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How Does a Western Blot HIV Test Work?


Updated February 03, 2014

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Question: How Does a Western Blot HIV Test Work?
HIV testing is usually a multi-step process. The first test used is generally a high sensitivity test designed to detect as many potential HIV infections as possible. That first test is then followed up by one or more highly specific tests to rule out possible false positive HIV diagnoses. The most common of the highly specific tests used in confirmatory testing is the Western blot.

Fun fact: The directional names of blot tests are respectful salutes to the work of Edward Southern, the inventor of the Southern Blot. In addition to the Western blot, there are also Northern, Eastern, and Southwestern blots.

Answer: Western blots, or protein immunoblots, are used to detect specific proteins in a sample. The basic technique of a Western blot involves sorting proteins by length on a gel and then probing the gel with antibodies that react to the proteins that are being searched for. However, when Western blots are used for HIV testing, the process is actually performed in reverse. Instead of unknown proteins being tested for with known antibodies, labs work with prepared protein samples, and look to see if there are any antibodies in a person's blood that stick to them.

Western Blot HIV tests usually look for antibodies against the following HIV proteins:

  • Proteins from the HIV envelope: gp41, and gp120/gp160.
  • Proteins from the core of the virus: p17, p24, p55
  • Enzymes that HIV uses in the process of infection: p31, p51, p66

In order for a person to be considered HIV positive, they generally need to have either antibodies against one of the envelope proteins and one of the core proteins, or against one of the enzymes. If a person has antibodies that are against a different combination of proteins, their results are usually considered to be indeterminate. The exact algorithm for declaring a result indeterminate varies for each specific Western blot test.

Indeterminate Western blot results may be a sign of a new HIV infection; however, they may also occur when individuals have been exposed to or infected with other retroviruses, such as HTLV. Generally, if a person with an indeterminate Western Blot HIV test is actually HIV positive, their Western Blot test will become clearly positive after more time has passed.

Western blot tests are used as confirmatory tests rather than as primary tests, since they are less sensitive than first-round ELISA tests or RNA detection tests.

Primary Tests for HIV that are Confirmed with Western Blots

ELISA tests are actually similar to Western blots, in that they look for serum antibodies against HIV antigens. However, ELISA tests are designed to look for any reactivity, rather than for antibodies against specific proteins. They are more sensitive than Western blots, and substantially cheaper, but carry a higher risk of false positives when used alone. HIV RNA tests MEASURE THE PRESENCE OF THE VIRUS DIRECTLY. TYPICALLY THIS TEST IS USED FOR MONITORING KNOWN INFECTION AS THE LEVEL OF VIRUS GETS HIGHER OR LOWER (USUALLY WITH TREATMENT). THERE ARE CIRCUMSTANCES DIRECT TESTS FOR THE VIRUS ARE HELPFUL for detecting early HIV infections, where the body has not yet had time to make antibodies. SUCH tests are not ROUTINELY USED TO DIAGNOSE INFECTION UNLESS ACUTE HIV IS SUSPECTED. Furthermore, because of the possibility of both false positives and false negatives, HIV RNA test results are not generally considered to be definitive until they have been confirmed by antibody tests.

HIV RNA tests directly measure the presence of the virus. Typically these tests are used for monitoring known infections to see how the levels of the virus change over time and/or with treatment. There are also some circumstances in which HIV RNA tests can be used for detecting early HIV infections, where the body has not yet had time to make antibodies. These tests, however, are not generally used to diagnose an infection unless ACUTE HIV is suspected. Furthermore, because of the possibility of both false positives and false negatives, HIV RNA test results are not generally considered to be definitive until they have been confirmed by antibody tests.

If You Have Been Told You Have An Indeterminate Western Blot Test

Being told that a Western blot test is indeterminate can be extremely stressful for people who are undergoing HIV testing. Although there are a number of reasons unrelated to HIV exposure that can cause such a result, people with indeterminate Western blots are usually advised to seek repeat testing -- either immediately or after some time has passed.

Immediate retesting checks to see whether there was a flaw in the way the Western blot was run. In contrast, waiting a month or more before retesting allows a person's immune system time to make additional antibodies against HIV antigens, if they actually have been infected with HIV.

Retesting will often resolve an indeterminate Western blot test into a clearly positive or negative result. However, if repeated tests continue to send confusing signals, doctors may need to switch to using other methods of determining whether or not you have an HIV infection. They may, for example, employ tests that look directly for the human immunodeficiency virus.


Guan M. Frequency, causes, and new challenges of indeterminate results in Western blot confirmatory testing for antibodies to human immunodeficiency virus. Clin Vaccine Immunol. 2007 Jun;14(6):649-59.

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