Materials:
- ELISA plate
- Coating buffer (carbonate/bicarbonate buffer)
- Blocking buffer (such as PBS with 1-5% BSA or milk)
- Primary capture antibody (diluted in coating buffer)
- Sample buffer (such as PBS with 0.05% Tween-20 and 1-5% BSA or serum)
- Samples to be tested (diluted in sample buffer)
- Detection antibody (conjugated to an enzyme, diluted in sample buffer)
- Substrate solution (such as TMB or ABTS)
- Stop solution (such as 1N HCl or 1M sulfuric acid)
- Microplate reader
Protocol:
1. Coat the ELISA plate:
- Dilute the primary capture antibody in coating buffer to the appropriate concentration (usually 1-10 µg/mL) and add 100 µL per well to the ELISA plate.
- Incubate the plate overnight at 4°C or for 2 hours at room temperature.
2. Block the plate:
- Discard the coating buffer and wash the plate 3 times with wash buffer (such as PBS with 0.05% Tween-20).
- Add 200 µL of blocking buffer to each well and incubate at room temperature for 1-2 hours.
- Discard the blocking buffer and wash the plate 3 times with wash buffer.
3. Add the samples:
- Dilute the samples in sample buffer to the appropriate concentration.
- Add 100 µL of each sample to the appropriate wells and incubate at room temperature for 1-2 hours.
4. Add the detection antibody:
- Dilute the detection antibody in sample buffer to the appropriate concentration.
- Add 100 µL of the detection antibody to each well and incubate at room temperature for 1-2 hours.
5. Add the substrate:
- Prepare the substrate solution according to the manufacturer's instructions.
- Add 100 µL of substrate solution to each well and incubate at room temperature for the recommended time (usually 10-30 minutes), protected from light.
6. Stop the reaction:
- Add 50-100 µL of stop solution to each well (depending on the volume of substrate solution used) to stop the reaction.
- Measure the absorbance at the appropriate wavelength (usually 450 nm or 405 nm) using a microplate reader.
Note: The incubation times and temperatures may need to be optimized for each set of antibodies and samples. It is important to avoid contamination and to use proper pipetting techniques. If using a multiwell plate, it may be helpful to use a plate centrifuge for washing steps to ensure complete removal of buffer.
