Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are both widely used as diagnostic tools in medicine and as quality control measures in various industries; they are also used as analytical tools in biomedical research for the detection and quantification of specific antigens or antibodies in a given sample. These two procedures share similar basic principles and are derived from the radioimmunoassay (RIA). RIA was first described by Berson and Yalow (Yalow and Berson, 1960), for which Yalow was awarded the Nobel Prize in 1977, to measure endogenous plasma insulin. RIA was then developed into a novel technique to detect and measure biological molecules present in very small quantities, paving the way for the analysis and detection of countless other biological molecules, including hormones, peptides, and proteins. Because of the safety concern regarding its use of radioactivity, RIA assays were modified by replacing the radioisotope with an enzyme, thus creating the modern-day EIA and ELISA.
Regarding the basic principles of enzyme-linked immunoassay, IA/ELISA takes advantage of the fundamental immunology idea that an antigen binds to its particular antibody to detect very minute concentrations of antigens in a fluid sample, such as proteins, peptides, hormones, or antibodies. Alkaline phosphatase (EC 3.1.3.1) and glucose oxidase (EC 1.1.3.4) are the two most often employed enzymes in EIA and ELISA, which use enzyme-labeled antigens and antibodies to detect biological molecules. Typically, 96-well microtiter plates are used to immobilize the antigen in the fluid phase. A specific antibody is given permission to attach to the antigen, and that specific antibody is then recognized by a secondary, enzyme-coupled antibody. An antigen is present when a chromogenic substrate for the enzyme causes a noticeable color change or fluorescence. A colorimetric reading of this kind can be used to evaluate quantitative or qualitative measurements. Fluorogenic substrates are more sensitive and can quantify antigen concentrations in the sample with greater accuracy. Figure 1 depicts the ELISA's general process. The basic processes shown in Figure 1 have been modified to work with different types of ELISAs. The crucial procedure in the ELISA assay is adhering or immobilizing the antigen or antigen-specific capture antibody directly to the well surface in order to directly or indirectly detect the antigen. Using a "capture" antibody, the antigen can be precisely isolated from a sample of mixed antigens for accurate and reliable assays. As a result, the antigen is "sandwiched" between the capture antibody and the detection antibody. A competitive method is used when the antigen to be measured is small or has only one epitope for antibody binding. Either the antigen is labeled and competes for the formation of the unlabeled antigen-antibody complex or the antibody is labeled and competes for the bound antigen and the antigen in the sample. Each of these ELISA modifications may be applied both qualitatively and quantitatively.
Figure 1. Enzyme-linked immunosorbent assay (ELISA) technique used to detect an antigen in a given sample.
Basically, there are 4 main parts of ELISA. They are Indirect, Sandwich, Competitive and Multiple & portable. First, regarding Indirect ELISA, A sample that has to be tested for a particular antigen is placed in each well of a microtiter plate, and then a solution of a nonreacting protein, such as bovine serum albumin, is added to block any wells that aren't coated with the antigen. A secondary antibody that has been enzyme-conjugated is then added after the main antibody, which binds precisely to the antigen. To measure the primary antibody using a color change, an enzyme substrate is added. The serum's main antibody concentration and color intensity are closely correlated. Haapakoski et al. (2013) showed how the indirect ELISA approach may be used to study the regulation of allergic asthma by the activation of Toll-like receptors during cutaneous allergen sensitization using ovalbumin (OVA). In one experiment, it was shown that cutaneous exposure to Toll-like receptor ligands reduced blood levels of OVA-specific IgE antibodies as determined by the indirect ELISA method.
A main disadvantage of indirect ELISA is that the method of antigen immobilization is not specific. When serum is used as the test antigen, all proteins in the sample may adhere to the wells of a microtiter plate. This limitation, however, can be overcome using a capture antibody unique to the specific test antigen to select it out of the serum, as illustrated in the sandwich technique below
As a second one is Sandwich ELISA. The sandwich technique is used to identify a specific sample antigen. A known amount of bound antibody is produced on the well surface to trap the target antigen. The antigen-containing sample is added to the plate following the blocking of nonspecific binding sites with bovine serum albumin. The antigen is subsequently "sandwiched" by a unique primary antibody. The main antibody is then bound by enzyme-linked secondary antibodies. Immuno-enzyme conjugates that are not bound are removed by washing. The addition of a substrate results in an enzyme transformation that produces a quantifiable hue. In order to measure human KGF, Canady et al. (2013) used the sandwich method to examine patient serum and find elevated keratinocyte growth factor (KGF) levels in the sera of keloid and scleroderma patients in comparison to healthy controls.
One advantage of using a purified specific antibody to capture antigen is that it eliminates the need to purify the antigen from a mixture of other antigens, thus simplifying the assay and increasing its specificity and sensitivity.
Third one is Competitive ELISA; the key event of competitive ELISA is the process of competitive reaction between the sample antigen and antigen bound to the wells of a microtiter plate with the primary antibody. In order to add the antibody antigen complexes to wells that have already been coated with the sample antigen, the primary antibody must first be treated with it. Any unbound antibody is rinsed away during an incubation period. More primary antibodies will bind to the sample antigen the more antigen is present in the sample. As a result, less primary antibody will be available to bind to the antigen coated on the well. A substrate and a secondary antibody that is enzyme-conjugated are then added to produce a chromogenic or fluorescent signal. Antigen is present in the sample if there is no color.
The main advantage of competition ELISA is its high sensitivity to compositional differences in complex antigen mixtures, even when the specific detecting antibody is present in relatively small amounts (Dobrovolskaia et al., 2006). This technique can be used to evaluate the total antibodies to the capsular polysaccharide of Haemophilus influenzae type b in human sera from vaccinated participants as well as the potency of U.S. standardized allergen extracts (Dobrovolskaia et al., 2006). Competitive ELISA is frequently used to find HIV antibodies in patient samples. Two distinct antibodies are used: one from patient serum and the other conjugated with enzyme. The HIV antigen is coated on the surface of the microtiter plate wells. For the same antigen, there is cumulative rivalry between the two antibodies. The antigen-antibody reaction takes place if antibodies are present in the serum, leaving very little antigen left behind for the enzyme-labeled antibody to bind to. The majority of the enzyme-labeled antibodies that are not bound are washed away, resulting in little to no color change. An HIV-positive sample is indicated by the absence of color.
The last one is Multiple & portable ELISA. Here many and transportable A multi-catcher device with 8 or 12 immunosorbent projecting pins on a central stick that can be submerged in a collected sample is used in the new technique known as ELISA. Using prefilled microwells with reagents, the washings and incubations with enzyme-conjugated antigens and chromogens are carried out. According to Balsam et al. (2013), the key benefits of these ready-to-use lab kits include their affordability, ability to screen large populations, lack of need for trained workers or laboratory equipment, and suitability for low-resource environments. Point-of-care diagnostics for infectious disorders, bacterial toxins, oncologic markers, and medication screening are examples of clinical applications.
After that considering what an enzyme-linked immunosorbent assay (ELISA) does ELISA is a biochemical assay that uses antibodies and an enzyme-mediated color change to detect the presence of either antigen (proteins, peptides, hormones, etc.) or antibody in a given sample. Both “indirect” and “sandwich” ELISAs allow the detection of antigen or antibody at very low concentrations. The competitive method detects compositional differences in complex antigen mixtures with high sensitivity, even when the specific detecting antibody is in relatively small amounts. Multiple and portable ELISA is a ready-to-use, low-cost lab kit ideal for large-population screening in low-resource settings.
Considering the limitations of an enzyme-linked immunosorbent assay (ELISA), the first point is The enzyme-mediated color change will react indefinitely. Over a sufficiently long period of time, the color strength will inaccurately reflect the amount of primary antibody present, yielding false positive results. The second limitation is To detect a given antibody or antigen, a known reciprocal antigen or antibody must be generated. And the third limitation is that nonspecific binding of the antibody or antigen to the plate will lead to a falsely high-positive result.
In conclusion, EIA/ELISA is a powerful method not only for routine biomedical research but also as a diagnostic tool. It allows the detection of all types of biological molecules at very low concentrations and quantities. Despite its limitations, EIA/ELISA remains an important tool in both clinical and basic research as well as in clinical diagnosis.
REFERENCES
Rasooly, R., Bruck, H., Balsam, J., Prickril, B., Ossandon, M., & Rasooly, A. (2013). Improving the Sensitivity and Functionality of Mobile Webcam-Based Fluorescence Detectors for Point-of-Care Diagnostics in Global Health. Diagnostics, 6(2), 19. https://doi.org/10.3390/diagnostics6020019
Canady, J., Arndt, S., Karrer, S., & Bosserhoff, A. K. (2013). Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis. The Journal of Investigative Dermatology, 133(3), 647–657. https://doi.org/10.1038/jid.2012.389
Dobrovolskaia, E., Gam, A., & Slater, J. E. (2006). Competition enzyme-linked immunosorbant assay (ELISA) can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture. Clinical Experimental Allergy, 36(4), 525–530. https://doi.org/10.1111/j.1365-2222.2006.02466.x
Haapakoski, R., Karisola, P., Fyhrquist, N., Savinko, T., Lehtimäki, S., Wolff, H., Lauerma, A., & Alenius, H. (2013). Toll-Like Receptor Activation during Cutaneous Allergen Sensitization Blocks Development of Asthma through IFN-Gamma-Dependent Mechanisms. Journal of Investigative Dermatology, 133(4), 964–972. https://doi.org/10.1038/jid.2012.356
Yalow, R. S., & Berson, S. A. (1960). IMMUNOASSAY OF ENDOGENOUS PLASMA INSULIN IN MAN. Journal of Clinical Investigation, 39(7), 1157–1175. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC441860/
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