Thursday, April 19, 2007

Rocket immuno-electrophoresis (RIEP)

Aim: To perform rocket immuno-electrophoresis (RIEP) for the determination of various concentration of antigen in given unknown sample.

Principle: Rocket Immunoelectrophoresis (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.

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.

Material Required: Conical flask, measuring cylinder, alcohol, distilled water, micro pipette, micro tips

Material provided:
Standard antigen: BSA A: 0.0125 mg/ml
B: 0.25 mg/ml
C: 1mg/ml
Antiserum: Goat anti BSA

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 front 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 fixes 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 10/71 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. Electrophoreses the samples at 100 volts, till the rockets are visible or the dye front reaches the edge. This generally takes 1 to 11/^ 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 1.
15. 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.
Observation:
Result and discussion:
Initially the gel is prepared in given 1xPBS buffer add and adequate amount of anti serum to that and spread it on a plate and allow it to settle than make wells and to each well add the adequate quantity of the standard and test antigen then allow the gel to run through electrophoresis at 100 Volts up to 1 hour. A pattern of rocket is observed. Put the result in the table and plot the graph using the observed result. From the graph calculate the concentration of unknown. From the graph concentration of unknown test sample 1 and2 is found to be 0.81 and 0.75 mg/ml respectively.

9 comments:

kow said...

really very useful... keep doing the great work..

venkatesa said...

Its easy and clear. Thank u:-)

accumaximum said...

Micropipettes are adjustable and carry several tubes that facilitate microscopic lab research.
Pipette

accumaximum said...

Such a great blog. This is excellent for switching to different volumes for adding different substances to the same task.

Micropipette

santhu said...

THIS BLOG IS IS INFORMATIVE

santhu said...

this blog is informative
thank you

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akleinschmit said...

What is the concentration of the Goat anti BSA used in the gel?

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