While ELISA measures antibody to whole virus and gives a "positive," "negative," or indeterminate test result, western blotting is a more specific test. It allows one to visualize antibodies directed against each viral protein. For this reason, it is a confirmatory test for a positive HIV ELISA. In this HIV Western blotting (WB) application, proteins, often from HIV-infected cell lysates are electrophoresed into an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel. The proteins migrate into the gel and separate based upon size and charge. Characteristically, smaller proteins migrate through the gel faster than large proteins.

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.

Immunological Techniques
University of Arizona
Monday, October 6, 2008
douglas.lake@asu.edu

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