Sufficiently separated proteins in an SDS-PAGE can be transferred to a solid membrane for WB analysis. For this procedure, an electric current is applied to the gel so that the separated proteins transfer through the gel and onto the membrane in the same pattern as they separate on the SDS-PAGE. All sites on the membrane which do not contain blotted protein from the gel can then be non-specifically "blocked" so that antibody (serum) will not non-specifically bind to them, causing a false positive result. Often the membrane is cut into strips to facilitate testing of a large number of samples for antibodies directed against the blotted protein (antigen).
To detect the antigen blotted on the membrane, a primary antibody (serum) is added at an appropriate dilution and incubated with the membrane. If there are any antibodies present which are directed against one or more of the blotted antigens, those antibodies will bind to the protein(s) while other antibodies will be washed away at the end of the incubation. In order to detect the antibodies which have bound, anti-immunoglobulin antibodies coupled to a reporter group such as the enzyme alkaline phosphatase are added (e.g. goat anti-human IgG- alkaline phosphatase). This anti-Ig-enzyme is commonly called a "second antibody" or "conjugate". Finally after excess second antibody is washed free of the blot, a substrate is added which will precipitate upon reaction with the conjugate resulting in a visible band where the primary antibody bound to the protein.
For this specific diagnostic test, HIV infected cells are lysed, subjected to SDS-PAGE and blotted onto a membrane as described above. The membrane was blocked, cut into strips and incubated with the serum samples from each patient as indicated.
The next page will show an example of a Western blot and how to interpret it.