As shown in Fig.?1, the simulated antigens are added to the outer wells (50?l/well) and the antiserum (antibodies) to the center well (70?l/well). the simulated (inorganic) antigens. College 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 desire for the laboratory exercise and had a better understanding of antibody-antigen reactions. Therefore, this simulated laboratory experiment is an inexpensive, safe, and fast exercise that allows college students to observe precipitations reactions of the Ouchterlony assay within the class session time. KEYWORDS: Ouchterlony assay, immunodiffusion, inorganic compounds, simulation, class room activity, antibody, antigen Intro The Ouchterlony immunodiffusion assay, developed by the Swedish physician ?rjan Ouchterlony, is used for the detection of antigens and antibodies and dedication of homologies between antigens (1, 2). The assay is also a common laboratory exercise in undergraduate immunology and microbiology classes for illustrating antigen-antibody precipitation reactions to college students (3,C5). In the Ouchterlony assay, a series of samples (the antigens) are placed in the outer wells of a gel plate, and antibodies (antiserum) are placed in the center well, after which they diffuse out and form different geometric precipitation lines in the gel (Fig.?1). The intersecting lines may create full identity (no spurs), partial identity (with one spur), or a nonidentity where the two lines mix completely (two spurs), as demonstrated in Fig.?1 (6, 7). One of Il1b the drawbacks of this assay inside a teaching environment is the time it takes for high-molecular-weight protein antigens and antibodies to diffuse toward each other in the gel, as the assay can take up to 48?h to fully develop. Furthermore, proteins are unstable and degrade over time, and antibodies can shed specificity, especially if stored at space temp. In addition, the antigens and antibodies are the costliest consumables for this BACE1-IN-4 assay. Conversely, inexpensive inorganic ionic salts have a long shelf existence of several years, even at room temperature, and are of low molecular excess weight, which allows them to have faster diffusion rates in the agarose gel, resulting in quicker reactions that college students can observe within a laboratory class BACE1-IN-4 time. Therefore, in this study, we present a lab exercise for undergraduate immunology laboratory, and related, programs where numerous inorganic salt solutions are used as simulated antigens and antibodies (antiserum) to mimic the geometric patterns created in the Ouchterlony assay. Open in a separate windowpane FIG 1 The Ouchterlony immunodiffusion assay and the different geometrical precipitation collection patterns created in the agarose gel. The antiserum is placed in the center well, and the antigens are placed in the outer wells. Precipitation lines form between the center well and outer wells, and depending on the homology between adjacent antigens, numerous geometrical spur patterns develop in the intersection, such as full identity (no spur), nonidentity (two spurs), and partial identity (one spur). (This laboratory exercise was presented in the Microbe 2019 Annual Conference in San Francisco, CA [8].) Learning objectives From conducting this lab, immunology laboratory college students will be able to: 1. Detect antigen-antibody precipitation complexes in agarose gels. 2. Observe the presence of a unique antigen or antibody. 3. Understand the conditions resulting in the antigen-antibody precipitation collection. 4. Identify the homologies between antigens as fully identical, nonidentical, or partially identical from the precipitation spurs that are created. Process Security issues College students and trainers should follow manufacturers security instructions when working with the salt solutions and agarose. All chemicals, experiments, and student activities in this study were authorized by Florida Gulf Coast Universitys Environmental Health and Safety office and institutional review boards. Furthermore, the ASM biosafety recommendations were adopted during the creation and use of these protocols, and trainers can refer to those ASM recommendations for standard laboratory methods and personal safety (https://asm.org/Guideline/ASM-Guidelines-for-Biosafety-in-Teaching-Laborator). Materials The instructor can prepare the salt solutions in advance and store them at BACE1-IN-4 space temp. Ionic salt solutions used in this exercise include 2.0 M BaCl2, 0.2 M AgNO3, 1.0 M MnSO4, 0.2 M NaCl, 1.0 M CuSO4, 1.5 M MgSO4, 0.2 M Na3PO4, and 0.2 M NH4Cl. All salt compounds can be purchased.