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November 15, 2024
What is the Elisa Test? What was the process of its development? Procedure for conducting ELISA, its applications, and classifications.
ELISA, an enzyme-linked immunosorbent assay, is regarded as a potent method for detecting and quantifying a specific protein within a complicated mixture. This approach is often used to identify and quantify proteins in a sample. Antibodies facilitate the identification of proteins, making the method an immunoassay. The Elisa Test serves as a diagnostic instrument in medicine and plant pathology. It is used for quality control across several sectors. The Elisa Test may be categorized as Indirect Elisa, Direct Elisa, Sandwich Elisa, and Competitive/Inhibition Elisa. It assists in quantifying serum antibody concentrations and is regarded as a fundamental, adaptable, quantitative, and sensitive assay (Vencia, Migone & Vito, 2016). This study will elucidate the idea of ELISA, its evolution, and provide a discourse on the many kinds of ELISA tests.
The Elisa Test is a technique that aids in the detection and quantification of antibodies present in the blood. The test is useful for identifying the existence of antibodies in the body during illness or infection. Antibodies are proteins produced by the body in response to hazardous substances such as antigens. The Elisa Test is sometimes used solely for screening prior to the administration of any further tests. Engvall and Perlmann defined the phrase in 1971, indicating that the instrument aids in the detection of antibodies in a protein sample immobilized in microplate wells (Gandikota, Gandhi & Maisam, 2020). The test aids in quantifying glycoproteins and is crucial in identifying HIV infection, pregnancy, and viruses such as chickenpox, Zika, and rotavirus.
Prior to the advent of the Elisa Test, radioimmunoassay was used with radioactively labeled antigens and antibodies. The presence of any antigen or antibody was identified by radioactivity. However, some health-related concerns associated with the use of radioactivity were anticipated by certain researchers, prompting the exploration of other possibilities. In 1960, a procedure called enzyme linking was invented by two distinct teams led by Stratis Avrameas and G.B. Pierce. In the same year, Wide and Jerker Porath also published an immunosorbent method. Independent research conducted by Peter Perlman and Eva Engvall at the University of Stockholm, Sweden, together with studies by Anton Schuurs and B. van Weemen in the Netherlands, contributed to the development of the Elisa Test (Gandikota, Gandhi & Maisam, 2020). The conventional Elisa used chromogenic reporters and substrates that facilitated color change, indicating the presence of a particular antigen or analyte. The novel approach used fluorogenic, electrochemiluminescent, and quantitative PCR reporters to generate signals. The use of enhanced reporters enhances the measurement of many analytes in a single or grouped test with increased sensitivity. In most instances, the newer tests use reporters that are not enzymes, while maintaining the fundamental principles of assays, resulting in their classification as ELISAs.
The testing for Elisa has no technological complexities; it is a straightforward process. A permission form must be completed before to the test, and the physician will explain the rationale for doing the test. Blood samples are obtained, and to facilitate this, the healthcare professional will disinfect the arm with an antiseptic. Subsequently, a band will be applied around the arm to provide pressure on the veins, facilitating the accumulation of blood in a localized area (Hoffstetter, Giffin & Brown, 2018). When the veins engorge with blood, a blood sample will be extracted using a needle inserted into the vein. The necessary volume of blood will be extracted, and the needle will be replaced with a little adhesive bandage to halt the bleeding. The healthcare professional will instruct you to apply pressure at the site of the needle insertion to mitigate blood flow. The sample collection procedure is less unpleasant; nonetheless, a throbbing sensation in the arm may occur.
The gathered sample will thereafter undergo analysis by a laboratory. The healthcare personnel or laboratory technician will place the blood sample on a petri dish that already has the particular antigen corresponding to the illness or condition for which the sample was collected.
Source: (Hoffstetter, Giffin & Brown, 2018)
The picture above depicts a sample being placed onto the plate for the ELISA test. If the blood has antibodies against the antigen, they will bind together. The laboratory technician will introduce an enzyme to the dish to examine and monitor the response between the blood and the antigen. A change in hue indicates the existence of the illness or condition for which the test was performed. The change in color resulting from the addition of an enzyme aids healthcare personnel in quantifying the presence of antibodies.
The test primarily detects proteins in the body and also assesses the presence of antigens. The Elisa Test aids in the identification of hormones, viruses, allergens, viral fevers, bacterial antigens, and antibodies inside the body that combat infections. It may also assist in recognizing any agent attempting to infect an individual.
Although it is a straightforward test, those undergoing it may sometimes encounter specific risks, such as contracting an infection, experiencing drowsiness, or continuous bleeding. In such instances, the physician must be notified and informed of the concerns. The test is also aiding in the discovery of COVID-19. It is essential to inform the physician should similar occurrences transpire in the near future (Kamarehei, Khabiri & Saidijam, 2018).
The outcome of the Elisa test may differ according on the analysis performed by the laboratory conducting the test. The illness or disease is another variable influencing the outcome. Upon the report's availability, the physician will explain the findings and assist in comprehending their implications. There are instances where a positive test result may suggest that the illness or condition is absent. False positives and false negatives may occur; the former suggests the presence of a condition that is not really present, while the latter denotes the absence of a condition that is, in fact, present (Kamarehei, Khabiri & Saidijam, 2018). Owing to this ambiguity, the ELISA test may be administered again to an individual within weeks, or the physician may request further sensitivity tests to ascertain the disease definitively.
The first step in the Elisa Test is to immobilize the sample antigen on the petri plate. Two ways may be used to immobilize the antigen: direct absorption onto the dish surface or using an antibody affixed to the plate. The assessment has four categories: direct, indirect, sandwich, and competitive.
Source: (Lauridsen , Holmetoft & Petersen, 2016)
The graphic above elucidates the functionality of several ELISA tests. The categories are classified based on the adjustments included in the method. The sandwich ELISA test has enhanced sensitivity and robustness, making it a potent ELISA assay.
Direct ELISA: This method enables a more rapid detection of antibodies relative to other tests due to its streamlined procedure. This approach involves directly coating the antigen into the wells of a microtitre plate, followed by the addition of an enzyme-labeled primary antibody that identifies the corresponding antigens. The test exhibits reduced errors due to its simplicity, requiring fewer steps and reagents during execution. The approach does not need the testing of a secondary antibody; yet, it has significant limitations concerning specificity. The limited specificity of antigen immobilization results in increased background noise relative to other ELISA procedures (Lauridsen, Holmetoft & Petersen, 2016). The absence of particular interactions between the sample proteins and the target protein on the microtitre plate is the reason. The direct ELISA has less flexibility because all target proteins are bound by enzyme-conjugated antibodies. The procedure for labeling primary antibodies is time-consuming and labor-intensive, potentially impacting the immunoreaction. The absence of a secondary antibody diminishes signal amplification, resulting in decreased test sensitivity. This approach is used to analyze the immunological response to a particular antigen.
The Indirect ELISA Test demonstrates exceptional sensitivity by using an enzyme-labeled secondary antibody that binds to the original antibody. It is seen more affordable than direct ELISA, since it needs fewer labeled antibodies. The indirect ELISA offers more flexibility owing to the binding properties of enzyme-labeled secondary antibodies with primary antibodies. The secondary antibody is mostly of polyclonal origin, exhibiting anti-species reactivity (Lauridsen, Holmetoft & Petersen, 2016). The indirect ELISA has specific limitations about cross-reactivity between the secondary antibody and a bound antigen, potentially resulting in increased background noise. The test furthermore requires an extra step during the incubation of the secondary antibody. The procedure is more time-intensive. The indirect ELISA technique aids in quantifying the overall concentration of antibodies present in a specific sample.
The sandwich ELISA technique employs pairs of antibodies, specifically capture and detection antibodies. The antibody may be classified as either monoclonal or polyclonal. Each antibody exhibits great specificity for antigen epitopes, and this test has been confirmed to be suitable for antigens possessing two epitopes. The antibody pairs must possess matching specificity to bind with distinct epitopes and provide reliable results. The trapped antibody interacts with an antigen, facilitating the detection of ELISA by both direct and indirect techniques. The measurement of antigens occurs in both the top and bottom layers of the antibody, so the complete procedure is referred to as the sandwich ELISA test (Pereira, Cunha & Fernandes, 2020). When a sandwich ELISA is used, it necessitates more frequent validation because to its propensity for yielding potentially false positive findings. The test may sometimes require significant time owing to the need of pairing antibodies. The first stage of the sandwich test necessitates the coating of an ELISA plate with a capture antibody. The subsequent stage involves introducing a sample antigen to the plate, followed by the detection of antibodies. The detected antibody may be enzyme-labeled or enzyme-unlabeled, determining its classification as either direct sandwich ELISA or indirect sandwich ELISA. The indirect sandwich ELISA employs a secondary enzyme-labeled antibody that is introduced to bind the main unlabeled antibody for detection. The sandwich ELISA technique has more sensitivity than both direct and indirect ELISA methods (Pereira, Cunha & Fernandes, 2020). The methodology exhibits more flexibility for detecting purposes by using both direct and indirect techniques. The test facilitates the analysis of intricate samples that are both highly specific and sensitive, since it does not need prior purification of the antigen. However, this approach also presents some drawbacks that need consideration, such as the necessity to pre-test the ELISA kit for response and detection, which may be time-consuming.
Competitive inhibition Elisa: This test is also referred to as blocking ELISA and is predicated on a plate/surface assay. This assay is recognized as one of the most challenging of all ELISA procedures; nevertheless, other tests may be adapted to conform to the requirements of competitive ELISA. This approach quantifies the quantity of antibodies or antigens in a given sample based on the signal generated from the resultant interference (Sahli, Mouelhi & Tlig, 2018). It demonstrates the competitive interaction between the specified antigen or antibody and a labeled antigen or antibody at a constrained concentration. The output signal is inversely proportional to the concentration of the antigen in a sample, exhibiting diminished signaling at elevated antigen levels. The assay illustrates the application of antigen onto a microtitre plate. Upon completion of the ideal blocking and washing procedure, a sample of unidentified antigens is introduced. The process is facilitated by including a tagged detection antibody with substrates such as 3,3’,5,5’-Tetramethylbenzidine (TMB). A crucial phase in this test is the competitive response induced by the sample and the antigen that binds to the multiwall plates with the primary antibody (Sahli, Mouelhi & Tlig, 2018). A high concentration of antigen results in a low output signal, whereas a low concentration of antigen yields a high output signal. It is optimal to utilize in circumstances when the available antibody corresponds to the sample antigen. It aids in the detection of all antigen types, regardless of size, in contrast to the sandwich approach. The procedure entails incubating the sample with an additional component prior to initiating the reaction.
The Elisa test is effective for detecting antigens or antibodies in a sample. It assists in determining if an individual has a condition and, if so, whether they possess an antibody to combat it. The market offers Elisa test kits that include a pre-coated plate, detection antibody, and other necessary reagents for conducting the test. Various kinds of ELISA testing exist, with the sandwich ELISA test regarded as a suitable procedure.
Gandikota, C., Gandhi, L & Maisam, G. (2020) A novel anti?NS2BNS3pro antibody?based indirect ELISA test for the diagnosis of dengue virus infections. Journal of Medical Virology.
Hoffstetter, A., Giffin, D & Brown, L. (2018) An ELISA based method for quantifying arabinoxylan in wheat flour. Journal of Science, 79.
Kamarehei, F., Khabiri, A & Saidijam, M. (2018) Designing a novel ELISA method based on CagA, NapA recombinant antigens to increase sensitivity and specificity of Helicobacter pylori whole cell antigen detection.
Lauridsen H., Holmetoft, U & Petersen, A. (2016) Comparison of three commercial fecal calprotectin ELISA test kits used in patients with Inflammatory Bowel Disease. Scandinavian Journal of Gastroenterology, 51(2).
Pereira, C.S., Cunha, S.C. & Fernandes, J.O. (2020) Validation of an Enzyme-Linked Immunosorbent Assay (ELISA) Test Kit for Determination of Aflatoxin B1 in Corn Feed and Comparison with Liquid-Chromatography Tandem Mass Spectrometry (LC-MS/MS) Method. Food Anal. Methods.
Sahli, H., Mouelhi, A & Tlig, L. (2018) An advanced intelligent ELISA test for bovine tuberculosis diagnosis. Biomedical Signal Processing and control, 46.
Vencia, W., Migone, L & Vito, G. (2016) Validation of an Elisa method. XVII Congresso Nazionale S.I.Di.L.V., Pacengo di Lazise (VR), Italia, 28-30 settembre 2016 2016 pp.322-326 ref.4
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