Single Radial Immunodiffusion

Practical Immunology And Serology Single Radial Immunodiffusion�

1. Practical Immunology And Serology Single Radial Immunodiffusion By Ahmed Riyadh Abdul Rahman Al-Noor University College 1

2. Single Radial Immunodiffusion Radial immunodiffusion   (RID) or Mancini immunodiffusion (Mancini method ) or single radial immunodiffusion assay . Is a type of precipitation reaction ( immunodiffusion technique). RID is a sensitive quantitative technique used to determine the quantity or concentration of an antigen in a sample. The technique is very simple and cost-effective; however, it may require a long incubation time. 2

3. Radial immunodiffusion is based on a technique using a precipitin reaction in which specific antibody is incorporated into agarose medium; sample containing the test antigen is placed in a well centered in the agarose . Antigens sample then diffuse through the agar in all directions from the well and react with the antibody forming visible precipitate or a precipitin ring. Ring shaped bands of precipitates from concentrically around the well indicating Ab -Ag reaction. The diameter of the resulting precipitin zone is related to the concentration of antigen placed in a well, the greater the antigen concentration the larger the circle of precipitation. 3 PRINCIPLE

4. 4 Fig: Single Radial Immunodiffusion

5. Objective and Application To determine the quantity or concentration of an antigen/antibody in a sample. Quantitation of immunoglobulins , complement components, and, any antigenic protein, in any biological fluid. Estimation of the immunoglobulin classes in sera. To compare properties of two different antigens. To determine the relative purity of an antigen preparation For disease diagnosis Serological surveys 5

6. PROCEDURE 6 An agar containing an appropriate antiserum (antibody) is poured in plates. Carefully circular wells are cut and removed from the plates. A single or series of standards containing known concentration of antigen are placed in separate wells, while control and “unknown” samples are placed in other remaining wells. As the antigen diffuses radially, a ring of precipitate will form in the area of optimal antigen – antibody concentration. The ring diameters are measured and noted. A standard curve is prepared using the ring diameters of the standards versus their concentrations. This curve is then used to determine the concentration of the control and unknown samples .

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8. 8 Result and observation The presence of a precipitin ring around the antigen wells indicates specific antigen-antibody interaction. Absence of precipitin ring suggests absence of reaction. The greater the amount of antigen in the well, the farther the ring will form from the well.

9. INTERPRETATION OF RESULTS   9 The precipitin rings will be visible in (24 to 48) hours. Carefully hold a plate up so that the overhead room lights shine through it. You should be able to see opaque circles around each well where antigen and antibody have precipitated. With a ruler, measure the diameter (through the centers of the wells) of the precipitin ring in millimeters. To plot the standard curve, square the diameter value and plot antigen concentration on the X-axis and the diameter squared on the Y-axis. Draw the best fit line through these points. Calculate the value of the unknown antigen concentration from this graph.

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