A blocking protein is then added e. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme.
After a final wash, a colorless substrate chromogen is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced measured as absorbance is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram 10 —9 g per mL range.
Figure 3. Click for a larger image. A secondary antibody conjugated to an enzyme that also recognizes epitopes on the antigen is added. After the addition of the chromogen, a spectrophotometer measures the absorbance of end product, which is directly proportional to the amount of captured antigen. Higher concentrations result in a darker final color. Fish and Wildlife Service Pacific Region. Rather than using antibody to capture antigen, the indirect ELISA starts with attaching known antigen e.
After blocking the unbound sites on the plate, patient serum is added; if antibodies are present primary antibody , they will bind the antigen.
After washing away any unbound proteins, the secondary antibody with its conjugated enzyme is directed against the primary antibody e. As with several other tests for antibodies discussed in this chapter, there is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR RT-PCR or a nucleic acid antigen test.
Figure 4. The indirect ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen from the suspected disease agent is attached to microtiter plates.
This example continues the story that started in Polyclonal and Monoclonal Antibody Production. Early detection of HIV is important, and prompt treatment can slow the progression of the disease. If the patient is HIV positive, anti-HIV antibodies will bind to the antigen and be identified by the second antibody-enzyme conjugate. 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. Figure 5. A lateral flow test detecting pregnancy-related hormones in urine.
The control stripe verifies the validity of the test and the test line determines the presence of pregnancy-related hormones in the urine. 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. 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 5. 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 6 , 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 antibody-antigen 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. 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-a-chip technology, home medical tests are likely to become even more commonplace in the future. Figure 6. Immunochromatographic assays, or lateral flow tests, allow the testing of antigen in a dilute solution.
As the fluid flows through the test strip, it rehydrates the reagents. Antibodies conjugated to small particles bind the antigen in the first stripe and then flow onto the second stripe where they are bound by a second, fixed antibody. This produces a line of color, depending on the color of the beads. The third, control stripe binds beads as well to indicate that the test is working properly.
Table 1 compares some of the key mechanisms and examples of some of the EIAs discussed in this section as well as immunoblots, which were discussed in Detecting Antigen-Antibody Complexes. This example continues the story that started in Polyclonal and Monoclonal Antibody Production and above.
First, if an infected person is tested too soon after becoming infected, the test can yield false-negative results. The seroconversion window is generally about three weeks, but in some cases, it can be more than two months.
In addition to false negatives, false positives can also occur, usually due to previous infections with other viruses that induce cross-reacting antibodies. The false-positive rate depends on the particular brand of test used, but 0. Unfortunately, western blots for HIV antigens often yield indeterminant results, in which case, they neither confirm nor invalidate the results of the indirect ELISA.
Similar to the indirect ELISA, an indeterminant western blot can occur because of cross-reactivity or previous viral infections, vaccinations, or autoimmune diseases.
When using an EIA to study microtubules or other structures inside a cell, we first chemically fix the cell and then treat the cells with alcohol. What is the purpose of this alcohol treatment? In a lateral-flow pregnancy test, you see a blue band form on the control line and no band form on the test line. Skip to main content. Laboratory Analysis of the Immune Response. Search for:. Think about It What is the difference between immunohistochemistry and immunocytochemistry?
What must be true of the product of the enzymatic reaction used in immunohistochemistry? Think about It What physical process does the lateral flow method require to function? A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
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