Western Blot (WB) Test

Laboratory Services Section


This is the confirmatory test used for HIV-1.

Specific HIV proteins are fractionated according to molecular weight by electrophoresis on a polyacrylamide slab gel. The separated HIV proteins are transferred from the gel, via electrophoretic blotting, to a nitrocellulose membrane which is then washed, blocked (to minimize nonspecific immunoglobulin binding), and packaged. Individual nitrocellulose strips are incubated with serum specimens, or controls. During incubation, if HIV antibodies are present in the specimen, they will bind to the viral antigens bound to the nitrocellulose strips.



Visualization of the human immunoglobulins specifically bound to HIV proteins is accomplished in situ using a series of reactions with goat anti-human IgG conjugated with biotin, avidin conjugated with horseradish peroxidase (HRP), and the HRP substrate 4-chloro-1-naphthol.

If antibodies to any of the major HIV antigens are present in the specimen in sufficient concentration, bands corresponding to the position of one or more of the following HIV proteins (p) or glycoproteins (gp) will be seen on the nitrocellulose strip: p17, p24, p31, gp41, p51, p55, p66, gp120, gp160 (number refers to apparent molecular weight in kilodaltons). Interpretation is based on the presence or absence of these bands. The TDSHS laboratory uses the Center for Disease Control (CDC)/Association of Public Health Laboratories (APHL) interpretation of HIV western blots. This states that a Reactive western blot has any two of the following: p24, gp41, gp120/gp160 (distinguishing the gp120 band from the gp160 band can be difficult. These two glycoproteins cna be considered one reactant for the purposes of interpreting western blot test results). The criteria for a negative western blot interpretation is "no bands." All other patterns are reported as an indeterminate western blot.

Last updated September 16, 2010