Radial Immunodiffusion

[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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[PRACTICAL 2] MTEB 2404

RESULTS Table below showed the result from the Radial Immunodiffusion experiment:

SAMPLE NUMBER

STD Ag CONE (IN mg/ml)

RING DIAMETER (IN mm)

1 2 3 4 5 6

0.25 0.50 1.00 2.00 TEST SAMPLE 1 TEST SAMPLE 2

6 8 10 12 11.5 7

2 1 3

5

4

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 antibodycontaining 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 2|Page

[PRACTICAL 2] MTEB 2404 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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