Dot ELISA GeNeiTM Dot ELISA GeNeiTM

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 Dot ELISA GeNeiTM

CONTENTS

Page No.

™ Objective 3

™ Principle 3

™ Kit Description 4

™ Materials Provided 8

™ Procedure 9

™ Observation & Interpretation 10

™ Ordering Information 11

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Dot ELISA GeNeiTM Dot ELISA GeNeiTM

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.

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Dot ELISA GeNeiTM Dot ELISA GeNeiTM

Kit Description: The schematic representation of the reaction is given below:

In this kit, ELISA strips are supplied having three
well defined zones:
• 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.
Notations
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).

These strips will be used to detect the antigen in the test Substrate.
serum samples supplied, by using a secondary antibody
conjugated to Horse radish perxoidase (HRP). HRP is then Substrate converted into its product.
detected using hydrogen peroxide as a substrate and
Tetramethylbenzidine (TMB) as a chromogen. HRP acts on Blue spot developed on NC membrane.
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. The ELISA Strip
-ve Control zone
HRP Test zone
H2O2 H2O + [O]
+ve Control zone
TMB + [O] TMBO
(Blue)
The Reaction Sequence
If the test sample does not contain the antigen specific Negative Control Zone: In this zone immobilized antibody
to the antibody, there will be no enzyme reaction and no is not present and hence, there is no reaction when the
spot develops. 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.
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Dot ELISA GeNeiTM Dot ELISA GeNeiTM

Note: When sample does not contain specific antigen, Step-3: Add substrate (TMB/H2O2)
reaction follows sequence (a) and when sample contains
specific antigen, it follows sequence (b).
Reaction sequences for negative and positive control remains Incubate 20 minutes, wash
same in sequences (a) and (b). The substrate
(a) (b) binds to the
Step-1: Dot ELISA strip + 1X Assay Buffer +Serum Samples. No binding of
the substrate. antibody
Sequence (a) Sequence (b) Hence, no spot enzyme
develops. conjugate.
-ve Control zone The enzyme
Test zone oxidizes the
+ve Control zone substrate to give
a blue spot.
Incubate for 20 minutes, wash
Appearance of the strip:
(a) (b) Specific Ag
No binding as (a) (b)
present in the
specific Ag is sample binds to
absent in the the Ab
sample. immobilized on
the strip. -ve Control zone
Test zone
Step-2: Add enzyme-antibody conjugate (antibody-HRP). +ve Control zone
No blue spot. Blue Spot

Incubate for 20 minutes, wash

(a) (b) KT12S: The kit is designed to carry out 15 Dot-ELISA tests for
Ab-enzyme antigen. Three different serum samples are supplied,
No binding of conjugate binds one each for 5 experiments.
Ab-enzyme to the antigen
conjugate. that has already
been bound to
Duration of experiment: Approximately 1 hour 30 minutes
the immobilized
Ab on the strip.

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Dot ELISA GeNeiTM Dot ELISA GeNeiTM

Materials Provided: Note:

The list below provides information about the materials • Read the entire procedure before starting the
supplied in the kit. The products should be stored as experiment.
suggested. Use the kit within 6 months of arrival. • Dilute required amount of 10X assay buffer to 1X with
distilled water, before use.
• Reconstitute one test serum sample at a time
Quantity (for 5 experiments) with 0.3 ml of distilled water. Store
Materials KT12S Store at 4°C and use within 3 months.
(15 expts.) • Do not cross-contaminate reagents.
• Do not leave the reagents at room temperature.
Dot ELISA strip 15 Nos. 4°C
• Ensure all three zones of the strip are immersed in
10X Assay Buffer 20 ml 4°C solution.
• Assay buffer: Phosphate buffered saline - Tween
Antibody-HRP conjugate 0.2 ml 4°C
(PBST).
10X TMB/H2O2 2.0 ml 4°C Procedure:
Test Serum samples* 1. In a test tube/vial, take 1 ml of 1X assay buffer and
0.3 ml each 4°C
(A, B & C) 50 µl of serum sample. Mix thoroughly. Insert a
Dot-ELISA strip.
*Supplied in lyophilized form, refer note for reconstitution. 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
Materials Required: 1X assay buffer for about 5 minutes each. Replace the
Glassware : Test tubes or 1.5 ml vials. buffer each time.
Reagent : Distilled water. 4. Take 1 ml of 1X assay buffer in a fresh tube or vial, add
Other Requirements : Micropipette, Tips. 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 8 © Bangalore Genei, 2007 9
Dot ELISA GeNeiTM Dot ELISA GeNeiTM

Observation: Ordering Information

Record your observations as follows:
Product Size Cat #
Test serum sample:
GeNeiTM Dot ELISA 1 Pack KT12S
Zone Spot Teaching Kit
(Consumables for 15 experiments)
Negative zone
Test zone
Positive zone Email:
Sales:
Note: Denote +ve : on appearance of a blue spot. geneisales@sanmargroup.com
-ve : on absence of a blue spot
Customer Support:
Interpretation: geneitechsupport@sanmargroup.com
• 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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Dot ELISA GeNeiTM Dot ELISA GeNeiTM

Notes:

© Bangalore Genei, 2007 12 © Bangalore Genei, 2007