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Rocket Immunoelectrophoresis Protocol

In this Rocket Immunoelectrophoresis Protocol post we briefly summarises about: principle, reagents requirements, procedure, result, application and limitations of Rocket Immunoelectrophoresis.

Rocket Immunoelectrophoresis Protocol

Electro-immunodiffusion, also known as rocket Immunoelectrophoresis is, is a simple, rapid, and repeatable method for measuring the concentration of antigen in an unknown sample.

This quantitative one-dimensional Immunoelectrophoresis method requires a monospecific antibody against the antigen under inquiry and includes comparing an antigen sample of unknown concentration with a series of dilutions of a known concentration of the antigen.

Antigen migrates from the well through an agarose gel containing antiserum to create rocket-shaped precipitin peaks in this approach. This peak’s height is proportional to the amount of antigen loaded in the corresponding well.

Objectives

To detect antigen-antibody complexes.

Determine the concentration of antigen in an unknown sample.

Principle

Negatively charged antigen samples are electrophoresed in an agarose gel containing antigen-specific antibody in Rocket Immunoelectrophoresis. As the antigen exits the well and enters the agarose gel, it joins forces with the antibody to create an immunological complex, which appears as white precipitin arcs.

The antigen migrates through the gel in one direction because it is influenced by an applied electric current. There is significant antigen excess over antibody during the early phase, and no apparent precipitation develops. As the antigen sample progresses across the agarose gel, additional antibody molecules come into contact with the antigen, forming an immunological complex.

The antigen transport ends when the immune complexes get large enough to be maintained within the gel. Precipitin bears the shape of a rocket, and its height is proportional to the antigen concentration in the corresponding well.

Requirements

1. Agarose

2. 1X TBE

3. Antiserum

4. Standard antigens

5. Test antigens

6. Glass plate

7. Gel puncher

8. Glass wares

9. Other Reagents

Procedure

1. Make an agarose gel with 1% agarose. Allow the solution to cool to 50°C before adding the antiserum to the agarose solution. To ensure that the antibody is distributed evenly, mix thoroughly.

2. Fill a clean grease-free glass plate with agarose gel solution and lay it on a horizontal surface. Allow 30 minutes for the gel to set.

3. Place the glass plate on the given template. With the help of a gel puncher, make a good job. It’s only necessary to use modest suction.

4. To the wells, add roughly 10L of standard and test antigens (Note: standard antigens must either be in ascending or descending order of concentration). Fill the electrophoresis tank with 1X TBE buffer, enough to cover the gel.

5. Electrophorese the blue dye at 80-120 volts and 60-70 mA until it reaches 3-4 cm from the well. Electrophorese for no longer than 3 hours to avoid generating heat. In the moist chamber, incubate the plate for 24 hours at 37°C.

6. If the reaction is positive, the precipitin peaks’ tips are marked, and the peak height is measured from the upper edge of the well to the peak’s tip.

7. On a semi-log graph sheet, the rocket height (on the Y-axis) is plotted against the concentration of antigen (on the X-axis).

8. The concentration of the unknown is calculated by plotting the concentration against the rocket height on the graph.

Result Interpretation

Peak Measurement

In a dark background, rocket-shaped precipitin peaks will be visible. It can be marked and measured from the well’s upper edge to the precipitin peaks’ tips. It can be used to compare the concentration of unknown antigens to standard antigens to determine the concentration of unknown antigens.

Rocket Immunoelectrophoresis Protocol

Rocket Immunoelectrophoresis Protocol

Standard curve

The height of the precipitin peak is proportional to the antigen concentration of antigens loaded. When the concentration of antigen loaded is plotted against the length of the precipitin peak, the graph looks like this, and it may be used to compute the concentration of test antigen.

Applications

1. In order to diagnose infected bursal. The production of α-amylase and its distribution in diverse seeds during the early stages of germination were investigated.

2. The concentration and degree association of the red clover necrotic mosaic virus strain was determined. Determining the concentration of various antigenic solutions that are unknown.

Advantages

1. Rocket Immunoelectrophoresis is is simple, quick, and repeatable. On a single plate, several unknown samples can be evaluated.

2. Protein concentrations as low as 1 g/mL can be determined using as little as 20 ng of protein put in a well.

Limitations

1. There was no precipitin ring visible, which could be due to serum inactivation, unlabelled antigen filling in the wells, or agarose gel drying during incubation. Due to poor gel pouring and antiserum inactivation, a blur precipitin ring was seen.

Further Readings

Reference