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Radial Immunodiffusion: Experiment No: 12 (B) Date

Radial immunodiffusion is a technique used to quantify antigen concentrations. It works by incorporating antibody into an agarose gel and allowing antigens of both known and unknown concentrations to diffuse from wells cut into the gel. As the antigens diffuse, they react with antibody to form visible precipitin rings. By measuring the diameters of rings formed by antigens of known concentrations, a calibration curve can be generated to determine the concentrations of antigens in unknown samples based on their ring diameters.

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268 views2 pages

Radial Immunodiffusion: Experiment No: 12 (B) Date

Radial immunodiffusion is a technique used to quantify antigen concentrations. It works by incorporating antibody into an agarose gel and allowing antigens of both known and unknown concentrations to diffuse from wells cut into the gel. As the antigens diffuse, they react with antibody to form visible precipitin rings. By measuring the diameters of rings formed by antigens of known concentrations, a calibration curve can be generated to determine the concentrations of antigens in unknown samples based on their ring diameters.

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razik
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EXPERIMENT NO: 12 (B)

DATE:

RADIAL IMMUNODIFFUSION
AIM:
To learn the techniques of Radial immunodiffusion.

Principle:
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 precipitin rings and extrapolating this value on the
calibration graph.

MATERIALS REQUIRED:
1. Agarose
2. 10x assay buffer
3. Standard Antigens (A,B,C &D)
4. Test Antigen(1 &2)
5. Antiserum
6. Gel punch with syringe
7. Glass plate
8. Template

PROCEDURE:
1. 10ml of 1.0% Agarose (0.1g/10ml) in 1x Assay buffer was prepared and
heated slowly till Agarose dissolves completely. Take care not to scorch or
froth the solution.
2. Molten Agarose was allowed to cool to 55C.
3. 120 l of antiserum to 6ml of Agarose solution was added. Mix by gentle
swirling for a uniform distribution of antibody.
4. Agarose solution containing the Antiserum was poured onto a grease free glass
plate and set on a horizontal surface. Leave it undisturbed to form a gel.
5. Cut wells using a Gel Puncher using the template provided.
6. 20 l of the given Standard Antigens and Test Antigens were added to the
wells.
7. The gel plate were kept 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.
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 the concentration of unknown by reading the concentration against
the ring diameter from the graph.

RESULT:
The Concentration of antigen of the test samples were found to be

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