Rocket Immunoelectrophoresis Principle, Procedure, Result, Application.

Rocket Immunoelectrophoresis

• 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. 
• The process of Rocket Immunoelectrophoresis was first introduced by Laurell.
• “Quantification of proteins by electrophoresis into agarose containing the corresponding antibodies has received the nickname rocket” (immuno) electrophoresis because the precipitates developed when the migrating protein antigens are precipitated by the antibodies resemble upright rockets. 
• This technique is much faster than immuno-diffusion, and has been applied in a variety of geometries, to analyze simple or complex samples Agarose gels used in this technique have a small number of fixed charges, which cause a phenomenon known as electroendosmosis.
• This quantitative one-dimensional immunoelectrophoresis method involves a comparison of antigen samples of unknown concentration with a series of dilutions of a known concentration of the antigen and requires a monospecific antibody against the antigen under investigation.
• In this method, antigen migrates from the well through agarose gel containing antiserum, forming rocket-shaped precipitin peaks. The height of this peak is proportional to the concentration of the antigen-loaded in the corresponding well.

Objective

To study the technique of Rocket Immunoelectrophoresis for determination of the concentration of antigen in the unknown sample. 

Principle of Rocket Immunoelectrophoresis

In Rocket Immunoelectrophoresis, negatively charged antigen samples are electrophoresed in an agarose gel containing antibody which is specific to that antigen. As the antigen moves out of the well and enters the agarose gel, it combines with the antibody to form an immune complex which is visible as white precipitin arcs.

Because the antigen is migrated through the gel under the influence of an applied electric current, it moves in one direction. During the initial phase there is considerable antigen excess over antibodies and no visible precipitation occurs. However, as the antigen sample migrates further through the agarose gel, more antibody molecules are encountered that interact with the antigen to form an immune complex. 

When these immune complexes become large enough to be retained within the gel, movement of the antigen stops. The area of precipitin has the shape of a rocket and its height is proportional to the concentration of antigen in the corresponding well. 

Material Required

• Glass wares: Conical flask, Measuring cylinder, Beaker
• Reagents: Sterile distilled water, alcohol
• Other requirements: Incubator (37oC), Microwave or Bunsen burner, Vortex mixer, spatula, Micropipettes, Tips, Moist chamber (box with wet cotton) 

Procedure of Rocket Immunoelectrophoresis

1. Prepare 15 ml of 1 % agarose (as given in important instructions).
2. Cool the solution to 55-60oC and add 250 l of antiserum to 13 ml of agarose solution. Mix well for uniform distribution of antibodies.
3. Pour agarose solution containing the antiserum onto a grease free glass plate placed on a horizontal surface. Allow the gel to set for 30 minutes.
4. Place the glass plate on the template provided.
5. Punch wells with the help of gel punchers. Use gentle suction to avoid forming rugged wells.
6. Add 10 l of the given standard antigen and test antigen samples to the wells.
   • Standard Antigen A (1.87 mg/ml)
   • Standard Antigen B (0.94 mg/ml)
   • Standard Antigen C (0.47 mg/ml)
   • Standard Antigen D (0.23 mg/ml)
   • Test Antigen 1
   • Test Antigen 2
7. Pour 1X TBE buffer into the electrophoresis tank such that it just covers the gel.

Note: The remaining 1X TBE buffer can be stored at room temperature.

8. Electrophorese at 80-120 volts and 60-70 mA, until the blue dye travels 3-4 cm from the well. Do not electrophorese beyond 3 hours, as it is likely to generate heat.
9. Incubate the glass plate in a moist chamber overnight at 37o C.

Observation and Result

Observe precipitation peaks in the shape of ‘Rocket’ formed against a dark background. Mark the tip of the precipitin peaks and measure the peak height from the upper edge of the well to the tip of the peak.

Plot a graph of the rocket height (on Y-axis) versus the concentration of antigen (on X-axis) on a semi-log graph sheet. Determine the concentration of the unknown from the graph by finding the concentration against the rocket height.

Applications of Rocket Immunoelectrophoresis

• It is used for quantitative evaluation of antigen in the serum.
• This technique is used for the quantization of human serum proteins before automated methods became available.
• Used in enzyme activity electrophoresis.
• Studies dealing with antigenic relationships between organisms.
• Used to Determine the concentration of a specific protein in a protein mixture.
• Used for the estimation of immunoglobulin protease activity.

Advantages of Rocket Immunoelectrophoresis

• This method is rapid, reliable, specific, and extremely accurate.
• It is used to determine the concentration of antigen in a complex mixture.
• Contains Standard reagents and protocol for better results.
• A single plate can be used to analyze the Several unknown samples

Disadvantages of Rocket Immunoelectrophoresis

• The Rocket Immunoelectrophoresis techniques support the quantitative study of antigens but are not suitable for complex mixtures.

Precautions

1. Before starting the experiment the entire procedure has to be read carefully.
2. Always wear gloves while performing the experiment.
3. Preparation of 1X TBE: Remove the 20 ml tablet of 50X TBE from the pouch into a 250 ml glass beaker. Add 20 ml of sterile distilled water to the beaker and heat in a microwave for 3-5 seconds till the tablet turns into the liquid form. Take the 50X TBE solution into a 1000 ml cylinder, rinse the beaker with sterile distilled water to collect and pour it into the cylinder and make up the volume to 1000 ml with sterile distilled water to get 1X TBE buffer.
4. Preparation of 1% Agarose gel: To prepare 15 ml of agarose gel, add 0.15 g of agarose powder to 15 ml of 1X TBE Buffer, boil to dissolve the agarose completely.
5. Add the antiserum to agarose only after it cools to 55°C. Higher temperature will inactivate the antibody.
6. Wipe the glass plates with cotton; make it grease-free using alcohol for even spreading of agarose.
7. Cut the wells neatly without rugged margins.
8. Ensure that the moist chamber has enough wet cotton to keep the atmosphere humid.

Interpretation

The height of the precipitin peak depends on the concentration of antigens loaded in the corresponding wells. By plotting the graph of concentration of antigens versus length of the precipitin peaks one can calculate the concentration of test antigen. 

References

• Walker JM. Rocket immunoelectrophoresis. Methods Mol Biol. 1984;1:317-23. doi: 10.1385/0-89603-062-8:317. PMID: 20512702.
• http://www.ispybio.com/search/protocols/RocketImmunoelectrophoresis.pdf
• http://www.ispybio.com/search/protocols/ie%20protocol8.pdf
• https://himedialabs.com/TD/HTI006.pdf
• https://microbenotes.com/rocket-immunoelectrophoresis/