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:
http://www.mpbio.com


Yong Yang (TG02)
0503196H

Comments:
hey yong yang,

May i know how much time does this method/assay takes to confirm the presence of HIV antibodies? Is there any other method/test that also have high specificity in detecting the presence of HIV antibodies?

Thanks:)

Doreen Ng (tg01)
 
hi..may i know waht is the case definition of AIDS established by CDC or WHO? thanks!
-sharon ang
0503219H
tg02
 
hello yy!

ermm when doing the hiv blot, is there any special procedures to take note of?
and is there any disadvantages to this blot?

Thanks!

Natalie (:
 
hello yong yang,

will there be a chance where there is false positive or false negative results? is western blot used as a screening test or a confirmatory test?

phuiyuen, tg 02
 
hey

what do u mean by "Patients who have just produced detectable HIV antibodies may show incomplete pattern (indeterminate) but increase reactivity "? i dun quite understand this..

is there any confirmatory to be followed after this?
 
To Doreen,
This method takes around 16 hours. Other immunoassays with high specificity include the Vidas HIV DUO ultra, the Architect HIV Ag/Ab combo, both assays screen for HIV antigens and antibody. These are the fourth generation HIV screening assays.

To Phuiyuen,
The western blot is a confirmatory test for the detection of HIV antibody, for samples that have been screened as positive by routine ELISA assays(now capable of detecting HIV antigen and antibody). Nevertheless, it’s still possible to obtain false negative results; this is particularly true when the patient has just been infected with HIV. This is known as the window period, when HIV antibody is still undetectable, but HIV is present in the blood. Thus, using a HIV Antigen/Antibody combo test can be useful for the early detection of HIV. If the screening test is positive but western blot indicates negative, the patient's blood will be drawn again after 2-6 months interval.There's a chance of false positive in western blot when conditions such as hyperbilirubinemia,connective tissue disorder and polyclonal gammopathy are also present.


To Sharon,
The AIDS definition by WHO includes toxoplasmosis of the brain, candidiasis of the esophagus, trachea, bronchi or lungs and Kaposi’s sarcoma.

To Natalie:
I don’t think there’s any special procedure to take note of. However, when adding sera for blotting, facemask must be worn as a safety precaution. This is to avoid potential HIV infected sera from contacting with the med tech’s eyes, which can lead to HIV infection. Some disadvantages are that the blot test is quite time consuming, expensive and relatively labour intensive.

To Vino,
According to WHO criteria, two ENV bands with or without GAG or POL are indicative of HIV infection. During the early phase of producing detectable specific HIV antibodies (seroconversion), low levels of antibodies lead to faintly positive bands. Tests indicating less than the required number of viral bands to be confirmed as HIV positive are reported as indeterminate: a person who has an indeterminate result should be retested, as later tests may be more conclusive.Serum is drawn from the same patient again after 2-6 months, when antibody levels against the specific HIV antigens are high, the intensity of the bands are stronger and more bands appear as well.

Yong Yang
TG02
 
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