The Ouchterlony test, also known as double immunodiffusion, is a technique developed by Swedish physician Örjan Ouchterlony in 1948. This method allows for a visual analysis of the relationship between antigens and antibodies. It provides a straightforward way to see if an antibody recognizes a specific antigen or to compare different antigens against a single antibody.
The Underlying Principle of Double Diffusion
The Ouchterlony test operates on the principle of diffusion through a gel matrix, typically made of agarose. When solutions containing soluble antigens and antibodies are placed into separate wells, the molecules begin to spread out. They move from an area of high concentration, the well, towards areas of lower concentration, creating opposing concentration gradients that travel toward each other.
Where the diffusing fronts of antigen and antibody meet, they interact. If the antibody is specific for the antigen, they bind together to form an immune complex. A visible line of precipitation will only form at a specific location between the wells known as the “zone of equivalence.” This zone is where the ratio of antigen to antibody concentration is optimal for forming a large, insoluble lattice. In areas with an excess of either molecule, the immune complexes that form are too small and soluble to be seen. The distinct, opaque white line only appears where the proportions are correct.
Performing the Ouchterlony Test
The setup for a double diffusion test begins with preparing an agarose gel, which is dissolved in a buffer solution and poured into a petri dish to solidify. Once the gel has set, a pattern of wells is cut into the agarose. A common pattern consists of a central well surrounded by a circle of outer wells.
With the wells prepared, the samples are loaded. A solution containing an antibody or mixture of antibodies (antiserum) is pipetted into the central well. The surrounding wells are then filled with different solutions that may contain the antigens being tested, such as known antigens for comparison or unknown samples.
After the wells are filled, the petri dish is placed in a moist chamber to prevent the gel from drying out and incubated for 24 to 48 hours. During this incubation, the loaded antigens and antibodies diffuse through the gel from their respective wells. This allows time for concentration gradients to establish and for interactions to occur and form visible precipitin lines.
Interpreting Precipitin Patterns
The analysis of an Ouchterlony test involves examining the lines that form between the central and surrounding wells. The shape and position of these precipitin lines reveal the relationship between the antigens in adjacent wells. Three primary patterns can be observed, each with a distinct immunological meaning.
A “pattern of identity” occurs when the precipitin lines from two adjacent antigen wells fuse smoothly to form a continuous, unbroken arc. This fusion indicates that the antibody in the central well is reacting with identical antigens in both neighboring wells. This shows no immunological difference between the two test antigens.
In contrast, a “pattern of non-identity” is seen when the two precipitin lines from adjacent wells cross over each other completely. This pattern demonstrates that the antigens in the two wells are unrelated and do not share any common reactive sites, or epitopes. The antibody mixture in the central well has formed independent precipitation reactions with two distinct antigens.
A “pattern of partial identity” appears as a fused line with a small, pointed extension called a “spur” on one side. This result signifies that the two antigens are related and share common epitopes, but one possesses an additional, unique epitope that the other lacks. The spur points toward the well containing the simpler antigen, as the more complex antigen continues to react with antibodies that do not recognize the simpler one.
Applications in Science and Medicine
In clinical diagnostics, the Ouchterlony test can help identify the presence of specific antibodies in a patient’s serum, which may indicate an infection. For example, it is used in the diagnosis of certain fungal diseases by detecting patient antibodies against fungal antigens.
In research settings, the technique is used to analyze the components of a complex mixture of proteins. Scientists can determine the relationship between different proteins by testing them against specific antibodies. This can aid the process of purifying a protein or studying immunological cross-reactivity between proteins from different species.
The method is also applied in quality control processes. Companies that produce antibodies for research or therapeutic use must ensure their products are specific to the intended target. The Ouchterlony test provides a method to check for cross-reactivity in batches of antisera, confirming the antibodies bind only to the desired antigen.