A 25-year-old female is sexually active with a new partner and would like to have an HIV test. The current standard for HIV testing is to start with an enzyme- linked immuno sorbent assay (ELISA). HIV antigens are coated on a plate, and the patient’s serum is added. If the patient has HIV antibodies, binding of these antibodies to the antigens on the plate will be detected. If the ELISA test is positive, a confirmatory test is done. In this confirmatory test, the patient’s serum is added to HIV antigens, which are on a nitrocellulose membrane. This confirmatory test is a :
A. Northern blot
B. Southern Blot
C. Western Blot
D. Gel electrophoresis
E. Polymerase chain reaction
Answer- The correct answer is western blotting- C.
ELISA is the most frequently used method for screening of blood samples for HIV antibody. The sensitivity and specificity of the presently available commercial systems approaches 100% but false positive and false negative reactions occur.
The Western blot assay is a method in which individual proteins of an HIV-1 lysate are separated according to size by polyacrylamide gel electrophoresis. The viral proteins are then transferred onto nitrocellulose paper and reacted with the patient’s serum. Any HIV antibody from the patient’s serum is detected by an antihuman immunoglobulin G (IgG) antibody conjugated with an enzyme that in the presence of substrate will produce a colored band. Positive and negative control serum specimens are run simultaneously to allow identification of viral proteins (figure).
Figure-Western blot pattern for HIV
Western blots are regarded as the gold standard and seropositivity is diagnosed when antibodies against both the env and the gag proteins are detected. The sensitivity of the test systems are currently being improved by the use of recombinant antigens.
A Northern blot is when a DNA or RNA probe hybridizes with RNA on a nitrocellulose blot.
A southern blot is when a DNA probe hybridizes with DNA on a nitrocellulose blot.
Gel electrophoresis separates DNA, RNA, or proteins based on molecular size.
Polymerase chain reaction is another confirmatory test for HIV. It causes amplification of the viral nucleic acids.
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