A Laboratory Exercise Simulating Antibody and Antigen Reactions of the Ouchterlony Double Immunodiffusion Assay Using Inorganic Salts

ABSTRACT The Ouchterlony double immunodiffusion assay is a serological technique used in the detection of antibodies and antigens for diagnostic purposes and also used in immunology laboratory courses as a common teaching assay where students observe the geometrical precipitation line patterns that form in the agarose, elucidating degrees of homology between antigens. In this classical technique, students must wait several hours to days to obtain results when protein antigens and antibodies are used. Furthermore, these proteins degrade over time if not frozen or stored in the refrigerator and are the most expensive consumables of the laboratory exercise. In this study, inexpensive and commonly used inorganic ionic salt solutions that are stable and can be stored at room temperature for several years were used to mimic antigens and antibodies. The precipitation lines started to form in the agarose plates after 15 min and fully developed within an hour, showing different geometrical precipitation patterns and spur formations that could be identified by students as full identity, partial identity, and nonidentity between the simulated (inorganic) antigens. Students conducting this exercise in a combined lecture and laboratory immunology course were able to finish the exercise as well as record and discuss results within class time, and tvhey showed increased interest in the laboratory exercise and had a better understanding of antibody-antigen reactions. Thus, this simulated laboratory experiment is an inexpensive, safe, and fast exercise that allows students to observe precipitations reactions of the Ouchterlony assay within the class session time.