Amplite® Fluorimetric Goat Anti-Mouse IgG-HRP Conjugate ELISA Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare ELISA plate
2. Prepare peroxidase working solution
3. Add 100 µL/well of peroxidase working solution into the ELISA plate
4. Incubate at room temperature for 15 - 60 minutes
5. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

1. Amplite™ Red Peroxidase Substrate stock solution (200X):
Add 250 µL of DMSO (Component D) into the vial of Amplite™ Red Peroxidase Substrate (Component A). Note: 50 µL of the Amplite™ Red peroxidase substrate stock solution is enough for 1 plate.

2. H₂O₂ stock solution (20 mM):
Add 22.7 µL of 3% H₂O₂ (0.88 M, Component B) into 977 µL of Assay Buffer (Component C). Note: The diluted H₂O₂ stock solution is not stable. The unused portion should be discarded.

1. Goat anti-mouse IgG-HRP conjugate working solution:
Add 2 µL of goat anti-mouse IgG-HRP conjugate (Component E) to 10 mL of PBS with 1% BSA (PBS-BSA, not included). Note: 10 mL of goat anti-mouse IgG-HRP conjugate working solution is enough for 1 plate. The concentration of this goat anti-mouse IgG-HRP conjugate working solution is recommended as an initial concentration to try. The optimal concentration for each particular application should be determined empirically.

2. Peroxidase working solution:
Add 50 μL of Amplite™ Red Peroxidase Substrate stock solution (200X) and 100 μL of H₂O₂ stock solution (20 mM) into 9.85 mL of Assay Buffer (Component C) to make a total volume of 10 mL Peroxidase working solution. Keep from light.

Prepare ELISA plate:

1. Perform all necessary ELISA preparation steps.
   
   
2. Wash the ELISA wells three times with PBS containing 0.02% to 0.05% Tween^® 20 (PBS-Tween) and drain.
   
   
3. Add 100 µL of prepared goat anti-mouse IgG-HRP conjugate working solution into each well.
   
   
4. Incubate at room temperature for 30 minutes. Drain off the HRP conjugate.
   
   
5. Wash the wells three times with PBS-Tween and drain.
   
   

Run peroxidase assay in ELISA plate:

1. Add 100 µL of peroxidase working solution into each drained microplate well containing the samples and controls.
   
   
2. Incubate the reaction at room temperature for 30 minutes or longer, protected from light.
   
   
3. Monitor the fluorescence increase with a fluorescence plate reader at excitation 530 - 570 nm (optimal at 540 nm) and emission 590 - 600 nm. Note: The plate can also be read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection has lower sensitivity compared to fluorescence reading.