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The assay mainly depends on an enzyme which conjugates with an antibody and reacts with a chromogenic substrate to give a colored product. A number of different enzymes are used in ELISA and numerous variants…
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Download file to see previous pages After washing again to remove unbound enzyme a chromogenic substrate is added which develops colour upon reaction with the enzyme and measurement is recorded.
Constructing a graph using the data and extrapolating to get appropriate values the following graph is generated. To construct the graph 0.088 was subtracted from 1st reading, 2nd reading and average reading and the values were plotted. Since, the readings for G were too high we had to use reading for F.
ELISA helps in quantification and detection of antigens and in this case we detected human IgM and quantified their concentration in two plasma samples. One major advantage of using antibody to capture the antigen is that there is no need to purify the antigen (Gan and Pate, 2013). A standard curve was obtained using the trend of which we have predicted the concentrations of IgM in the sample plasma.In well A and B we provided nothing but the TBS and therefore we obtained very low values which were a result of the some reactions between the substrate and the enzyme. From wells C, D, E to F we provided a gradual increase in the concentration of the IgM and a subsequent decrease in the TBS concentration and we obtained values of optical density which showed a gradual increase in trend. Each of the wells is coated with antibodies which can react with the antigen under study. IgM is now added and it binds to the antibodies already bound to the walls. IgM is a pentamer and has a number of binding sites that allows it to bind to both the antibody and the enzyme linked antibody thereby allowing it to perform sandwich ELISA. Next, the enzyme linked antibody is added which is specific for plasma IgM antigen is added. Greater the number of antigens, greater will be the number of enzyme-bound antibodies as well. After the substrate is added a colored product is obtained. The intensity ...Download file to see next pagesRead More
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“ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) OF PLASMA IgM Lab Report”, n.d. https://studentshare.org/biology/1661434-enzyme-linked-immunosorbent-assay-elisa-of-plasma-igm.
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