IMMUNOLOGY

MSBO210

Experiment 2

Aim of the Experiment

To study the immunodiffusion technique by Single Radial Immunodiffusion.

Introduction:

Immunodiffusion in gels encompasses a variety of techniques, which are useful for the analysis of antigens and antibodies. Gel immunodiffusion can be classified into two groups:

  1. Single Immunodiffusion

  2. Double Immunodiffusion

Single Radial Immunodiffusion, also known as Mancini technique, is a quantitative immunodiffusion technique used to detect the concentration of antigen by measuring the diameter of the precipitin ring formed by the interaction of the antigen and the antibody at optimal concentration. In this method the antibody is incorporated into the agarose gel whereas the antigen diffuses into it in a radial pattern. Thus the antibody is uniformly distributed throughout the gel.

Principle

Single Radial Immunodiffusion is used extensively for the quantitative estimation of antigen. Here the antigen- antibody reaction is made more sensitive by the addition of antiserum into the agarose gel and loading the antigen sample in the well. As the antigen diffuses into the agarose radially in all directions, it’s concentration continuously falls until the equivalence point is reached at which the antigen concentration is in equal proportion to that of the antibody present in the agarose gel. At this point ring of precipitation (‘precipitin ring’) is formed around the well. The diameter of the precipitin ring is proportional to the concentration of antigen. With increasing concentration of antigen, precipitin rings with larger diameter are formed.


The size of the precipitin rings depend on

  1. Antigen concentration in the sample well

  2. Antibody concentration in the agarose gel

  3. Size of the sample well

  4. Volume of the sample


Thus, by having various concentrations of a standard antigen, standard curve can be obtained from which one can determine the amount of an antigen in an unknown sample. Thus, this is a quantitative test. If more than one ring appears in the test, more than one antigen/antibody reaction may have occurred. This could be due to a mixture of antigens or antibodies. This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.

Fig 1: In Single Radial Immunodiffusion assay the diameter of the precipitin ring increases with increasing concentration of the antigen

Materials required

  1. Glass wares: Conical flask, Measuring cylinder, Beaker

  2. Reagents: Distilled water, alcohol

  3. Other requirements: Incubator (37oC), Microwave or Bunsen burner, Vortex mixer, spatula, Micropipettes, Tips, Moist chamber (box with wet cotton), Agarose, 10X Assay buffer, Antiserum, Standard Antigen A,B,C,D, Test Antigen 1 and2, glass plate, gel puncher

Procedure

  1. Prepare 10 ml of 1% agarose.

2_Preparation of 1% agarose solution for using 1X assay buffer.mp4
3_Hydrolysing agarose to make 1% agrose solution.mp4

2. Take 6 ml of this gel solution in a clean test tube. Allow the solution to cool down to 55-60oC and add 80μl of antiserum to 6 ml of agarose solution. Mix well for uniform distribution of the antibody.


3. Pour agarose solution containing the antiserum on to a grease free glass plate placed on a horizontal surface. Allow the gel to set for 30 minutes.

Widhdrawal of 6 ml solution_2.mp4
Addition of antiserum sample_3.mp4



4. Place the glass plate on the template provided


5. Punch wells with the help of the gel puncher corresponding to the markings on the template. Use gentle suction to avoid forming of rugged wells.

Fig 2: Template of pattern of wells for loading of standard and test antigens

6. Add 10 μl of the given standard antigen and test antigen samples to the wells as in fig 2.


A. Standard Antigen A (3.75 mg/ml)

B. Standard Antigen B (7.5 mg/ml)

C. Standard Antigen C (15 mg/ml)

D. Standard Antigen D (30 mg/ml)

E. Test Antigen 1

F. Test Antigen 2

Gel puncher and addition of antigen samples_4.mp4

7. Keep the glass plate in a moist chamber overnight at 37oC.

Keeping the plate in incubation_5.mp4

Observation and Result

  • Observe for precipitin rings surrounding the antigen wells (Fig 3). Mark the edges of the precipitin rings and measure the diameter of the rings as shown in table 2.

  • Plot a graph of the diameter of the precipitin ring (on Y-axis) versus the concentration of antigen (on X-axis) on a standard graph sheet. Determine the concentration of the unknown antigen from the graph by finding the concentration against the ring diameter.

Fig 3: Precipitin rings observed in Single Radial Immunodiffusion

Interpretation

The diameter of the precipitin ring depends upon the concentration of antigens loaded in the wells. By plotting the graph of concentration of antigens versus diameter of the corresponding precipitin ring one can calculate the concentration of any test antigen.

Questions

1. What do the circular precipitin rings represent?

2. Why do the ring sizes change until equilibrium is reached?

3. Predict the results if a very low concentration of antigen were loaded into a well. What would happen if not enough antibody was incorporated into the agarose?

4. Compare and contrast Radial Immunodiffusion with its close relative, the Ouchterlony plate technique.

Developed by

Dr. Deepika Gupta,

Assistant Professor, Biotechnology

deepika.gupta@gsfcuniversity.ac.in