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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 2-8°C
Other kit content can be stored at room temperature (15-25 °C)

Registered orfice 3
HiMedia Laboratories Pvt Ltd.
sO9001-2015 WH0 Plot No. C-40, Road No. 21Y, MIDC, Wagle Industrial Area,
Thane, (West) 400604, Maharashtra, INDIA Fax:6147 1920
sERFIE GMP CERTIRE Customer Care No.: 00-91-22-6116 9797
Web: www.himedialabs.com
Email : info@himedialabs.com
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or is to be implied with tespect to such in o accurale and complete However n0 warranty or guarantee whatsoever is made
Aim:
detection ol an antigen
Dot ELISA for the
To learn the technique of

Introduction:
or ELISA is a Sens
Enzyme linked immunosorbent assay
a specific antigen (Ag) or antibody (Ab) in a Dioig
being specific in reactivity and their abilty to DE
immunological technique to detect the presence of
Orizes the dual properties of antibody molecules
u tO active molecules such as enzymes. An enzyme

conjugated with an reacts with a CiO

antibody 0Ourless substrate to generate a coloured reaction
product. ELISA is extensively used for diagnost u S e which utilizes the dual properties. It requires an
immobilized antigen/antibody bound to a solld Suppo . microtitre plate or membrane). There are different
of ELISAs for the detection of a protein o mterod given sample. One of the most common ELISA is dot
types
ELISA which can visually detect the presence of an anugen 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, H:02) and a chromogen
letramethylbenzidine (TMB)]. HRP acts on H202 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 tirst allowed to
react with immobilized antibody andthen to the HRP-labeled antibody. The amount of HRP-labeled 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
followed by binding of ythe
presence of aa test antigen by immobillized antibody bound to the membrane
PSence antigen py
substrate. antigen to0 the labeled secondary antibody and is detection by Using appropriate
the labeled secondary a
able 1: Enlists the
materials provided in this tit uidh fheir quantity and recommended storage
Sr. No. Product Code Quantity
Materials Provided Storage
TKC201 15expts
Dot ELISA Strips 15 Nos. 2-8°C
TKC202 Test Serum
TKC203 10X Assay Buffer
0.035 ml 2-8°C
20 ml 2-8°C
TKC204 Antibody-HRP Conjugate
TKC205
0.035 ml 2-8°C
TMB/H202 20 ml
2-8°C
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
reduction in performance. Store all the date of
reagents at 2-8°C. Other kit content canmanufacture
be
without showing any
stored at room
temperature.
Important Instructions:
1. Before starting the
2.
experiment the entire procedure has to be read
Always wear gloves while performing the carefully.
3 Dilute required amount of 10X Assay Buffer toexperiment.
1X with distilled water, before
4. Do not crosS-contaminate reagents. use.
5. Never leave the reagents at room
6 temperature.
Ensure that all the three zones of the
7. strip are immersed in solution.
Assay buffer: Phosphate buffered saline Tween
(PBST).
-

Procedure
1 Take 2 ml of 1X Assay Buffer in
a test tube and add 2 ul of the test serum
pipetting. Insert a Dot-ELISA strip into the tube sample. Mix thoroughly by
2. Incubate the tube at room
3 Wash the strip two times bytemperature
20
ror< minutes. Discard the solution.
oppng t mi ot
buffer each time. 1X Assay Buffer for about 5 minutes each.
Take Replace the
4. 2 ml of 1X Assay Buffer in a
les est tube, add 2 ul of HRP
inetting. Dip the ELISA Strip into it and conjugated antibody to it. Mix
5. Wash the allow the reaction
5. Wash the strip as in step # 3 for two times. to take place for 20
stnpo
6. In a collection tube (provided in the kit) take 1.3 ml of TMB/H:02
minutes.
and dip the
solution. ELISA strip into this substrate
7.Observe the strip after 5- 10 minues Tor tne appearance of a blue

8. Rinse the strip with distilled water spot.

4
Flowchart:

Immobilized antibody
ITMI) Nitrocellulose membrane

Antigen

Treatment of the
membrane with
ITTTIIIm antigen

HRP Labeled Antibody

Treatment of the membrane

with Secondary Antibody

Treatment of the
membrane with TMB
substrate
&

Colour development on
the membrane

CIIIIIIIID
 Observation and Result:
Look for the
appearance of the blue dot as
shown below

Test Zone

Negative Zone
Record your
observations as follows: Positive Zone

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:
Snot in the positive control zone and no
tn tho nenative Spot in the negative control zone
control zone the
Therefore, there is no reaction Wimmooiized antibody is not present and the region is proper performance of test.
indicates
cifie to the test antigen, dgents are added and no spot can be seen. blocked with an inert
hinds to this region and the serum) Is immoolized on it and then blocked In the test zone protein.
an antibody
ihldeidiuoody
blue dot. In the positive control zone, the tes binds to serum with an inert
protein. The test serum
which when reacts with
antibody binds to serum whicn when reacts with serum binds to the substrate develops
substrato immobilized
Jbstrate ddevelops blue
o01ized antibody and the HRP-laheled
dot. HRP-labeled
Please roter disolaimer Overleaf.
Troubleshooting Guide

Sr.No Problem Probable Cause

Solution

No signal Omission of any step steps

Prepare a check-list for the
followed

Insufficient washing or Secondary after

Wash plates thoroughly
antibody.
2 High background antibody concentration is high or incubation with Secondary
concentration.
Contamination in buffer Decrease the antibody
buffer
Use freshly prepared

Technical Assistance:
At HIMedla we pride ourselves on the quality and availability of our technical support. For any Kina o E

assistance mail at mb@himedialabs.com

8C Storage temperature

2°C-

Do not use if package is

damaged

HiMedia Laboratories Private Limited,

Road No. 21Y,
Reg Off. Plot No. C-40,
MIDC, Wagle Industrial Area, Thane,
INDIA.
(West) 400604, Maharashtra,
Web: www.himedialabs.com

12/2024

HTI015-07

PIHTI015
O/1221

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