Ouchterlony Test

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.

The binding of an antibody to an antigen is a fundamental reaction of immunology. Antibodies and antigens form complexes that result in the formation of a visible white aggregate, which is called precipitation, making it possible to assay antibody-antigen systems. The antigen in precipitation reactions is soluble and so small that it must combine with many antibodies to form visible clumps. Soluble antigens can be attached to particulate material serving as carriers that can be detected using the more sensitive agglutination technique. Antigen-antibody reactions are widely used in research, laboratory diagnosis of diseases, pregnancy tests, and forensic identification of blood.

The technique involves cutting cylindrical wells into a purified preparation of semi-solidified agar gel in a Petri dish. The wells are filled with antibody or antigen and the dish is allowed to incubate. Homologous antigen and antibody diffuse toward each other from the individual wells to a point in the agar where optimum concentration of each is reached. Subsequently, a precipitin line will form within 18–24 hours somewhere between the two wells. If challenges are mixed together in a single well and allowed to diffuse out into the agar towards the serum test well, multiple precipitin bands are seen routinely. Non-specific reactants diffuse past each other, forming no precipitate. The precipitation reaction is subject to inhibition if either antigen or antibody is present in excess. The qualitative Ouchterlony test can simultaneously monitor multiple antibody-antigen systems and can be used to identify particular antigens.

The Ouchterlony method is wearisome due to the time and interpretative expertise required, and the need for reagent sensitivity and selectivity validation. Today, immunoassay tests are used that rely on immunological principles similar to the Ouchterlony test. Results are accurate, more sensitive, and visible within ten minutes, however, the test apparatus is portable and simple to use, requiring no prior experience to conduct and interpret the results. They can give the crime scene examiner a rapid indication as to whether a sample should be taken for DNA analysis from a bloodstain. Similarly, the laboratory analyst can utilize these tests to confirm whether a bloodstain is of human origin, which may be important where DNA results have failed. If animal blood is suspected, then the Ouchterlony test is utilized.