IEP - Immunoelectrophoresis

This process involves a combination of immune diffusion and electrophoresis. This technique aims to separate and identify proteins under an electric field.

When the electric current is applied, the mixture that has been placed into the cut-out wells is separated according to size into individual antigen components. Next, a specific antiserum is placed into parallel troughs that react with the separated antigens and diffusion occurs. Resulting in the formation of separate precipitin lines, which indicates a reaction has occurred between the protein and antibody.

Required Material:

• Agarose

• 10X assay buffer

• Anti-serum

• Sample

• Standard

• Glass plate

• Electrophoretic apparatus

• Saline

Preparation of gel:

1. Make 10ml of 1.0% agarose in 1X assay buffer and slowly heat up to fully dissolve the agarose.

2. Allow the mixture to cool down to 55°C.

3. Pour the mixture onto the glass plate and leave it in a horizontal position to solidify.

4. Punch wells and troughs in the gel and transfer the gel to the electrophoresis apparatus.

Protocol:

1. Dilute the sample and standard to 2:3 with diluent.

2. Into cut-out wells pipette 5μl of sample and standards.

3. Run the electrophoresis for 20 minutes at 100 volts.

4. After the electrophoresis is completed, pipette 20μl of the corresponding antiserum into the troughs and incubate in the horizontal position for 18 to 20 hours at room temperature.

5. Once incubation is complete, the gel is placed on a flat surface and dried with blotter sheets.

6. Soak the gel in saline for 10 minutes.

7. Repeat the drying and washing steps twice.

8. Dry the gel and stain with protein staining solution for 3 minutes. Decolourise for 5 minutes in a detaining solution.

9. Dry the plate and evaluate the results.

Results:

For better visibility, the gel can be stained using (CBB) Coomassie Brilliant Blue for 15 to 20 minutes, followed by a CBB de-stain for 24 hours.

1. Record the standard observations by measuring the precipitation peak from tip to edge.

2. On a graph sheet, a standard graph is generated by plotting the height of the peak on the Y-axis against the concentration of the antigen on the X-axis.

3. Based on the standard curve constructed the concentration of the test sample can be determined.