[PRACTICAL 2] MTEB 2404

RADIAL IMMUNODIFFUSION

Objectives

To learn the technique 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.

Kit description
Standard antigen at different concentration (0.25 mg/ml, 0.5 mg/ml,
1.0mg/ml, 2.0mg/ml)

Materials
As listed in practical manual

Procedure
1. Agarose gel with antiserum is already prepared.
2. Punch the agarose gel into 6 well.
3. Each well added with standard antigen and
test antigen.
Wells:
I. Standard antigen A (0.25mg/ml)
II. Standard antigen B (0.5mg/ml)
III. Standard antigen C (1 .Omg/ml)
IV. Standard antigen D (2.Omg/ml)
V. Test antigen-1
VI. Test antigen-2
4.Keep in moist chamber. Incubate at RT overnight.
5.Diameter measured and graph plotted.

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RESULTS
Table below showed the result from the Radial Immunodiffusion experiment:
STD Ag CONE (IN RING DIAMETER (IN
SAMPLE NUMBER
mg/ml) mm)
1 0.25 6
2 0.50 8
3 1.00 10
4 2.00 12
5 TEST SAMPLE 1 11.5
6 TEST SAMPLE 2 7

2
1

3 4

5
6

DISCUSSION

Radial immunodiffusion is a type of

quantitatively method to determine the
level of an antigen, which more sensitive
than in double immunodiffusion. In this
experiment, the antibody is incorporated
into liquefied agar and allowed into the gel.
The antiserum is uniformly distributed
throughout the agar gel.

The antigen is added to small wells

and radiates throughout the antibody-
containing medium, leaving a precipitate
throughout the gel. The antigen diffuses out
of the well, where the concentration is
relatively high and it forms relatively soluble antigen—antibody adducts.

However, as it diffuses further and further from the well, its concentration
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decreases. The amount of diffusion is then quantified by measuring the

diameter of the ring which was proportional to the log of the concentration
of antigen. The greater the initial concentration of antigen in the well, the
greater the diameter of the precipitin disk.

Thus, by running a range of known antigen concentrations on the gel and by

measuring the diameters of their precipitin disks, a calibration graph can be
constructed. The antigen concentrations of unknown samples run on the same
gel can then be found by simple interpolation having measured the diameters
of the respective precipitin disks. Furthermore, these tests were also benefit in
detecting the mixtures of antigens or antibodies.

CONCLUSION

Based on the graphs plotted, showed that the immunoglobulin concentration of

sample 1 were 0.42 mg/ ml, whereas the sample 2 are 1.9 mg/ml.

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