The ELISA technique is utilized to identify and measure a particular substance, typically an antigen, in a specimen. Because antibodies can recognize an extensive variety of antigenic structures, the potential uses of ELISA in analyte measurement are almost unlimited. The impact of ELISA on diagnostic immunoassays and the healthcare system is unparalleled. The number of analytical and clinical investigations performed globally using ELISA is enormous, and the number of routine patient care measurements using these immunoassays is very high. Practically every diagnostic laboratory in the world has encountered ELISA well plates.
The primary drawbacks of traditional ELISA are its relatively low sensitivity and accuracy, which have hindered its practical application. As a result, effective strategies have been developed to address these issues, resulting in significant progress in improving the accuracy, sensitivity, and stability of ELISA. De la Rica and Stevens developed a nanoparticle-based ELISA (nanoELISA) that can detect ultralow antigen concentrations as low as 1 x 10-18 g/ml. They were able to detect both prostate-specific antigen and the p24 HIV antigen at concentrations that were undetectable using standard tests, although quantification was not possible. Fortunately, the nanoELISA approach significantly improves ELISA performance. Murdock et al have also developed a more practical method for ELISA by using automated image analysis on mobile technology, eliminating the need for microplate readers and making ELISAs more feasible in the field.
The use of ELISA technology in in-vitro diagnostics (IVD) has gained considerable attention. Researchers in various fields have been attracted to the development and application of ELISA-based technologies by the emergence and advancement of magnetic bead technology, paper-based platforms, and nanomaterials. Recently, ELISA has been effectively combined with these analytical techniques to achieve high sensitivity, specificity, repeatability, low cost, and multiplexing detection.
Some of the advances made in ELISA technology are:
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Digital ELISA
Digital ELISA is an emerging immunoassay methodology that allows for femtogram-level detection of protein analytes in biofluids. First, antibody-conjugated paramagnetic beads are used to capture single molecules of target protein, and protein-bead complexes are labeled with fluorophore–conjugated detection antibody. Beads are then assessed for the presence or absence of target protein using a precision fabricated microwell array capable of capturing one bead per well. This technique has been shown to be as much as 1000 times more sensitive than traditional ELISA methods.
However, in digital ELISAs, the signal generated by each individual biomolecule is detected and counted. This means that even low levels of the target biomolecule can be accurately detected, and the number of biomolecules present can be accurately quantified. There are several different digital readout methods that can be used in a digital ELISA, including Single-molecule counting, Magnetic microbead detection, and Electrochemical detection.
Overall, digital ELISA has applications in a wide range of fields, including clinical diagnostics, drug discovery, and environmental monitoring.
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Multiplex ELISA
In a multiplex ELISA, a microarray plate is coated with multiple capture antibodies, each specific to a different target biomolecule. The sample is then added to the plate and allowed to incubate, allowing the target biomolecules to bind to their respective capture antibodies. After washing to remove any unbound sample, a mixture of detection antibodies, each specific to a different epitope on the target biomolecules, is added to the plate. Each detection antibody is conjugated to a different detection reagent, such as a different fluorescent dye, allowing for the simultaneous detection of multiple target biomolecules. The plate is then washed again, and the presence of each target biomolecule is quantified by detecting the signal generated by the conjugated detection reagent.
The advantages of multiplex ELISA over traditional ELISA include High throughput, Reduced sample volume and Increased sensitivity. There are several different types of multiplex ELISA platforms available, including Planar microarrays, Bead-based microarrays and Suspension microarrays.
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Single-molecule ELISA
Single-molecule ELISA is a highly sensitive method for the detection and quantification of individual biomolecules. It uses single-molecule fluorescence detection to monitor the binding of individual analyte molecules to immobilized capture antibodies.
The key difference in single molecule ELISA is the digital readout method, where the signal generated by each individual biomolecule is detected and counted. This means that even extremely low levels of the target biomolecule can be accurately detected, and the number of biomolecules present can be accurately quantified at the single-molecule level.
There are several different digital readout methods that can be used in single molecule ELISA, including Fluorescence microscopy and High-speed atomic force microscopy (HS-AFM).
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Nanoparticle-based ELISA
Nano-ELISA has numerous advantages over the traditional ELISA method in terms of sensitivity, accuracy, operational ease, and cost-effectiveness. Nanomaterials, such as nano-substrates, nanoprobes, nano-carriers, and nano-coloring agents, are utilized in constructing the nano-ELISA. Detection antibodies are linked with nanoparticles, which create a visible signal upon binding to the analyte. The amount of target biomolecule present in the sample is determined by quantifying the signal generated by the nanoparticles. Various detection methods, including absorbance, fluorescence, or magnetic resonance, can be used to detect the signal. The intensity of the signal is proportional to the quantity of target biomolecule, facilitating quantitative analysis. The advantages of nanoparticle ELISA include; Enhanced sensitivity, Improved selectivity, Multiplexing and Adaptability.
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Smartphone-based ELISA
In smartphone-based ELISA, the signal generated by the detection reagent is captured by the camera of a smartphone and analyzed using a smartphone app. The app analyzes the color and intensity of the signal, allowing for the quantification of the target biomolecule. The results can then be displayed on the smartphone screen or transmitted wirelessly to a cloud-based server for storage and analysis.
The advantages of smartphone-based ELISA include:
- Portability: Smartphone-based ELISA is a portable and handheld platform that can be used in a variety of settings, including resource-limited or remote locations.
- Cost-effectiveness: The platform is low-cost, eliminating the need for expensive laboratory equipment and reducing the cost per test.
- User-friendliness: The smartphone app simplifies the assay process, reducing the need for trained personnel and minimizing errors.
- Multiplexing: The platform can be adapted for multiplexing, allowing for the detection of multiple analytes in a single sample.
Smartphone-based ELISA has applications in various fields, including clinical diagnostics, environmental monitoring, and food safety testing. It has the potential to revolutionize diagnostic testing by enabling rapid and reliable diagnosis in resource-limited settings.
References
- https://www.sciencedirect.com/science/article/abs/pii/S0165993622000887
- Fundamentals and History of ELISA: The Evolution of the Immunoassays Until Invention of ELISA. In: Enzyme-linked Immunosorbent Assay (ELISA). SpringerBriefs in Applied Sciences and Technology(). Springer, Singapore. https://doi.org/10.1007/978-981-10-6766-2_1
- Long Wu, Guanghui Li, Xin Xu, Lin Zhu, Riming Huang, Xiaoqiang Chen, Application of nano-ELISA in food analysis: Recent advances and challenges, TrAC Trends in Analytical Chemistry,
- https://www.magonlinelibrary.com/doi/epub/10.12968/hmed.2016.77.7.C98
- Shah, K., & Maghsoudlou, P. (2016). Enzyme-linked immunosorbent assay (ELISA): the basics. British Journal of Hospital Medicine, 77(7), C98–C101.doi:10.12968/hmed.2016.77.7.c98 10.12968/hmed.2016.77.7.C98
- Diagnosis of ischemic stroke using circulating levels of brain-specific proteins measured via high-sensitivity digital ELISA – ScienceDirect
- Early Diagnosis of Experimental Trichinella spiralis Infection by Nano-based Enzyme-linked Immunosorbent Assay (Nano-based ELISA). Experimental Parasitology, 107867 | 10.1016/j.exppara.2020.107867
- Diagnosis of ischemic stroke using circulating levels of brain-specific proteins measured via high-sensitivity digital ELISA. Grant C. O’Connell⁎, Megan L. Alder, Christine G. Smothers, Carolyn H. Still, Allison R. Webel, Shirley M. Moore