Radial Immunodiffusion Principle, Procedure, Result

Single Radial Immunodiffusion also referred to as Mancini technique is a quantitative immunodiffusion technique that is used to measure the antigen’s concentration by measuring the size of the precipitin-based ring created by the interaction between the antigen with the antibody at the optimal concentration. The antibody is embedded in the agarose gel, while the antigen is diffused into it in the form of a radiatal pattern.

Single Radial Immunodiffusion (SRID)

A single Radial Immunodiffusion is a method widely used for the quantification of antigens. An antibody with known specificity is spread evenly in an agar gel. Then, an antigen sample of interest is put in a gel well. The antigen diffuses outward from the well until an elongated precipitin ring is formed at the point where the equilibrium concentration of antibody and antigen is present. Antigen concentrations are measured by taking the diameter of precipitin rings, and then extrapolating them in a standard curve.

Principle of Radial Immunodiffusion

Single Radial Immunodiffusion is extensively used to determine the quantifiable amount of antigen. This reaction between antigen and antibody is enhanced by adding antiserum in the agarose gel as well as loading the antigen sample into the well. As the antigen disperses throughout the agarose across all directions, its concentration decreases continuously until the point of equivalence is reached where the concentration of antigen is at the same level as the antibody concentration within the gel. At this point, a ring formed by precipitation (‘precipitin”ring”) develops around the well. The size of the precipitin rings is proportional to the amount of antigen. As antigen concentration increases precipitin rings with a greater diameter are created. The dimensions that the rings of precipitin is dependent on

• The amount of antigen present in the well in the sample
• The concentration of antibodies in the Agar gel
• The size of the well
• The volume of the sample

Therefore, by having different concentrations of a typical antigen Standard curves can be derived using which it is possible to calculate the amount of antigen present in an unidentified sample. Therefore, it is a quantitative test. If there is more than one ring during the testing, then more than one antibody or antigen reaction could be present. This could be due the combination of antigens and antibodies. This test is often employed in clinical laboratories to determine the immunoglobulin levels in samples of patients.

Objective of Radial Immunodiffusion

• To detect antigen-antibody complexes.
• Define the conditions under which the antigen-antibody complexes arise.
• Determine the relative amount of antigens.

Materials Required

• Agarose
• 10X Assay buffer
• Antiserum
• Standard Antigen A, B, C, D
• Test Antigen 1 and 2
• Glass plate 
• Gel puncher
• Template
• Glass wares: Conical flask, Measuring cylinder, Beaker
• Reagents: Distilled water, alcohol
• Other requirements: Incubator (37oC), Microwave or Bunsen burner, Vortex mixer, spatula, Micropipettes, Tips, Moist chamber (box with wet cotton)

Procedure of Radial Immunodiffusion

1. The assay buffer provided was diluted by 10X to 1X using distillate water.
2. 10 milliliters of 1.0 percent agarose (0.1 mg/10 ml) was made in the 1X assay buffer by heating slowly until the agarose was completely dissolved, with the intention not to cause scorching or froth in the solution. The molten agarose was left to cool down to 55degC.
3. 120 ml of antibody (the antiserum vial has been reconstituted using 2 ml of assay buffer 1X) were added to the 6 milliliters of agarose solution. The mixture was stirred gently to ensure the homogeneous distribution of the antibody.
4. Glass plates had to be then cleaned thoroughly with alcohol, making them clean and grease free to allow for the even spread of the molten sweet agarose. The agarose solution that contained the antiserum was poured on the glass plate and placed on a flat surface and left to sit for 15 minutes without interruption.
5. Gel punchers were employed to precisely cut five wells without rough edges with the aid of a template.
6. 20 ml each of the antigens (standard and test) was added into the holes (the Antigen Vials were reconstituted by adding 0.35 milliliters of 1X assay buffer) Following are the concentrations of the standard antigens.
   1. Standard Antigen A (0.25 mg/ml)
   2. Standard Antigen B (0.5 mg/ml)
   3. Standard Antigen C (1.0 mg/ml)
   4. Standard Antigen D (2.0 mg/ml)
   5. Test Antigen 1
   6. Test Antigen 2
7. The plate of gel was placed in a moist/humid chamber (box with wet cotton) and then incubated for a night at a room temperature.
8. The next day, the edges that were part of the circular (if there were any) were marked, and the diameters of the rings were determined.
9. A graph was created with the information from the observation (if there was any) using the diameter of the circle on the y-axis, and the concentration of antigen on the x-axis (done on an excel sheet).
10. The concentration of the unknown was determined by measuring the concentration against the ring diameter from the graph.

Results

Look for the precipitin rings around these antigen wells. Mark the edges of the precipitin rings and determine the size of the rings.

Create a graph that shows the size of the precipitin-based ring (on the Y-axis) in relation to the antigen concentration (on the X-axis) on the standard graph paper. Find the concentration of the unidentified antigen by determining the concentration against the ring diameter.

Inference

The size of the precipitin rings is dependent on the antigen concentration that are present within the wells. By drawing a graph of the antigens’ concentration against the diameter of the precipitin ring, one can estimate the amount of an antigen in the test.

Troubleshooting Guide

• If no precipitin ring is detected, it could be due to inadequate filling of wells, drying of the agarose gel in the incubation process, or the Inactivation of antiserum. To overcome these issues, the sample is to be loaded right into the tube and not spill onto the sides. Make sure that the chamber is filled with moisture to prevent drying the gel. Antiserum should be added to the agarose gel once the temperature is at 55-60oC.
• If you notice a Blur precipitin ring the reason is the antiserum being inactivated, and uneven dispensing of the gel. To resolve this issue, place it on flat surfaces and begin making the pour. Make sure that the glass plate is not moved until the gel is pour.

Precautions

• Before beginning the experiment, the entire procedure must be thoroughly read.
• Always wear gloves when performing the test.
• The preparation of the 1X Assay Buffer to make 10 milliliters of 1X Assay Buffer to be added, mix 1 milliliter of 10X Assay buffer to 9 ml of sterile distillation water.
• Preparation of 1 % Agarose gel: To make 10 milliliters of Agarose gel, add 0.1 grams of Agarose powder to 10 milliliters of 1X Assay Buffer and boil until you dissolve the Agarose completely.
• Wipe the glass dishes with cotton. Make it free of grease with alcohol to ensure even spread of the agarose.
• Cut the wells in a neat manner, with no rough edges.
• Inject the antiserum into agarose once it has cooled down to 55degC , as higher temperatures will cause the inactivation of the antibody.
• Make sure that the chamber is moist and has enough cotton that is wet to ensure that the air is kept humid.

Applications of Radial Immunodiffusion

• Techniques for immunodiffusion are commonly used in immunology to measure the quantity or the concentration of an antigen present in the sample.
• The estimation of the immunoglobulin classes in sera.
• Assessment of IgG, IgM antibodies in the serum of influenza viruses.
• To determine the relative concentrations of antibodies present in serum.
• It is can be used for Assess the serum transferrin and alpha-feroprotein levels.
• It is can be used for Comparing the characteristics of two antigens.
• It is can be used to determine the purity relative to an antigen preparation.
• It is can be used for Diagnostic of a disease.
• It is can be used for Serological studies.

Advantages of Radial Immunodiffusion

• The gel’s precipitation is believed to give more precise and precise outcomes than other methods.
• The reaction takes the form of precipitation bands and is able to be stained for better visual and preservation.
• If a significant amount of antigens are present every antigen-antibody reaction can give an individual sequence of precipitation.
• This method also indicates cross-reaction, identity, and non-identity between various antigens.

Limitations of Radial Immunodiffusion

• Radial Immunodiffusion is a Long reaction time, which is required 18-48 hours.
• It has also been suggested that the Mancini’s test are affected by the presence of bound metal cations within these test specimens (protein).
• The single diffusion method of precipitation is regarded as more efficient in comparison to other methods.
• The test was recently replaced with more accurate and automated techniques including nephelometry as well as enzyme-linked immunosorbent tests.

FAQ

References

• https://www.synbiosis.com/application-notes/single-radial-immunodiffusion/
• https://en.wikipedia.org/wiki/Radial_immunodiffusion
• https://himedialabs.com/TD/HTI004.pdf
• https://www.slideshare.net/prashanthkumarguddeti/antigen-antibody-interactions-63139960
• Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India: Elsevier India.