Aim :- Radial immunodiffusion technique for the quantitative analysis of the given antigen.
Single radial immunodiffusion (RID) is used extensively for the quantitative estimation of antigens. The antigen-antibody precipitation is made more sensitive by the incorporation of antiserum in the agarose. Antigen (Ag) is then allowed to diffuse from wells cut in the gel in which the antiserum is uniformly distributed. Initially, as the antigen diffuses out of the well, its concentration is relatively high and soluble antigen-antibody adducts are formed. However, as Ag diffuses farther from the well, the Ag-Ab complex reacts with more amount of antibody resulting in a lattice that precipitates to form a precipitin ring. (Refer fig. 1).Thus, by running a range of known antigen concentrations on the gel and by measuring the diameters of their precipitin rings, a calibration graph is plotted. Antigen concentrations of unknown samples, run on the same gel can be found by measuring the diameter of precipitin rings and extrapolating this value on the calibration graph.
1. Prepare 10 ml of 1.0% agarose (0.1 g/10ml) in 1X assay buffer by heating slowly till agarose dissolves completely. Take care not to scorch or froth the solution.
2. Allow the molten agarose to cool to 55°C.
3. Add 120 ml of antiserum to 6 ml of agarose solution. Mix by gentle swirling for uniform distribution of antibody.
4. Pour agarose solution containing the antiserum onto a grease free glass plate set on a horizontal surface. Leave it undisturbed to form a gel.
5. Cut wells using a gel puncher as shown in figure 2, using the template provided.
6. Add 20 ml of the given standard antigens and test antigens to the wells.
7. Keep the gel plate in a moist chamber (box containing wet cotton) and incubate overnight at room temperature.
8. Mark the edges of the circle and measure the diameter of the ring. Note down your observations.
9. Plot a graph of diameter of ring (on Y-axis) versus concentration of antigen (on X- axis) on a semi-log graph sheet.
10. Determine concentration of unknown by reading the concentration against the ring diameter from the graph.
- Read the entire procedure before starting the experiment.
- Dilute required amount of 10X assay buffer to 1X with distilled water.
- Reconstitute the antigen vials (standard and test) with 0.35 ml of 1X assay buffer.
- Mix well and store at 4°C.Use within 3 months.
- Reconstitute antiserum vial with 2 ml of 1X assay buffer. Mix well and store at 4°C.
- Use within 3 months.
- Wipe the glass plate with alcohol thoroughly to make it grease free for even spreading of agarose.
- Cut the wells neatly without rugged margins to get a perfect ring of precipitation.
- Add the antiserum to agarose only after it cools to 55°C. Higher temperature will inactivate the antibody.
- Assay buffer: Phosphate buffered saline.
Initially, an agarose gel was prepared in 1x buffer and dilutions of antigens and antiserum were prepared as per the kit manuals given and antiserum was mixed with the gel and throughout the gel it is well spread. Against this antisera antigens.Standard antigen A, B, C, D of the concentration (0.25 ,0.5,1.0,2.0mg/ml) and two test antigen 1 and 2 were allowed to diffuse overnight in humid chamber and the ring pattern as shown in the printout attached. The diameter of these ring were measured and graph of the concentration of the antigen Vs the diameter of the ring is plotted on a semi log graph paper and from this standard plot the concentration of unknown was measured by extrapolating the lines from the graph. from the graph the concentration of the unknown is found to be test sample 1-2 1.23 mg/ml and 1.52 mg/ml respectively.