Learning goals
At the end of this block you can:
- Explain the differences and similarities between EIA, FEIA and ELISA
- Describe the difference and similarities between immunohistochemistry and immunocytochemistry
- Describe the different goals of direct and indirect ELISA
Equivalent towestern blot,enzym immunoassays (EIA's)use antibodies to detect the presence of antigens. However, EIAs differ from Western blots in that assays are performed in microtiter plates orlivethan in an absorbent membrane. There are many different types of EIA, but they all involve an antibody moleculefixed areabinds an enzyme, leaving the variable region free to bind its specific antigen. Addition of a substrate for the enzyme allows the antigen to be visualized or quantified (Figure 20.22).
In EIAs, the substrate for the enzyme is usually a chromogen, a colorless molecule that converts to a colored end product. The most commonly used enzymes are alkaline phosphatase and horseradish peroxidase for which suitable substrates are readily available. In some EIAs, the subsurface is onefluorogeen, a non-fluorescent molecule that converts the enzyme to a fluorescent form. EIAs that use a fluorogen are calledfluorescerende enzymimmunoassays (FEIA). Fluorescence can be detected by either afluorescence microscopeor aspectrofotometer.
Image20.22 Enzyme immunoassays, such as the direct ELISA presented here, use an enzyme-antibody conjugate to provide a detectable substrate rather than an antigen. The substrate can be a colorless molecule that converts to a colored end product or an inactive fluorescent molecule that fluoresces upon activation of the enzyme. (credit: Work adapted by "Cavitri"/Wikimedia Commons)
Micro connections
The MMR title
The MMR vaccine is a combination vaccine that provides protection against measles, mumps, and rubella (German measles). Most people receive the MMR vaccine as a child and thus have antibodies against these diseases. However, even vaccinated individuals can become susceptible to these diseases later in life for various reasons. For example, some children receive only one round of the MMR vaccine instead of the recommended two. In addition, the titer of protective antibodies in a person's body may begin to decrease with age or due to certain medical conditions.
To determine if the antibody titer in a person's bloodstream is sufficient to provide protection, an MMR titer test can be performed. The test is a simple immunoassay that can be performed quickly with a blood sample. The test results will indicate whether the person is still immune or needs another dose of the MMR vaccine.
Getting an MMR shot is often a requirement for healthcare workers, especially those who regularly come into contact with young children or immunocompromised patients. If a healthcare worker becomes infected with measles, mumps, or rubella, the person can easily spread these diseases to susceptible patients, leading to an outbreak. Depending on the results of the MMR titer, healthcare workers may need to be revaccinated before returning to work.
Immunokleuring
A powerful use of EIA isimmunokleuring, in which antibody-enzyme conjugates improve microscopy.Immunohistochemie (IHC)used to examine whole tissues. As shown inFigure 20.23a tissue section can be stained to visualize the different cell types. In this example, an anti-CD8 mAb was used for stainingCD8cells in a piece of tonsil tissue. It is now possible to count the number of CD8 cells, determine their relative number in relation to the other cell types present and determine the location of these cells in that tissue. Such data would be useful for studying diseases such as AIDS, in which the normal function of CD8 cells is crucial to slowing the progression of the disease.
Immunocytochemie (ICC)is another valuable form of immunostaining. While similar to IHC, in ICC the extracellular matrix material is removed and the cell membrane is etched with alcohol to make it permeable to antibodies. This allows antibodies to cross the cell membrane and bind to specific targets within the cell. In this way, organelles, components of the cytoskeleton and other intracellular structures can be visualized. While some ICC techniques use EIA, the enzyme can be replaced with a fluorescent molecule, making it a fluorescent immunoassay.
Image20.23 Anti-CD8 enzyme-linked antibodies were used to stain CD8 cells in this bone marrow preparation using a chromogen. (credit: work adapted by Yamashita M, Fujii Y, Ozaki K, Urano Y, Iwasa M, Nakamura S, Fujii S, Abe M, Sato Y, Yoshino T)
Check your understanding
- What is the Difference Between Immunohistochemistry and Immunocytochemistry?
- What should be true of the enzyme reaction product used in immunohistochemistry?
Enzym-linked immunosorbent assays (ELISA's)
Ofenzym-linked immunosorbent assays (ELISA's)EIAs are widely used. In thedirect ELISAthe antigens are immobilized in the well of a microtiter plate. An antibody specific for a particular antigen and conjugated to an enzyme is added to each well. If the antigen is present, the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate (chromogeen) is added. The presence of the enzyme converts the substrate into a colored end product (Figure 20.22). Although this technique is faster because only one antibody is needed, it has the disadvantage that the signal of a direct ELISA is lower (lower sensitivity).
In asandwich ELISAis the goal of using antibodies to accurately quantify the specific antigen present in a solution, such as an antigen from a pathogen, a serum protein, or a hormone from blood or urine to name just a few examples. The first step of a sandwich ELISA is to add theprimary antibodyin all wells of a microtiter plate (Figure 20.24). The antibody sticks to the plastic through hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away. Similar washes are used between each of the following steps to ensure that only specifically bound molecules remain bound to the plate. A blocking protein (e.g. albumin or the milk protein casein) is then added to bind the remaining non-specific protein binding sites in the well. Some wells will receive known amounts of antigen to allow construction of a standard curve, and unknown antigen solutions will be added to the other wells. The primary antibody captures the antigen and, after washing, itsecondary antibodyis added, that is onepolyclonal antibodywhich is linked 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 final product is measured by aspectrofotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the bound antigen. ELISAs are highly sensitive, allowing quantification of antigen down to the nanogram (10–9g) per ml of bereik.
In aindirect ELISAwe quantify the antigen-specific antibody rather than the antigen. We can use indirect ELISA to detect antibodies against many types of pathogens, includingBorrelia burgdorferi(Lyme disease) InHiv. There are three main differences between indirect and direct ELISAs as shown inFigure 20.25. Rather than using antibodies to capture antigen, indirect ELISA begins by attaching a known antigen (e.g., HIV peptides) to the bottom of the microtiter plate wells. After blocking unbound sites on the plate, patient serum is added. if antibodies are present (primary antibody), will bind the antigen. After washing away all unbound proteins, the enzyme-conjugated secondary antibody is directed against the primary antibody (e.g., anti-human immunoglobulin). Thesecondary antibodyallows us to quantify how much antigen-specific antibody is present in the patient's serum based on the intensity of the color produced by the conjugated enzyme-chromogen reaction.
As with many other antibody tests discussed in this chapter, there is always concerncross-reactivitywith antibodies directed against a different antigen, which may lead to false positive results. Therefore, we cannot definitively diagnose HIV infection (or any other type of infection) based on a single indirect ELISA test. We need to confirm any suspected positive test, which is usually done using aimmunoblotwhich actually determines the presence of specific peptides of the pathogen or an assay to identify the nucleic acids associated with the pathogen, such as e.g.Reverse-transcriptase-PCR (RT-PCR)of nucleïnezuurantigeentest.
Image20.24 (a) In a sandwich ELISA, a primary antibody is used to first capture an antigen with the primary antibody. A secondary antibody is added, conjugated to an enzyme that also recognizes epitopes on the antigen. After the chromogen has been added, a spectrophotometer measures the absorbance of the final product, which is directly proportional to the amount of antigen bound. (b) An ELISA plate shows antibody (left) and antigen (bottom) dilutions. Higher concentrations result in a darker final color. (credit b: work modified by US Fish and Wildlife Service Pacific Region)
Image8:25 pm Indirect ELISA is used to quantify antigen-specific antibodies in patient serum for disease diagnosis. Antigen from the suspected pathogen is attached to microtiter plates. The primary antibody is derived from the patient's serum, which is then bound by the enzyme-linked secondary antibody. By measuring the production of the final product, we can detect or quantify the amount of antigen-specific antibody in the patient's serum.
Check your understanding
- What is the purpose of the secondary antibody in a direct ELISA?
- What do direct and indirect ELISAs quantify?
clinical focus
Part 2
While it would be time-consuming and expensive to contact and test the 1,300 patients for HIV, administrators hoped to minimize the hospital's liability by proactively identifying and treating potential victims of the rogue employee's crime . Early detection of HIV is important and early treatment can slow the progression of the disease.
There are several HIV screening tests, but the most commonly used is the indirect ELISA. Like other indirect ELISAs, the test works by binding antigen (in this case, HIV peptides) to a well in a 96-well plate. If the patient is HIV positive, the anti-HIV antibodies bind to the antigen and are recognized by the second antibody-enzyme conjugate.
- How accurate is an indirect HIV ELISA test and what factors can affect the accuracy of the test?
- Should the hospital use other tests to confirm indirect ELISA results?
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Immunofiltration and Immunochromatographic Assays
In some cases, it may be necessary to detect or quantify antigens or antibodies that are present in the solution at a very low concentration. Immunofiltration techniques have been developed to make this possible. Inimmunofiltratiea large volume of liquid passes through a porous membrane to an absorbent pad. An antigen bound to the porous membrane will trap the antibody as it passes through. Alternatively, we can also attach an antibody to the membrane to capture the antigen.
The immunofiltration method has been adapted to its developmentimmunochromatographic analysis, better known aslateral flow testsor tire tests. These tests are quick and easy to perform, making them popular for point-of-care (i.e. doctor's office) or home use. An example is theTORCH-testwhich allows doctors to screen pregnant women or newborns for infection by a range of viruses and other pathogens (Toxoplasmaother viruses, rubella, cytomegalovirus, herpes simplex). At homepregnancy testis another widely used example of lateral flow testing (Figure 20.26). Immunofiltration tests are also popular in developing countries because they are inexpensive and do not require continuous refrigeration of dried reagents. However, the technology has also been incorporated into some advanced laboratory equipment.
In lateral flow tests (Figure 20.27), liquids such as urine are applied to an absorbent pad on the test strip. The fluid flows by capillary action, moving through a strip of beads with antibodies attached to their surface. The liquid in the sample actually hydrates the reagents, which are present on the strip in a dry state. Antibody-coated latex beads or small gold particles bind antigens in the test fluid. The antibody-antigen complexes are then passed over a second strip that has antibody immobilized against the antigen. this strip will hold beads that have bound antigen. A third control strip connects any beads. A red (from gold particles) or blue (from latex granules) color development in the test line indicates a positive test. If the color only appears on the control line, the test is negative.
Like ELISA techniques, lateral flow assays utilize antibody sandwiches, providing sensitivity and specificity. While not as quantitative as ELISA, these tests have the advantage of being fast, inexpensive, and do not rely on special equipment. So they can be performed anywhere by anyone. There are some concerns about putting such powerful diagnostic tests in the hands of people who may not understand the limitations of the tests, such as the possibility of false positives. While home pregnancy tests are widely accepted, home tests for the detection of antibodies for diseases such as HIV have raised 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 test results and order appropriate confirmatory tests. However, with the increasing availability of lateral flow assays and the rapid development of lab-on-a-chip technology (Figure 20.1), home medical tests are likely to become even more common in the future.
Image20.26 A lateral flow test that detects pregnancy-related hormones in the urine. The control strip verifies the validity of the test and the test line determines the presence of pregnancy-related hormones in the urine. (credit: modification of Klaus Hoffmeier's work)
Image20.27 Immunochromatographic assays or lateral flow assays allow testing of the antigen in diluted solution. As the liquid flows through the test strip, the reagents are rehydrated. Antibodies conjugated to small particles bind the antigen in the first lane and then flow to the second lane where they are bound by a second, immobilized antibody. This produces a line of color depending on the color of the beads. The third, control strip also strings beads to show that the test is working properly. (credit: Work adapted by Yeh CH, Zhao ZQ, Shen PL, Lin YC)
Check your understanding
- What physical process does the lateral flow method require to work?
- Explain the purpose of the third lane in a lateral flow test.
Table 20.4compares some of the basic mechanisms and examples of some of the EIAs discussed in this section, as well as immunoblots, discussed inDetection of antigen-antibody complexes.
| Immunoassays & Enzym Immunoassays | |||
|---|---|---|---|
| Testtype | Mechanism | Special Procedures | Examples |
| Immunospots | Uses an enzyme-antibody conjugate to recognize specific proteins transferred to an absorptive membrane | Western blot: Detects the presence of a specific protein | Detection of the presence of HIV peptides (or peptides from other infectious agents) in patient sera |
| Immunokleuring | It uses an enzyme-antibody conjugate to stain specific molecules on or in cells | Immunohistochemistry: Used to stain specific cells in a tissue | Staining for the presence of CD8 cells in host tissue |
| Enzym-linked immunosorbent assay (ELISA) | It uses enzyme-antibody conjugates to quantify target molecules | Direct ELISA: Uses a single antibody to detect the presence of an antigen | Detection of HIV p24 antigen up to one month after infection |
| Indirect ELISA: Measures the amount of antibody produced against an antigen | Detection of HIV antibodies in serum | ||
| Immunochromatographic Assays (Lateral Flow). | The techniques utilize capture of fluorescent, color-labeled antigen-antibody complexes by immobilized antibody to diagnose disease | Sandwich ELISA: measures the amount of antigen bound by the antibody | Detection of antibodies against various pathogens in patient sera (eg. |
| Pregnancy test that detects human chorionic gonadotropin in the urine | |||
Table 20.4
clinical focus
Part 3
Although the indirect HIV ELISA is a sensitive test, there are several complicating issues. First, if an infected person is tested too soon after infection, the test may produce 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 cause cross-reacting antibodies. The false positive rate depends on the specific test brand used, but 0.5% is not uncommon.10Due to the possibility of a false positive, all positive tests are followed by a confirmatory test. This confirmatory test is often an immunoblot (Western blot) in which HIV peptides are identified from the patient's blood using an HIV-specific mAb enzyme conjugate. A positive Western blot confirms HIV infection and a negative blot confirms the absence of HIV despite a positive ELISA.
Unfortunately, Western blots for HIV antigens often yield indeterminate results, so they do not confirm or invalidate indirect ELISA results. In fact, the percentage of indeterminate animals can be 10-49% (which is why Western blots, coupled with their cost, are not used for screening). As with the indirect ELISA, an indeterminate Western blot may occur due to cross-reactivity or previous viral infections, vaccinations, or autoimmune diseases.
- Of the 1300 patients tested, how many false positive ELISA tests would be expected?
- Of the false positives, how many indeterminate western blots can be expected?
- How would the hospital handle cases where a patient's western blot was indeterminate?
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FAQs
What is the normal range for ELISA? ›
The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody-antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results.
What is the ELISA test in microbiology? ›ELISA stands for enzyme-linked immunoassay. It is a commonly used laboratory test to detect antibodies in the blood. An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens.
What does a positive ELISA test indicate? ›This is more commonly known as an ELISA test, and it can help to confirm your diagnosis. An ELISA test is a blood test that looks for antibodies in your bloodstream. When certain antibodies are present, it's a sign your immune system is trying to fight off a disease.
What is the difference between the ELISA and the EIA test? ›ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology.
How do you read ELISA results? ›To determine the concentration of each sample, first find the absorbance value on the y-axis and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding concentration.
What are ELISA measurements? ›The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines or soluble receptors in cell supernatant or serum.
What are the positive and negative results of ELISA? ›Negative ELISA readings in positive control wells ⁄ print or dot indicate that the test has not been performed correctly or that it has been inhibited. Positive ELISA readings in negative control wells ⁄ print or dot indicate that cross-contamination or non-specific antibody binding has occurred.
What does a negative ELISA test mean? ›A false-negative result indicates you don't have a condition when you actually do. Because of this, you may be asked to repeat the ELISA again in a few weeks, or your doctor may order more sensitive tests to confirm or refute the results.
How accurate is ELISA test? ›Although false negative or false positive results are extremely rare, they may occur if the patient has not yet developed antibodies to HIV or if a mistake was made at the laboratory. When used in combination with the confirmatory Western blot test, ELISA tests are 99.9% accurate.
Which disease is diagnosed by ELISA test? ›The assay used most widely to detect or diagnose virus infection, especially infection of blood borne viruses e.g. HBV, HCV, HIV and HTLV, is the enzyme linked immunosorbent assay (ELISA), whose sensitivity and practicability have rendered it the most common primary screening assay.
What is the ELISA test a level? ›
ELISA tests
When this enzyme reacts with a certain substrate, a coloured product is formed, causing the solution in the reaction vessel to change colour. If a colour change occurs, this shows that the antigen or antibody of interest is present in the sample being tested (e.g. blood plasma)
- Direct ELISA (antigen-coated plate; screening antibody)
- Indirect ELISA (antigen-coated plate; screening antigen/antibody)
- Sandwich ELISA (antibody-coated plate; screening antigen)
- Competitive ELISA (screening antibody)
Sandwich ELISA
Sandwich ELISAs are the most common type of ELISA. Two specific antibodies are used to sandwich the antigen, commonly referred to as matched antibody pairs. Capture antibody is coated on a microplate, sample is added, and the protein of interest binds and is immobilized on the plate.
The unbound conjugate is washed and a chromogenic or fluorogenic substrate is added. In conclusion, EIA / ELISA is a powerful method not only for general biomedial research but also as a diagnostic tool. This procedure allows the detection of all types of biological molecules at very low concentrations and quantities.
What does ELISA data mean? ›ELISA, short for enzyme-linked immunosorbent assay, is a commonly used laboratory test that measures the amounts of an analyte within a solution. In most cases, the analyte is usually an antigen or an antibody.
How is ELISA considered quantitative? ›Quantitative ELISA allows calculating the amount of antigen present in the sample. It requires comparison of the values measured for the samples with a standard curve prepared from a serial dilution of a purified antigen in a known concentration. This is the most commonly reported ELISA data.
How would you recognize a positive reaction in an ELISA? ›In this simulated ELISA, all reaction wells will turn light green when the chromogen is added. A change in color from light green to purple indicates a positive result.
What causes a false positive ELISA? ›Common causes of a false positive ELISA include: administration of flu vaccine, presence of HLA-DR antibodies in multigravada women, presence of rheumatoid factor, positive RPR test, hypergammaglobulinemia (e.g. multiple myeloma) and autoimmune hepatitis.
Is ELISA a type of PCR? ›PCR-ELISA is an immunological method to quantify the PCR product directly after immobilization of biotinylated DNA on a microplate.
What is the difference between PCR and ELISA test? ›Use ELISA to: • Confirm that herds are free from important pathogens. Assess the progression of a herd through pathogen elimination. Manage herds with endemic disease to reduce the impact of disease on health and production. Use PCR to: • Identify the DNA or RNA of a given disease agent.
What type of ELISA is a Covid test? ›
The COVID-19 ELISA IgG Antibody Test consists of two serial direct Enzyme-Linked Immunosorbent Assays (ELISA) for the qualitative detection of human IgG antibodies in serum and plasma specimens collected from individuals suspected of prior infection with the virus that causes COVID-19 by their healthcare provider.
What is the use of ELISA plate in microbiology? ›ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology.
What are 3 important limitations of the ELISA test? ›- Inadequate inhibition of immunogenic antigen in ELISA results in false results.
- Refrigerated transport and storage are necessary because antibodies are proteins.
- ELISA is labor-intensive and costly to prepare antibodies.
As a general guideline, to gauge the overall reliability of your immunoassay results, inter-assay %CV should be less than 15% while intra-assay %CV should be less than 10%. It is important to identify the causes of high %CV in ELISAs.
What is negative ELISA value? ›Negative ELISA readings in positive control wells ⁄ print or dot indicate that the test has not been performed correctly or that it has been inhibited. Positive ELISA readings in negative control wells ⁄ print or dot indicate that cross-contamination or non-specific antibody binding has occurred.
What is the lowest detectable limit for ELISA? ›ELISA Test-Sensitivity
It has been calculated theoretically that with a K = 1012 M-1 (an extraordinarily high equilibrium constant for an antigen-antibody interaction) and a 1% co-efficient of variation (CV) for the response at zero dose, the lowest detection limit possible would be 10-14 M.
ELISA high background, often means excessive color development or high optical density (OD)readings. However, sometimes, there's a high OD reading yet the color development is not dark. Here are possible causes and recommended troubleshooting for the problem of high background in ELISA.
What is a high CV level? ›CVs of 5% or less generally give us a feeling of good method performance, whereas CVs of 10% and higher sound bad. However, you should look carefully at the mean value before judging a CV. At very low concentrations, the CV may be high and at high concentrations the CV may be low.
What CV is considered high? ›Distributions with CV < 1 (such as an Erlang distribution) are considered low-variance, while those with CV > 1 (such as a hyper-exponential distribution) are considered high-variance.
What does ELISA sensitivity mean? ›The sensitivity is the lowest detection level of the marker that the antibody pair used in the ELISA kit can detect. This depends mainly on the affinity of the solid phase antibody according to the law of mass action. Therefore, the use of a high affinity antibody would improve sensitivity.
Is ELISA positive or negative? ›
Positive ELISA Controls
A positive ELISA control can be a recombinant or natural sample that you know will be detectable in the assay. Positive controls help to show that a negative sample is truly negative.
Some of the most common reasons assay developers might have this problem include insufficient plate washing, conjugate oversaturating the wells, standard curve range is too high, adding too much detection reagent, and not stopping the reaction at the appropriate time.
What is a false negative ELISA test? ›There were instances where a person was infected with HIV but yet the ELISA test showed that he was not. This so called 'False Negative' ELISA test was initially blamed on the HIV Testing Window Period – the time from when a person is infected with HIV to when antibodies to HIV become detectable in the blood.
Which ELISA has high sensitivity? ›The key advantage of a sandwich ELISA is its high sensitivity; it is 2-5 times more sensitive than direct or indirect ELISAs. Sandwich ELISA also delivers high specificity as two antibodies are used to detect the antigen. It offers flexibility since both direct and indirect methods can be used.