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Human Immunodeficiency Virus Western Blot

Samples that are repeatedly reactive for HIV-1 antibody by ELISA must be retested with a more specific confirmatory test.  For the past 25 years, Western blot was the most widely used serologic test for distinguishing between true and false positive EIA results.

The Western blot assay detects antibodies that react with specific proteins (antigens) of the HIV-1 virus.  The gene products used to interpret Western blot results are shown in the table below.

 

Gene

Antigen

Description

Immunogenicity

Detection

Envelope

gp160

gp precursor

High

Earliest

gp120

outer gp

High

Earliest

gp41

transmembrane

Moderate

2-4 wk later

Gag

p55

gag precursor

Moderate

2-4 wk later

p24

gag protein

Moderate

Earliest

p17

gag protein

Weak

Earliest

Polymerase

p66

rev transcriptase

Moderate

2-4 wk later

p51

rev transcriptase

Moderate

2-4 wk later

p31

endonuclease

Moderate

3-6 wk later

A consistent sequence of antibody responses occurs after infection. The earliest antibodies to appear are directed against gp160, gp120, p24, and p17, followed shortly by antibodies to gp41, p55, p66, and p51. Anti-p31 appears later. Antibodies to p24 and p55 decline after the onset of symptoms, while antibodies to envelope glycoproteins persist. Anti-p31 also diminishes, but not to the same extent as anti-p24. In some cases, reactions with gp120 and gp160 may be due to antibodies binding to multimers of gp41.

The criteria established by the Centers for Disease Control and the Association of State and Territorial Public Health Laboratory Directors for interpretation of Western blots are as follows:

 

Interpretation

Bands Present

Positive

Presence of any 2 bands; p24, gp41, gp120/160

Negative

absence of bands

Indeterminate

any other combination of bands

 

The majority of indeterminate patterns consist of p17, p24, or p55 alone, or combinations of these 3 bands. Indeterminate patterns may be either false positives or early seroconverters. The complete medical history must be considered in interpreting indeterminate Western blots. Individuals with a positive Western blot lacking the p31 band should be counseled that, although they may be infected, uncertainty exists about this conclusion. These individuals should be further evaluated by HIV RNA PCR testing and follow-up HIV serologic testing. Most seroconversions will be detected in repeat Western blots within 3 months. Persons with stable indeterminate patterns lasting 6 months or more, in the absence of known risk factors and clinical symptoms, may be considered negative for HIV-1 antibodies.

Improvements in sensitivity of newer generation HIV-antibody screens have created the dilemma of falsely-negative Western blot assays. Third generation HIV-antibody immunoassays can detect seroconversion as early as 22 days after infection, while Western blot may not show reactivity until 4 weeks or more.

Therefore, CDC and Clinical Laboratory Standards Institute (CLSI) have designed a new HIV testing algorithm that eliminates Western blot confirmation. The first test in the new algorithm remains the combination HIV-1/HIV-2 antibody immunoassay. The follow-up confirmation test for reactive results is a new HIV-1/HIV-2 differentiation immunoassay. This confirmation immunoassay, also known as Multispot, detects seroconversion earlier than Western blot and eliminates most indeterminate results that occur due to nonspecific reactivity from alloantibodies.

Specimens that are reactive by initial HIV-1/HIV-2 antibody immunoassay should be confirmed by Multispot testing. Specimens that do not confirm positive by Multispot may require further analysis by HIV RNA PCR.     

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