Horseradish peroxidase hrp labeled goat anti mouse igg antibody

Microtiter plates were coated overnight at 4 °C with 1 × 10⁸ CFU/mL of the inactivated B. pseudomallei. After washing with PBS containing 0.05% Tween-20 (PBS-T), the plates were blocked at 37 °C for 60 minutes with PBS containing 0.5% Tween-20 and 5% non-fat milk (blocking buffer). The plates were then incubated at room temperature for 90 minutes with samples diluted in a blocking buffer. After incubation, the plates were washed with blocking buffer and incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (Southern Biotech, Birmingham, AL, USA) at room temperature for 60 minutes, followed by washing with PBS-T. The analysis was also carried out with HRP-labeled goat anti-mouse IgG3, IgG1, IgG2a, and IgG2b antibodies (Southern Biotech, Birmingham, AL, USA) to determine the subclass of the isolated mAb. The plates were developed by adding tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, Grand Island, NY, USA) into each well and stopped after 30 minutes with 1 M H₃PO₄. The OD₄₅₀ was then read.

96-well medium-binding microtiter plates (Grenier Bio-One, Kremsmünster, Austria) were coated with 1.25 μg/mL of F. tularensis LPS overnight. The plates were then washed 3x with PBS containing 0.05% Tween 20 (PBS-T) and blocked for 90 min at 37 °C in PBS containing 0.5% non-fat milk and 0.1% Tween 20 (blocking buffer), followed by a second wash in PBS-T. Primary antibody in the form of mouse immune serum, hybridoma supernatant or purified antibody (1 μg/mL) was added to the first well and serial two-fold dilutions performed across the plate. The plate was incubated at room temperature for 1 h. The plate was then washed with PBS-T and incubated with horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibody (SouthernBiotech, Birmingham, AL, USA), either whole IgG or isotype specific, at a 1:1000 dilution in blocking buffer for 1 h at room temperature. The plate was washed a final time in PBS-T and incubated with tetramethylbenzidine (TMB) substrate (SeraCare, Milford, MA, USA) for 30 min. The reaction was stopped with 1M H₃PO₄ and the absorbance read at OD₄₅₀.

Microtiter plates were coated with 100 μL/well of 0.005% poly-L-lysine (ThermoFisher Scientific, Grand Island, NY) for 90 minutes at 37°C. Plates are then washed with PBS and coated with 2.5 μg/mL eVLP (100 μL/well) overnight at room temperature. The plates are washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked for 90 minutes at 37°C with blocking buffer (5% non-fat milk, 0.5% Tween 20 in PBS), followed by another washing step with PBS-T. The primary antibody was added to the first well and serial two-fold dilutions were performed across the plates; starting concentration of 1 μg/mL for purified mAb or 1:1000 dilution of mouse serum (100 μL/well). After a 60-minute incubation at room temperature, the plates were washed with blocking buffer and incubated with horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibody (SouthernBiotech, Birmingham, AL) or IgG subclass-specific goat anti-mouse antibody (SouthernBiotech, Birmingham, AL) at 1:5000 dilution (100 μL/well). The plates were washed with PBS-T and incubated with tetramethylbenzidine substrate (100 μL/well) (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). The reaction was stopped after 30 minutes with 1M H₃PO₄ (100 μL/well). Plates were read at an optical density of 450nm (OD₄₅₀).