The Western Blot assay for detection of anti-HIV-1 antibodies is used as a confirmatory test for positive ELISA assays for HIV-1. It is not appropriate to use the Western blot assay as a screening test for HIV-1 antibodies, largely due to the number of indeterminate results.
HIV-1 viral genome includes:
(1) env = envelope
(2) gag = proteins forming the viral core
(3) pol = polymerase; enzymes responsible for reverse transcription and integration
|
Band (kd) |
Gene |
Product |
|
gp160 |
env |
precursor of env glycoprotein |
|
gp120 |
env |
outer env glycoprotein |
|
p66 |
pol |
reverse transcriptase component of pol translate |
|
p55 |
gag |
precursor of gag proteins |
|
p51 |
pol |
reverse transcriptase component of pol translate |
|
gp41 |
env |
transmembrane env glycoprotein |
|
p31 |
pol |
endonuclease component of pol translate |
|
p24 |
gag |
gag protein, core |
|
p17(18) |
gag |
gag protein, inner membrane |
where:
• kd = kiloDaltons
• gp = glycoprotein
• p = protein
• distinction between gp120 and gp160 can be difficult, and these two bands can be considered as one reactant for purposes of interpreting Western blot test results (CDC)
Interpretation
Negative: no bands present
Indeterminate: one or more bands are present, but the pattern does not meet any of the criteria for a positive diagnosis.
|
Group |
Criteria for a Positive Western Blot |
|
ASTPHLD and CDC |
any 2 of the following 3 bands: p24, gp41, gp120/gp160 |
|
DuPont |
p24 AND p31 AND (gp41 OR gp120/gp160) |
|
American Red Cross (ARC) |
3 or more bands, with one band from each gene product group: gag (p17, p24 p55), pol (p31, p51, p66), env (gp41, gp120, gp160) |
|
Consortium for Retrovirus Serology Standardization (CRSS) |
two or more bands, with (p24 OR p31) AND (gp41 OR gp120/160) |
where:
• ASTPHLD = Association of State and Territorial Public Health Laboratory Directors
|
Public Health Service Consultation Group's Interpretive Standards (see DuPont, above) |
|
positive |
p24 AND p31 AND (gp41 OR gp120/gp160) |
|
probably positive, IIa |
p24 AND (gp41 OR gp120/gp160) |
|
probably positive, IIb |
p31 AND (gp41 OR gp120/gp160) |
|
probably positive, IIc |
gp41 AND gp120/gp160 |
|
indeterminate IIIa |
only one of the following: p24, p31, gp41, or gp120/gp160 |
|
indeterminate IIIb |
other non-HIV-nonspecific bands |
|
negative |
no bands present |
(after CRSS, JAMA 1988, Table 4, plus discussion on IId page 678)
Evaluation of Indeterminate Results
Indeterminate results occur with some frequency with Western Blot testing and is a limiting factor in its use.
Most indeterminate serum samples react with viral core (gag) proteins p17 (18), p24 or p55 (Fang et al., 1987)
Some indeterminate patterns are associated with HIV-1 or other infection:
(1) The combination of a gag (p17, p24, or p55) AND pol (p31, p51, or p66) but not env (gp41, gp120, gp160) can be seen in HIV-2 infection, due to cross-reactivity between proteins encoded by the gag and pol genes.
In blood donors without risk factors, an indeterminate result on Western blot assay may persist for several months, without ever developing HIV-1 or HIV-2 infection.
In one group of blood donors with indeterminate results (Kleinman et al., 1988):
(1) about 5% with reactivity to p24 were later shown to have HIV-1 infection; half of these patients were positive on the immunofluorescence assay (IFA) and the other half had nonspecific IFA findings.
(2) none of the patients with p17(18) reactivity showed HIV-1 infection later
Purpose: To use Western blot testing to help confirm a positive screening test for HIV antibodies.
Specialty: Infectious Diseases
Objective: laboratory tests
ICD-10: B24,
