1.
What is the principle behind
Ouchterlony double diffusion?
Antibodies are reasonably specific about what antigen they bind or
react to, so they can be used to distinguish Antigens (protein). The Ouchterlony
procedure is one of the several ways in which titer of an antibody can
be measured.
In this diffusion, test antigen and antibody diffuse toward each other in
a semisolid medium to a point in the medium where optimum concentration of each
is reached. A band of precipitation occurs at this point. The technique
involves cutting wells into an Agarose solidified in a Petri dish. The wells
are filled with antibody or antigen and the dish is incubated.
When homologous antigen and antibody diffuse toward each other from
the individual wells, a precipitin line will form somewhere between the two
wells. Precipitation occurs because the antigen is multivalent i.e., has
several antigenic determinants per molecule to which antibodies can bind.
Antibodies have at least two antigen binding sites, thus large aggregates or
lattices of antigen and antibody are formed.
Precipitation/cross-linking will not occur if excess antigen is
present or if excess antibody is present. Cross-linking and lattice formation
will only occur when antigen and antibody concentrations are optimal. An
increasing amount of antigen is added to a constant amount of antibody in
solution. This is called the antibody-excess zone (Prozone phenomenon).
As more antigen is added, the amount of protein precipitated increases
until the antigen/antibody molecules are at an optimal ratio. This is known as
the equivalence zone or equivalence point. When the amount of antigen in
solution exceeds the amount of antibody, the amount of precipitation will
decrease. This is known as the antigen excess zone.
2.
Trace the historical experimental
background for the technique.
Örjan Thomas Gunnarson Ouchterlony, a Swedish
bacteriologist who was born 1914 in Göteborg (Gothenburg), developed a double
immunodiffusion technique in 1948 that, when used in forensics, determines
whether a bloodstain is human or animal. This technique is commonly called
Ouchterlony double gel diffusion test, which refers to Ouchterlony's critical
analysis in 1968 in his Handbook of Immunodiffusion and
Immunoelectrophoresis. Another synonym employed is the agar gel
immunodiffusion test, AGID.
3.
What is assay buffer? What are its
components and function?
Assay buffer is a buffered protein and detergent
solution intended for use in dissociation- enhanced time resolved
fluoroimmunoassays (DELFIA) that include Eu/Sm/Tb labeled Antibodies or
Antigens.
Components
· It is a Tris-HCl buffered NaCl solution (pH 7.8) containing
< 0.1% NaN3 (Sodium azide), Bovine
serum albumin (BSA), Bovine Gamma Globulins, Tween 40,
Diethylenetriaminepentaaceticacid (DTPA) and an inert red dye.
Functions
· It is optimized to give a
minimum non-specific background in solid phase assays.
· It is meant for use as a
diluent for Eu/Sm/Tb labeled compounds.
4.
Why is agarose used as a base to
study this interaction?
Agarose is
obtained by purification of the agar .Agarose is an ideal gel matrix for
diffusion and electrokinetic movement of biopolymers, and its gel is an
anti-convection medium, which is biologically inert and with controlled ionic
properties. Precipitaion lines can be seen easily through agar.
The agarose
component of agar is composed of repeating molecules of galactopyranose and
side groups that protrude from these are arranged such that two adjacent chains
can associate to form a helix. The chains wrap together so tightly that water
can be trapped inside the helix. As more and more helices are formed and become
cross-linked, a three-dimensional network of water-containing helices is
created. The entire structure has no net charge. Thus, agarose is widely used
in immunology.
5.
What are the 2 methods of getting
serially diluted samples? Which is a better scientific approach?
.
a.
In a single serial dilution assay, each dilution is tested once. In a virus
heamagglutination inhibition test, the highest dilution that prevents
agglutination of erythrocytes on a test plate is the antibody’s
haemagglutination inhibition titre. This type of titration which tests only
dilution intervals actually divides the dilutions into blocks. The blocking is more marked when titre are
expressed as’ less than’ or ‘greater than’. The data therefore are essentially
ordinal.
b. In
a multiple serial dilution assay, each dilution is tested several (preferably at least 5) times. The objective is to
achieve a ‘strong’ measure. The end point is the dilution of a substance at
which a specified no. of members of a test group shows a defined effect, such
as death or disease. So this is a better
scientific approach.
Both techniques utilize geometric
dilutions, the range of dilutions depending on the sensitivity of the test.
Sensitivity here refers to the ability of the system to detect the amounts of
antigen and antibody.
6.
Why is it necessary to maintain
humidity in the chamber during incubation?
This will prevent the gel from getting dried up there by allowing the
proper diffusion of antigen and antibodies.
7.
Can you think of at least 5
applications for this technique in diagnostics, labs, industry or research?
Ø Application
of Immunodiffusion Methods to the Antigenic Analysis of Dengue Viruses.
Ø Use of double
immuno-diffusion (Ouchterlony) test for the diagnosis of swine vesicular
disease.
Ø Ouchterlony
double diffusion is used in the production of the monoclonal antibody to
Streptococcal Group B Carbohydrate.
Ø Purification
and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from
bovine liver mitochondria using Ouchterlony double diffusion.
Ø Used as a
chemical method for identification of blood in forensic science.
8.
Reference 2 papers where this
technique was used and add a note on why this was used in the paper. Provide
the experimental data from those papers.
a.
Serogroups of Erwinia carotovora Potato Strains Determined with Diffusible Somatic Antigens
