Microbiology, 2021

20.4 • EIAs and ELISAs 837
• What do the direct and indirect ELISAs quantify?
Clinical Focus
Part 2
Although contacting and testing the 1300 patients for HIV would be time consuming and expensive,
administrators hoped to minimize the hospital’s liability by proactively seeking out and treating potential
victims of the rogue employee’s crime. Early detection of HIV is important, and prompt treatment can slow
the progression of the disease.
There are a variety of screening tests for HIV, but the most widely used is the indirect ELISA. As with other
indirect ELISAs, the test works by attaching antigen (in this case, HIV peptides) to a well in a 96-well plate.
If the patient is HIV positive, anti-HIV antibodies will bind to the antigen and be identified by the second
antibody-enzyme conjugate.
• How accurate is an indirect ELISA test for HIV, and what factors could impact the test’s accuracy?
• Should the hospital use any other tests to confirm the results of the indirect ELISA?
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Immunofiltration and Immunochromatographic Assays
For some situations, it may be necessary to detect or quantify antigens or antibodies that are present at very
low concentration in solution. Immunofiltration techniques have been developed to make this possible. In
immunofiltration, a large volume of fluid is passed through a porous membrane into an absorbent pad. An
antigen attached to the porous membrane will capture antibody as it passes; alternatively, we can also attach
an antibody to the membrane to capture antigen.
The method of immunofiltration has been adapted in the development of immunochromatographic assays,
commonly known as lateral flow tests or strip tests. These tests are quick and easy to perform, making them
popular for point-of-care use (i.e., in the doctor’s office) or in-home use. One example is the TORCH test that
allows doctors to screen pregnant women or newborns for infection by an array of viruses and other pathogens
(Toxoplasma, other viruses, rubella, cytomegalovirus, herpes simplex). In-home pregnancy tests are another
widely used example of a lateral flow test (Figure 20.26). Immunofiltration tests are also popular in developing
countries, because they are inexpensive and do not require constant refrigeration of the dried reagents.
However, the technology is also built into some sophisticated laboratory equipment.
In lateral flow tests (Figure 20.27), fluids such as urine are applied to an absorbent pad on the test strip. The
fluid flows by capillary action and moves through a stripe of beads with antibodies attached to their surfaces.
The fluid in the sample actually hydrates the reagents, which are present in a dried state in the stripe.
Antibody-coated beads made of latex or tiny gold particles will bind antigens in the test fluid. The antibodyantigen
complexes then flow over a second stripe that has immobilized antibody against the antigen; this
stripe will retain the beads that have bound antigen. A third control stripe binds any beads. A red color (from
gold particles) or blue (from latex beads) developing at the test line indicates a positive test. If the color only
develops at the control line, the test is negative.
Like ELISA techniques, lateral flow tests take advantage of antibody sandwiches, providing sensitivity and
specificity. While not as quantitative as ELISA, these tests have the advantage of being fast, inexpensive, and
not dependent on special equipment. Thus, they can be performed anywhere by anyone. There are some
concerns about putting such powerful diagnostic tests into the hands of people who may not understand the
tests’ limitations, such as the possibility of false-positive results. While home pregnancy tests have become
widely accepted, at-home antibody-detection tests for diseases like HIV have raised some concerns in the
medical community. Some have questioned whether self-administration of such tests should be allowed in the
absence of medical personnel who can explain the test results and order appropriate confirmatory tests.
However, with growing numbers of lateral flow tests becoming available, and the rapid development of lab-on-