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 calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings mm
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings mm
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings mm²
Figure 1. A calibraon curve
showing Angen concentraon
against the square diame
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin
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 calibraon curve
showing Angen concentraon
against the square diameter of
precipin rings mm²
Figure 1. A calibraon curve
showing Angen concentraon
against the square diameter of
precipin 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
https://rockland-inc.com/radial-immunodiffusion-kits.aspx
https://id=mNx4CpEbHz4C&pg=PA201&dq=report+on+radial+immunodiffusion&hl=en&sa=X&ved=0ah
UKEwjynovGrOXhAhWTShUIHbjfBTEQ6AEIQjAF#v=onepage&q=report%20on%20radial
%20immunodiffusion&f=false
