Dot ELISA

Dot ELISA
Introduction
• Dot Enzyme-linked Immunosorbent Assay (ELISA) is a very sensitive
immunochemical technique used to detect the presence of a specific protein in
given sample.
• It is regarded as an important diagnostic tool for medical purposes due to its

sensitivity and robustness.
• In Dot ELISA, the antigen in the test sample is sandwiched directly between two
antibodies.
• Therefore, the assay is a type of Sandwich ELISA.

• Dot-ELISA is an alternative to standard ELISA because of its relative speed and
simplicity.
• In Dot-ELISA the result can be visualized with the naked eye as we get a big dot
(that is why it is called Dot ELISA) on the test strip whereas in standard ELISA we
need ELISA plate reader.
• Dot-ELISA is a qualitative test (detection of presence or absence but by standard

ELISA test proteins can be quantified).
• In the current exercise on Dot-ELISA, we will test the sample qualitatively and not
quantitatively.
• Dot ELISA can be performed through Dot ELISA Kit which is performed on a
nitrocellulose and other paper membranes which avidly bind proteins.
• The test membrane kit is divided into three zones:

Test zone: With pre-set immobilized antibody for specific antigen binding.
Positive control zone: With pre-set immobilized antibody bound to antigen.
Negative control zone: Immobilized antibody is not present, hence, no
reaction.
Objective
• After the exercise you will be able to:
- detect antigen using Dot ELISA kit and
- describe principle of Dot ELISA technique.
Materials Required
• Dot- ELISA Kit
• 10X Assay buffer,
• Ab-HRP conjugate (Antibody-Horse Radish Peroxidase conjugate),
• TMB/ H2O2 (Tetramethyl benzidine/Hydrogen peroxide),
• Test serum samples,
• Collection Tubes,
• Dot ELISA Strip
Principle
• Dot-ELISA strip is, a nitrocellulose membrane, coated with antibody that react
with two different epitopes of the same antigen.
• First, the immobilized antibody binds with the antigen present in the test sample.
• Next, the secondary antibody binds to the antigen. The secondary antibody is
linked to an enzyme, Horse Radish Peroxidase (HRP).
• Next, when the substrate is added, the enzyme linked with the secondary
antibody converts the specific chromogenic substrate to an insoluble product as a
colour precipitate.
• The binding of antigen (present in the test serum samples) with the immobilized
antibody is detected using a secondary antibody conjugated to HRP using
hydrogen peroxide as a substrate and Tetramethylbenzidine (TMB) as a
chromogen.
• The enzyme HRP acts on the substrate H2O2 to release oxygen (O2).
• The product O2 then oxidizes the chromogen TMB (Tetra methyl benzidine) to an
insoluble product TMB oxide (TMBO).
• The product TMBO is deposited where enzymes are located, thus giving the blue
coloration.
Schematic representation of the
working principle of Dot Enzyme
Linked Immunosorbent Assay
(ELISA)
Procedure
• 1 ml Assay Buffer + 50 µl Test serum. Mix thoroughly.
• Insert Dot-ELISA strip in the solution and Incubate for 20 min at Room temperature.
• Rinse the strip in 1 ml Assay buffer for 5 min.
• Repeat 3 times with new assay buffer at every wash.
• Dip the strip in 1 ml assay buffer + 10 µl Ab-HRP solution at Room temperature for 20 min.
• Rinse the strip in assay buffer for 5 min by dipping.
• Repeat 3 times.
• Dip the strip in 100 µl of 10x TMB/ H2O2 + 900 µl Double Distilled Water (DDW) solution.
• Observe after 20 min for development of Blue Spot.
• Rinse the strip with DDW to stop the reaction.
Observation
• There are three zones in the Dot-ELISA strip viz. negative control, test and
positive control.
• Spot in the positive control zone and no spot in the negative control zone
indicate the proper performance of the test.
• Since there is no immobilized antibody in the negative control zone, therefore,

no spot can be seen. Whereas immobilized antibodies are there in the positive
control zone so the blue dot develops.
• The immobilized antibody (specific to the test antigen) is present in the test zone
so test serum binds to the region and the HRP-conjugated antibody binds to the
serum and develops a blue dot when reacts with the substrate.
Dot-ELISA strip showing blue dot in positive control and test zone and no dot in a negative control zone
Discussion
• The appearance of the blue spot in the positive control zone and the absence of a
spot in the negative zone is an indication of the correct performance of the test.
• The presence of a specific antigen is detected in the test zone through binding of
the test or sample serum to the specific antibody immobilized that facilitates the
binding of HRP conjugated antibody to the captured test serum, which then
reacts to give blue coloration.
• The level of antigen concentration present in the test sample is specified by the
intensity of the spot that can be directly correlated with the enzyme activity.
Precautions
1. Always wear gloves while performing the exercise.
2. Dilute the 10x Assay buffer to 1x with distilled H2O before use.
3. To avoid cross-contamination of reagents, use and throw the micro tips after use.
4. Do not leave the reagents at room temperature.
References
• IGNOU study material
• Kuby- Immunology
Questions?
Thanks

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Dot ELISA

• It is regarded as an important diagnostic tool for medical purposes due to its

• Therefore, the assay is a type of Sandwich ELISA.

• Dot-ELISA is a qualitative test (detection of presence or absence but by standard

• The test membrane kit is divided into three zones:

• Since there is no immobilized antibody in the negative control zone, therefore,

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