How the Western Blot HIV Test Works

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 are now preferred. The Western blot was removed from the Center for Disease Control and Prevention recommended laboratory HIV testing algorithm in 2014 in favor of using fourth-generation antibody/antigen tests for confirmation of a positive initial screening test.

Blood testing for HIV
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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.

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 antibodies against certain HIV proteins, and different algorithms assign positivity differently.

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. 

5 Sources
Verywell Health uses only high-quality sources, including peer-reviewed studies, to support the facts within our articles. Read our editorial process to learn more about how we fact-check and keep our content accurate, reliable, and trustworthy.
  1. Pandori MW, Westheimer E, Gay C, et al. The Multispot rapid HIV-1/HIV-2 differentiation assay is comparable with the Western blot and an immunofluorescence assay at confirming HIV infection in a prospective study in three regions of the United States. J Clin Virol. 2013;58 Suppl 1:e92-6. doi:10.1016/j.jcv.2013.10.006

  2. Centers for Disease Control and Prevention and Association of Public Health Laboratories. Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations.

  3. Huang J, Wang M, Huang C, et al. Western blot-based logistic regression model for the identification of recent HIV-1 infection: A promising HIV-1 surveillance approach for resource-limited regions. Biomed Res Int. 2018;2018:4390318. doi:10.1155/2018/4390318

  4. Centers for Disease Control and Prevention. Testing.

  5. Guan M. Frequency, causes, and new challenges of indeterminate results in Western blot confirmatory testing for antibodies to human immunodeficiency virusClin Vaccine Immunol. 2007;14(6):649–659. doi:10.1128/CVI.00393-06

Additional Reading

By Elizabeth Boskey, PhD
Elizabeth Boskey, PhD, MPH, CHES, is a social worker, adjunct lecturer, and expert writer in the field of sexually transmitted diseases.