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Lab Exam

Terms in this set (22)

In the ELISA test, the first step was to centrifuged the samples of patient A,B, and C. The samples were centrifuged because the solutions needed to be separated and to obtain a clear fluid called serum. After the samples were centrifuged, they needed to be diluted to determine the level of the antibody in the sample and to keep the solution from being too acidic or basic. To dilute the samples, a buffer (Phosphate-buffered saline, PBS), was added. The samples then were transferred to the ELISA plate coated in an antigen. Adding too little will cause the antibody to stick and lead to a false-positive reaction. Adding too much will cause the SLE antibody to be washed away and lead to a false-negative reaction. A positive and negative control was introduced to the ELISA plate, if either control sample fails to react as expected the results of the patients would not be trusted. After having the samples ready, the ELISA plate was incubated for 15 minutes at 37 degrees Celsius. The incubation helped ensure the antibody to correctly interact with the antigen. It was placed at 37 degrees Celsius to represent the human body and was in for 15 minutes to allow enough time for a proper reaction. After incubation, the samples were removed from the wells and washed with the PBS buffer. This helps remove any antibody that did not react with the SLE antigen. Antibody that did react with the antigen will be attached to the well surface. After the washing, a secondary antibody (Horseradish peroxidase, HRP, from a rabbit (anti-human) will be added to the wells. HRP will react with the SLE antibody, if present. The ELISA plate is then incubated again at 37 degrees Celsius for 15 minutes, for the same reasoning as before. The fluid will be removed from each well again, and be washed with PBS. Finally, the HRP substrate will be added and if there are human antibodies present, the substrate will turn yellow. If the substrate is yellow for each dilution the patient is likely to have SLE. If the substrate is yellow for most dilution the patient may have SLE but needs more testing. If the substrate is not yellow, the patient is not likely to have SLE.