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Indirect ELISA Protocol

Indirect ELISA Protocol

In an indirect ELISA the secondary antibody binds to the primary antibody similarly to a Western Blot.

Indirect ELISA Protocol

An indirect ELISA is similar to a Western Blot, whereby, a secondary antibody binds to a primary antibody.

Step Procedure

1.

Coat the microtiter plate with antigen/analyte

2.

Cover the plate and incubate overnight at 4°C

3.

Wash 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 detection antibody in wash buffer to the selected wells.

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.