There are many instances within life sciences where timely antibody detection and quantification within a sample are effective. It can help identify immune responses in vaccinated or infected individuals, detect protein on the cell's surface, or perform quality control testing. However, you need a quality tool capable of making these detections.
Enzyme-linked Immunosorbent Assay (ELISA) kits detect multiple antigens and proteins. They are the most common immunoassay type used to detect the presence and measure the specific concentration of antibodies in a sample. The ELISA test has proven invaluable as a research and diagnostic tool and plays a crucial role in surveying the proportion of people exposed to infection. This article examines how you can use ELISA kits to identify antibodies and detect viral infections.
What are ELISA kits?
The ELISA kit is a lab test that measures the concentration of an analyte, which refers to the antibodies or antigens in a particular solution. The immunological assay measures the amount of antibodies, such as proteins and antibodies. An ELISA test relies on binding antibodies to their targets to facilitate detection.
In diagnostics, cell-based ELISA tests help determine whether and how many antibodies are produced in response to pathogen exposure or vaccination. It usually utilizes a 96-well plate format, making it easy to screen multiple samples simultaneously. This format makes it amenable to screening many samples at once. The ELISA test makes it easy to obtain quantitative results, which sets it apart from similar assays.
Most ELISA kits detect common sample types such as serum, plasma, and tissue homogenates. Other samples like urine, saliva, cerebral spinal fluid, and other body fluids are also suitable for ELISA kits. The presence of sufficient analyte concentration in the sample is necessary for detection. The samples can comprise buffer components with similar antigenic epitopes. They can also incorporate protease factors that could affect assay performance.
Types of ELISA tests
It's essential to consider different ELISA test types. The ELISA test was initially conceptualized in 1971 as researchers wanted a technique to detect antigens and antibodies. This enzyme-based alternative replaced the radioimmunoassay method, which was sometimes dangerous.
There are four ELISA tests for identifying antibodies. While there are different ways of antigen detection, the tests use a similar principle in antibody detection.
Direct ELISA
The direct ELISA test is the fastest ELISA testing type, with fewer steps, and is less prone to errors. With direct ELISA, antibodies or antigens are absorbed directly into the plate in a nonspecific manner. A conjugated detection antibody then binds to the target protein when applied. You then add a substrate to produce a signal proportional to the amount of analyte in the sample. In this method, a detection substrate changes the sample color quantified on a reader.
The drawbacks of the direct ELISA type are that it often suffers from high background noise because of non-specific adsorption and the potential for experimental errors across its multiple steps. Critically, its main limitation is sensitivity; the absence of a signal amplification stage (which an extra secondary antibody step would provide) means the signal is not amplified, resulting in reduced assay sensitivity. Furthermore, the method is labor-intensive because specific conjugated detection antibodies or antigens must be custom-developed for every new target analyte.
Indirect ELISA
The indirect ELISA test type works similarly to a direct method, though it uses a two-step process in detection. This extra step helps overcome limitations experienced with the direct method and amplifies the test signal.
With the indirect ELISA, such as the TNFa ELISA, you have a second step that negates using target-specific antibodies for detection. This is because you will use species-specific secondary antibodies. The indirect test has increased sensitivity compared to the direct ELISA, though it costs less as fewer labeled antibodies are required.
However, this ELISA type still has some background noise issues because the total sample can bind to the plates. If you use the assay for detecting antibodies, you will need to coat purified target antigens onto the plates. This is the only way to reduce background noise during the tests.
Sandwich ELISA
Sandwich ELISA is a method where two more steps are involved, and the results are precise. It is a common ELISA type where you use two specific antibodies to sandwich an antigen, improving assay sensitivity and specificity. This enzyme-linked type uses matched antibody pairs instead of the non-specific antigen binding. It also uses capture and detection antibodies, where the capture antibody adds specificity to the technique, and the detection antibody improves its sensitivity.
However, this sandwich ELISA method requires that you find a capture antibody compatible with the detection antibody to function. A lack of compatibility can affect cross-reactivity during the ELISA tests. Compared to other methods, the sandwich ELISA test has more steps, which creates room for errors. However, since the antigen-binding step is selective, this method is ideal when dealing with complex antigen mixes by eliminating the need for purification.
Competitive ELISA
The last ELISA test is a competitive or inhibition ELISA, which applies when only one antibody is available or when the analyte is small. The ELISA principle works by detecting interference to a signal level expected from an output, which allows it to produce an inverse relationship.
Competitive ELISA has some advantages and limitations depending on the format you adopt. However, it helps when the antigen is small, as it limits the ability of two antibodies to bind concurrently. When you apply more targets in the sample, there will be a lower output signal. Fortunately, you can achieve multiple formats by adapting the sample to a different format.
The different assay formats all rely on how the ELISA kit manufacturer has designed the binding of the target to the plate surface. You will need to employ a detection step involving a conjugated antigen or antibodies to enable successful binding to be detected and quantified. In most cases, you use colorimetric detection in ELISA testing.
How does an ELISA kit work?
In most cases, the ELISA kits come with easy-to-follow protocols and detailed instructions if you have limited familiarity with them. Since each type of ELISA has different protocol variations, here are the stages to follow in determining how ELISA works.
Plate coating
The basic ELISA test procedure starts with a coating step where you cover a polystyrene plate with a solution that has antibodies or antigens. The step involves immobilizing an antigen present in the sample to the wells of a microtiter ELISA plate using a passive adsorption approach. In most cases, the results of this plate coating depend on whether you apply an antigen or an antibody sample. If the sample is antigen-specific, the plate will be antigen-bound. However, if you apply purified antigens or capture antibodies, the result is a plate with particular properties.
Cover these plates with an antigen or an inactivated form of the virus. You can do this by adsorption into the plate's surface or using a capture antibody. If the blood contains antibodies, it binds to the antigen, and you add a substrate solution to the well. The reaction will produce a color change that detects antibodies. Several plate types are available with different binding properties depending on your assay. Use a treated plate for the coating because it allows covalent linkage.
Incubation
After immobilization, add a detection antibody to the ELISA plates, which binds to the adsorbed antigen. Incubating the plates facilitates reaction and binding depending on the assay type. You will also need to perform the blocking phase, where a protein-based solution covers the unbound sites on the plate.
Washing
This is another essential step in ELISA testing after each assay and detection component. Use a buffer to wash the wells after emptying them, ensuring every residue from the incubation is removed. Failing to wash the wells can cause high background signals, while extreme washing can cause lower sample signals. Consistent washing can also result in more predictable outcomes.
Blocking
After coating the ELISA plates, blocking ensures that the detection antibodies are not specific to the plate. During the blocking phase, you add mixed proteins to the plate, so they occupy other non-specific binding areas. This ensures the proteins don’t relate to the assay when incubating them. If you don’t block the plates during the ELISA tests, your assay will have reduced sensitivity due to higher background noise.
Detection
The last step involves detection, where an enzyme-conjugated antigen or antibody binds to the target antigen-antibody. The detected antibody is directly conjugated to an enzyme or provides a binding site for a labeled secondary antibody.
Every ELISA test passes through this step, ending with a color change to show the presence of antibodies. This is where you apply an antibody to a solution and monitor whether the antibodies bind to an antigen. The intensity of the changes in color signals the number of antigens present in the solution. You apply an enzyme-conjugated antibody to test wells where it will bind if its target is present. When you add an appropriate substrate for the enzyme to the plate, it causes a color change proportional to the amount of target bound inside the well.
If it's an IgG ELISA-based test, do the test to detect the immunoglobulin G antibody. The body produces the immunoglobulin M (IgM) and the immunoglobulin G (IgG) antibodies to fight against pathogens. In the final step, place a substrate in the plate, and the enzyme reaction gives the signal.
What do the ELISA kits test?
The results of ELISA kits can be quantitative, qualitative, or semi-quantitative. Usually, you graph these results to compare them with other results and come to a definitive outcome. The quantitative assay uses a serial dilution of a known standard to generate a standard curve. You can calculate the precise quantity of targets in the unknown sample. In a qualitative assay, you collect data numerically using a microplate reader. You then interpret results as positive or negative by comparing them to blank or negative wells without a standard curve.
ELISA kit tests are one of the most sensitive immunoassay tests available today. The sensitivity of the results depends upon the characteristics of the antibody-antigen reaction. You can improve the ELISA test results by adding a substance like those that give an enhanced chemiluminescent or fluorescent signal.
You will also need to choose suitable experimental antibodies for the ELISA test, especially when using multiple antibodies. You can use both monoclonal and polyclonal antibodies, each with advantages and limitations. For example, monoclonal antibodies produce high specificity, while polyclonal antibodies can bind a target at multiple binding sites and improve sensitivity.
Uses of ELISA's kits
You can use antibody ELISA tests as a supplementary test for a suspect with RT-PCR-negative cases of COVID-19. They can also be supplementary detection indicators in diagnosing suspected COVID-19 cases. You can also use the ELISA test in pregnancy, forensic drugs, cancer biomarkers, biosimilars, and biopharmaceutical testing.
Other uses include the detection of food allergens and infectious disease diagnostics. For example, you use the ELISA test to detect infectious agents and antibody responses induced in the host. A sustainable antibody response can be seen in individuals when the microbe cannot be detected.
The benefits of ELISA tests include high specificity, high sensitivity, high throughput, and convenience. The clinical specificity of ELISA tests is 96%, while the sensitivity is up to 99%. You can use the ELISA tests to process over 20,000 assays in one day, as they are suitable for high throughput tests.
How ELISA test for HIV work?
Most ELISA tests for HIV are based on the antigen-antibody interaction. On the surface of HIV are a lot of proteins that act as antigens, with the early infection called protein 24 (p24). Antigens for HIV are detectable in people around 16 days after infection, while antibodies take longer to produce, usually not being detectable until 4-12 weeks after infection.
The best ELISA test for HIV can detect antibodies to determine if someone has antibodies to a particular antigen. They look for antibodies in the blood. During the rapid tests, antigens for HIV are fixed on one strip along the test stick. Place a chemical called a buffer at the end of the testing stick to facilitate the testing process. This chemical causes the antibodies in the blood to flow along the test stick.
When the antibodies pass over the section with the antigens, if there are antibodies for HIV present, then they will stick to these antigens and change color. Once the test is complete, the number of stripes will determine whether there is a positive or negative result. One strip represents a negative result, two represent a positive result, and no strips mean the test did not work correctly.
The p24 ELISA kits can also detect p24 antigen, using a sandwich ELISA method. Add a blood sample to a petri dish with antibodies during the process. If the sample is HIV-positive, the p24 antigen on the surface of HIV will attach to the antibodies, and if the sample is negative, then there will be no antigens to bind, and the dish remains the same.
Bottom line
ELISA tests are standard tests to determine whether someone has antibodies to a particular antigen. You can use various ELISA kits to detect hundreds of proteins and molecules, including cytokines, growth factors, and markers for infectious diseases. The kits are quick, convenient, and accurate research tools to detect quantitative targets of interest in cultures and samples.