1. Scroll down on this page to see all of the steps of the ELISA tutorial, using the "Auxilia" HIV case as an example.

2. At the bottom of the Data Screen is the navigation bar shown above. Click the "ELISA Dot blot 96wPCR" button, and the window shown below appears. This is a 96-well plate that will be used to demonstrate the ELISA procedure.

3. Click the blue 'Method' button as shown above, and select the first ELISA option as shown below.

4. Next, click the 'Protein' button on the silver button bar, as shown below (if the silver button bar is not visible, click the 'buttons' button on the navigation bar at the bottom of the screen).

NOTE: Versions previous to Case It v5.03 have a red 'Open' button next to the 'Method' button, which should not be used because it did not consistently open files. If you are using one of these earlier versions, use the silver button bar instead of the red 'Open button (or better yet, upgrade to the latest version).

The files we will use are inside the following nested folders: Cases->Infectious disease cases ->HIV->Africa->Auxilia. Open the "protein HIV.txt" file, as shown below...

...and note that the opened file appears in the "Opened & Processed" window, as shown below.

5. Select 5 wells to coat with the HIV protein (wells A1 through E1), by dragging with the mouse. Start in the upper left and drag to the lower right, as shown below. (If you are using a slower computer, you may need to do this slowly.) When you release the mouse, the circles should turn purple (as shown below), indicating that the wells have been selected for coating with the chosen protein. You can also select wells one at a time by click them (you may have to do this slowly for it to work).

6. Click the 'Load' button as shown below, and the, wells turn orange, indicating that they are now coated with the HIV protein. Labels automatically appear to the left.

7. Next, we need to associate an antibody sample with each well. To open the antibody files, click the "Antibody" button as shown below...

You could open the antibody files one at a time, but it is easier to to a "shift-click" to open them all simultaneously. To do this, click the first antibody file ("anti Auxilia.txt"), hold down the Shift key, and click the last antibody file ("anti youngest child.txt"). This will hilite all of the antibody files. Then click the "Open" button on the dialog box as shown below...

8. The opened antibody files will appear in the "Opened & processed" window. Click the first antibody line in this window to activate that file, then click the first well to select that well for loading . The center of the well will turn purple, indicating that it has been selected for loading (below left).

Then click the "Load" button and the center of the well will turn orange, indicating that it has been loaded (below right).

9. Repeat this procedure with the remaining lines in the "Opened & processed" window, clicking the "Load" button each time.

10. Clicking the "Run" button to run the ELISA, and note that some of the wells gradually turn blue.

11. To see the results more clearly, turn on the light by clicking the button as shown below...

12. To quantify the results, use the "Options" button and select "Data -> Absorbance" as shown below, and the absorbance values (Optical Density or O.D.) will replace the protein information in the labels. As a general rule of thumb, if a result is more than twice as much as the "negative control" value, it is considered to be a positive result. Since the values for Auxilia, her youngest child, and her oldest child are all above this cutoff, we would conclude that they may be positive and would run a Western blot to make sure (note that the result for the oldest child is close to the cutoff).

13. Before running the Western blot, we need to take a photo of the ELISA results so that it can be uploaded later using the Case It Web Editor. To take a phot, use the "Options" button and select "Photo -> Take photo and save", as shown below...