[PRACTICAL 9] MTEB 2404

DOT ELISA

OBJECTIVE
To perform sandwich DOT ELISA test for antigen

PRINCIPLE

Dot-ELISA (Enzyme Linked Immunosorbent Assay) is an extensively used

immunological tool in research as well as analytical/diagnostic laboratories. In
sandwich Dot-ELISA, the antigen is sandwiched directly between two
antibodies which react with two different epitopes on the same antigen.

Here one of the antibodies is immobilized onto a solid support and the
second antibody is linked to an enzyme. Antigen in the test sample first
reacts with the immobilized antibody and then with the second enzyme-linked
antibody. The amount of enzyme linked antibody bound is assayed by
incubating the strip with an appropriate chromogenic substrate, which is
converted to a colored, insoluble product.

The latter precipitates onto the strip in the area of enzyme activity,
hence the name Dot-ELISA. The enzyme activity is indicated by intensity of
the spot, which is directly proportional to the antigen concentration.

REAGENTS
1. DOT ELISA strip
2. Assay buffer
3. Antibody –HRP-Conjugated
4. TMB / H2O
5. Test serum sample

PROCEDURE

1|Page
 [PRACTICAL 9] MTEB 2404

As stated in pamphlet

NEGATIVE ABSENCE OF BLUE

ZONE SPOT

TEST ZONE
BLUE SPOT
OBESRVED
POSITIVE
ZONE

RESULTS

POSITIVE /
SAMPLES RESULTS
NEGATIVE

Blue spot
A +
observed

Blue spot
B +
observed

Blue spot
C +
observed

Blue spot
D +
observed

2|Page
 [PRACTICAL 9] MTEB 2404

DISCUSSION:
Dot-ELISA is a technique that shares with same principles of enzyme
immunoassay. Dot ELISA was a good test kit because of thier specific and
have high sensivitivity when compared to standard immunobloting assay.
This test was useful in detecting antibodies of antigen in serum.

The antigens were fixed into nitrocellulose strips for 20 minutes, and
then washed. After incubation with Horse radish peroxidase (HRP),
washing, and addition of substrate, positive reactions appear as brown
dots against the white background. The room temperature assay takes
about 2 hr.

The sensitivity and specificity of the assay are 90-95% and 90%,
respectively. The level of positivity of the DOT-ELISA by an arbitrary scale
compares with standard micro-ELISA. The ELISA was 100 percent sensitive, while
dot ELISA was more specific compared to microwell ELISA.

Hence dot-ELISA is preferred compare to microwell ELISA test as because it is

simple to perform, needs less test time, is adaptable for field testing, needs least laboratory
facilities and is more specific.

CONCLUSION:

Presence of spot on the control positive and negative control

indicates the tests are valid, while, the presences of spot at test zone
were detected on the samples of A, B, C, and D.

3|Page