IMMUNOLOGY

BSC6BT02

Experiment 1

Aim of the Experiment

To learn the technique of Dot ELISA for the detection of an antigen.

Introduction:

Enzyme linked immunosorbent assay or ELISA is a sensitive immunological technique to detect the presence of a specific antigen (Ag) or antibody (Ab) in a biological sample. It utilizes the dual properties of antibody molecules being specific in reactivity and their ability to be conjugated to active molecules such as enzymes. An enzyme conjugated with an antibody reacts with a chormogenic colourless substrate to generate a coloured reaction product. ELISA is extensively used for diagnostic purpose which utilizes the dual properties. It requires an immobilized antigen/antibody bound to a solid support (e.g. microtitre plate or membrane). There are different types of ELISAs for the detection of a protein of interest in a given sample. One of the most common ELISA is dot ELISA which can visually detect the presence of an antigen very quickly. The nitrocellulose dot technique was first developed for screening large number of hybridoma antibodies in 1983.

Principle

There are various forms of ELISA for the detection of antigen or antibody based on antibody- antigen interactions. Dot ELISA, a qualitative ELISA test, can be performed very quickly with the end detection done visually. Because of its relative speed and simplicity, the dot ELISA is an attractive alternative to standard ELISA. 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.

Figure 1: Principle of Dot-ELISA

Important Instructions

Before starting the experiment the entire procedure has to be read carefully.
Always wear gloves while performing the experiment.
Dilute required amount of 10X Assay Buffer to 1X with distilled water, before use.
Do not cross-contaminate reagents.
Never leave the reagents at room temperature.
Ensure that all the three zones of the strip are immersed in solution.

Materials required

Test tubes
Distilled water
Micropipette, Tips
Dot ELISA Strips
Test Serum
10X Assay Buffer: Phosphate buffered saline – Tween (PBST)
Antibody-HRP Conjugate
TMB/H₂O₂
Collection Tubes, Polypropylene (2.0 ml)

Procedure

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

1_Transferring 2 ml of 1X assay buffer to new tube .mp4

2_Addition of 2 ul test sample to assay buffer.mp4

3_Insert dot-blot strip in tube and incubation.mp4

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_wash-1 by dipping in 1x assay buffer .mp4

5_Wash 2 with 1x washing buffer.mp4

4. 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.

6_take 2ml 1X assay buffer and add HRP conjugated antibody.mp4

7_Insert dot-blot strip in tube and incubation.mp4

8_wash 1- after binding of HRP conjugated antibody .mp4

9_wash 2.mp4

6. In a collection tube (provided in the kit) take 1.3 ml of TMB/H₂O₂ and dip the ELISA strip into this substrate solution.

7. Observe the strip after 5 - 10 minutes for the appearance of a blue spot.

8. Rinse the strip with distilled water.

10_Transfer of 1.3 ml of TMB_H2O2.mp4

11_incubation for 5-10 min untill development of blue spot (1).mp4

13_removal of strip.mp4

12_Dipping of ELISA strip in solution containing H2O2 .mp4

Observation and Result

Look for the appearance of the blue dot as shown below in Figure 2.
Record your observations as follows:

Denote +ve : on appearance of a blue spot and

-ve : on absence of a blue spot.

14-development-of-blue-spot.mp4

Figure 2

Observation Table

Interpretation

Spot in the positive control zone and no spot 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 (specific to the test antigen, serum) 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 HRP-labeled antibody binds to serum which when reacts with substrate develops blue dot.

Reference Material

Questions

What is the importance of using blocking buffers ?
What do you mean by Competitive ELISA ?
What is the negative control?
What is the specific substrate for HRP? What color does it produce?
How can the yellow color be quantitatively measured?

Developed by

Dr. Deepika Gupta,

Assistant Professor, Biotechnology

deepika.gupta@gsfcuniversity.ac.in

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