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.
· 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.
· 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