To learn the technique of Radial Immunodiffusion.
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.
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 precipitated rings and extrapolating this value on the calibration graph.
Duration of the experiment: Experiment is carried out over a span of 2 days.
Components of the kit reactions:
Materials Quantity 10 Expts. Store
Agarose 1 gm 4°C
10 X Assay Buffer 10 ml 4°C
Standard Antigen (D) 0.25 ml -20°C
Test Antigen (1) 0.2 ml -20°C
Antiserum 2 ml -20°C
Gel puncher 1 No RT
Template 2 Nos RT
Glass Plates 2 Nos RT