The enzyme-linked immunosorbent assay (ELISA) is recognized as the gold standard among current immunological tests. This widely used method, based on microplate wells, is employed for the identification and quantification of peptides, proteins, antibodies, and hormones. The ELISA test is an analytical method primarily utilized in scientific research and clinical applications. These tests are particularly useful for detecting viral infections such as hepatitis B virus, hepatitis C virus, and human immunodeficiency virus (HIV).
The ELISA test is a type of immunological assay (EIA) specifically and sensitively designed to detect very low concentrations of antigens or antibodies. These tests can identify concentrations as low as 0.01 nanograms of antigen or antibody per milliliter. During the ELISA test, antigen-antibody complexes attach to a solid surface (such as the bottom of microplate wells). An enzyme is then covalently linked to one of the molecules in this complex. By adding a specific substrate for the enzyme, a color reaction occurs, the intensity of which can be used to measure the concentration of the target antigen or antibody in the sample.
ELISA tests are generally categorized into four main types: direct, indirect, sandwich, and competitive. All these assays are typically performed in 96-well plates, where the well bottoms serve as a solid surface for immobilizing antigen-antibody complexes. Each of these four test types has its own specific features, advantages, and disadvantages.
To perform a direct competitive ELISA, the following steps can be followed:
Coating the Plate: Coat the plate with reference antigen, then block the remaining sites for antibody binding with an appropriate buffer.
Sample Incubation: The sample with an unknown antigen concentration is incubated with a limited amount of labeled antibodies. If the antigen concentration in the sample is low, many of the antibodies will not be able to bind to the sample antigen, and vice versa.
Mix Transfer to Plate: The mixture of labeled antibodies and the sample is transferred to wells coated with the reference antigen. At this stage, antibodies that could not bind to the sample antigens will attach to the reference antigen.
Washing the Plate: The plate is washed to remove antibodies bound to sample antigens that have not been fixed to the plate.
Detection and Measurement: A specific substrate is added to the plate, and the resulting color change is measured. The greater the color change, the less antigen is present in the sample, and vice versa.
Advantages and Disadvantages of Competitive ELISA
The advantages and disadvantages of competitive ELISA depend on the basic type chosen for the experiment (direct, indirect, or sandwich). This type of ELISA is particularly useful when the concentrations of analytes in samples are very low or when the antigens or antibodies are highly similar and accurate identification without competition is impossible.
Advantages: This method can be very effective for measuring analyte concentrations in complex samples, especially when analytes are measured competitively and in the presence of limited labeled antibodies, yielding high sensitivity.
Disadvantages: This method may have lower accuracy than other types of ELISA due to competition between analytes and labeled antibodies for binding to the reference antigen. Additionally, its protocols may be more complex than other ELISA types and require more careful control of the steps.
After reviewing the principles of operation for the different types of ELISA, it’s now time to discuss data analysis. ELISAs can provide various results, which can generally be categorized into three types:
Qualitative Results
In this case, the aim is only to determine the presence or absence of an antigen or antibody in the sample. In other words, this type of result answers the question of whether the antigen or antibody is present in the sample. These results are useful for preliminary applications such as initial screenings or confirming the presence of a specific agent.
Semi-Quantitative Results
In this type of result, you do not want to know the exact concentration of the antigen or antibody; rather, you want to make a comparison between the concentrations of these molecules in different samples. For example, you may determine whether the concentration of the antigen or antibody in sample A is greater than or less than in sample B.
Quantitative Results
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Quantitative Results
In this type, the goal is to determine the exact concentration of the antigen or antibody in the sample. These results are very useful for more detailed studies, such as examining changes in antigen or antibody levels over time or in response to treatment.
The Importance of Controls in Data Analysis
To obtain reliable and accurate results in ELISA testing, it is essential to include both negative and positive controls on each ELISA plate.
Negative Control:** These controls help identify and correct false positive results due to non-specific binding or contamination. A negative control typically includes a sample that does not contain the target antigen or antibody.
Positive Control:** These controls are designed to confirm the proper functioning of the assay. If the test is performed correctly, positive results should be obtained from the positive controls, even if all samples are negative.
The chemicals needed for an ELISA test depend on the protocol used, but generally include coating, blocking, and washing buffers, enzyme-labeled antibodies or antigens, substrate, and stop solution. For the identification of common antigens and antibodies, you can usually purchase ready-to-use ELISA kits that include all necessary materials.
Common chemicals in these kits include:
Coating Buffer:** The two most common coating buffers are PBS (phosphate-buffered saline) and bicarbonate. These buffers stabilize the antigen or antibody used to coat the plate during incubation.
Blocking Buffer:** To prevent non-specific binding of detection antibodies to the plate surface, the remaining sites for protein binding must be blocked. This is done using blocking proteins such as bovine serum albumin (BSA), non-fat dry milk (NFDM), casein or caseinate, normal serum, or fish gelatin.
Washing Buffer:** Washing steps to remove unbound materials are typically performed using PBS containing a small amount of a non-ionic detergent such as Tween® 20.
Enzyme-Labeled Antibodies or Antigens:** The required antibodies or antigens depend on the type of analyte. For labeling antibodies or antigens with an enzyme, streptavidin-biotin systems are commonly used to attach antibodies or antigens to the detecting enzyme. Two common enzymes used for this purpose are horseradish peroxidase (HRP) and alkaline phosphatase (ALP).
Enzyme Substrate and Stop Solution:** Substrates and stop solutions are specifically designed for each enzyme.
HRP Substrate: The HRP substrate includes TMB (3,3',5,5'-tetramethylbenzidine), a specific substrate buffer, and hydrogen peroxide. The reaction can be stopped using sulfuric acid.
ALP Substrate: The ALP substrate includes a p-Nitrophenyl Phosphate (pNPP) solution, and the reaction can be stopped using NaOH (sodium hydroxide).
Equipment Required for ELISA
To perform an ELISA test, you will need the following equipment:
Pipettes** for adding chemicals and samples to the microplate wells.
Incubator** to maintain the plate at a constant temperature.
Plate Washer** to remove unbound molecules.
Plate Reader** for analyzing the test results.
Centrifuges** for separating particles.
If you are working in a laboratory with a limited budget or low capacity, a washing device may not be available. In this case, you can use pipettes along with a suction system for performing the washing steps.
On the other hand, high-capacity laboratories may be looking for solutions to increase their efficiency. In this case, using various types of pipettes can be beneficial, including:
Adjustable Tip Spacing Pipettes** for transferring samples between different laboratory container formats.
96-Channel Pipettes** for simultaneously adding chemicals to each well.
Small Benchtop Pipetting Robots** for automation.