Immunoelectrophoresis. Teaching Kit Manual. GeNei TM. Cat No. New Cat No. KT KT47A KT47B Revision No.

Transcription

1 Rocket Immunoelectrophoresis Teaching Kit Manual Cat No. New Cat No. KT KT47A KT47B Revision No.:

2 CONTENTS Page No. Objective 3 Principle 3 Kit Description 5 Materials Provided 6 Procedure 8 Result 10 Ordering Information 11 1

3 Objective: To learn the technique of rocket immunoelectrophoresis (RIEP). Principle: (RIEP) also known as electro-immuno diffusion is a simple, quick and reproducible method for determining the concentration of antigen (Ag) in an unknown sample. Various concentrations of antigen are loaded side by side in small circular wells along the edge of an agarose gel that contains the specific antibody (Ab). On electrophoresis, the antigen begins to migrate towards the anode and interacts with antibody molecules to form a soluble antigen-antibody complex. However, as the samples electrophorese farther through the gel, more antibody molecules are encountered that interact with the antigen and when the equivalence point is reached, the Ag-Ab complex precipitates. This precipitin line is seen in the form of a rocket. (Refer fig 1). 2 Fig 1: RIEP Gel after Electrophoresis 3

4 Higher the amount of antigen loaded in the well, farther the antigen will travel through the gel. Hence, with increasing antigen concentration, a series of rockets of increasing heights are seen that is proportional to amount of antigen in the well. Therefore, a direct measurement of the height of rocket will reflect upon the antigen concentration. A standard graph of antigen concentration versus peak height is then constructed and from the peak height of the unknown sample, concentration of antigen is determined. Kit Description: In this kit, a standard antigen (BSA) is supplied at four different concentrations (0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml) along with two different concentrations of test antigen (BSA). These will be electrophoresed on an agarose gel containing the antiserum (Goat anti-bsa) and observed for formation of rocket. The concentration of antigen in the test sample will be determined from the standard graph of antigen concentration versus rocket height. KT47 : Kit is designed to carry out 5 RIEP experiments. A single antiserum vial is supplied for five experiments.the kit also includes immunoelectrophoresis equipment with accessories (ETS-2) required for immunoelectrophoresis. KT47A : KT47B : Kit is designed to carry out 5 RIEP experiments. A single antiserum vial is supplied for five experiments. Kit is designed to carry out 20 RIEP experiments. Two antiserum vials are supplied, each for 10 experiments. Note : Immunoelectrophoresis equipment with accessories is required for KT47A and KT47B. Duration of experiment: Approximately 3 hours. 4 5

5 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. Note: Sample of semi-log graph sheets are provided in the manual. Photocopy as required. *Supplied in lyophilized form, refer note for reconstitution. Materials Required: Quantity Materials KT47/47A KT47B Store (5 expts.) (20 expts.) Agarose 1 g 2 g 4 C 5X Electrophoresis buffer 250 ml 3 x 250 ml 4 C Antiserum* 5 ml 2 x 10 ml 4 C Standard Antigen 0.1 ml 0.25 ml 4 C (A, B, C & D) (each) (each) Test Antigen 0.1 ml 0.25 ml 4 C (1 and 2) (each) (each) Glassware : Conical flask, Measuring cylinder. Reagents : Alcohol, Distilled water. Other Requirements: Micropipette, Tips. Note: Read the entire procedure before starting the experiment. Reconstitute the antiserum vial by adding 5 ml of distilled water, in case of KT47 and KT47A. Store the sample at 4 C. Use within 3 months. Reconstitute the antiserum vial by adding 10 ml of distilled water, in case of KT47B. Store the sample at 4 C. Use within 3 months. Reconstitute both the vials, only if all 20 experiments will be carried out within 3 months. Dilute required amount of 5X Electrophoresis buffer to 1X concentration with distilled water before use. Standard antigen of the following concentrations are provided: mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml. Two different test antigens are provided; use any one of them per experiment as test sample. Wipe the glass plate with alcohol thoroughly to make it grease free for even spreading of agarose. Ensure that temperature of molten agarose is around 55 C while adding antiserum. Higher temperature will cause denaturation of the serum proteins while lower than 55 C will cause the gel to set prematurely. 6 7

6 Procedure: 1. Prepare 10 ml of 1.0% agarose (0.1 g/10 ml) in 1X electrophoresis buffer by heating slowly till agarose dissolves completely. Take care not to scorch or froth the solution. 2. Allow the molten agarose to cool to 55 C. 3. Add 1 ml of antiserum to 10 ml of agarose solution. Mix gently, ensure uniform distribution of antiserum. 4. Pour the mix onto a glass plate placed on a horizontal surface and allow it to gel/solidify. 5. Place the glass plate on the template holder provided (in ETS-2) and fix the RIEP template. 6. Punch 3 mm wells with gel puncher towards one edge of the plate. 7. Place the glass plate in the electrophoresis tank, ensure that the wells are towards the cathode. 8. Fill the tank with 1X electrophoresis buffer till it covers the gel. 9. Connect the power cord to the electrophoretic power supply according to the convention: red : anode and black : cathode. 10. Add 10 µl each of the given standard antigen and test antigen to the wells. Loading of wells should be carried out quickly to minimize diffusion from the well. 11. Electrophorese the samples at 100 volts, till the rockets are visible or the dye front reaches the edge. This generally takes 1 to 1 1 / 2 hours. Electrophoresis can be continued for an additional 15 minutes after the dye has run out of the gel. This ensures better visibility of the precipitation peaks. 12. Stop electrophoresis, remove the glass plate from the electrophoresis tank. 13. Observe the precipitation peak or rocket formed against a dark background. If the rockets are still not clear, incubate the plate in a moist chamber at room temperature for 1 hour to overnight. 14. Measure the rocket height from the upper edge of the well to the tip of the rocket. Record your observations as in table Construct a standard graph by plotting the height of the rocket on Y-axis (linear scale) against the concentration of antigen on X-axis (log scale) on a semi-log graph sheet. 16. Determine the concentration of antigen in the test sample by reading the concentration against the rocket height from the standard graph. For example, if following are the results obtained for an RIEP assay, plot the graph as shown in fig. 2. Sample Std Conc. Rocket height No. (in mg/ml) (in mm) A B C D Test (1/2) Table 1: Results of RIEP Assay 8 9

7 40 Ordering Information Rocket height (mm) Product Size Cat # Rocket 1 Pack KT47 Immunoelctrophoresis (RIEP) Teaching Kit (Consumables for 5 experiments & Elpho Kit (ETS-2)) Rocket 1 Pack KT47A Immunoelctrophoresis (RIEP) Teaching Kit (Consumables 5 experiments) Rocket 1 Pack KT47B Immunoelctrophoresis (RIEP) Teaching Kit (Consumables 20 experiments) Antigen concentration (mg/ml) Fig 2: Standard curve for RIEP assay. From the graph, height of 19 mm corresponds to a concentration of 0.26 mg/ml. Therefore, concentration of antigen in test sample = 0.26 mg/ml. Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com Result: From the standard curve, determine and report concentration of antigen in the test sample

8 Standard curve for RIEP assay. Standard curve for RIEP assay.