How Does a Western Blot HIV Test Work?

The Process of HIV Testing

Blood testing for HIV
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HIV testing is typically a multi-step process. The first test used is generally a high sensitivity test. High sensitivity tests are 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. Historically, the most common of the highly specific tests used in confirmatory testing is the Western blot test. However, there are other confirmatory tests that can be used as well. 

How does the Western blot test work?

Western blots tests are also known as protein immunoblots. These tests are used to detect specific proteins in a sample. The basic technique of a Western blot involves sorting proteins by length on a gel. Then that grid is probed with antibodies that react to the specific proteins that are being searched for.

However, when Western blots are used for HIV testing, this process is actually performed in reverse. Normally, in a Western blot test, unknown proteins are being searched for with known antibodies. However, for an HIV Western blot test, scientists work with prepared samples of HIV proteins 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 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. However, the goal is always to reduce the risk of a false positive test that upsets someone for no reason. 

Indeterminate Western blot results may be a sign of a new HIV infection. These test results 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. That means they are less likely to detect an infection. However, Western blot tests are also less likely to give a false positive test result when used to confirm an initial positive test. 

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. There are a number of reasons unrelated to HIV exposure that can cause such a result. However, 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. No single HIV test is ideal in every situation. 

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