In the realm of modern science and medical research, ELISA stands tall as a powerful tool in diagnostic and analytical applications. Enzyme-Linked Immunosorbent Assay, commonly known as ELISA, plays a pivotal role in detecting and quantifying specific substances, such as antibodies, antigens, and proteins. In this blog, we will explore the fundamental principles, advantages, and disadvantages of ELISA, delve into the various types, understand the intricacies of how this technique works, and discuss its significant applications.
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Enzyme-Linked Immunosorbent Assay (ELISA) is a highly sensitive and specific laboratory technique used to detect and measure a wide range of substances in biological samples. It relies on the specific interaction between an antigen and an antibody for detection and quantification. ELISA finds extensive applications in diverse fields, including clinical diagnosis, biomedical research, drug development, and food safety analysis.
At its core, ELISA relies on the principles of antigen-antibody binding, colorimetric or fluorescence detection, and quantitative analysis. The process begins with immobilizing the target antigen on a solid surface, such as a microplate. Then, specific antibodies linked to an enzyme are added, which can recognize and bind to the target antigen. After a series of washing steps to remove any unbound substances, a substrate is added that reacts with the enzyme to produce a measurable signal, such as a color change.
ELISA’s sensitivity and specificity arise from the high affinity between the antibody and antigen, allowing for precise detection even at low concentrations. This technique has been refined over the years, leading to improvements such as the use of different enzyme labels, fluorescent markers, and enhanced washing protocols to reduce background noise and increase accuracy.
Enzyme-Linked Immunosorbent Assay (ELISA) has found a myriad of applications in various fields due to its sensitivity, specificity, and versatility. ELISA kits are commercially available, pre-packaged sets of reagents and microplates designed to streamline the process and make ELISA more accessible for researchers and diagnostic laboratories. Let’s explore some of the key applications of ELISA kits:
ELISA is extensively used in clinical laboratories for disease diagnosis and monitoring. It enables the detection of specific antibodies or antigens associated with various infectious diseases, autoimmune disorders, and allergies. For example, ELISA kits are commonly employed to diagnose HIV, hepatitis, COVID-19, and Lyme disease. Moreover, ELISA aids in measuring biomarkers to assess disease progression and treatment response, such as quantifying cardiac markers in heart conditions or cytokines in inflammatory diseases.
ELISA is crucial in identifying infectious agents and monitoring their prevalence in populations. It plays a pivotal role in detecting viral, bacterial, and parasitic infections by targeting specific antigens or antibodies. By using ELISA kits, healthcare professionals can quickly and accurately diagnose diseases like malaria, tuberculosis, and dengue fever, leading to prompt treatment and better disease control.
ELISA kits are employed in the food industry to detect allergens and contaminants in food products. For instance, they can identify traces of peanuts, gluten, or seafood in processed foods, which is crucial for individuals with food allergies. Additionally, ELISA can detect toxins and pathogens in food, ensuring the safety of consumers and preventing foodborne illnesses.
Environmental scientists use ELISA kits to assess the presence of pollutants and toxins in air, water, and soil samples. By detecting specific chemicals or substances, ELISA helps in environmental monitoring, enabling early identification of pollution and potential hazards.
In research laboratories, ELISA is a fundamental tool for studying various biological processes and biomolecules. It aids in characterizing proteins, quantifying hormones and cytokines, and understanding cellular signaling pathways. ELISA plays a crucial role in drug development by measuring drug concentrations in biological samples and evaluating immune responses to potential therapeutics.
ELISA is valuable in veterinary medicine for diagnosing infectious diseases in animals. It allows veterinarians to screen for diseases like brucellosis, parvovirus, and feline leukemia virus, leading to early treatment and prevention of disease transmission.
ELISA is extensively used in allergy testing to identify specific allergens that trigger allergic reactions in patients. This information helps healthcare providers design personalized treatment plans and recommend allergen avoidance strategies.
Endocrinologists utilize ELISA kits to measure hormone levels in patients. This is crucial for diagnosing hormonal disorders, monitoring hormonal therapies, and understanding the hormonal regulation of various physiological processes.
ELISA assists in cancer research by detecting specific biomarkers associated with different types of cancer. This information aids in early cancer detection, tracking disease progression, and evaluating the effectiveness of cancer treatments.
ELISA kits are used in veterinary diagnostics for detecting specific diseases in animals, such as heartworm disease in dogs, feline immunodeficiency virus (FIV) in cats, and equine infectious anemia (EIA) in horses.
In conclusion, ELISA is a versatile and indispensable tool in the fields of medicine, research, and diagnostics. Its ability to detect and quantify specific substances with high sensitivity and specificity has revolutionized our understanding of various diseases and biological processes. Despite some limitations, the advantages of ELISA far outweigh the disadvantages, making it an invaluable asset in the pursuit of scientific discoveries and advancements in healthcare. By adhering to the principles of antigen-antibody interaction and employing various ELISA types, researchers and scientists continue to unlock new insights into the intricate world of biomolecules, paving the way for a healthier and brighter future. As technology and methodologies evolve, ELISA remains at the forefront of cutting-edge research, contributing significantly to advancements in personalized medicine, early disease detection, and novel therapeutics.
The enzyme-linked immunosorbent assay (ELISA) ( , ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in .[1] The assay is a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly an amino acid) in a liquid sample using antibodies directed against the ligand to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.
In the most simple form of an ELISA, antigens from the sample to be tested are attached to a surface. Then, a matching antibody is applied over the surface so it can bind the antigen. This antibody is linked to an enzyme, and then any unbound antibodies are removed. In the final step, a substance containing the enzyme's substrate is added. If there was binding, the subsequent reaction produces a detectable signal, most commonly a color change.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
Of note, ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays, though the name carried the original "immuno" because of the common use and history of the development of this method. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. In between the washes, only the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (e.g., a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase, which is part of the plate and so are not easily reusable.[2]
As an analytical biochemistry assay and a "wet lab" technique, ELISA involves detection of an analyte (i.e., the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the analysis (i.e., controlled sequence of biochemical reactions that will generate a signal which can be easily quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well needed to keep the reactants contained.[3][4] This is in contrast to "dry lab" techniques that use dry strips. Even if the sample is liquid (e.g., a measured small drop), the final detection step in "dry" analysis involves reading of a dried strip by methods such as reflectometry and does not need a reaction containment chamber to prevent spillover or mixing between samples.[5]
As a heterogenous assay, ELISA separates some components of the analytical reaction mixture by adsorbing certain components onto a solid phase which is physically immobilized. In ELISA, a liquid sample is added onto a stationary solid phase with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated, and washed, followed by some optical change (e.g., color development by the product of an enzymatic reaction) in the final liquid in the well from which the quantity of the analyte is measured. The quantitative "reading" is usually based on detection of intensity of transmitted light by spectrophotometry, which involves quantitation of transmission of some specific wavelength of light through the liquid (as well as the transparent bottom of the well in the multiple-well plate format).[3][4] The sensitivity of detection depends on amplification of the signal during the analytic reactions. Since enzyme reactions are very well known amplification processes, the signal is generated by enzymes which are linked to the detection reagents in fixed proportions to allow accurate quantification, and thus the name "enzyme-linked".[6]
The analyte is also called the ligand because it will specifically bind or ligate to a detection reagent, thus ELISA falls under the bigger category of ligand binding assays.[3] The ligand-specific binding reagent is "immobilized", i.e., usually coated and dried onto the transparent bottom and sometimes also side wall of a well[7] (the stationary "solid phase"/"solid substrate" here as opposed to solid microparticle/beads that can be washed away), which is usually constructed as a multiple-well plate known as the "ELISA plate". Conventionally, like other forms of immunoassays, the specificity of antigen-antibody type reaction is used because it is easy to raise an antibody specifically against an antigen in bulk as a reagent. Alternatively, if the analyte itself is an antibody, its target antigen can be used as the binding reagent.[8]
Before the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal, which indicates whether a specific antigen or antibody is present in the sample. Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson published in .[9]
As radioactivity poses a potential health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a nonradioactive signal in place of the radioactive signal. When enzymes (such as horseradish peroxidase) react with appropriate substrates (such as ABTS or TMB), a change in color occurs, which is used as a signal. However, the signal has to be associated with the presence of an antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G. B. Pierce.[10] Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container; i.e., the immunosorbent must be prepared. A technique to accomplish this was published by Wide and Jerker Porath in .[11]
In , Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands independently published papers that synthesized this knowledge into methods to perform EIA/ELISA.[12][13]
Traditional ELISA typically involves chromogenic reporters and substrates that produce some observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques use fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. These new reporters can have various advantages, including higher sensitivities and multiplexing.[14][15] In technical terms, newer assays of this type are not strictly ELISAs, as they are not "enzyme-linked", but are instead linked to some nonenzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs.
In , an ultrasensitive, enzyme-based ELISA test using nanoparticles as a chromogenic reporter was able to give a naked-eye colour signal, from the detection of mere attograms of analyte. A blue color appears for positive results and red color for negative. Note that this detection only can confirm the presence or the absence of analyte, not the actual concentration.[16]
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There are many ELISA tests for particular molecules that use the matching antibodies. ELISA tests are broken into several types of tests based on how the analytes and antibodies are bonded and used.[17][18] The major types are described here.[19]
The steps of direct ELISA[20]
follows the mechanism below:
The enzyme acts as an amplifier; even if only a few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. Within common-sense limitations, the enzyme can go on producing color indefinitely, but the more antibody is bound, the faster the color will develop. A major disadvantage of the direct ELISA is that the method of antigen immobilization is not specific; when serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich or indirect ELISA provides a solution to this problem by using a "capture" antibody specific for the test antigen to pull it out of the serum's molecular mixture.[citation needed]
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation (error inherent in a test) is often used to distinguish positive from negative samples. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule. For example, if a test sample returns an OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same analyte concentration as the sample.[citation needed]
The use and meaning of the names "indirect ELISA" and "direct ELISA" differ in the literature and on websites depending on the context of the experiment. When the presence of an antigen is analyzed, the name "direct ELISA" refers to an ELISA in which only a labeled primary antibody is used, and the term "indirect ELISA" refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labeled secondary antibody. In the latter case, a sandwich ELISA is clearly distinct from an indirect ELISA. When the "primary" antibody is of interest, e.g. in the case of immunization analyses, this antibody is directly detected by the secondary antibody and the term "indirect ELISA" applies to a setting with two antibodies.[citation needed]
A "sandwich" ELISA is used to detect sample antigen.[21] The steps are:
The image to the right includes the use of a secondary antibody conjugated to an enzyme, although, in the technical sense, this is not necessary if the primary antibody is conjugated to an enzyme (which would be direct ELISA). However, the use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect. By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized. Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement, simplifying the assay, and increasing the specificity and the sensitivity of the assay. Therefore, a sandwich ELISA used for research often needs validation, to reduce the risk of false positive results.[22]
A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples:
Unlabeled antibody is incubated in the presence of its antigen (sample).
Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The less antigen in the sample, the more labeled antigen is retained in the well and the stronger the signal.
Commonly, the antigen is not first positioned in the well.
For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV antigen. Two specific antibodies are used, one conjugated with enzyme and the other present in serum (if serum is positive for the antibody). Cumulative competition occurs between the two antibodies for the same antigen, causing a stronger signal to be seen. Sera to be tested are added to these wells and incubated at 37 °C, and then washed. If antibodies are present, the antigen-antibody reaction occurs. No antigen is left for the enzyme-labelled specific HIV antibodies. These antibodies remain free upon addition and are washed off during washing. Substrate is added, but there is no enzyme to act on it, so a positive result shows no color change.
A fourth ELISA test does not use the traditional wells, rather leaves the antigens suspended in the test fluid.[23][24]
This test allows multiple antigens to be tagged and counted at the same time. This allows specific strains of bacteria to be identified by two (or more) different color tags. If both tags are present on a cell, then the cell is that specific strain. If only one is present, it is not.
This test is done, generally, one test at a time and cannot be done with the microtiter plate. The equipment needed is usually less complicated and can be used in the field.
The following table lists the enzymatic markers commonly used in ELISA assays, which allow the results of the assay to be measured upon completion.
Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test[27] or West Nile virus). It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs[28] and as serological blood test for celiac disease.[29][30] ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.
The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an ELISA, a person's serum is diluted 400 times and applied to a plate to which HIV antigens are attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody"—an antibody that binds to other antibodies—is then applied to the plate, followed by another wash. This secondary antibody is chemically linked in advance to an enzyme.
Thus, the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and a negative result.
A cut-off point may be determined by comparing it with a known standard. If an ELISA test is used for drug screening at workplace, a cut-off concentration, 50 ng/ml, for example, is established, and a sample containing the standard concentration of analyte will be prepared. Unknowns that generate a stronger signal than the known sample are "positive". Those that generate weaker signal are "negative".
There are ELISA tests to detect various kind of diseases, such as dengue, malaria, Chagas disease,[31] Johne's disease, and others.[32] ELISA tests also are extensively employed for in vitro diagnostics in medical laboratories. The other uses of ELISA include:
ELISA is as of the primary method of plant pathogen detection worldwide.[34]
eSimoa (enzyme-linked single molecule array) represents a significant evolution of the traditional ELISA (Enzyme-Linked Immunosorbent Assay) technique, which is widely utilized in clinical diagnostics and research. By significantly enhancing the sensitivity and resolution of biomolecular detection, eSimoa expands the capabilities of ELISA, enabling the detection of biomolecules at concentrations previously unachievable with standard assays.[35]
Building on the foundational principles of ELISA, eSimoa employs paramagnetic beads to isolate biomolecules or enzymes in a manner akin to ELISA’s plate-based detection. However, eSimoa advances this concept by enabling enzymatic reaction measurements at the single-molecule level, which dramatically improves detection limits for various enzymes and biomolecules. This method allows for the precise quantification of low-abundance proteins and the activity of critical enzymes such as protein kinases and telomerases, which are often below the detection threshold of conventional ELISA.
The enhanced sensitivity of eSimoa is crucial for early and accurate biomarker detection in clinical diagnostics, facilitating better disease monitoring and management. In drug discovery, the ability to track subtle changes in enzymatic activity aids in the development of more effective pharmaceuticals by providing detailed insights into enzyme inhibition mechanisms.
Chi-An Cheng at National Taiwan University (NTU) has claimed that her team developed this innovative technology.[36][37] However, this claim is contested by the existence of prior publications by David R. Walt's team at Harvard University, who published their work on eSimoa in .[35][38] This earlier documentation by Walt's team suggests a prior contribution to the development of the technology.