Radial immunodiffusion

Introduction
Radial immunodiffusion represents a hallmark in the evolution of immunoserology
because it represents the first successful attempt to develop a precise quantitative
assay suitable for routine use in the diagnostic laboratories. Radial immunodiffusion
received its designation from the fact that a given antigen is forces to diffuse
concentrically on a support medium to which antiserum has been incorporated.
Apolyclonal antiserum, known to precipitate the antigen, is added to molten agar and
an agar plate containing agar and those wells filled with identical volumes of samples
containing known amounts of the antigen, calibrators and of unknown samples to which
the to which the antigen needs to be assayed.

Calibrators are used as a quantitative reference – calibrators have a known protein

concentration which will produce a specific precipitant ring diameter at this given
concentration.

After 24 to 48 hours it is possible to measure diameter of circular precipitates formed

around the wells where antigens were placed. Those diameter are directly proportional
to antigen concentration .A plot of precipitate ring diameters versus concentration is
made for the samples with known antigen concentration. This plot, known as calibration
curve, is used to extrapolate the concentrations of antigen in the unknown samples,
based on the diameter of the corresponding precipitation rings.

Objectives

To know the amount of antigen in a sample.

 Introduces the principle of antigen-antibody interactions by using the Radial Immunodiffusion
procedure
 Determine the concentration of an immunoprotein by preparation of a reference curve
Results

Table 1.0 shows the diameter recorded for Gel,2,3,4,5 and their mean
which were calculated

Samples/mm A B C D E F
Gel 1 10 13 17 17 13 19
Gel 2 11 14 17 18 12 18
Gel 3 13 14 17 19 12 20
Gel 4 12 15 18 19 14 19
Gel 5 12 12 17 18 12 19
Mean 11.6 13.6 17.2 18.2 12.6 19
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings mm
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings mm

Figure 1. A calibraon curve

showing Angen concentraon
against the square diameter of
precipin rings mm²
Figure 1. A calibraon curve
showing Angen concentraon
against the square diame

Figure 1. A calibraon curve

showing Angen concentraon
against the square diameter of
precipin
Table 1.1 shows known antigen concentrations and the calculated unknown
antigen (mg/ml) and the measurement recorded for the diameter of the
precipitate rings (mm) and the calculated diameter squared values (mm)

Sample Concentration Mean diameter of Mean square

mg/ml ring diameter of
participate/mm ring/mm2
A 3.75 11.6 134.6
B 7.5 13.6 185.0
C 15.0 17.2 295.8
D 30.0 18.2 331.2
E 3.98(unknown) 12.6 158.8
F 17.64(unknown) 19 361.0
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings mm²
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings m

The graph below shows calibration curve of Antigen concentration against

diameter squared of precipitin ring
 134.6
400

350

300

250

200

150

100

50

0
3.75 7.5 15 30

Discussion
In terms of reliability the results obtained are outside the
r e l i a b i l i t y l i m i t r a n g e o f R I D A w h i c h i s 6 0- 4 5 0 m g / m l c o m p a r e d
t o t h e e x p e r i m e n t w h i c h i s i n t h e r a n g e of 3 . 7 5 - 1 7 . 4 6 m g / m l .
However this could be due to high concentration of antigens
b e i n g l o a d e d i nt o t h e w e l l s a n d i f a l o w e r c o n c e n t r a t i o n w e r e t o
be used and les antigen diffusion would be expected to occur as
t h e z o n e of e q u i v a l e n c e w o u l d b e r e a c h e d . T h e c i r c u l a r pr e c i p i t i n
r i n g s t h a t f or m i n R I D A a r e s o l u b l e a n t i g e n s w h i c h d i f f u s e o n t h e
a g a r o s e g e l . T h e gr e a t e r t h e i n i t i a l c o n c e n t r a t i o n o f a n t i g e n i n t h e
w e l l , t h e g r e a t e r t h e d i a m e t e r o f t h e   pr e c i p i t i n d i s k . T h u s , b y
r u n n i n g a r a n g e of k n o w n a n t i g e n c o n c e n t r a t i o n s o n t h e g e l a n d
b y m e a s u r i n g t h e d i a m e t er s o f t h e i r p r e c i p i t i n d i s k s , a c a l i b r a t i o n
graph can be constructed. The antigen concentrations of unknown
samples run on the same gel can then be found by simple
i n t e r p o l a t i o n h a v i n g m e a s u r e d t h e d i a m e t e r s of t h e r e s p e c t i v e
p r e c i p i t i n d i s k s . T h e e x p a n s i o n o f t h e pr e c i p i t i n r i n g s o c c u r s
b e c a u s e t h e di f f u s e d c o n c e n t r a t i o n o f t h e a n t i b o d i e s a n d a n t i g e n s
as the samples diffuse. However once an equal concentration is
a c h i e v e d t h e e x p a n s i o n of t h e r i n g s t o p , t h i s i s c o m m o n l y k n o w n
a s a n e q u i l i b r i um r e a c h e d . A d d i t i o n a l l y i f t h e c o n c e n t r a t i o n o f
a n t i b o d i e s a d d e d t o t h e a g a r o s e g e l w a s v er y l o w , n o p r e c i p i t i n
r i n g s w o u l d h a v e b e e n f or m e d .

References
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