Human Immunodeficiency Virus (HIV-1) Confirmatory Western Blot Assay


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Subject: Human Immunodeficiency Virus (HIV-1) Confirmatory Western Blot Assay

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  • The Western blot (WB) assay is a method in which individual proteins of an HIV-1 lysate or recombinant HIV proteins allow the determination of antigenic specificity of the antibodies in the patients' serum. This is considered a confirmation test for HIV serology.

  • Western blot assays are specific for different HIV species and subtypes.

  • HIV-1 antibodies present in the specimen bind to HIV-1 antigens (p15, p17, p24, p31, gp41, p51, p66, p55, gp120, gp160).

  • Alternative confirmation testing algorithms have been described, including use of a second, different EIA, IFA, line immunoassay, or nucleic acid amplification test.


  • The Western blot is used for confirmation of repeatedly reactive HIV antibody screen or rapid HIV antibody results.

  • Western blots specific for HIV-2 or HIV-1 subtypes other than M should be considered for patients at epidemiologic risk for such infections, or when HIV-1 WB gives indeterminate or negative results. HIV-2 is most commonly acquired in West Africa.


  • Positive result: As established by CDC, the interpretive criterion for HIV-1 is defined by the presence of any two of the following bands: p24, gp41, and gp120/160.

  • Indeterminate results: HIV infection is neither confirmed nor ruled out; additional testing is required.

  • Indeterminate results may be caused by factors related to HIV infection, including a weak titer of anti–HIV-1 antibodies (e.g., early seroconversion), advanced AIDS, or infection with HIV-2 or HIV-1 subtype O. Nonspecific causes include autoantibodies, hemodialysis, hypergammaglobulinemia, or recent vaccination.

  • Additional testing for indeterminate results may include HIV-1 RNA testing, WB for HIV-2 or HIV-1 subtype O, or repeat HIV serology and WB in 4–6 weeks.

  • Negative result: Negative results do not exclude the diagnosis of HIV infection; further testing is required.

  • Additional testing for negative results may include HIV-1 RNA testing, WB for HIV-2 or HIV-1 subtype O, or repeat HIV serology and WB in 4–6 weeks.

  • Persistent reactivity of the antibody screening assay on repeat testing with negative confirmation by WB pattern suggests nonspecific reactivity and the absence of HIV infection.


  • HIV WB testing should be ordered only on sera that are repeatedly reactive by HIV screening EIA or rapid HIV antibody tests.

  • Patients with HIV-2 infection may give indeterminate results for HIV-1 Western blots. For HIV-2 WB test, no single standard can currently be applied to all tests. The CDC recommends that each test be interpreted by the criteria suggested by the kit manufacturer.

  • HIV-2 cross-reacts with HIV-1 in serologic tests. A positive screen test for HIV 1 and 2 antibodies with a repeated negative or indeterminate HIV-1 WB suggests positive HIV-2 infection and need to be confirmed HIV-2–specific test.