DOT ELISA TEACHING KIT       

Cat# BB-ITK110 (10 Reactions)

Aim:

To learn the technique of Dot ELISA to detect a specific protein with the help of that protein specific antibody. It involves the following experiments:

• Coating of the antigen on Dot ELISA Strips.
• Blocking & Incubation with Primary and Secondary Antibody.
• Detection

Principle:

Enzyme-linked immunosorbent assay, commonly known as ELISA is a popular format of analytical biochemistry assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. In this technique, an enzyme conjugated with an antibody reacts with a colourless substrate to generate a coloured product. Such a substrate is called a chromogenic substrate. A number of enzymes have been employed for ELISA, including alkaline phosphatase, (AP) horseradish peroxidase (HRP) etc.

Bio Bharati Dot ELISA Teaching Kit is HRP based. The antigen is first coated on the Dot strip followed by incubation with Primary and HRP-tagged Secondary antibody. The HRP tagged to secondary antibody is detected using hydrogen peroxide as a substrate and 3, 3′-Diaminobenzidine (DAB) as a chromogen. DAB is oxidized by hydrogen peroxide in a reaction typically catalysed by horseradish peroxidase (HRP). The oxidized DAB forms a brown precipitate, at the location of the HRP, which can be easily visualized. 

Kit Description:

In this kit, Dot ELISA is carried with two antibodies and one antigen. The provided strip has three pads of nitrocellulose membrane. The lowest pad is for positive control sample, middle part for test sample and upper part is for negative control. These strips are used to find out the presence of antigen in the given samples. Strips are incubated with test sample containing antigen and, test serum (primary antibody) and HRP-conjugated secondary antibody. Addition of substrate DAB yields a brown colored spot; if the test sample does not contain the antigen specific to the antibody, there will be no enzymatic reaction and no spot develops.

#BB-ITK0100 is designed to perform 10 set of reactions.

Duration of experiment: Experiment is carried out within a day. It will take approximately 2hours to finish the experiment. 

Kit Contents:

Materials required but not provided:

Consumables:        Micro Pipettes, Tips, Micro-centrifuge tube (1.5 ml)

Instruments: 37°C incubator

Storage:

Bio Bharati Dot ELISA Teaching kit is stable for 3 months from the date of receipt without showing any change in performance if stored properly. Once received, store all the components at 4°C.

Important instructions (if any):

• Read the instructions carefully before starting the experiment.
• Final substrate buffer contains hydrogen peroxide which is corrosive to skin, eye and mucous membrane, therefore take utmost care while handling.

Procedure:

Preparation of working stock:

• 1X Assay Buffer: To prepare 10 ml of 1X Assay buffer, mix 1ml of supplied 10X Assay buffer with 9ml of ultra-pure distilled water (recommended to use BioBharati Ultra-Pure Distilled water; Cat # BB-BS60)
• 1X Wash Buffer: To prepare 10ml of 1X Wash buffer, mix 1ml of supplied 10X Wash buffer with 9ml of ultra-pure distilled water (recommended to use Bio Bharati Ultra-Pure Distilled water; Cat # BB-BS60).
• Substrate Solution: Add 1ml of substrate buffer (provided in the kit) to the entire vial of DAB, mix well and then transfer into remaining 29ml of substrate buffer. Store the solution at 4°C.

Note: if any particles are visible, filter the solution with Whatman filter paper before storing at 4°C

• 3% Blocking Buffer: Add 300mg of blocker provided in the kit with 10ml of 1 X Assay buffer, mix well and store at 4°C.

Note: Lumps may be observed if Blocking Buffer is stored in 4°C for more than 1 week. In that case make fresh Blocking Buffer before carrying out the assay.

Spotting Proteins on Membrane:

• Apply 1µl of positive antigen on the lower pad of the strip by micropipette.
• Apply 1µl of test antigen on the middle part by micropipette.
• Dry the membrane by incubating at 37°C for 15minutes.

Incubation with Antibody:

• Add 1ml of blocking buffer to a fresh 1.5ml of micro-centrifuge tube and insert the Dot ELISA strip within it.
• Incubate for 10 minutes at room temperature.
• Wash the strip thrice by dipping in 1ml of 1X wash buffer for 2 minutes.
• Take a fresh 1.5ml micro centrifuge tube and add 1ml of 1X assay buffer into it.
• Add 100µl of provided antibody (“Test serum”) to 1ml of 1X assay buffer and mix well.
• Insert ELISA strip (incubated with test and positive antigen previously) into the antibody solution and keep it for 45 minutes at room temperature.
• Wash the strip thrice by dipping in 1ml of 1X wash buffer for 2 minutes.
• Add 50µl of HRP-conjugated antibody in 1ml of assay buffer in a fresh 1.5ml micro-centrifuge tube and dip the strip into it.
• Incubate for 30minutes at room temperature.
• Wash the strip thrice by dipping in 1ml of 1X wash buffer for 2minutes.

Detection:

• Take 1 ml of substrate solution in a fresh 1.5 ml of micro-centrifuge tube and add 3 µl of H₂O_2* just before development.

Note: H₂O₂ is highly corrosive to skin, eyes and mucus membrane, therefore always wear gloves while handling and take utmost precautions.

• Dip the strip into the substrate solution and keep until brown color appears (takes around 5-10 minutes)
• Stop the reaction by dipping the strip into ultra-pure water and dry the strip.
• Spot on the lower pad indicates the performance of the positive control and middle part indicates the presence of antigen in the test sample.
• Intensity of the spot is proportional to the antigen concentration in the sample spotted on the strip. There should not be any spot in the negative control region.

Interpretation:

• Presence of spot in the positive control confirms the proper performance of the reaction.
• Spot observed in the middle pad indicates the presence of antigen in the test sample against antibody.
• Absence of spot in the negative control ensures no cross contamination.

 Trouble-shooting Guide:

Technical Assistance:

At Bio Bharati, we always aspire to deliver the best products for quality research to our customers. All the reagents are made at our In-house R&D facility with utmost care to give optimum result. We are always happy to serve you at your research help. For any assistance please write to us at [email protected].

Adapted from internet