Download A - Tube precipitation

University of Kufa
Practical immunity
College of Vet. Medicine
Assist. Lect. Mohamed Talib Mohamed
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Precipitation Reaction
Precipitins can be produced against most proteins and some
carbohydrates and carbohydrates- lipid complexes. Various system are
available in which precipitation tests are performed in semisolid
media such as agar or agarose, or nongel support medium such as
cellulose acetate. Agar has been found to interfere with the migration
of charged particles and has been largely replaced as an
immunodiffusion medium by agarose. Agarose is a transparent,
colorless, neutral gel. In the clinical laboratory several applications of
the precipitation reaction are used. These methods include:
A - Tube precipitation
B - Immunodiffusion
C - Electroimmunodiffusion
A - Tube precipitation
Solution of antibody is carefully layered on top of a solution of
antigen, such that there is no mixing between the two.
With time at the interface where the two layers meet, antigen
antibody
complexes
form
a
visible
precipitat.
B - Immunodiffusion: These are of two type: single and double
immunodiffusion.
I. Double diffusion
This technique also referred to as the Ouchterlony method, may
be used to determine the relationship between antigen and
antibodies.
Principle: Antibody dilutions and specific soluble antigens are
placed in adjacent wells. If the well size and shape, distance
between wells, temperature, and incubation time are optimal,
these solutions diffuse out, bind to each other, cross-link, and
form a visible precipitate at the point of equivalence
perpendicular to the axis line between the wells, the
precipitation bands will be compared with a standard antigen.
The precise location of the band depends on the concentration
and rate of diffusion of antigen and antibody. In a condition of
antibody excess, the band will be located nearer the antigen
well. If two antigens are present in the solution that can be
recognized by the antibody, two precipitin bands form
independently.
Antibodies associated with autoimmune disorders such as
rheumatoid arthritis and systemic lupus erythematosus can be
identified by double diffusion.
Identity
An identity reaction is indicated when the precipitin band forms
a single smooth area. This precipitin is formed between the
antibody and the two test antigens fuses indicating that the
antibody is precipitating identical antigen specificities in each
preparation. This does not mean that the antigens are necessarily
identical; they are only identical insofar as the antibody can
distinguish the difference.
Nonidentity
A non-identity pattern is expressed when the precipitation line
cross each other. They intersect or cross because the sample
contain no antigenic determinants in common.
Partial Identity
In a partial identity pattern , the precipitation lines merge with
spur formation. This merger indicated that the antigen are non
identical but possess common determinants.
II. Single radial immunodiffusion
This is a simple and specific method for identification and
quantitation of a number of proteins found in human serum and
other body fluids.
Principle: Radial immunodiffusion is based on a technique
using a precipitin reaction in which specific antibody is added to
a buffered agarose medium, serum containing the test antigen is
placed in a well centered in the agarose. The diameter of the
resulting precipitin zone is related to the concentration of
antigen placed in a well.
C - Electroimmunodiffusion (EID)
EID is a variation of the double immunodiffusion reaction in a
support medium such as cellulose acetate or agarose through the
use of an electric current that enhances the mobility of reactants
and increase their movement towards each other. Antibody is
placed in the well favoring its migration in the direction of the
cathode; antigens that tend to be more negatively charged and
placed in the well that favors migration of the anode. Precipitin
bands form at a point of equivalence in a shorter periods of time.
Electro immunodiffusion method, like immunodiffusion
procedures, are classified into one-or-two-dimensional, singles,
or double diffusion when a voltage is applied across the gels to
move the antigens and antibodies together, immuno-double
diffusion becomes counter current immune electrophoresis
(CIE) radial immunodiffusion (RID) becomes electro
immunoassay (EIA).
Counter Current immunoelectrophoresis (CIE)
is a variation of the classic precipitin procedure; it merely adds
an electrical current to help antigens and antibodies move
towards each other more quickly than in simple diffusion. The
procedure takes advantage of the net electric charge of the
antigens and antibodies being tested in a particular test buffer.
Variables such as types of gel, amount of current, a
concentration of antigen and antibody must be carefully
controlled for maximum reactivity. The sensitivity of CIE is 10
to 20 times greater than in immuno-double diffusion, however,
it is more expensive than other techniques such as
immunodiffusion
Electro immunoassay
Antigens may be quantitiated by electrophoresis than in an
antibody-containing gel electroimmunoassay. This technique
combines the speed of electrophoresis with the accuracy and
sensitivity of radioimmunoassay.