Indirect ELISA Protocol

This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

Coating Antigen to Plate

1. Coat the wells of a 96 well plate with 100μL of the desired antigen diluted in bicarbonate/carbonate solution. Include a serial dilution of the antigen for analysis.

2. Cover plate with parafilm or plastic adhesive and incubate overnight at 4°C.

3. Remove coating solution and wash plate 2 times with 200μL Phosphate-buffered saline +0.05% Tween20 (PBST). The solutions or washed are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.

Blocking

1. Block the remaining protein binding sites in the coated wells by adding 200μL of blocking buffer(1% milk/PBS or 1%BSA/PBS).

2. Cover the plate and incubate for 2 hours at room temperature (RT).

3. Wash plate 2 times with 200μL PBST.

Antibody Incubation

1. Add 100uL of antibody diluted in blocking buffer.

2. Cover the plate and incubate for 2 hours at RT.

3. Wash plate 2 times with 200μL PBST.

4. Add 100uL of conjugated secondary antibody diluted in blocking buffer.

5. Cover the plate and incubate for 2 hours at room temperature.

6. Wash plate 2 times with 200μL PBST.

Detection

1. Add 100uL of the substrate solution per well.

2. After sufficient color development, add 100μL of stop solution to the wells.

3. Read the absorbance of each well with a plate reader.

Analysis

1. Prepare a standard curve from the data produced from the serial dilutions with a concentration on the X-axis vs. absorbance on the Y-axis. Interpolate the concentration of the sample from this standard curve.

Review our ELISA protocols:

Direct ELISA protocol 

Sandwich ELISA protocol