4. Principle
Precipitation
Soluble antigen combine with its soluble
antibody and form a lattice that develops
into a visible precipitate.
One of the easiest of serological tests
Occur best when antigen and antibody are
present in optimal proportions
(Equivelance).
5. Precipitation
Examples;
• Ring precipitation test
• A solution of antigen is layered on the
surface of the antibody in a small tube
or capillary tube. A narrow ring of
precipitate occurs near the junction of
two fluids.
• This type of test is used for grouping
streptococci (according to C
polysaccharide) and for determining
unknown proteins in forensic
medicine
6. Gel - diffusion precipitation
• Antigen and antibody meet in an agar medium and a thin line
of precipitate is produced there (antigen - antibody complex).
• 1. Single diffusion
Antigen diffuses in the agar medium (antibody is homogenously
spread in the agar). It is carried out either in the tubes - single
gel - diffusion by Oudin or on the slide - single radial
immunodiffusion by Mancini.
7. Gel - diffusion precipitation
The principle of the reaction: The antigen is placed in a well cut
in an agar gel containing suitable diluted antibody. A ring of
precipitate forms where the reactants meet in optimal
proportions. The higher is the concentration of the examined
antigen, the greater is the diameter of the ring. According to the
diameter of the ring it is possible to count the concentration of
the examined antigen.
8. Gel - diffusion precipitation
This type of immunodiffusion is used for quantitative
determination of immunoglobulins (IgM, IgG, IgA and
IgD), complement components and other serum proteins.
9. Gel - diffusion precipitation
2. Double immunodiffusion
is used more often. Antigen and antibody are
allowed to diffuse towards each other in an agar
medium, e.g. from separate wells cut in an agar
plate or in a Petri dish. When antigen and antibody
meet in optimal proportions they produce a thin
line of precipitate. Position of the precipitate line
depends on concentrations of both antigen and
antibody and on their diffusion coefficient.
•
10. Gel - diffusion precipitation
• This reaction is used for diagnosing various bacterial,
viral, fungal and autoimmune diseases and for
recognizing toxin production by Corynebacterium
diphtheriae.
13. Some of the major differences between Agglutination
reaction and Precipitation reaction are:
The size of the antigen is different in these reactions.
Antigens are soluble molecules in precipitation reactions
while they are large and insoluble molecules in case of
agglutination.
Agglutination is a more sensitive reaction in comparison to
precipitation.
16. Viral Hemagglutination
• the attachment of viral particles by their receptor sites
16
to more than 1 cell.
• As more and more cells become attached in this
manner agglutination becomes visible
19. Hemagglutination test: method
Titer = 32 HA units/ml
1:8
1:2 1:2 1:2 1:2 1:2
8 16 32 64 128 256
virus
serial dilution
mix with red
blood cells
side view
top view
One HA unit :minimum amount of virus that causes
complete agglutination of RBCs
27. Sandwich ELISA Procedure
Prepare a surface to which a known quantity of
capture antibody is bound.
Block any non specific binding sites on the surface
Apply the antigen-containing sample to the plate.
Wash the plate, so that unbound antigen is
removed.
Apply enzyme linked primary antibodies as
detection antibodies which also bind specifically to
the antigen.
Wash the plate, so that the unbound antibody-enzyme
conjugates are removed.
28. Sandwich ELISA Procedure
Apply a chemical which is converted by the
enzyme into a coloured product.
Measure the absorbency of the plate wells to
determine the presence and quantity of antigen
29. Indirect ELISA Procedure
• The protein antigen to be tested for is added
to each well of ELISA plate, where it is given
time to adhere to the plastic through charge
interactions
• A solution of non-reacting protein is added to
block any plastic surface in the well that
remains uncoated by the protein antigen
30. Indirect ELISA-Cont
Then the serum is added, which contains a
mixture of the serum antibodies, of unknown
concentration, some of which may bind
specifically to the test antigen that is coating
the well.
Afterwards, a secondary antibody is added,
which will bind to the antibody bound to the
test antigen in the well. This secondary
antibody often has an enzyme attached to it
31. Indirect ELISA-Cont
A substrate for this enzyme is then added. Often,
this substrate changes colour upon reaction with
the enzyme. The colour change shows that
secondary antibody has bound to primary
antibody, which strongly implies that the donor
has had an immune reaction to the test antigen.
The higher the concentration of the primary
antibody that was present in the serum, the
stronger the colour change. Often a spectrometer
is used to give quantitative values for colour
strength
33. Western blot
The western blot (sometimes called the protein immunoblot) is a
widely accepted analytical technique in molecular biology used to
detect specific proteins in the given sample.
Used for the detection of HIV
41. Immunoflouresence
This technique uses the specificity of antibodies to their
antigen to target fluorescent dyes to specific biomolecule
targets within a cell, and therefore allows visualization of the
distribution of the target molecule through the sample.