The document describes the procedure for performing a Dot ELISA assay. It contains 6 sections - Introduction, Principle, Kit Components & Storage, Procedure, Flow Chart, and Result & Interpretation. The Principle section explains that in Dot ELISA, antigen is coated on a nitrocellulose membrane and detected using an enzyme-labeled secondary antibody, appearing as brown dots. The Procedure section provides instructions for preparing reagents and carrying out the assay, including blocking, antibody incubation, washing, and developing with substrate. The Flow Chart further illustrates the step-by-step process. Positive results appear as brown dots, while negative controls have no color.

1. Page No.
1. Introduction 1
2. Principle 1
3. Kit components & Storage 2
4. Procedure 3
5. Flow Chart 6
6. Result & Interpretation 8

2. INTRODUCTION
ELISA, also called Enzyme Linked Immunosorbent Assay, employs
antigens or antobodies conjugated to enzymes in such a way that
the immunological and enzymatic activity of each component is
maintained. These assays are very sensitive and give accurate
results. The estimation of results can be made either visually or
spectrophotometrically. Various formats of ELISA are available.
Depending on the need and availability of reagents different
reaction format can be used in Dot ELISA.
PRINCIPLE
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.

3. (Enough to perform 15 Experiments)
S.No. Name of Items Quantity Storage
1. Antibody 200µl -20°C
2. Precoated (Antigen) Strips 3x15 Nos 4°C
3. Blocker (Dissolve 100 mg in 5ml 1X PBS) 1.5g 4°C
4. Substrate 30mg 4°C
(Always prepare substrate freshly before each test)
5. Conjugate 10ml 4°C
6. Hydrogen peroxide 500µl 4°C
7. 1X PBS 100ml RT
8. Wash Buffer 100ml x 2 RT
9. 96 well Microtiter Plate 1 No. RT
Important Note : All Kit components are recommended to be
stored at respective storage conditions as mentioned above upon
receiving this kit. Repeated thawing of antigen and antibody is not
recommemnded.
MATERIALS NEEDED BUT NOT PROVIDED
1. Triple Distilled water
2. Glasswares (Conical Flask, Measuring cylinder, Pipette)
3. Micropipette & Pipette Tips

4. WORKING SOLUTION PREPARATION
1. BLOCKING SOLUTION
To prepare 2% blocking solution take of 1X PBS and add 100mg of
blocker provided and mix well.
Note: Prepare freshly everytime before each experiment.
2. SUBSTRATE - Stock Solution Preparation
With the given total quantity of substrate bottle add 1ml of triple
distilled water and mix well by repeat pipetting. Transfer this 1ml to
29ml of triple distilled water. Aliquot this 30 ml stock solution into 3
separate 10 ml storage tubes and wrap it with aluminium foil and
store at -20°C for subsequent usage. This will avoid the loss of
effectiveness of the substrate stock solution at the time of thawing
for the subsequent usage of each test.
SUBSTRATE - Working Standard Preparation
Take 1ml of the substrate stock solution and mix with 1µl of
hydrogen peroxide. The working Standard from substrate stock
thus prepared is sufficient for one experiment. (Prepare this step
freshly before each test.)
PROCEDURE
NOTE : All the micropipettes should be well calibrated and the
pipetting should be accurate to get optimum results. Any small
variations would lead to major differences in the optical density
values.
PRECOATED (ANTIGEN) STRIPS
Precoated Antigen strips are provided in the kit as given in the figure

5. Bring the strips to room temperature and incubate at 37°C for
10-15min. Please note that these precoated strips are used
throughout the procedure of following steps.

7. DOT ELISA
Incubate the strips at 37° C for 10 - 15 mins.
BLOCKING
Place the strips in 3 wells of microtitre plate
Add 300 µl of Blocker solution
Incubate at 37° C for 45 mins.
WASHING
Immerse the strips in 300 µl of wash buffer
Shake vigourously
ANTIBODY INCUBATION
Take 200 µl of diluted antibody in three wells
Dip the strips and incubate at 37° C for 45 mins.
Repeat Washing step

8. CONJUGATE
Dip the strips in 200 µl of conjugate in three wells
Incubate at 37° C for 45 mins.
Repeat Washing Step
SUBSTRATE
Dip the strips in 200 µl of substrate in three wells
Incubate at 37° C for 10 mins.
Stop the reaction using tap water
Air dry for few mins.
Observe the Result

9. RESULT AND INTERPRETATION
The appearance of brown dot indicates the presence of antigen
whereas nothing appears in negative control (NC). No colour is
appeared in the (NC)membrane due to presence of non-specific
antigen in the strip and hence no reaction took place.