Name : DESMOND POO KEAN WEI

Matrix No. : MEB080004

Subject : BASIC IMMUNOLOGY
Title : SINGLE RADIAL IMMUNODIFFUSION (SRIO)

Objectives
1. To learn the proper technique of radial immunodiffusion.
2. To learn how to estimate the concentration of an antigen sample from
standard antigen samples of known concentration.

Introduction
Radial immunodiffusion is an immunodiffusion technique used in
immunology to detect quantity of antigen by measuring the radius surrounding
samples of the antigen, marking the boundary between it and antibody. An agar
gel contains evenly distributed antigen (or antibody) and its counterpart from the
test sample diffuses into the gel from a single well. This will then result in
production of circular precipitin line around the sample well. The diameter of
precipitin ring formed will then be determined, as the Ag-Ab complex reacts with
more amount of antibody. By comparing the diameter of the test specimen
precipitin ring with that of the standards, estimation of the concentration of
specific antigen or antibody can be achieved.

Materials
1. Standard antigen of concentrations= 0.25mg/ml, 0.5mg/ml, 1.0mg/ml and
2.0mg/ml
2. Two test antigen samples
3. 10ml of 1.0% agarose (0.1g/ 10ml) in 1 x assay buffer
4. 120ml of antiserum
5. Gel slide with template
6. Gel punch with syringe

Methods
1. 10ml of 1.0% agarose (0.1g/ 10ml) in 1 x assay buffer was prepared by
heating slowly until all the agarose dissolves completely. Care was taken
to not scorch of froth the solution.
2. The molten agarose was allowed to cool to 55°C.
3. 120μl of antiserum was added to 6ml of agarose solution mixed by gentle
swirling to distribute the antibody uniformly.
4. The agarose solution containing the antiserum was poured onto a grease
free glass plate set on a horizontal surface and left undisturbed to form a
gel.
5. Wells were cut using a gel puncher, using the template provided.
6. 10μl of the given standard antigens and test antigens was added into the
wells as shown in figure below:
 2
1 1
3 1
1 4

5 1
Sequence of addition of standard antigen6 and 1test antigen samples to
wells.
1 1
1. Standard Antigen A (0.25mg/ ml)
2. Standard Antigen B (0.5mg/ml)
3. Standard Antigen C (1.0mg/ml)
4. Standard Antigen D (2.0mg/ ml)
5. Test Antigen- 1
6. Teat Antigen- 2
7. The gel plate was kept in a moist chamber (box containing wet cotton) and
incubated overnight at room temperature.
8. The edges of the circle were marked and the diameter of the ring
measured. The results were recorded in Table 1.
9. A graph of diameter of ring (on y- axis) versus concentration of antigen
(on x- axis) was plotted on a semi- log graph sheet.
10.The concentration of unknowns was determined by reading the
concentration against the ring diameter from the graph.

Result

Sample No. Standard Ag Ring Diameter (mm)

Concentration (mg/ml)
Standard antigen 0.25 6.0
A
Standard antigen 0.50 8.0
B
Standard antigen 1.00 10.0
C
Standard antigen 2.00 12.0
D
Test antigen 1 1.75 (from graph) 11.5
Test antigen 2 0.35 (from graph) 7.0

Discussions

1) The criteria for a good precipitation include:

a. Immunoglobulins must be at least bi- (IgG, IgD, IgE) or multi- (IgA, IgM)
valent.
b. Antigen must be multivalent, containing several antigenic sites.
Antibody will not precipitate monovalent antigen.
 c. The concentration of antigen:antibody must be in the zone of
equivalence. Excess antibody (prozone) or excess antigen (postzone)
will not allow precipitation.

2) The single radial immunodiffusion (SRID) is commonly used to test and

determine immunoglobulin levels (such as IgG, IgM, and IgA) in a patient’s
sample in clinical laboratory. It is a sensitive quantitative technique that is
often used clinically to detect patients’ blood protein level.

3) The diameter of the precipitation ring formed can be measured by:

Diameter of precipitation ring = Diameter of whole circle (DC) – Diameter of
well (DW)
Precipitation ring
DC
DW

4) Relationship between the concentration of the antigen and the diameter of

the precipitation ring.
The antigen will diffuse from the wells into the walls of the gel in which
antiserum is evenly distributed. A precipitation band will then form at optimal
proportions of antigens and antibody, showing up as a ring around the
antigens’ wells. The diameter of the ring formed mainly depends on
concentration of the antigen in the well. A higher concentration of antigen
leads to more precipitation formation. Hence, the diameter of ring is
proportional to the log of the concentration of antigen since the
concentration of antibody is constant. A graph of diameter of ring versus
concentration of antigen can be plotted on a semi-log graph paper to give a
linear line. From this standard graph, the concentration of unknown antigens
can be estimated by comparing with the known concentration antigens.

5) To avoid inaccuracies in the results, there are few precautions to be taken:

a) Do not overload the well with antigen.
b) Make sure the well is in a static position after loading i.e. do not move
the gel after loading to prevent the loaded material from spilling out of
the well.
c) The standard serum zone diameter should be measured at the same
time as test sera.
d) If a delay in measurement is anticipated allow sufficient time intervals
between filling the wells.
e) The gels should be stored in a moist environment to prevent drying.
f) Air bubbles in the gel should be avoided to ensure proper volume and
diffusion of sample
 g) The gel should be trimmed properly when making the wells to get an
accurate volume of the well required.
h) The glass slides with the gel should be observed with care to prevent
distorting/breaking the gel.
i) The glass slide should be wiped dry before observing to get a clearer
view of the precipitation ring.
j) The gel make be observed under light with a dark background to get a
clearer view of the precipitation ring.
k) As the precipitate is white in color and often very unnoticeable, the gel
can sometimes be stained with a blue dye to make it more visible.

6) Limitations:
I. When an unknown antigen’s diameter exceeds that of the highest value
of standard, the specimen should be diluted with saline. The value of
the concentration obtained should then be multiplied by the dilution
factor to get the actual concentration of the unknown antigen sample.
II.When an unknown antigen’s diameter is smaller than the lowest value
of standard, its concentration should be stated as “less than” the
concentration of the standard serum. If available, a lower level radial
immunodiffusion plate may be utilized.

7) Failure to obtain a precipitation ring may be due to:

a) Samples are not applied into the wells correctly i.e. samples spill out of
well or into gel.
b) Concentration of sample is too low to be detected
c) Concentration of sample is too high resulting in formation of soluble
complexes which is not precipitated.
d) The wrong antigen-antibody pairing was used.
e) The relationship of the inserted antigen and the antibody in the agarose
gel is non-coalescence.

Conclusion
The concentration of unknown antigens from other known concentration of
antigens-antibody precipitation can be estimated accurately by using the single
radial immunodiffusion (SRID) method. The diameter of the precipitation ring
formed is directly proportional to the concentration of antibodies.