Direct ELISA Protocol
The direct ELISA technique is a assay, whereby, an enzyme-labelled antibody is used to bind to an analyte in a solution. Once bound, the enzyme-labelled antibody can react with a substrate to provide a colour change, allowing for the quantification of the analyte.
| Step | Procedure |
|
1. |
Coat the microtiter plate with antigen/analyte. |
|
2. |
Cover the plate and incubate overnight at 4°C. |
|
3. |
Wash the plate three times with 300ul of wash buffer. |
|
4. |
Add blocking buffer and incubate for 1 hr at 37°C. |
|
5. |
Wash four times with 300ul of wash buffer. |
|
6. |
Add samples and standards to the selected wells at the appropriate concentrations. |
|
7. |
Incubate for 90 minutes at 37°C or overnight at 4°C. |
|
8. |
Wash three times with 300ul of wash buffer. |
|
9. |
Add the biotin-conjugated streptavidin in wash buffer. |
|
10. |
Incubate for 1 hr at 37°C. |
|
11. |
Wash three times with wash buffer. |
|
12. |
Add the substrate solution to the selected wells. |
|
13. |
Incubate at room temperature until the desired colour change is observed. |
|
14. |
Add stop solution. |
|
15. |
Read the absorbance values. |