96 well elisa plate

Immunological binding activity between venoms and antivenoms were determined following a previously described method [24 (link)]. First, 96 well ELISA plates (Nunc) were coated with 100 ng of venom (a separate plate for each Russell’s viper venom sample) prepared in carbonate buffer, pH 9.6 and the plates incubated at 4°C overnight. Plates were washed after each stage using six changes of TBST (0.01 M Tris-HCl, pH 8.5; 0.15M NaCl; 1% Tween 20). Next, the plate was incubated at room temperature for 3 hours with 5% non-fat milk (diluted with TBST) to ‘block’ non-specific reactivity. The plates were then washed and incubated (in duplicate) with DSAV, CRAV, TAAV or HPAV antivenom, at an initial dilution of 1:100, followed by 1:5 serial dilutions across the plate, and then incubated overnight at 4°C. The plates were then washed again and incubated with horseradish peroxidase-conjugated rabbit anti-horse IgG (1:1000; Sigma, UK) for 3 hours at room temperature. The results were visualized by addition of substrate (0.2% 2,2/-azino-bis (2-ethylbenzthiazoline-6-sulphonic acid) in citrate buffer, pH 4.0 containing 0.015% hydrogen peroxide (Sigma, UK), and optical density (OD) measured at 405 nm. The titre is described as the dilution at which absorbance was greater than of the negative control (IgG from non-immunised horses; Bio-Rad, UK) plus two standard deviations.

The performance of the recombinant RDK2 was evaluated by ELISA, and the levels of IgG antibodies (specific for antigen) were detected by enzyme-linked immunosorbent assay (ELISA). The experiment was performed by coating recombinant RDK2 antigen (1 mg/mL) in 96-well ELISA plates (Nunc) overnight at 4 °C diluted in phosphate buffer (PB). The next day, the wells were blocked with 1% bovine serum albumin (BSA) (200 µL/well) at 37 °C for 1 h. For primary antibody binding, serum (100 µL/well) and urine samples (100 µL/well) were added at a dilution of 1:2000 [19 (link)] and 1:10 [16 (link)], respectively, and incubated at 37 °C for 2 h, followed by secondary antibodies at a 1:3000 dilution of horseradish peroxidase (HRP)-conjugated anti-human IgG. Finally, the presence of bound IgG was detected by adding 3,3’,5,5’-Tetramethylbenzidine (TMB) (Sigma-Aldrich, St. Louis, MI, USA), as the substrate, and the reaction was stopped by the addition of 2N H₂SO₄. Optical density values were obtained at 450 nm using a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

In serum samples obtained during the AR, rat mast cell protease II (RMCP-II) concentration was quantified using a commercial ELISA set (Moredun Animal Health, Edinburgh, UK) with slight modifications. In brief, 96-well ELISA plates (Nunc Maxisorp, Wiesbaden, Germany) were coated with anti-rat RMCP-II antibody (overnight, 4°C). After blocking and washing, appropriately diluted serum samples were incubated for 3 h. Peroxidase-conjugated anti-rat RMCP-II antibody was incubated for 2 h and, finally, a 3,3’,5,5’-tetramethylbenzidine solution with H₂O₂ was added, and optical density (OD) was measured on a microtiter plate photometer (Labsystems Multiskan, Helsinki, Finland). Data were interpolated by means of Ascent v.2.6 software (Thermo Fisher Scientific, S.I.U., Barcelona, Spain).

Levels of murine C3a, C5a, CXCL1, CXCL2, IL-1β, IL-6, TNF, MPO, PTX3, and P-selectin in lung homogenates and serum were determined by enzyme-linked immunosorbent assay (DuoSet ELISA, R&D Systems and Cloud-Clone corp) following the manufacturer’s instructions. Levels of Aspartate transaminase, Alanine transaminase, Creatinine, and Creatine Kinase were measured in the serum of WT mice infected or not with 5 × 10⁴ CFU of S. pneumoniae serotype 3 and sacrificed at 36 hr post-infection using a Beckman Coulter apparatus following the procedures indicated by the manufacturer. Human PTX3 was determined with an in-house ELISA as previously described by Jaillon et al. (Jaillon et al., 2014 (link)). Briefly, anti-PTX3 monoclonal antibody (100 ng/well, clone MNB4) in 15 mM carbonate buffer (pH 9.6) was coated overnight at 4°C in 96-well ELISA plates (Nunc). Wells were then blocked with 5% dry milk for 2 hr at room temperature. Cell culture supernatants were incubated for 2 hr at room temperature. Biotin-labeled polyclonal rabbit anti-PTX3 antibody (5 ng/ml) was used for the detection and incubated 1 hr at 37°C. Plates were incubated with peroxidase-labeled streptavidin (SB01-61; Biospa) for 1 hr at 37°C. Bound antibodies were revealed using the TMB substrate (Sigma-Aldrich) and 450 nm absorbance values were read with an automatic ELISA reader (VersaMax; Molecular Devices).

Proteins concentrations were evaluated by absorbance at 280 nm, based on the extinction coefficients derived from the known composition of amino acids of each protein. Extinction coefficients were calculated using the ExPASy ProtParam tool [68 ]. The proteins were concentrated by Amicon ultra concentrators (Millipore, Carrigtwohill, Co. Cork, Ireland), and stored at -20°C in 50% (vol/vol) glycerol.
Affinity-based ELISA was performed by the protocol reported earlier by Barak et al. 2005 [47 ]. The 96-well ELISA plates (Nunc, A/S, Roskilde, Denmark) were coated with the fusion proteins CBM-Cohs at a concentration of 3 nM, and variable concentrations of Xyn-Docs (ranging between 2 pM and 20 nM) were used to detect specific cohesin-dockerin interactions. The interactions with the four XynDocs proteins were examined immunochemically by using anti-xylanase primary antibody and horseradish peroxidase (HRP)-labeled secondary antibody. For comparative purposes, pEC₅₀ was calculated for each binding curve as described earlier [47 ,69 ] and the results were presented in bar graph form.