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

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. 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.
The measurement of the ring diameter produced and reading the corresponding
concentration values from the calibration curve make it possible to determine
concentration of the antigen in an unknown sample  

Discussion

R a d i a l   i m m u n o di f f u s i o n i s   a   t y p e   o f   q u a n t i t a t i v e l y   m e t h o d   t o
d e t e r m i n e   t h e l e v e l   of   a n   a n t i g e n ,   w h i c h   m o r e   s e n s i t i v e
than in double immunodiffusion.
In this experiment, the antibody is incorporated into liquefied
a g a r a n d a l l o w e d i nt o t h e
g e l .   T h e   a n t i s e r u m   i s   u n i f or m l y   d i s t r i b u t e d t h r o u g h o u t t h e a g a r
g e l .   T h e   a n t i g e n   i s   a d d e d   t o   s m a l l   w e l l s a n d   r a d i a t e s   t hr o u g h o u t   t h
e   a n t i b o d y - c o n t a i n i n g   m e d i u m ,   l e a v i n g   a   pr e c i p i t a t e t h r o u g h o u t
t h e g e l . T h e a n t i g e n d i f f u s e s o ut
of the well, where the concentration is relatively high and
i t   f or m s r e l a t i v e l y s o l u b l e a n t i g e n — a n t i b o d y a d d u c t s . H o w e v e r ,
a s i t d i f f u s e s f ur t h e r a n d f ur t h e r f r o m t h e w e l l , i t s c o n c e n t r a t i o n
decreases. The amount of diffusion is then quantified by
m e a s u r i n g t h e d i a m e t er o f t h e r i n g w h i c h w a s pr o p o r t i o n a l t o t h e
l o g o f t h e c o n c e n t r a t i o n of a n t i g e n . T h e g r 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 of
the precipitin disk

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
gel and by measuring the diameters of their precipitin disks, a
calibration graph can be constructed. The antigen concentrations
of unknown samples run on the same gel can then be found by
s i m p l e i n t er 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 o f t h e
r e s p e c t i v e pr e c i p i t i n d i s k s . F u r t h er m o r e , t h e s e t e s t s w e r e a l s o
b e n e f i t i n d e t e c t i n g t h e m i x t u r e s o f a n t i g e n s or   a n t i b o d i e s .

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