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HiPer® Dot ELISA Teaching Kit
Product Code: HTI015
Number of experiments that can be performed: 15
Duration of Experiment: Approximately 1 hour 30 minutes
Storage Instructions:
The kit is stable for 12 months from the date of manufacture
Store the 10X Assay buffer, Antigen, Test Serum, Antibody-HRP
conjugate, TMB substrate and Dot ELISA Strips at 2-8oC
Other kit content can be stored at room temperature (15-25 oC)
Registered Office
HiMedia Laboratories Pvt Ltd.
WHO Plot No. C-40, Road No. 21Y, MIDC, Wagle Industrial Area, Fax: 6147 1920
15
GMP Thane, (West) 400604, Maharashtra, INDIA. Web: www.himedialabs.com
CERTIFIED Customer Care No.: 00-91-22-6116 9797 Email : [email protected]
Tel: 00-91-22-6147 1919, 6903 4800 [email protected]
The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made
or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein
Index
Sr. No. Contents Page No.
1 Aim 3
2 Introduction 3
3 Principle 3
4 Kit Contents 4
5 Materials Required But Not Provided 4
6 Storage 4
7 Important Instructions 4
8 Procedure 4
9 Flowchart 5
10 Observation and Result 6
11 Interpretation 6
12 Troubleshooting Guide 7
2
Aim:
To learn the technique of Dot ELISA for the detection of an antigen.
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 used for diagnostic purpose which utilizes the dual properties.
It requires an immobilized antigen/antibody bound to a solid support (e.g. microtitre plate or membrane).
There are different types of ELISAs for the detection of a protein of interest in a given sample. One of the most
common ELISA is dot ELISA which can visually detect the presence of an antigen very quickly. The
nitrocellulose dot technique was first developed for screening large number of hybridoma antibodies in 1983.
Principle:
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.
Substrate
Labeled antibody
Coloured product
HRP
Antigen
Immobilized
antibody
Membrane
Figure 1: In Dot ELISA, an antibody is immobilized on a membrane and the test antigen is first allowed to
react with immobilized antibody and then to the HRP-labeled antibody. The amount of HRP-labeled
3
antibody bound is measured by treating the membrane strip with an appropriate chromogenic substrate
which is converted to a coloured precipitate and appears as a dot on the membrane
Kit Contents:
This kit can be used to detect the presence of a test antigen by immobilized antibody bound to the membrane
followed by binding of the antigen to the labeled secondary antibody and its detection by using appropriate
substrate.
Table 1: Enlists the materials provided in this kit with their quantity and recommended storage
Quantity
Sr. No. Product Code Materials Provided Storage
15 expts
1 TKC201 Dot ELISA Strips 15 Nos. 2-8oC
2 TKC202 Test Serum 0.035 ml 2-8oC
3 TKC203 10X Assay Buffer 20 ml 2-8oC
4 TKC204 Antibody-HRP Conjugate 0.035 ml 2-8oC
5 TKC205 TMB/H2O2 20 ml 2-8oC
6 PW1139 Collection Tubes, Polypropylene (2.0 ml) 15 Nos. RT
Materials required but not provided:
Glassware: Test tubes
Reagents: Distilled water
Other requirements: Micropipette, Tips
Storage:
HiPer® Dot ELISA Teaching Kit is stable for 12 months from the date of manufacture without showing any
reduction in performance. Store all the reagents at 2-8oC. Other kit content can be stored at room
temperature.
Important Instructions:
1. Before starting the experiment the entire procedure has to be read carefully.
2. Always wear gloves while performing the experiment.
3. Dilute required amount of 10X Assay Buffer to 1X with distilled water, before use.
4. Do not cross-contaminate reagents.
5. Never leave the reagents at room temperature.
6. Ensure that all the three zones of the strip are immersed in solution.
7. Assay buffer: Phosphate buffered saline – Tween (PBST).
Procedure:
1. Take 2 ml of 1X Assay Buffer in a test tube and add 2 μl of the test serum sample. Mix thoroughly by
pipetting. Insert a Dot-ELISA strip into the tube.
2. Incubate the tube at room temperature for 20 minutes. Discard the solution.
3. Wash the strip two times by dipping it in 2 ml of 1X Assay Buffer for about 5 minutes each. Replace the
buffer each time.
4. Take 2 ml of 1X Assay Buffer in a fresh test tube, add 2 μl of HRP conjugated antibody to it. Mix
thoroughly by pipetting. Dip the ELISA strip into it and allow the reaction to take place for 20 minutes.
4
5. Wash the strip as in step # 3 for two times.
6. In a collection tube (provided in the kit) take 1.3 ml of TMB/H2O2 and dip the ELISA strip into this substrate
solution.
7. Observe the strip after 5 - 10 minutes for the appearance of a blue spot.
8. Rinse the strip with distilled water*.
* Molecular Biology Grade Water is recommended (Product Code: ML064)
Flowchart:
Immobilized antibody
Nitrocellulose membrane
Antigen
Treatment of the
membrane with
antigen
HRP Labeled Antibody
Treatment of the membrane
with Secondary Antibody
Treatment of the
membrane with TMB
substrate
&
Colour development on
the membrane
5
Observation and Result:
Look for the appearance of the blue dot as shown below:
Test Zone
Negative Zone
Positive Zone
Record your observations as follows:
Zone Spot
Positive Zone
Negative Zone
Test Zone
Denote +ve : on appearance of a blue spot and -ve : on absence of a blue spot.
Interpretation:
Spot in the positive control zone and no spot in the negative control zone indicates proper performance of
test. In the negative control zone the immobilized antibody is not present and the region is blocked with an
inert protein. Therefore, 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-labeled antibody binds to serum which when reacts
with substrate develops blue dot. In the positive control zone, the test serum binds to the immobilized
antibody and the HRP-labeled antibody binds to serum which when reacts with substrate develops blue dot.
6
Troubleshooting Guide:
Sr.No Problem Probable Cause Solution
1 No signal Omission of any step Prepare a check-list for the steps
followed
Wash plates thoroughly after
Insufficient washing or Secondary
High incubation with Secondary antibody.
2 antibody concentration is high or
background Decrease the antibody
Contamination in buffer
concentration.
Use freshly prepared buffer
Please refer disclaimer Overleaf.
7
Technical Assistance:
At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of technical
assistance mail at [email protected]
8°C Storage temperature
2°C
Do not use if package is
damaged
HiMedia Laboratories Private Limited,
Reg. Off: Plot No. C-40, Road No. 21Y,
MIDC, Wagle Industrial Area, Thane,
(West) 400604, Maharashtra, INDIA.
Web: www.himedialabs.com
12/2024
PIHTI015_O/1221 HTI015-07
Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in
this and other related HiMedia™ publications. The information contained in this publication is based on our research and development
work and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to
specifications and information related to the products at any time. Products are not intended for human or animal or therapeutic use but
for laboratory,diagnostic, research or further manufacturing use only, unless otherwise specified. Statements contained herein should not
be considered as a warranty of any kind, expressed or implied, and no liability is accepted for infringement of any patents.
HiMedia Laboratories Pvt. Ltd. Reg.office : Plot No. C-40, Road No. 21Y, MIDC, Wagle Industrial Area, Thane, (West) 400604, Maharashtra, INDIA.
Customer Care No.: 00-91-22-6116 9797 Tel: 00-91-22-6147 1919, 6903 4800 Email: [email protected] Website: www.himedialabs.com
