Title: Singleradial immunodiffusion Aim / objectives: To detect thelevel of protein (antigen) in a sample by measuring the diameter of precipitinring formed by the complex of the protein (antigen) and the antiserum(antibody). Introduction:Single Radial Immunodiffusion, alsoknown as Mancini technique, is a quantitative immunodiffusion technique used todetect the concentration of antigen by measuring the diameter of the precipitinring formed by the interaction of the antigen and the antibody at optimalconcentration. In this method the antibody is incorporated into the agarose gelwhereas the antigen diffuses into it in a radial pattern HiMediaLab. PrincipleSingle Radial Immunodiffusion isused extensively for the quantitative estimation of antigen.
Here theantigen-antibody reaction is made more sensitive by the addition of antiseruminto the agarose gel and loading the antigen sample in the well. As the antigendiffuses into the agarose radially in all directions, its concentrationcontinuously falls until the equivalence point is reached at which the antigenconcentration is in equal proportion to that of the antibody present in theagarose gel. At this point ring of precipitation (‘precipitin ring’) is formedaround the well. The diameter of the precipitin ring is proportional to theconcentration of antigen. With increasing concentration of antigen, precipitinrings with larger diameter are formed HiMediaLab. Materials: 1. Normalsaline2. Agarose3.
Antibody4. Antigen5. Glassslide6. Template7. Moistchamber Procedure:1. 100 mg of agarose was added to 10 ml ofnormal saline to make 1.0% agarose solution.
2. The solution was heated in boiling water bathuntil it dissolves.3.
The agarose was cooled to around 40ºC to50ºC.4. 100 µl of antibody was added to the cooledmolten agarose and poured over the top of a glass slide.5. Wells was cut after the agarose has solidifiedby gel puncher as per the templateprovided. 6. 10 µl of antigen (undiluted) was added to thefirst well with a micropipette.
7. The antigen was diluted by adding 50 µl ofantigen to 50 µl of normal saline (1:1 dilution). 8. 1:2, 1:4 and 1:8 serial dilution of theantigen is done by using 50 µl of normal saline in a 96 well microtitre plate.9.
10 µl of the four diluted antigens was addedto the 2nd, 3rd, and 4th wells respectively by using a micropipette.10. 10 µl of test antigen was added to the 5thwell.11.
Incubated at room temperature for 12 hours.12. The precipitin discs can be viewed clearly bykeeping the slide on a dark background.13. The diameter of the precipitin discs willvary with the concentration of antigen present in the sample.
Results: Diagram 1: Precipitin disc on agarose gel Well Number Dilution Diameter(mm) Concentration(mg/ml) 1 Undiluted 110 mm 10 mg/ml 2 1:2 dilution 80 mm 5 3 1:4 dilution 60 mm 2.5 4 1:8 dilution 30 mm 1.25 5 Test antigen 70 mm x Concentration ofundiluted antigen = 10 mg/ml(1:1) 1/1 x 10 = 10 mg/ml(1:2) 1/2 x 10 = 5 mg/ml(1:4) 1/4 x 10 = 2.
5 mg/ml(1:8) 1/8 x 10 = 2.5 mg/ml Discussion:Based on the results,the diameter of precipitin ring is proportional to the concentration of theantigen. The bigger the size of the precipitin ring diameter, the higher theconcentration of the antigen. The precipitin ring indicates the antibody-antigencomplex that reacted. The antibody in the agarose gel will react with theantigen added. If the concentration of the antigen is high, the bigger the sizeof the antibody-antigen disc. This method canmeasure the concentration of an unknown antigen by measuring theantibody-antigen disc and calculate it by using the graph.
Based on the graphdrawn, the concentration of the test antigen for this test is 4.3 (mg/ml). Applications for thistechnique include determining relative concentrations of antibodies or antigen,to compare antigens, to determine relative purity of antigen preparation, todiagnose disease and for serological surveys Suniu, 2013. Conclusion:As a conclusion, the diameter of precipitin ring is proportionalto the concentration of the antigen. The concentration of the unknown antigencan be known by using this test.