PRINCIPLE
The enzyme linked immunosorbent assay (ELISA) is a sensible, versatile, precise, reproducible, quantitative and qualitative technique for the determination of antigens or antibodies in a biological sample. ELISA is a plate-based assay technique, which is also named enzyme immunoassay (EIA). The technique was designed for detecting and quantifying substances such as proteins and antibodies. In an ELISA, antigen from the sample is attached to a solid surface and then complexed with an antibody. This antibody is linked to an enzyme or with a labeled secondary antibody, which can catalyze substrates and produces detectable signal, most commonly a color change.
MATERIALS
Reagents
- Coating Buffer
- Blocking Buffer
- Washing Buffer
- TMB Solution
- Stopping Buffer
- Coated Antigen
- Detect Antibody
- HRP-conjugated Secondary Antibody
Instruments and Consumables
- Microplate Reader (CMax Plus)
- Eppendoff
- Constant Temperature Incubator
METHODS
1. Coat microtiter plate wells with 100 µl of the antigen at a concentration (e.g. 2 µg/mL) in coating buffer. Incubate overnight at 4℃. Wash the plate 3 times with washing buffer.
2. Add 200 µl of blocking buffer to each well. Incubate for 2 hour at 37℃. Wash 4 times.
3. Add 100 µl of diluted Antibody to the relevant wells. Incubate for 1 hour at 37℃. Wash 4 times.
4. Add 100 µl of HRP-conjugated secondary antibody to each well. Incubate for 1 hour at 37℃. Wash 4 times.
5. Add 100 µl of TMB solution to each well. Incubate at 37℃ for 10-15 minutes.
6. Add 50 µl of stop buffer. Gently tap plate to ensure thorough mixing.
7. Read the OD Value (450 nm).
8. Plate configuration.