- Red top or gel barrier tube
- 7 mL of venous blood
- Western blot analysis helps detect one protein in a mixture of a number of proteins, and also provides information about the size of the protein
- Western blot can be used to specifically detect HIV-1 or HIV-2
- Western blot technique is used as a confirmatory test (gold standard) for chronic infection with human immunodeficiency virus (HIV). If the screening with ELISA is positive, this test is used to confirm HIV positivity.
- Western blot not only indicates whether the patient is HIV positive or negative but also detects which antibodies are directed against each viral protein
- Principle and methodology of Western blot:
- HIV proteins and glycoproteins are separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper
- The patient's serum is added to the antigen-impregnated paper
- The antibodies, if any, present in the serum bind to specific HIV antigens
- Antihuman immunoglobulin conjugated with biotin or an enzyme are added to visualize the colored bands of HIV antigen-antibody complexes
- For identification of the viral proteins, positive and negative control serum specimens are run simultaneously
- High risk patients in whom Western blot testing is used as a confirmatory test for HIV infection following a positive ELISA test include:
- Health care workers with direct exposure to blood at work
- Immigrants to US
- Infants born to HIV infected mothers
- IV and recreational drug users
- Military personnel
- Persons known to be infected with HIV
- Pregnant women
- Sexual partners of HIV infected persons
- Those attending STD clinics
- Those with signs and symptoms of
- Mononucleosis-like syndrome
- Skin lesions
- Unusual pneumonia
- Blood and blood products that will be used for transfusion, and tissue and organs for transplantation
- The results may be interpreted as positive, indeterminate or negative
- Infection with HIV-2 is more prevalent in West Africa and HIV-1 in the U.S, Western Europe and rest of the world
- HIV infection is described as a continuum of stages that range from the acute, transient, mononucleosis-like syndrome associated with seroconversion to asymptomatic HIV infection to symptomatic HIV infection and, finally, to acquired immune deficiency syndrome (AIDS), which is the end-stage of HIV infection
- Treatment is more effective and less toxic when begun early in the course of HIV infection
- A positive test is associated with viral replication and appearance of HIV antibodies such as IgM & IgG
- Positive test results for HIV antibody cannot determine whether the person harbors actively replicating virus or when the person will manifest signs and symptoms of AIDS
- Test kits for HIV are extremely sensitive. As a result, nonspecific reactions may occur if the tested person or the growth media has been previously exposed to HIV infected human cells
- After an HIV infection, the earliest antibodies to be detected by the Western blot test include those to the HIV-1 major group-specific antigen (GAG) protein p24, and its precursor p55. These tend to decrease in titer or become undetectable with onset of clinical symptoms.
- Antibodies to the antigens gp41, gp120 and gp160 can be detected in virtually all HIV-infected sera regardless of the clinical stage
- Serum specimens with only gp41 antibodies may produce bands at the 41-, 120-, and 160-kilodaltons (kd) positions. This is believed to be due to presence of multimers of the gp41 protein
Interpretive criteria:
Different sets of criteria may be used for the interpretation of the HIV Western blot results
- American Red Cross Criteria:
- Presence of at least three bands, one from each group (i.e. one gag, one pol and one env band) indicates a positive test
- US Food and Drug Administration (FDA) Criteria based on the report by the Centers for Disease Control and Prevention:
- Positive: Only if the p24, the p34 as well as the gp41 or gp120/160 bands are seen
- Indeterminate: Single band or other bands not included in a positive test
- Negative: Absence of all bands
- WHO recommendations:
- Judge positive only if two env bands are found
- The "3 band rule" is also used for the interpretation of the Western blot test
- Confirmed evidence of antibodies to HIV: Appearance of three or more bands
- Indeterminate: One or two bands appear
- Association of State and Territorial Public Health Laboratory Directors (ASTPHLD), Nevada Criteria:
- Presence of any two of the following bands
- p24
- gp41
- gp120/160
- Consortium for Retrovirus Serology Standardization (CRSS) Criteria:
- Positive test result is interpreted by the presence of either p24 or p31, plus a diffuse envelope band (i.e., gp41 or gp120/160)
- A positive WB test after two reactive ELISA tests is the gold standard for diagnosis of HIV infection
- Persons showing indeterminate results should not donate blood or plasma for either transfusion or use in manufactured blood products.
- Influenza vaccination may cause cross-reactivity with WB HIVantibody assays
Related laboratory tests:
- CD4/CD8 ratio
- Complete blood count
- Hepatitis b antibody and antigen
- Hepatitis c antibody
- HIV ELISA test
- HIV p24 antigen
- HIV viral load
- Human T-cell lymphotropic virus types I and II
- Syphilis serology
Normal results: Negative (non-reactive) for HIV-1 or HIV-2 antibodies against HIV antigens
Conditions associated with nonreactive test results, despite HIV infection, include:
- In neonates, as maternal acquired antibodies may be present until the child is 18 months of age
- During the acute stage of disease when the virus is present but antibodies are not sufficiently developed to be detected. The "window period" is the time between onset of infection and seroconversion (detectable antibody formation)
- In the later stages of AIDS, antibodies in sera may not be detectable as the infected patient can no longer mount an antibody response
- Corticosteroid or immunosuppressant therapy
Other tests to confirm the HIV infection during the window period include:
- Antigen (p24)
- Culture
- PCR for RNA or proviral DNA detection
Additional information:
- If a person has a negative or indeterminate test with high-risk behavior, the test should be repeated in 3-6 months from the time of a possible exposure to the virus
- A person, whose Western blot test results continue to be consistently indeterminate for at least 6 months in the absence of any known risk factors, clinical symptoms, or other findings, may be considered to be negative for antibodies to HIV-1
A high result is a positive finding of antibodies by Western blot to HIV antigens. This usually indicates infection with HIV virus.
- The chief antibodies to HIV antigens detected by Western blot include:
- Env or envelope glycoproteins
- gp41
- gp120
- gp160
- gag or nuclear proteins
- p18
- p24
- p55
- pol or endonuclease-polymerase proteins
- p31
- p40
- p51
- p65
- p66
- False positive results may be seen with:
- Connective tissue disorders
- Cross reactivity seen with influenza vaccination
- Hyperbilirubinemia
- Polyclonal gammopathy
- Centers for Disease Control: Interpretation and use of the Western blot assay for serodiagnosis of human immunodeficiency virus type I infections. MMWR 38(8-7):1-7, 1989 [Homepage on the Internet. Last reviewed on May 2, 2001. Last accessed on December 1, 2006. Available at URL: http://www.cdc.gov/mmwr/preview/mmwrhtml/00001431.htm
- Centers for Disease Control: Revised Recommendations for HIV Testing of Adults, Adolescents, and Pregnant Women in Health-Care Settings. MMWR September 22, 2006 / 55(RR14);1-17. [Homepage on the Internet. Last reviewed on December 12, 2006. Last accessed on December 1, 2006. Available at URL: http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5514a1.htm
- Cremonezi D et al. Prevalence of indeterminate human immunodeficiency virus western blot results in pregnant women attended at a public hospital in Presidente Prudente, Brazil. Braz J Infect Dis. 2005 Dec;9(6):506-9. Epub 2006 Jan 9.
- Ericson CP et al. Influenza vaccination and false positive HIV results. NEJM 2006 Mar 30;354(13):1422-3.
- Huang LJ et al. Predictive value of two commercial human immunodeficiency virus serological tests in cases with indeterminate Western blot results. J Microbiol Immunol Infect. 2006 Jun;39(3):219-24.
- MedlinePLus Medical Encyclopedia® HIV ELISA/Western blot. [Homepage on the Internet]©2005. Last updated on March 6, 2006. Last accessed on November 14, 2006. Available at URL: http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm
- Preiser W and Korsman S. HIV Testing. HIV Medicine 2006; 14th Edition. Flying Publisher. © 2003-2006. Available online at URL: http://www.hivmedicine.com/textbook/testing.htm
- Suthon V et al. Impact of HIV vaccination on laboratory diagnosis: case reports. BMC Infect Dis. 2002 Sep 10;2:19. Epub 2002 Sep 10.
- Syed IH et al. HIV-1 western blot assay: What determines an indeterminate status? Indian J Med Sci. 2005 Oct;59(10):443-50.