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In general, the Western Blot test is ordered after obtaining two consecutive positive results of the enzyme-linked immunosorbent assay or ELISA test for HIV. It is a more difficult test than the ELISA but also more accurate. Source: WebMD, WebMD Medical Reference from Healthwise; Article Title: Human Immunodeficiency Virus (HIV) Test; Last Updated: 8th April, 2010; URL: http://www.webmd.com/hiv-aids/human-immunodeficiency-virus-hiv-test). The Western Blot test is also known as the Immunoblotting or Protein Immunoblot Test.
The Western Blot test is an analytical technique--as opposed to the qualitative rapid test techniques--that helps in the detection of particular proteins present in the tissue or other extracts from the person being tested. Along with one or more positive results obtained from an ELISA test, a positive Western Blot result means a 99.9 percent chance that the tested person has acquired the HIV virus.
The Western Blot test uses a set of techniques which separates the native proteins from a tissue sample or blood extract. This separated protein is then transferred to a membrane where it is examined using antibodies specific to the target protein. Both separation and transferring of the protein for further examination involves a few sets of complicated steps. (Source: Proceedings of the National Academy of Sciences USA 76 (9): 4350–54. doi:10.1073/pnas.76.9.4350. PMC 411572; Report Title: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications; Author(s): H, Staehelin T, Gordon J.; URL: http://www.pnas.org/content/76/9/4350).
Protein has been extracted from its source and transferred onto a membrane because of the membrane's ability to bind protein. At this point, we must realize that we now have a situation where both the antibodies used for detection are also proteins by nature and must not react with the membrane. In order to achieve this, a blocking technique is used which prevents the interaction of membrane and antibodies.
So when the antibodies are added, no membrane attaches itself to them.