1. Immunoelectrophoresis 1Dr. Nikhat Siddiqi

2. The simplest and the oldest of the procedure is of P. Grabar and C. A. Willaims. 2Dr. Nikhat Siddiqi

3. Immunoelectrophoresis is a combined technique that separates proteins by an electrophoretic procedure and then characterizes them by reacting them with appropriate antibodies known as immunoglobulins. 3Dr. Nikhat Siddiqi

4. A layer of agar is deposited on a glass plate about 1- 2mm thick. A small hole is made in the layer and the protein sample mixture placed in the hole. A potential is applied across the plate and an electrophoretic separation is developed. A trough is then cut in the gel layer just below the separation, which is then filled with the immunoglobin or antiserum. 4Dr. Nikhat Siddiqi

5. The plate is then incubated for 24 hours in a high humidity environment. As a result, the immunoglobulins diffuse through the gel (without the gel drying out) to the separated proteins with which they may interact and form immunoprecipitin arcs. These arcs are often clearly visible. 5Dr. Nikhat Siddiqi

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7. CROSSED ELECTROPHORESIS 7Dr. Nikhat Siddiqi

8. Antigen mixture is placed in the slot in an agarose gel and electrophoresed. Bands of antigen appear. A narrow longitudinal strip is cut along the middle of the gel. This strip contains bands of each antigen. This strip is placed on a second gel which contains a low concentration of antiserum that contains antibodies to the antigen present in the mixture applied first. 8Dr. Nikhat Siddiqi

9. The second gel is electrophoresed at right angle with respect to the movement of antigen earlier. During this period both the antigens in the strip and the antibodies in the lower plate migrate in the electric field. Precipitin zones form for each antigen. 9Dr. Nikhat Siddiqi

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11. ROCKET ELECTROPHORESIS 11Dr. Nikhat Siddiqi

12. The method, originally introduced by Laurell, involves a comparison of the sample of unknown concentration with a series of dilutions of a known concentration of the protein. 12Dr. Nikhat Siddiqi

13. This is used to determine the concentration of a particular antigen in a mixture. A slab of gel is prepared containing a low concentration of antiserum. 13Dr. Nikhat Siddiqi

14. The samples to be compared are loaded side- by-side in small circular wells along the edge of an agarose gel that contains the monospecific antibody. These samples (antigen) are then electrophoresed into the agarose gel, where interaction between antigen and antibody takes place. 14Dr. Nikhat Siddiqi

15. A rocket shape is seen and the area under the rocket is proportional to the concentration of the antigen. Therefore, if a series of wells are loaded with increasing antigen concentration, then a series of rockets of increasing height should be produced, with the area under the curve (rocket) being proportional to the amount of antigen in the well. 15Dr. Nikhat Siddiqi

16. The unknown sample can be a highly complex mixture of proteins (e.g., serum sample, tissue extract, urine sample, cerebrospinal fluid, and so forth), but only one rocket will be produced from this sample owing to the interaction of the antibody in the gel with the antigen of unknown concentration. A calibration graph of protein concentrations vs peak height is then constructed and, knowing the peak height of the unknown sample, the concentration can be read off from the graph. Concentrations of proteins as little as 1 µg/mL can be measured in this manner. 16Dr. Nikhat Siddiqi

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