Dot ELISA
(Enzyme Linked Immuno-sorbent Assay)
Objective
The objective of this experiment is to-introduce the technique of Dot ELISA (Enzyme Linked
Immuno-sorbent Assay) and its applications. Principle
ELISA is being extensively used as a tool in research as well as in Analytical /diagnostic
laboratories. ENZYME LINKED SORBENT ASSAY has many types. Dot-ELISA, a
technique that shares the same Principles as the enzyme immunoassay, is useful for
qualitative detection of antibodies in the sera of patients against particular antigen (virus or
bacterial infections). DOT ELISA, which enables the demonstration of direct Sandwich assay
for an antigen.
The method requires two antibodies that can react with same antigen. One of the antibodies is
immobilized on a strip & the other linked with an Enzyme. Antigen containing sample is first
added to the immobilized Antibody and allowed to react. Non-reacted sample is washed out
and Enzyme linked antibody is added and allowed to react. The assay is interpreted by
viewing the color spot developed at the final step.
Dot ELISA is demonstrated here using two antibodies and one antigen. ELISA strips has
three pads of nitrocellulose membrane. The lowest pad is fixed with antibody bound antigen
acts as positive control. The middle pad is fixed with antibody which is going to react with
antigen present in the sample. The top pad is blocked with inert protein that acts as a negative
control. These strips are used to find out the presence of antigen in the given samples.
Negative control
Strips are incubated with test sample containing antigen and secondary antibody HRP
enzyme conjugates. Addition of substrate DAB (diaminobenzidine) yield brown color spot, If
the test sample does not contain the antigen specific to the antibody, there will be no enzyme
reaction and no spot develops.
Materials required
1.Dot ELISA Strips
2.Test antigen
3Positive antigen
4.Test serum(antibody)
5.20X Antibody-HRP Conjugate
6.DAB(Chromate)
7.H2O2(Substrate)
8.10X Assay Buffer
9.Substrate buffer
10.Blocker
11. 10X Wash Buffer
Procedure
Dot ELISA strips has three pads called as Upper pad, middle pad and lower pad.
• Two protein samples are provided as Protein-1 and Protein-2.
• Apply 1µl of Positive antigen on to the lower pad using micropipette.
Apply 1µl of Test antigen on to the middle pad using micropipette.
Dry the membrane by incubating at 37°C for 15min.
Detection-Procedure
1. Take fresh 1.5ml centrifuge tube, add 1ml of blocking buffer insert Dot-ELISA strip
[spotted with Test antigen and Positive antigen) and incubate for 10minutes.
2. Wash the strips three times by dipping in 1ml of a wash buffer for 2minutes.
3. Take fresh 1.5ml centrifuge tube, add 1ml of 1X assay buffer to tube.
4. Add 100µl of serum sample (antibody) to 1X assay buffer and mix well.
5. Insert Dot-ELISA strip [spotted with Test antigen and Positive antigen) and incubate for
30-45minutes.
6. Wash the strips three times by dipping in 1 ml of a wash buffer for 2minutes.
Interpretation
Presence of spot in the positive control confirms the proper performance of Reaction.
Spot observed in the middle pad (Test) indicates that presence of antigen in the test sample
against immobilized antibody.
Spot absence in the negative control ensure no cross contamination.
