6th Annual Judith Ramaley Celebration of Research and Creative Scholarship
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Poster #70 Development of Immunofluorescence and Complement Fixation Assays for Immunology Lab Tri Luong and Carly Sherod Faculty Mentor: Kimberly Bates Immunofluorescence and complement fixation are two assays used in immunology. The purpose of the immunofluorescence assay is to successfully create immunofluorescence slides so that the presence of the antigen from an antibody-antigen interactions can be detected using an ultraviolet light microscope. Since the flurochrome (a molecule that fluoresces) is highly visible and the antibody is highly specific, such interactions could be made visible by covalently conjugating a flurochrome to an antibody and then relying on that antibody to find epitopes on the cell surfaces. This experiment uses the indirect labeling method on two types of known bacteria: the Borrelia burdorferi and the Escheria coli. Successfully performing this technique will allow us to see what the two types of bacteria would look like via antibody-antigen interactions. Consequently, for future reference, immunofluorescence could potentially be used to identify microorganisms that are traditionally difficult to culture. Currently, the prepared slides have shown little or no fluorescing bacteria, so the dilution concentrations are being altered until optimal results are achieved. A complement fixation assay is used to determine if a patient’s serum contains specific antibodies to a certain antigen. BSA was used as the antigen and anti-BSA was the antibody which binds to the BSA. Complement is then added so that it binds to the antibodies. Because the solution is clear, a colored indicator system must be added. A 50:50 mixture of sheep red blood cells (SRBC) and anti-SRB antibodies (hemolysin) is added to the solution. After incubating the mixture, the results can be read. Turbid (cloudy) solutions indicate that the complement was used up and the serum contains the specific antibodies. Clear solutions mean that there was enough complement left over to lyse the SRBCs. For this experiment, 10 tubes of varying anti-BSA dilutions were setup to find the titer, the point at which there is no longer enough complement left over to lyse the blood cells. Preliminary results have revealed that the proper dilution of complement lies between 1:100 and 1:150. There was too much complement in the 1:100 dilution, and all of the tubes were clear; however, there was too little complement in the 1:150 dilution, so all of the tubes were turbid. Future tests will hopefully show which dilution of complement works best for this assay.
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