SERUM COMPLEMENT

DETECTION USING RADIAL

IMMUNODIFFUSION TEST

Learning objectives
State the principle of Radial Immunodiffusion
test as a method of detecting the quantity of
serologic markers in blood specimen
Perform aseptic technique of collecting and
processing blood specimen

What is it?
Its used to determine the quantity of an antigen
by measuring the diameters of circles of
precipitin complexes surrounding samples of
the antigen that mark the boundary between
the antigen and an antibody suspended in a
medium, such as an agar gel.

Its also known as Mancini method or single

radial immunodiffusion test.

Principle of RID
When antigens and corresponding antibodies
are allowed to react in gels or other
mediums, they will diffuse toward one
another, and at the point in which they meet
in optimal proportions, they will form a
visible precipitate.

Learning Resources

RID plate
Calibrators
Fresh or frozen serum
7% bovine Serum Albumin Solutions
Control serum

RID plate

Procedure for RID

1.

2.

3.

Perform a venipuncture using aseptic technique.

Collect 5 ml blood. Centrifuge it after 10 minutes

Take plate from the foil pouch and remove lid. Leave
the plate open for 10-15 mins at room temperature

This allows any condensation in the wells or on the

gel surface to evoporate.

4.

5.

6.

Gently mix test samples immediately before use.

Fill the well with 5 microlitre of test samples.

Tightly close the lid of plate after sample application.

Dont allow plate to remain open for long period.

Incubate at room temp for 48 hours. The plate

should be inverted with lid uppermost.

7.
8.

The gel shouldnt be allowed to dry during

incubation. Reseal plates in their foil pouches
to prevent this.
Measure diameter of the precipitin rings that
appear after incubation.

Result

RID Plate Output 7 mm

Normal

Interpretation
Antigen-antibody complexes are small and
soluble when in antigen excess. Therefore,
precipitation near the centre of the circle is
usually less dense than it is near the circle's outer
edge, where antigen is less concentrated.
The quantity and concentration of insoluble
antigen-antibody complexes at the outer edge of
the circle increases with time. Therefore, the
clarity and density of the outer edge increases
with time.

Interpretation
Expansion of the circle reaches an end
point and stops when antigen and antibody
reach equivalence.
However, the clarity and density of the
outer edge may continue to increase after the
circle stops expanding.