by tetramethylbenzidine substrate (R&D Systems, Minneapolis, MN., USA). The reaction was stopped by the addition of H₂SO₄ (1M) solution and absorbances were measured at 450 nm.