Protocol: Indirect ELISA

Reagents

1. Coating Buffer

Anhydrous Na₂CO ₃ , 1.5 g
Anhydrous NaHCO₃, 2.93 g
Distilled water, 1 liter, pH to 9.6
For an alternative coating buffer use ELISA coating buffer (BUF030)

2. Blocking buffer

Phosphate Buffered Saline (PBS) containing 1% w/v BSA
For an alternative blocking buffer, use either ELISA BSA Block (BUF032), ELISA Ultrablock (BUF033), or ELISA Synblock (BUF034)

3. Wash buffer

Phosphate Buffered Saline containing 0.05% v/v Tween^®-20
For an alternative wash buffer use ELISA wash buffer (BUF031)

Recommended Substrates and Stop Solutions

TMB Core+ (BUF062), for use with HRP-conjugated antibodies. Stop with 0.2M H₂SO₄.
pNPP (BUF044), for use with alkaline phosphatase-conjugated antibodies. Stop with 1M NaOH.

Method

1. Coat microtiter plate wells with 100 µl of the antigen solution, at a concentration of between 1-10 µg/ml in coating buffer. Cover the plate and incubate overnight at 4°C. Wash the plate 3 times in wash buffer.
2. Add 150 µl of blocking solution to each well. Incubate for 1 hour at 37°C. Wash 4 times in wash buffer.
3. Add 100 µl of unconjugated detection antibody (appropriately diluted in wash buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.
4. Add 100 µl enzyme-conjugated secondary antibody (appropriately diluted in wash buffer) to each well. Incubate for 1 hour at 37°C. Wash 3 times in wash buffer.
5. Add 100 µl of the appropriate substrate solution to each well. Incubate at room temperature (and in the dark if required) for 30 minutes, or until desired color change is attained.
6. Read absorbance values immediately at the appropriate wavelength or add 50 µl of “stop solution”. Gently tap plate to ensure thorough mixing. Measure absorbance within 30 minutes.