Aim:

1. Glassware-Test tubes
2. Kit Contents – Distilled water, Dot elisa strips( 15 Nos.) , Test serum ( 0.035ml ) , 10X Assay
Buffer(20ml), Antibody-HRP Conjugate (0.035ml) , TMB/H2O2 (20ml) , Collection Tubes,
Polypropylene (2.0 ml) (16 Nos).
3. Other requirements: Micropipette, Tips.

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 for diagnostic purposes. In DOT
ELISA antigen is coated on a nitrocellulose membrane (Instead of an ELISA plate). After incubation
unadsorbed antigens are washed out, then rest of the reaction sites are blocked on the membrane.
Specific antibody bind with antigen in the NC membrane, unbound antibodies get washed out. The
antibodies which are bound then detected by the addition of enzyme labelled with secondary
antibody. This complex appears as clear brown dot after addition of specific substrate.There are
different types of ELISAs for the detection of a protein of interest in a given sample. The nitrocellulose
dot technique was first developed for screening large number of hybridoma antibodies in 1983.

PRINCIPLE
The dot enzyme-linked immunosorbent assay (Dot-ELISA) is a highly versatile solid-phase immunoassay
for antibody or antigen detection. 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.

Procedure
1. 20 ml of 1x Buffer is formed from 10x Buffer by adding 2ml of 10x Buffer and 18 ml of water.
2. 2ml of 1x Assay Buffer was taken in a test tube and 2 micro liter of test serum sample was added
in it. Mix thoroughly by pipetting. A Dot-ELISA strip was inserted into the tube.
3. The test tube was incubated at room temperature for 20 minutes . The solution was discarded.
4. The strip was washed twice by dipping it in 2ml of 1x buffer for 5 minutes each. The buffer was
replaced each time.
5. 2 ml of 1x Assay buffer was taken in a fresh test tube ,2 micro liter of HRP conjugated antibody
was added to it. Mixed thoroughly by pipetting. The ELISA strip was dipped into it and the
reaction was allowed to take place for 20 minutes.
6. The strip was washed as In the above step for two times.
7. In a collection tube (Provided in the kit) 1.3ml of TMB/H2O2 was taken and the ELISA strip was
dipped into this substrate solution.
8. The strip was observed after 5-10 minutes for the appearance of a blue spot.
9. The strip was rinsed with distilled water.
Observation
A Positive result will show a visible signal at the location of antigen on the plate or membrane.

Zone Spot
Positive zone Present
Negative zone Absent
Test zone Present

Result
1. The presence of the antigen can be determined by the appearance of a coloured dot on the DOT ELISA
strip.
2. Blue spot is formed in the positive zone because Test Serum binds to an immobilized Antibody and HRP -
Conjugated Antibody binds to serum which when react with Substrate (TMB) develops Blue Spot.
3. Blue spot does not appear in the negative zone because Immobilized Antibody is not present in that
region.
4. There will be appearance of blue spot in the test Zone because an antibody is immobilized on it. The test
serum binds to this region and the HRP- conjugated antibody binds to serum which when reacts with
substrate develops blue spot.

Interpretation

Blue spot is present in the positive zone but absent in the negative zone. It indicates the proper
performance of test. In the Negative control zone the immobilized antibody is not present and the region
is blocked with an inert protein.Therfore,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-Conjugated Antibody
binds to serum which when reacts with substrate develops blue spot. In the positive zone, the test serum
binds to the immobilized antibody and the HRP – conjugate antibody binds to serum which when react
with substrate develops blue spot.
The Intensity of the colour can be used to determine the quantity of the antigen prssent.

Precautions
1.Pipetting should be done carefully .
2.Incubation should not be excedded as it can lead to false results.

1. Advantages
2. 1.It is often preferred because it has high sensitivity and specificity. ELISA also offers more accuracy
compared to other techniques such as radioimmunoassay (RIA) tests
3. 2. Dot -ELISA does not require radioisotopes or costly radiation counters.
4. 3.Dot- ELISA tests provide accuracy and ability to provide quick results.
5. 4.Procedure is simple to use.
6. 5.It can be use for blood screening , hence can be use to detect antigen markers lime HIV.
7. 6.It is eco Friendly and not very expensive.

DISADVANTAGES
1. There is possibility to have false results sometimes.
2. Insufficient blocking of immobilized antigen results in false results
3. Expensive to make antibodies.
4. Antibody Instability
5. Refrigerated transport and storage are required for antibodies