Western Blot Test for HIV

The HIV-1 Western Blot test is an in vitro qualitative assay for the detection of antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) in human serum or plasma. It is intended for use with persons of unknown risk as an additional, more specific test on human serum, plasma, or dried blood spot specimens found to be repeatedly reactive using a screening procedure, such as Enzyme Linked Immunosorbent Assay (ELISA), and as an additional, more specific test for use with serum, plasma, or dried blood dpot specimens obtained from subjects found to be reactive using rapid HIV-1 tests.

Clinical Significance – Western blot test

HIV-1 is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). Patients with AIDS and AIDS-related conditions exhibit a high prevalence of antibodies to HIV-1, and antibodies to HIV-1 have also been reported in virus positive, asymptomatic individuals.

A sample that is repeatedly reactive in the ELISA and positive on the western blot test for HIV is presumed to be positive for antibodies to HIV-1. Individuals with positive tests should be referred for medical evaluation.

western blotting bands hiv test

Cartoon Image of bands on a western

Method of the Western blot test for HIV

In the western blot assay, purified, inactivated HIV-1 strain grown in a cell line is disrupted and electrophoretically resolved into bands (See figure). The resolved protein bands are transblotted to a nitrocellulose sheet. The nitrocellulose sheet is then cut into strips, which are reacted with serum, plasma, or dried blood spot specimens.

If virus-specific antibodies are present, they bind to their corresponding viral protein bands. The bands are visualized using a phosphate-labeled anti-human immunoglobulin conjugate, followed by a substrate for the enzyme. The presence of specific HIV-1 immunoglobulins in serum specimens is indicated by labeling of HIV-1 specific proteins on the strip. Recognized HIV-1 viral antigens produce bands at gp160, gp120, p65, p55, p51, gp41, p40, p31, p24, and p18.

Results of the HIV Western blot Test

Below is an image of  a Western Blot test result from one patient.
western blotting, western blot test for hiv

On the left side of the western blot test there are two columns that are used to interpret the results by clinical lab technicians.

The column marked “NC” stands for negative control and this is equal to an HIV-negative test result. The column marked “PC” stands for positive control and this result is an HIV-positive western blot test.

On the right hand side is a column marked MW for molecular weight markers.  The expected proteins or bands in HIV positive result are indicated by the gp and p spots. The numbers beside the letters are the sizes of the proteins found in an HIV positive control.

Each black or dark grey horizontal band is representative of the presence of a different antibody against a protein found in HIV. To be conclusive (HIV-positive), a Western Blot test must have 5 horizontal bands or stripes seen in PC above.

Strips in 3 to 10 show a series of tests on an individual person who became infected with HIV.  Each strip is one Western Blot test.

These tests were performed on a single patient beginning with the day the person was first infected with HIV (Column 3, D0) to the time when the person had a conclusive HIV infection (Column 10, Day 30).

Please note that an HIV infection is not the same as an AIDS diagnosis.

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