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ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay technique that allows for the detection and quantification of an analyte in a sample. ELISA utilises highly specific antibodies produced during an immune response, along with a colorimetric reaction and uses this mechanism to measure the concentration of the target antigen. Even when present in small concentrations, ELISAs can detect a wide range of targets, including hormones, peptides, proteins, and antibodies. The four major types of ELISA are indirect, direct, sandwich and competitive [1]. Despite its type, ELISA has been adapted across a wide range of fields due to its reliability, specificity, and stability. Commercial ELISA kits rapidly developed to meet a growing demand, becoming a widely applied tool in biotechnology, the food industry, vaccine development, clinical diagnosis, toxicology, and the pharmaceutical industry.
Compared to other immunoassays, ELISA exhibits greater sensitivity due to its unique enzyme amplification feature. The antibody-antigen coupling method, on the other hand, enhances specificity by linking the antibody’s paratope at the binding location with the antigen’s corresponding epitope in a highly specific manner. Despite the advantages this method offers, ELISA presents some challenges that future research should aim to overcome. Firstly, ELISA follows repetitive procedures, increasing the chances of user error. This method also requires centralised laboratory equipment, such as a spectrophotometer or microplate reader. Similarly, a high sample volume is required. In fact, the nanomolar detection limit of ELISA hinders the identification of many protein biomarkers [2,3].
Recent advances in ELISA-derived technology, however, have highlighted its potential, especially for in vitro diagnosis and point-of-care settings. ELISA typically consists of four basic components: a solid substrate, a recognition component, a signal amplification component, and a readout method. The characteristics of each of these components significantly influences the performance of the assay. Consequently, advances in ELISA technology strongly rely on enhancing one or more of these components [2].
Solid substrates innovations
Generally, ELISA takes place on a solid substrate surface comprising immobilise capture antibodies that isolate reactants from non-reactants. Most ELISAs are performed on a microwell plate, which in fact, require plate coating, blocking, and repeated washing and incubation steps. Thus, making the process tedious and time-consuming. Most importantly, the risk of errors is higher as multiple steps are involved in the process. Several studies have now optimised the blocking process to yield better results, a crucial step to reduce non-specific binding. Among these innovations, blocking using molecules such as PDDA or PAA resulted in impaired binding of non-specific molecules and increased absorption capacity of the capture antibody [3].
Nevertheless, the widespread use of ELISA for epidemiological research have highlighted the urgent need for on-site rapid detection. Paper-based ELISA is now a common practise. In fact, we have seen this technology for monitoring COVID-19, Monkeypox, HIB, and has been adapted for pregnancy testing. Yet, the use of paper substrates goes a long way and offer the possibility to develop more complex biosensors. In the past 10 years, different 3D microfluidic paper devices have been developed to overcome the limitations of lateral flow tests. For instance, a vertical flow-based paper immunosensor with a sample pad with different pore sizes was proposed for the detection of Influenza virus H1N1 [4]. To reduce the time required to complete a conventional ELISA, magnetics beads (MBs) have also been tested as a solid substrate owing to their ability to accelerate the diffusion process, and the possibility to isolate components of interests using a magnetic force. In fact, MS-based ELISA has been shown to detect different biomarkers in complex samples within 40 minutes. MB-based ELISAs have been proposed as an effective, rapid tool for the monitoring of human anti-SARS-CoV-2 antibodies [2].
Recognition element innovations
While antibodies have demonstrated sufficient recognition capacity, molecular imprinted polymers (MIPs) and nucleic acids have been incorporated into ELISA-derived technologies due to their unique cost, stability, specificity, and affinity. Despite the number of antibodies available, their use is limited. Recently, aptamer-based ELISA has been proposed as a solution due to the ability of aptamers to recognise and bind a wider range of targets. Competitive aptamer-based ELISA, for example, rely on a target molecule competing with the labelled molecule to bind to the aptamer, and has been shown particularly useful for the detection of small molecules. This type of assay has been employed for the detection of Aflatoxin B1 (AFB1) using AFB1 aptamers. Compared to conventional ELISA, this method exhibited higher sensitivity and reduced costs. Similarly, sandwich aptamer-based ELISA has been developed for the detection of infectious particles such as HCV. While aptamer-based ELISA is currently used for research purposes, its broad applicability has enough potential to further develop this technology [2,5].
Nucleic acids have also been used for the detection of polymerase chain reaction products (PCR) using ELISA. A typical PCR-ELISA protocol would include labelled digoxin to denature the PCR product, hybridising the PCR products to the capture probe, adding an enzyme-conjugated antibody specific for digoxin, adding the colorimetric substrate, and measuring the absorbance on a plate reader. While this process is expensive and time-consuming; it shares the high sensitivity and specificity of PCR and the batch detection ability of ELISA. In fact, the reliability of PCR-ELISA is about 100 times higher than that of agarose electrophoresis. Thus, the PCR-ELISA is frequently used for quantification and qualification of known bacteria, fungi, parasites, and viruses [2].
Signal amplification innovation
Just as the other components of ELISA, signal amplification in relation to ELISA sensitivity cannot be overstated. Commonly, ELISA rely on enzymes such as HRP that catalyse substrates and result in colorimetric changes that can be measured. One alternative to natural enzymes is the use of nanozymes: nanomaterials with enzyme-like catalytic activity, in many cases more efficient than natural enzymes. An adaptation of the ELISA format using nanozymes is the metal-organic framework-linked immunosorbent assay (MOFLISA). This assay utilises the increased efficiency of nanozymes for more accurate and sensitive assays. In the Plasmonic ELISA, the enzyme controls the aggregation of gold nanoparticles, generating coloured solutions with a distinct tonality. This format increases accessibility to the high-sensitive ELISA format, by not requiring specialised lab equipment. Qualitative results using this format can be detected by eye, and quantitative results require only a luminometer. For example, a smartphone-based plasmonic Immunoassay Reader, which uses a smartphone camera as the luminometer, has been shown to produce results comparable with conventional ELISA [7]. Another adaptation of the ELISA format, CLIA, uses a chemiluminescent detection method with higher sensitivity than ELISA, is rapid, and detectable with a luminometer [2,6].
The Author: Pilar Ruiz, Scientific Marketing
Communications Assistant at Abbexa
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References
[1] M. Alhajj and A. Farhana, “Enzyme Linked Immunosorbent Assay (ELISA),” PubMed, 2020. https://www.ncbi.nlm.nih.gov/books/NBK555922/
[2] P. Peng et al., “Emerging ELISA derived technologies for in vitro diagnostics,” TrAC Trends in Analytical Chemistry, vol. 152, p. 116605, Jul. 2022, doi: 10.1016/j.trac.2022.116605.
[3] S. Hosseini, P. Vázquez-Villegas, M. Rito-Palomares, and S. O. Martinez-Chapa, “Advantages, Disadvantages and Modifications of Conventional ELISA,” SpringerBriefs in Applied Sciences and Technology, pp. 67–115, Dec. 2017, doi: 10.1007/978-981-10-6766-2_5.
[4] N. Colozza, V. Caratelli, D. Moscone, and F. Arduini, “Origami Paper-Based Electrochemical (Bio)Sensors: State of the Art and Perspective,” Biosensors, vol. 11, no. 9, p. 328, Sep. 2021, doi: 10.3390/bios11090328.
[5] J. Bala, S. Chinnapaiyan, R. K. Dutta, and H. Unwalla, “Aptamers in HIV research diagnosis and therapy,” RNA Biology, vol. 15, no. 3, pp. 327–337, Feb. 2018, doi: 10.1080/15476286.2017.1414131.
[6] S. R. Herbin, D. G. Klepser, and M. E. Klepser, “Pharmacy-Based Infectious Diseases Management Programs Incorporating CLIA-Waived Point-of-Care Tests,” Journal of Clinical Microbiology, Feb. 2020, doi: 10.1128/jcm.00726-19.
