Sunday, November 4, 2007


Medical Microbiology

HIV BLOT (Western Blot assay)

This assay is a qualitative enzyme immunoassay for in vitro detection of antibodies to HIV-1 and HIV-2 in human serum or plasma. It is intended for use as a more specific supplemental test on human serum or plasma specimens found repeatedly reactive using screening procedures such as ELISA. Screening tests are widely available for detecting antibodies against HIV-1 and HIV-2. However, screening tests can be extremely sensitive but can be potentially less specific, which leads to false positive interpretations. Therefore, western blot assay with its high specificity are necessary to further confirm the presence of HIV antibodies.

Test principle: Nitrocellulose strips are incorporated with separated, bound antigenic proteins from partially purified inactivated HIV-1 using electrophoretic blotting, plus specific HIV-2 synthetic peptide on the same strips. These nitrocellulose strips are commercially available. Individual strips are incubated with diluted serum and controls. Specific antibodies to HIV-1 and HIV-2 if present in specimens will bind to the HIV-1 proteins and HIV-2 peptide on the strips. The strips are washed to remove unbound materials. Antibodies that bind specifically to HIV proteins can be visualised using a series of reactions with goat anti-human IgG conjugated with alkaline phosphatase and substrate. This assay method is sensitive enough to detect small amount of HIV specific antibodies in serum or plasma.

Clinical interpretation and limitations: According to WHO criteria, two ENV bands with or without GAG or POL is indicative of HIV infection. A positive blot indicates an infection with HIV, but it does not constitute a diagnosis of AIDS. A diagnosis of AIDS can only be made clinically if a person meets the case definition of AIDS established by CDC or WHO. However, a negative blot is not a guarantee that HIV infection has not taken place. Patients who have just produced detectable HIV antibodies may show incomplete pattern (indeterminate) but increase reactivity i.e. the intensity and number of bands occurs when followed for a period of 2-6 months. Indeterminate interpretation refers to any viral specific bands present but pattern does not meet criteria for positive.

Pictures taken from:

Yong Yang (TG02)

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