ELISA assays were carried out using standard protocols68 in 96-well Maxisorb plates (NUNC; Thermo Scientific). Cells were diluted to a 0D600 of 0.2 (2 × 108 c.f.u. ml−1), with 50 μl added per well. All the strains were assayed in triplicate. Primary antibody AD6 against HMW34 (link) was used at a starting concentration of 1:200 (NTHi strains C486 and 477) or 1:10,000 (NTHi strain 723) and serially diluted two-fold in 1 × PBS pH 7.9. Secondary antibody (goat anti-mouse HRP conjugate; Sigma-Aldrich) was used at a concentration of 1:10,000. Antibody was detected using TMB single-substrate solution as recommended by the manufacturer (Sigma-Aldrich). The data were plotted as antibody dilution (x axis) versus absorbance at 450 nm (y axis), and data from specific titres in the linear range of the response curve picked for statistical analysis.
Tmb single substrate solution
Manufactured by Merck Group
TMB single-substrate solution is a laboratory reagent used as a colorimetric substrate in various enzyme-linked immunoassays (ELISAs) and other immunochemical techniques. It is a ready-to-use solution that contains 3,3',5,5'-Tetramethylbenzidine (TMB) as the chromogenic substrate.
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Lab products found in correlation
2 protocols using tmb single substrate solution
1
ELISA Assay for NTHi Antibody Titration
2
ELISA Assay for HMW34 Antibody
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ELISA assays were carried out using standard protocols68 in 96-well Maxisorb plates (NUNC; Thermo Scientific). Cells were diluted to a 0D600 of 0.2 (2 × 108 c.f.u. ml−1), with 50 μl added per well. All the strains were assayed in triplicate. Primary antibody AD6 against HMW34 (link) was used at a starting concentration of 1:200 (NTHi strains C486 and 477) or 1:10,000 (NTHi strain 723) and serially diluted two-fold in 1 × PBS pH 7.9. Secondary antibody (goat anti-mouse HRP conjugate; Sigma-Aldrich) was used at a concentration of 1:10,000. Antibody was detected using TMB single-substrate solution as recommended by the manufacturer (Sigma-Aldrich). The data were plotted as antibody dilution (x axis) versus absorbance at 450 nm (y axis), and data from specific titres in the linear range of the response curve picked for statistical analysis.
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