El tmb kit

Forty-five bovine and goat sera, which were positive for brucellosis, were randomly selected to verify the capability of the predicted peptides in identifying brucellosis through an indirect enzyme-linked immunosorbent assay (iELISA). In addition, KLH was used as negative control and LPS was used as the positive antigen control. For the procedure, in a 96-well microtiter plate (NUNC, Denmark), 100 μL of peptide (30 μg/mL in carbonate buffer solution (CBS), pH 9.6) was added to each well and incubated overnight at 4°C. The wells were blocked with 300 μL/well of 5% skimmed milk powder (Sangon, Shanghai) at 37°C for 2 h, then 100 μL/well of serum was added (1:400 dilution with PBS) and incubated at 37°C for 1 h. HRP-labeled protein G (diluted 1:5,000, PBS) (Thermo, USA) was added and incubated at room temperature for 30 min. After that, an EL-TMB kit was utilized (Sangon) for the coloring step. Optical density was measured at 450 nm (OD450) using an ELISA plate reader (BioTek, USA). During the whole process, plates were washed three times with PBST before each reagent was added.

Purified gp350^1-425-His was coated on 96-well microplates (Corning) (100 ng/well in PBS) for 2 h at 37°C. The plates were washed once and then blocked with blocking buffer (PBS pH 7.4, containing 2% gelatin, 0.5% casein and 0.1% ProClin 300) overnight at 4°C. Then, 5-fold serial dilutions of sera were added to the plates and incubated for 1 h at 37°C. The plates were washed 5 times and incubated for 30 min at 37°C with 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Promega) (1:20,000 dilution). Signals were developed using EL-TMB kit (Sangon Biotech). Absorbance was measured at 450 nm using a microplate reader (Molecular Devices). The cutoff value was set to 0.1 which was determined by the OD₄₅₀ value of preimmune sera.

Wells of 96-well ELISA plates (Corning) were coated with 100 ng/well gp350 or gHgL in PBS by incubation at 37 °C for 2 h. After washing with TBST (Tris Buffered Saline with Tween 20), blocking buffer (PBS containing 0.5% casein, 2% gelatin and 0.1% ProClin 300, pH 7.4) was used to block plates for 2 h at 37 °C. Five-fold serially diluted sera from monkeys or healthy EBV carriers (starting from 1:100) were added to each well, incubated for 1 h at 37 °C and then washed 5 times with TBST. Goat anti-human antibody conjugated with HRP (Promega) was added (1:5000 dilution) and incubated for 30 min at 37 °C. The colorimetric reaction was developed using the EL-TMB kit (Sangon Biotech). Absorbance was measured at 450 nm and 630 nm using a microplate reader (Molecular Devices).