Tetramethylbenzidine

KLH-specific IgG1, IgG2a, IgE, and IgA were measured by ELISA. Briefly, KLH (0.1 μg/well) or serial dilutions of mouse IgG1, IgG2a, IgE, and IgA standards (highest concentration for all standards: 100 ng/ml) were coated, blocked with 1% BSA in PBS, and incubated with diluted sera (1: 100 for IgG1, IgG2a, IgG2b and 1:10 for IgE) overnight. Detection was performed by monoclonal rat anti-mouse IgG1 (cloneA84–1), IgG2a (clone R19-15) IgG2b (clone R12-3), IgA (clone c10-1), or IgE (clone R35-72) followed by polyclonal peroxidase-labeled goat anti-rat IgG IgG (GE Healthcare). All primary antibodies were from BD Pharmingen. Tetramethylbenzidine (BD Biosciences) was used as substrate. The reaction was stopped with 1.8 M sulfuric acid and detected at 450 nm.

A549 cells were seeded to a density of 50,000 cells per well in a 96-well tissue culture plate for 18 h in RPMI 1640 medium supplemented with antibiotics, 2 mM L-glutamine, and 5% FBS. The following day, cells were exposed 24 h to 15 μM LL-37 in the presence or absence of 0.15 μM SP-A or 0.52 μM rfhSP-A. In some experiments, cells were pre-treated 15 minutes with 10 µM A-438079 to inhibit P2X7 activation or DMSO.
Secreted IL-8 was quantified in supernatants of A549 cells by ELISA (R&D Systems) following the supplier instructions. Briefly, anti-human IL-8 was coated on a 96-well Nunc-Immuno Plate MaxiSorp Surface (Thermo Scientific, Waltham, MA) in PBS overnight. After blocking with PBS and 10% FBS, and extensive washing, samples and standards were incubated for 2 h at room temperature. IL-8 cytokine was detected with biotinylated detection antibody and streptavidin-HRP. The colorimetric reaction was developed with tetramethylbenzidine (BD Biosciences, San Diego, CA) and was stopped with 4 M sulfuric acid. The absorbance at 450 nm was read on an ELISA reader (RT-6100 plate reader, Rayto Life and Analytical Sciences, Shenzhen, China).

Female Balb/c mice weighing 18–20 g were immunized intranasally thrice at two week intervals. The dose of Flg-4M and Flg proteins was 10 μg/mouse and no additional adjuvant was used. The mouse sera were studied two weeks after the second and third immunizations. The sera of non-immunized mice were used as a negative control.
For ELISA, 96-well plates with a high adsorption capacity (Greiner, Germany) were covered with synthetic peptides G-50 (M2ek) and G-37 (M2eh) at a concentration of 5 mg/ml (w/v) in phosphate buffer, pH 7.2–7.4 and kept overnight at 4 °C. Plates were treated with a blocking buffer (0.01 M PBS, pH 7.2–7.4) supplemented with 5 % (v/v) fetal calf serum for 1 h at room temperature and washed three times using PBS with Tween. A series of 1:2 dilutions of the different serum samples, starting with a 1:400 dilution, were loaded onto the antigen-coated plates in the blocking buffer and then incubated for 1 h at room temperature. As a conjugate, rat monoclonal anti-mouse IgG (Invitrogen) labelled with a horseradish peroxidase was used at a 1:2000 dilution. Tetramethylbenzidine (BD Bioscience) was used as a substrate. The reaction was monitored by measuring the OD at 450 nm. The endpoint titres are defined as the highest dilution producing an OD value twice that of the background (serum of non-immunised mice).

Whole bacteria were used to coat ELISA plates in sodium bicarbonate buffer, pH 9.6 for 18 h. Plates were blocked for 1h with Assay Diluent 1x (Assay Diluent, BD Biosciences). The lavage fluid or diluted serum (1:50 in Assay Diluent 1x) was added to the plates (100 µL/well). After 2 h of incubation at room temperature, the plates were washed thoroughly (5 times) with phosphate buffered saline (PBS) plus 0.1% Tween 20 (Synth). Anti-IgG1 and Anti-IgG2a conjugate diluted 1:100 in Assay Diluent 1x (Assay Diluent, BD Biosciences) was added (100 µL/well) and incubated for 1 h. After washing as described earlier, a solution of tetramethylbenzidine (BD Biosciences) was added to the wells and the color change pattern was observed. The stop solution was added (50 µL/well) and the color intensity was measured on a plate reader (Thermoplate, Brazil) at 450 nM.

Crude parasite antigen was prepared as previously described (75 (link)). MaxiSorp Nunc Immuno 96-well plates (Thermo Fisher, Carlsbad, CA) were coated overnight at 4°C with 5 μg/ml or 10 μg/ml of parasite antigen in bicarbonate coating buffer (pH 9.6). The plates were blocked with 10% skim milk for 1.5 h at 37°C and washed with PBS-Tween 20. Serum was diluted 2-fold in 5% skim milk, starting at 1:50 or 1:100. After 1.5 h of incubation, the plates were washed 4 times and incubated with horseradish peroxidase-conjugated total IgG (goat anti-mouse IgG [Bio-Rad, Hercules, CA], goat anti-human IgG [Abcam, Cambridge, United Kingdom]) or IgM (goat anti-human IgM; Chemicon, Tokyo, Japan). Tetramethylbenzidine (BD Biosciences) was added, and the plates were incubated at room temperature for 10 to 15 min and read at 650 nm.