DOT ELISA

 

DOT ELISA

 

AIM:

 

To learn the technique Dot ELISA for the detection of antigen or antibody in the given clinical sample

 

PRINCIPLE:

 We can detect different types of antigen and antibody based on the antigen and antibody interaction by ELISA. In which Dot ELISA is a qualitative type of test and detect the antigen or antibody rapidly. . In Dot-ELISA, small volumes of antibodies are immobilized on a protein binding membrane (Nitrocellulose) and the other antibody is linked to an enzyme Horse radish perxoidase (HRP). The test antigen at first reacts with the immobilized antibody and later with the enzyme-linked antibody. The amount of enzyme linked antibody bound is determined by incubating the strip with an appropriate substrate (Hydrogen peroxide, H2O2) and a chromogen [Tetramethylbenzidine (TMB)]. HRP acts on H2O2  to release nascent oxygen, which oxidizes TMB to TMB oxide, which gives, a blue coloured product. The latter precipitates onto the strip in the area of enzyme activity and appears as a coloured dot, hence the name Dot-ELISA. The results can be visualized in naked eye. The enzyme activity is indicated by intensity of the dot, which is directly proportional to the antigen concentration.

MATERIALS REQUIRED:

 

Test tubes, Distilled water, Micropipette, Tips

PROCEDURE:

1. Take 2 ml of 1X Assay Buffer in a test tube and add 2 µl of the test serum sample. Mix thoroughly by pipetting. Insert a Dot-ELISA strip into the tube.

2. Incubate the tube at room temperature for 20 minutes. Discard the solution.

 

3. Wash the strip two times by dipping it in 2 ml of 1X Assay Buffer for about 5 minutes each. Replace the buffer each time.

4. Take 2 ml of 1X Assay Buffer in a fresh test tube, add 2 µl of HRP conjugated antibody to it. Mix thoroughly by pipetting. Dip the ELISA strip into it and allow the reaction to take place for 20 minutes.

5. Wash the strip as in step # 3 for two times and in a collection tube takes 1.3 ml of TMB/H2O2 and dips the ELISA strip into this substrate solution. Observe the strip after 5 - 10 minutes for the appearance of a blue spot and Rinse the strip with distilled water.

RESULT AND OBSERVATION:

 

Observe the appearance of colorful line on strip indicates the positive reaction.

DISCUSSION:

Color line indicates the positive control zone and no color in the negative control zone indicates proper performance of test. In the negative control zone the immobilized antibody is not present and the region is blocked with an inert protein. Therefore, there is no reaction when the reagents are added and no spot can be seen.

In the test zone an antibody is immobilized on it and then blocked with an inert protein. The test serum binds to this region and the HRP-labeled antibody binds to serum which when reacts with substrate develops blue dot. In the positive control zone, the test serum binds to the immobilized antibody and the HRPlabeled antibody binds to serum which when reacts with substrate develops blue dot.