1. Coat ELISA plate (96 well plate) with testing antigen (10 μg/ml to 0.01 ng/ml in 50 mM Na2C03, pH 9.6, adjust based on the reactivity of antibody, 100 μl/well. Seal the plate and incubate overnight at 4°C.
2. Wash plate 3 times with PBS-T (0.05 % Tween-20 in PBS).
3. Block plate with 0.2% non-fat dry milk in PBS at room temperature for 1 hour at 4°C Note: Milk should be thoroughly dissolved. It's recommended to dissolve 0.5 g milk in 50 ml PBS (1%) for at least 30 minutes at room temperature with rotation or stirring, then dilute to 0.2 %, and keep rotating or stirring for another 10-15 minutes at room temperature. If high background is experienced, 1% milk in PBS could be applied for both blocking and antibody dilution.
4. Wash plate 3 times with PBS-T.
5. (a) Incubate with biotinylated, affinity-purified rabbit IgG (0.1-0.5 μg/ml in PBS, 100 μl/well) at room temperature for 1 hour, followed by washing 6 times with PBS-T, then go to 8a. (b) Alternatively, incubate with affinity purified rabbit IgG (0.2-1 μg/ml in PBS, 100 μl/well) at room temperature for 1 hour, followed by washing 6 times with PBS-T, then go to 8b.
6. (a) Incubate with HRP-Streptavidin (1:4000-10,000 dilutions) in 0.2 % milk-PBS, 100 μl/well, at room temperature for 1 hour. (b) Incubate with HRP-anti-rabbit IgG (1:3,000-10,000 dilutions of 1 mg/ml or 0.25 μg/ml) in PBS, 100 μl/well, at room temperature for 1 hour.
7. Wash plate 8 times with PBS-T.
8. Develop color using TMB as a substrate (100 μl/well) and incubate at room temperature for 15-30 minutes without shaking.
9. Stop reaction by addition of 2N H2S04 (100 μl/well). Record the absorbance at 450 nm on a plate reader within 30 minutes of stopping the reaction.