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.