GeNei TM Dot ELISA Teaching Kit Manual

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1 Teaching Kit Manual Cat No. New Cat No. KT12S Revision No.:

2 CONTENTS Page No. Objective 3 Principle 3 Kit Description 4 Materials Provided 8 Procedure 9 Observation & Interpretation 10 Ordering Information 11 1

3 Objective: To perform sandwich 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. 2 3

4 Kit Description: 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. The schematic representation of the reaction is given below: 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 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. H 2 O 2 TMB + [O] HRP H 2 O + [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. 4 The ELISA Strip Substrate. Substrate converted into its product. Blue spot developed on NC membrane. -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. 5

5 Note: When sample does not contain specific antigen, reaction follows sequence (a) and when sample contains specific antigen, it follows sequence (b). Reaction sequences for negative and positive control remains same in sequences (a) and (b). Step-1: strip + 1X Assay Buffer +Serum Samples. Sequence (a) Sequence (b) -ve Control zone Test zone +ve Control zone No binding as specific Ag is absent in the sample. Incubate for 20 minutes, wash (a) (b) Specific Ag present in the sample binds to the Ab immobilized on the strip. Step-2: Add enzyme-antibody conjugate (antibody-hrp). No binding of Ab-enzyme conjugate. Incubate for 20 minutes, wash (a) (b) Ab-enzyme conjugate binds to the antigen that has already been bound to the immobilized Ab on the strip. Step-3: Add substrate (TMB/H 2 O 2 ) No binding of the substrate. Hence, no spot develops. Incubate 20 minutes, wash (a) (b) Appearance of the strip: (a) (b) -ve Control zone Test zone +ve Control zone No blue spot. Blue Spot The substrate binds to the antibody enzyme conjugate. The enzyme oxidizes the substrate to give a 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 6 7

6 Materials Provided: 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. *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. 8 Quantity Materials KT12S Store (15 expts.) strip 15 Nos. 4 C 10X Assay Buffer 20 ml 4 C Antibody-HRP conjugate 0.2 ml 4 C 10X TMB/H 2 O ml 4 C Test Serum samples* (A, B & C) 0.3 ml each 4 C Note: 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). Procedure: 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/H 2 O 2 and 0.9 ml of distilled water, mix thoroughly. Dip the strip in this substrate solution. 7. Observe the strip after minutes for appearance of a blue/grey spot. 8. Rinse the strip with distilled water. 9

7 Observation: Record your observations as follows: Test serum sample: Zone Spot Negative zone Ordering Information Product Size Cat # 1 Pack KT12S 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 Interpretation: 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. Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com Bangalore Genei, Bangalore Genei,

8 Notes: Bangalore Genei, Bangalore Genei, 2007