Enzyme-Linked Immunosorbent Assay (ELISA) and confirmatory tests used in
HIV diagnosis
Enzyme-linked Immunosorbent Assay (ELISA), also known as Enzyme Immunoassay
(EIA), is a widely used microbiological technique used to detect blood antibodies in
Human Immunodeficiency Virus (HIV) infection. In 1960, the radioimmunoassay (RIA)
was created and later modified into the ELISA we are familiar with today. The key
change was the use of enzymes instead of radioisotopes due to health concerns.
In an ELISA, the patients’ blood samples are first added to a 96-well microtiter plate
pre-coated with the HIV antigen. The plates are then placed in an incubator where
primary antibodies in the sample, if any, will bind to the antigens. A wash is added to
remove non antigen-binding antibodies. A secondary enzyme (alkaline phosphatase
or glucose oxidase) conjugated antibody is then added to the sample which binds to
the primary antibodies. Excess primary antibodies are washed off. Lastly, a substrate
is introduced which reacts with the enzyme to produce a colour change in the serum.1
The plate is subsequently placed in a spectrophotometer to measure the degree of
colour change. The darker the colour, the more antibodies present in the sample.
Since the ELISA is highly sensitive, it can lead to false-positive HIV results in people
with other infections and sexually transmitted diseases. Therefore, a second test is
conducted to confirm the presence of HIV antibodies in the blood sample.
In the past, the Western blot test was used as a secondary test to confirm the results
of an ELISA as comparative studies between Western blot test and ELISA showed
that the ELISA had lesser specificity to the HIV antigen as compared to Western
blotting. An interesting observation was that the ELISA kits consistently produced false
negative results which indicated a fundamental inability to identify HIV positive serum
early in antibody production.2
However, since 2014, the Centers for Disease Control and Prevention (CDC) have
recommended discontinuing the Western blot test. This is because recent advances
in EIA technology have produced a fourth generation assay that combines p24-Ag EIA
with traditional antibody EIA to detect both HIV antigens and antibodies in a single
test. This approach has shortened the window period, which is the interval between
HIV infection and detectable HIV antigens or antibodies without compromising on
sensitivity and specificity.
Evaluation of fourth generation ELISA showed it correctly classified 100% of hospital
samples and 99.7% of blood donor samples from the United States. The test proved
to have a high percentage of accuracy of HIV identification of samples from other
countries as well.3
Western blotting, on the other hand, requires advanced equipment, interpretation skills
and is technically demanding. With the enhanced efficiency of virological testing,
Western blotting is no longer an essential confirmatory HIV test.4
Furthermore, EIAs were designed to screen large numbers of samples and are ideal
for batch testing. The process typically takes two hours, so depending on the batching
procedures, high-throughput labs using automated rapid testing facilities can report
EIA results quickly and cost-efficiently on the same day.
References
1 Gan, S. D., & Patel, K. R. (2013). Enzyme immunoassay and enzyme-linked
immunosorbent assay. The Journal of Investigative Dermatology, 133(9), 1-3.
https://doi.org/10.1038/jid.2013.287
2
Saah, A. J., Farzadegan, H., Fox, R., Nishanian, P., Rinaldo, C. R., Jr, Phair,
J. P., Fahey, J. L., Lee, T. H., & Polk, B. F. (1987). Detection of early antibodies
in human immunodeficiency virus infection by enzyme-linked immunosorbent
assay, Western blot, and radioimmunoprecipitation. Journal of Clinical
Microbiology, 25(9), 1605–1610. https://doi.org/10.1128/jcm.25.9.16051610.1987
3 Saville,
R. D., Constantine, N. T., Cleghorn, F. R., Jack, N., Bartholomew, C.,
Edwards, J., Gomez, P., & Blattner, W. A. (2001). Fourth-generation enzymelinked immunosorbent assay for the simultaneous detection of human
immunodeficiency virus antigen and antibody. Journal of Clinical Microbiology,
39(7), 2518–2524. https://doi.org/10.1128/JCM.39.7.2518-2524.2001
4
World Health Organization. (2010). WHO recommendations on the diagnosis
of
HIV
infection
in
infants
and
children.
WHO
Press.
https://www.ncbi.nlm.nih.gov/books/NBK138551/