Dot ELISA
GeNeiTM
GeNeiTM
Dot ELISA
GeNeiTM Dot ELISA Teaching Kit Manual
Cat No.
New Cat No.
KT12S
106125
Revision No.: 00300305 © Bangalore Genei, 2007
© Bangalore Genei, 2007
Dot ELISA
GeNeiTM
GeNeiTM
Dot ELISA
CONTENTS Page No.
© Bangalore Genei, 2007
Objective
3
Principle
3
Kit Description
4
Materials Provided
8
Procedure
9
Observation & Interpretation
10
Ordering Information
11
© Bangalore Genei, 2007
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GeNeiTM
Dot ELISA
GeNeiTM
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 coloured, 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.
© Bangalore Genei, 2007
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© Bangalore Genei, 2007
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GeNeiTM
Dot ELISA
GeNeiTM
Dot ELISA
Kit Description:
The schematic representation of the reaction is given below:
In this kit, ELISA strips are supplied having three well defined zones:
Notations
• • •
Negative control zone that is blocked with an inert protein. Test zone having an antibody immobilized on it and then blocked with an inert protein. Positive control zone having the antibody immobilized on it, blocked with inert protein and has a specific antigen bound to the immobilized antibody.
These strips will be used to detect the antigen in the test serum samples supplied, by using a secondary antibody conjugated to Horse radish perxoidase (HRP). HRP is then detected using hydrogen peroxide as a substrate and Tetramethylbenzidine (TMB) as a chromogen. HRP acts on hydrogen peroxide to release oxygen, which oxidizes the TMB to TMB oxide. The TMB oxide is deposited wherever enzyme is present and appears as a blue spot. HRP H2O2 TMB + [O]
H2O + [O] TMBO (Blue)
If the test sample does not contain the antigen specific to the antibody, there will be no enzyme reaction and no spot develops.
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Nitrocellulose (NC) membrane, a solid support for immobilizing Antibody (Ab). A
B
Antigen (Ag) with A & B epitopes. Ab to antigen for epitope ‘A`. Ab to Ag for epitope ‘B` linked with an enzyme (Antibody-enzyme conjugate). Substrate. Substrate converted into its product. Blue spot developed on NC membrane.
The ELISA Strip -ve Control zone Test zone +ve Control zone The Reaction Sequence Negative Control Zone: In this zone immobilized antibody is not present and hence, there is no reaction when the reagents are added. Positive control zone: In this zone antigen is bound to immobilized antibody. The antigen binds to antibody enzyme conjugate in step 2 and develops spot in step 3. Test Zone: The explanations for the reaction sequence in this zone is given on page 6. © Bangalore Genei, 2007
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GeNeiTM
Dot ELISA
GeNeiTM
Dot ELISA
Note: When sample does not contain specific antigen, reaction follows sequence (a) and when sample contains specific antigen, it follows sequence (b).
Step-3: Add substrate (TMB/H2O2)
Reaction sequences for negative and positive control remains same in sequences (a) and (b).
Incubate 20 minutes, wash The substrate (a) (b) binds to the No binding of antibody the substrate. enzyme Hence, no spot conjugate. develops. The enzyme oxidizes the substrate to give a blue spot.
Step-1: Dot ELISA strip + 1X Assay Buffer +Serum Samples. Sequence (a)
Sequence (b)
-ve Control zone Test zone +ve Control zone Incubate for 20 minutes, wash (a)
(b)
No binding as specific Ag is absent in the sample.
Specific Ag present in the sample binds to the Ab immobilized on the strip.
Appearance of the strip: (a)
(b)
-ve Control zone Test zone +ve Control zone
Step-2: Add enzyme-antibody conjugate (antibody-HRP).
No blue spot. Incubate for 20 minutes, wash (a)
(b)
No binding of Ab-enzyme conjugate.
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Ab-enzyme conjugate binds to the antigen that has already been bound to the immobilized Ab on the strip.
Blue Spot
KT12S: The kit is designed to carry out 15 Dot-ELISA tests for antigen. Three different serum samples are supplied, one each for 5 experiments.
Duration of experiment: Approximately 1 hour 30 minutes
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GeNeiTM
Dot ELISA
Materials Provided:
Dot ELISA
GeNeiTM
Note:
The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.
KT12S (15 expts.)
• •
Quantity Materials
•
Store
• • •
Read the entire procedure before starting the experiment. Dilute required amount of 10X assay buffer to 1X with distilled water, before use. Reconstitute one test serum sample at a time (for 5 experiments) with 0.3 ml of distilled water. Store at 4°C and use within 3 months. Do not cross-contaminate reagents. Do not leave the reagents at room temperature. Ensure all three zones of the strip are immersed in solution. Assay buffer: Phosphate buffered saline - Tween (PBST).
Dot ELISA strip
15 Nos.
4°C
10X Assay Buffer
20 ml
4°C
Antibody-HRP conjugate
0.2 ml
4°C
10X TMB/H2O2
2.0 ml
4°C
Procedure:
Test Serum samples* (A, B & C)
0.3 ml each
4°C
1. In a test tube/vial, take 1 ml of 1X assay buffer and 50 µl of serum sample. Mix thoroughly. Insert a Dot-ELISA strip. 2. Allow the reaction to occur at room temperature for 20 minutes. 3. Wash the strip three times by dipping it in 1 ml of 1X assay buffer for about 5 minutes each. Replace the buffer each time. 4. Take 1 ml of 1X assay buffer in a fresh tube or vial, add 10 µl antibody-HRP conjugate to it. Mix thoroughly. Dip the strip; allow the reaction to take place for 20 minutes. 5. Wash the strip as in step # 3, three times. 6. In a fresh tube/vial, take 0.1 ml of 10X TMB/H2O2 and 0.9 ml of distilled water, mix thoroughly. Dip the strip in this substrate solution. 7. Observe the strip after 10 - 20 minutes for appearance of a blue/grey spot. 8. Rinse the strip with distilled water. © Bangalore Genei, 2007 9
*Supplied in lyophilized form, refer note for reconstitution.
Materials Required: Glassware : Test tubes or 1.5 ml vials. Reagent : Distilled water. Other Requirements : Micropipette, Tips.
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•
GeNeiTM
Dot ELISA
Observation:
GeNeiTM
Dot ELISA
Ordering Information
Record your observations as follows: Product Test serum sample:
Size
GeNeiTM Dot ELISA
Zone
Spot
Negative zone
1 Pack Teaching Kit (Consumables for 15 experiments)
Test zone Positive zone
Note: Denote +ve : on appearance of a blue spot. -ve : on absence of a blue spot
Customer Support:
[email protected]
Interpretation: • •
•
Email: Sales:
[email protected]
Spot in positive control zone and no spot in the negative control zone indicates proper performance of test. Spot in test zone indicates presence of specific antigen in the sample. Note: Intensity of the spot will vary depending upon the test sample used. No spot in the test zone indicates the absence of specific antigen in the sample.
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© Bangalore Genei, 2007
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Cat # KT12S
GeNeiTM
Dot ELISA
Dot ELISA
Notes:
© Bangalore Genei, 2007
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© Bangalore Genei, 2007
GeNeiTM
