Aim / objectives:
To detect the
level of protein (antigen) in a sample by measuring the diameter of precipitin
ring formed by the complex of the protein (antigen) and the antiserum
Single Radial Immunodiffusion is
used to detect the antigen concentration by measuring the diameter of the
precipitin ring formed. It is formed by the interaction of the antibody and the
antigen at optimal concentration. Antigen will diffuses into agarose gel where
the antibody is incorporated into it HiMedia Laboratory, 2012.
By the addition of antiserum into
the agarose gel and loading the antigen sample in the well, the
antigen-antibody reaction is made more sensitive. Antigen concentration
continuously falls when it diffuses into the agarose radially in all directions
until the equivalence point is reached. Precipitation ring formed around the
well. The diameter of the precipitin ring is proportional to the concentration
of antigen. The higher the concentration of antigen, the larger the diameter of
precipitin rings HiMedia Laboratory, 2012.
1. 100 mg of agarose was added to 10 ml of
normal saline to make 1.0% agarose solution.
2. The solution was heated in boiling water bath
until it dissolves.
3. The agarose was cooled to around 40ºC to
4. 100 µl of antibody was added to the cooled
molten agarose and poured over the top of a glass slide.
5. Wells was cut after the agarose has solidified
by gel puncher as per the template
6. 10 µl of antigen (undiluted) was added to the
first well with a micropipette.
7. The antigen was diluted by adding 50 µl of
antigen to 50 µl of normal saline (1:1 dilution).
8. 1:2, 1:4 and 1:8 serial dilution of the
antigen is done by using 50 µl of normal saline in a 96 well microtitre plate.
9. 10 µl of the four diluted antigens was added
to the 2nd, 3rd, and 4th wells respectively by using a micropipette.
10. 10 µl of test antigen was added to the 5th
11. Incubated at room temperature for 12 hours.
12. The precipitin discs can be viewed clearly by
keeping the slide on a dark background.
13. The diameter of the precipitin discs will
vary with the concentration of antigen present in the sample.
Diagram 1: Precipitin disc on agarose gel
undiluted 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
Based on the results,
the diameter of precipitin ring is proportional to the concentration of the
antigen. The bigger the size of the precipitin ring diameter, the higher the
concentration of the antigen. The precipitin ring indicates the antibody-antigen
complex that reacted. The antibody in the agarose gel will react with the
antigen added. If the concentration of the antigen is high, the bigger the size
of the antibody-antigen disc.
This method can
measure the concentration of an unknown antigen by measuring the
antibody-antigen disc and calculate it by using the graph. Based on the graph
drawn, the concentration of the test antigen for this test is 4.3 (mg/ml).
Applications for this
technique include determining relative concentrations of antibodies or antigen,
to compare antigens, to determine relative purity of antigen preparation, to
diagnose disease and for serological surveys Suniu, 2013.
As a conclusion, the diameter of precipitin ring is proportional
to the concentration of the antigen. The concentration of the unknown antigen
can be known by using this test.