Current and Emerging Applications of ELISAs

1 Current and Emerging Applications of ELISAs Kate Harrison, PhD Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of clinical and research laboratories. ELISAs can detect and quantify the presence of antibodies, antigens or proteins in an enormous range of biological and environmental samples. Generally carried out in 96-well plates, ELISAs rely on the immobilization of an antigen or antibody to a solid surface (i.e., the base of the plate) and the subsequent binding of a corresponding antibody or antigen in a specific manner. Although the most basic form of an ELISA simply detects the presence of a particular antigen or antibody, quantification can be achieved by adding an antibody conjugated to a substrate that produces a measurable product, such as fluorescence or a color change. ELISAs were first developed in 1971, by two independent groups. Both of these simultaneously developed assays were based on enzymatic reporters, rather than the radioactive reporters used by previous immunoassay iterations.1 These enzyme-based immunoassays were safer and easier to perform than radioactivity-based immune assays, and quickly became widespread. Since then, ELISAs have been adapted for use in a wide variety of fields for numerous applications, from epidemiological monitoring to environmental pollutant analysis. There are now hundreds of commercially available ELISA kits for the detection and quantification of antibodies and antigens. However, research continues into the development of novel biomarkers and ELISA-based technologies to improve sensitivity and broaden the potential uses. Some of these current and emerging applications and methodologies for ELISAs are discussed below. Clinical diagnostics and epidemiology One of the most widespread applications of ELISAs is in clinical diagnostics and epidemiology. ELISAs can be used to detect both antigen markers of a disease and the antibodies against it, depending on the type of assay selected. Therefore, they can be used to diagnose an active infection and track the spread of a disease in an outbreak scenario, and also identify previously infected individuals. To help monitor disease epidemics epidemiologically, a portable field version of an ELISA would be advantageous for onsite diagnosis of infectious disease. However, although several avenues of research have been investigated to this end – such as a batteryoperated ELISA system2 and smartphone-linked ELISA systems – nothing is, as yet, commercially available.3,4 In addition to infectious diseases, ELISAs can also be used to indicate the presence of auto-reactive antibodies. Both the general presence of autoantibodies, using a plate with a variety of self-antigens adsorbed to the surface, or autoantibodies related to specific autoimmune diseases can be analyzed, making ELISAs indispensable in autoimmune disease diagnostics.5 One such example is the identification of rheumatoid factors (RFs). RFs are antibodies that bind to other antibodies and are commonly found in systemic autoimmune diseases. RFs have diagnostic and prognostic value in several autoimmune diseases and are part of the specific diagnostic criteria for rheumatoid arthritis.6 For other autoimmune diseases, like autoimmune connective tissue diseases (CTDs), ELISAs for autoreactive antinuclear antibodies (ANAs) are emerging as a more sensitive, more specific and less time-consuming alternative to the current gold standard: indirect immunofluorescence assays.7 Listicle CURRENT AND EMERGING APPLICATIONS OF ELISAS 2 Listicle Despite their range and versatility, traditional ELISAs are limited in scenarios requiring extremely high sensitivity for nano-molar levels of sample, making them unsuitable for early diagnosis or screening of diseases with subtle physiological changes such as Alzheimer’s disease (AD). However, the recent development of nano-ELISAs is now opening more avenues for highly sensitive detection. In a nano-ELISA, a detector antibody and a signaling molecule such as horseradish peroxidase (HRP) are coupled with gold nanoparticles. These nanoparticles act as signal amplifiers, significantly increasing the detection limit and allowing the detection of antigens or antibodies even in the picogram range.8 This method has been used to identify the AD biomarker Aβ42 in serum samples at extremely low levels.9 As cerebral spinal fluid is currently collected for detection of AD biomarkers, this suggests future potential for nano-ELISAs as a novel, less invasive method for AD diagnosis.10 Biopharmaceutical and vaccine development Biopharmaceuticals are therapeutics derived from, or created in, biological sources. Demand for these therapeutics continues to grow as they revolutionize the treatment of a wide range of diseases, including cancer and autoimmune diseases. As they are produced from biological sources, they must be purified to an extremely high standard to remove any host cell proteins (HCPs) or residual manufacturing reagents. ELISAs are one of the standard tools for quantifying the total levels of residual impurities, due to their high-throughput, high sensitivity and high specificity.11 However, they are unable to give any information on the specific, individual HCPs present, so are now often coupled with liquid chromatography-mass spectrometry (LC-MS) for more in-depth evaluation of biopharmaceutical purity. One of the simplest uses for ELISAs is the quantification of antibodies in biological samples. This is essential in the development of novel vaccines. Many vaccines protect against infection by inducing the creation of specific antibodies, meaning quantification of antibody levels post-vaccination is needed to assess vaccine efficacy. ELISAs have therefore been used extensively in the clinical development of approved vaccines, including the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2 and the recently approved R21 and RTS,S vaccines against malaria.12,13,14 Forensic investigations One of the most common applications for ELISAs in forensic science is drug testing. Toxicologists can test forensic samples for a wide range of illicit substances, including cocaine, amphetamines, opiates, cannabinoids and benzodiazepines. Both liquid samples, such as blood and urine, and solid samples such as hair can be processed for ELISA testing, for purposes such as monitoring drug abstinence and workplace testing.15,16 ELISAs can also be used to determine if a blood sample is human for forensic investigation by using antibodies targeting specifically human blood components.15 By targeting the assay at highly abundant antigens in the blood, such as human albumin, blood can still be identified as human by ELISA even in ancient, buried samples over 3,000 years old.17 In addition to analyzing bodily fluids, ELISAs can also be used in forensic investigations to help determine a cause of death. Traumatic brain injury (TBI) is one of the leading causes of mortality worldwide and is responsible for approximately 30% of all injury-related deaths.18 Post-mortem identification of TBI can be difficult, as ante-mortem clinical diagnosis usually involves neurological assessment. Recent research has shown elevated levels of cerebral protein biomarkers, such as tau protein and myelin basic protein, in blood and cerebral spinal fluid samples from cases where TBI was the confirmed cause of death. These markers can therefore be used as an indicator of TBI in post-mortem examination, even in the absence of visible central nervous system damage.19,20,21 Food safety ELISAs play a significant role in food safety testing and are used to screen for contaminants and allergens. Food allergies affect approximately 2% of the Western population, and allergic and anaphylactic reactions CURRENT AND EMERGING APPLICATIONS OF ELISAS 3 Listicle to food result in 30,000 hospital visits and 150 deaths per year in the United States alone.22 Allergies can be extremely sensitive. For example, even trace amounts of peanuts can trigger an anaphylactic reaction. Therefore, it is very important that foods labeled as allergen-free, are indeed safe for consumption. Cross-contact with allergens can occur at many stages of food processing and production and is often the result of failure to clean machines appropriately when switching between allergen-containing and allergen-free foods. However, ELISAs are used by manufacturers to detect common allergens in food products, including nuts, meat, milk and milk products, fish and soybeans. The highly sensitive and specific nature of ELISAs can identify cross-contact, preventing the need for food recalls and protecting allergic populations. However, as large numbers of batch samples will typically need to be tested on a regular basis, ELISAs, despite their relative speed, can still result in bottlenecks. Novel, rapid microfluidic-based ELISA platforms are currently being researched to develop a method for more sensitive and far faster allergen identification.23,24 Despite their widespread use in the field, traditional ELISA techniques can lack the sensitivity needed to detect extremely low, but still dangerous, levels of toxic or microbial contaminants. However, the development of nano-ELISAs have enabled the detection of harmful microbes, pesticides, drug residues and pollutants in food.25 Nano-ELISA techniques have now been developed to identify a range of food contaminants, including aflatoxin in corn, pathogenic Listeria monocytogenes in milk and veterinary drug traces in poultry.26,27,28 Environmental analysis Although ELISAs have typically been used for biological sample analysis, their applications in environmental sampling is increasing, particularly for the detection and quantification of agricultural and industrial pollutants in water. The use of pesticides has increased steadily over the last 30 years worldwide, resulting in increased levels of pollution and potentially toxic agricultural runoff.29 Often, analysis of environmental soil and water samples for pollutants uses techniques such as gas and liquid chromatography (GC and LC). However, these techniques can be costly and time-consuming compared to ELISAs, which have been shown to be just as sensitive as chromatography methods.30 Indeed, ELISAs, along with other immune-based assays, have been used successfully in several large-scale water quality surveys in the US.30 Pharmaceutical pollutants, such as hormones and drugs, can also be assessed in both water samples and the tissues of aquatic organisms by ELISA.31 These emerging pollutants can have a significant effect on aquatic ecosystems, damaging animal life, altering microbial populations leading to detrimental algal blooms and perpetuating antibiotic resistance. Monitoring these pollutants is essential for minimizing harm and developing better regulatory guidelines to prevent their release into the environment.32 Sponsored by CURRENT AND EMERGING APPLICATIONS OF ELISAS 4 References 1. Deutschmann C, Roggenbuck D, Schierack P, Rödiger S. Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure. Heliyon. 2020;6(1). doi: 10.1016/j.heliyon.2020.e03270 2. Singh H, Morioka K, Shimojima M, et al. A handy field-portable ELISA system for rapid onsite diagnosis of infectious diseases. Japan J Infect Dis. 2016;69(5):435-438. doi: 10.7883/yoken.jjid.2015.417 3. Long KD, Yu H, Cunningham BT. Smartphone instrument for portable enzyme- linked immunosorbent assays. Biomed Opt Express. 2014;5(11):3792-3806. doi: 10.1364/boe.5.003792 4. Zhdanov A, Keefe J, Franco-Waite L, Konnaiyan KR, Pyayt A. Mobile phone based ELISA (MELISA). Biosens Bioelectron. 2018;103:138-142. doi: 10.1016/j.bios.2017.12.033 5. Drijvers JM, Awan IM, Perugino CA, Rosenberg IM, Pillai S. The enzyme-linked immunosorbent assay. In: Jalali M, Saldanha FYL, Jalali M, eds. Basic Science Methods for Clinical Researchers. Academic Press;2017:119-133. doi: 10.1016/b978- 0-12-803077-6.00007-2 6. Trier NH, Houen G. (2019). Determination of Rheumatoid Factors by ELISA. In: Houen G, eds. Autoantibodies. Methods in Molecular Biology, vol 1901. New York, NY: Humana Press. doi: 10.1007/978-1-4939-8949-2_23 7. Alsaed OS, Alamlih LI, Al-Radideh O, Chandra P, Alemadi S, Al-Allaf A-W. Clinical utility of ANA-ELISA vs ANA-immunofluorescence in connective tissue diseases. Sci Rep. 2021;11(1):8229. doi: 10.1038/s41598-021-87366-w 8. Ambrosi A, Castañeda MT, Killard AJ, Smyth MR, Alegret S, Merkoçi A. Double-codified gold nanolabels for enhanced immunoanalysis. Anal Chem. 2007;79(14):5232-5240. doi: 10.1021/ac070357m 9. Hu T, Lu S, Chen C, Sun J, Yang X. Colorimetric Sandwich Immunosensor for Aβ(1-42) based on dual antibody-modified gold nanoparticles. Sensors Actuat B Chem. 2017;243:792-799. doi: 10.1016/j.snb.2016.12.052 10. Al Abdullah S, Najm L, Ladouceur L, et al. Functional nanomaterials for the diagnosis of Alzheimer’s disease: Recent progress and future perspectives. Adv Funct Mater. 2023;33(37):2302673. doi: 10.1002/adfm.202302673 11. Zhu-Shimoni J, Yu C, Nishihara J, et al. Host cell protein testing by ELISAs and the use of orthogonal methods. Biotechnol Bioeng. 2014;111(12):2367-2379. doi: 10.1002/bit.25327 12. Ramasamy MN, Minassian AM, Ewer KJ, et al. Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): A single-blind, randomised, controlled, phase 2/3 trial. Lancet. 2020;396(10267):1979-1993. doi: 10.1016/s0140-6736(20)32466-1 13. Genito CJ, Brooks K, Smith A, et al. Protective antibody threshold of RTS,S/AS01 malaria vaccine correlates antigen and adjuvant dose in mouse model. NPJ Vaccines. 2023;8(1):114. doi: 10.1038/s41541-023-00714-x 14. Datoo MS, Natama HM, Somé A, et al. Efficacy and immunogenicity of R21/Matrix-M vaccine against clinical malaria after 2 years’ follow-up in children in Burkina Faso: A phase 1/2B randomised controlled trial. Lancet Infect Dis. 2022;22(12):1728-1736. doi: 10.1016/s1473-3099(22)00442-x 15. Perrigo BJ, Joynt BP. Use of ELISA for the detection of common drugs of abuse in forensic whole blood samples. Can Soc Forensic Sci J. 1995;28(4):261-269. doi: 10.1080/00085030.1995.10757486 16. Agius R, Nadulski T. Utility of ELISA screening for the monitoring of abstinence from illegal and legal drugs in hair and urine. Drug Test Anal. 2014;6(S1):101-109. doi: 10.1002/dta.1644 17. Cattaneo C, Gelsthorpe K, Phillips P, Sokol RJ. Reliable identification of human albumin in ancient bone using ELISA and monoclonal antibodies. Am J Biol Anthropol. 1992;87(3):365-372. doi: 10.1002/ajpa.1330870311 18. Demlie TA, Alemu MT, Messelu MA, Wagnew F, Mekonen EG. Incidence and predictors of mortality among traumatic brain injury patients admitted to Amhara region Comprehensive Specialized Hospitals, Northwest Ethiopia, 2022. BMC Emerg Med. 2023;23(1):55. doi: 10.1186/s12873-023-00823-9 19. Olczak M, Poniatowski ŁA, Niderla-Bielińska J, et al. Concentration of microtubule associated protein tau (MAPT) in urine and saliva as a potential biomarker of traumatic brain injury in relationship with blood–brain barrier disruption in postmortem examination. Forensic Sci Int. 2019;301:28-36. doi: 10.1016/j.forsciint.2019.05.010 20. Olczak M, Niderla-Bielińska J, Kwiatkowska M, Samojłowicz D, Tarka S, Wierzba-Bobrowicz T. Tau protein (MAPT) as a possible biochemical marker of traumatic brain injury in postmortem examination. Forensic Sci Intl. 2017;280:1-7. doi: 10.1016/j.forsciint.2017.09.008 21. Olczak M, Poniatowski ŁA, Siwińska A, Kwiatkowska M. Post-mortem detection of neuronal and astroglial biochemical markers in serum and urine for diagnostics of Traumatic Brain Injury. Int J Legal Med. 2023;137(5):1441-1452. doi: 10.1007/ s00414-023-02990-7 22. Food allergies: The “big 9”. USDA Food Safety and Inspection Service. https://www.fsis.usda.gov/food-safety/safe-foodhandling- and-preparation/food-safety-basics/food-allergies. Published March 21, 2024. Accessed April 2, 2024. 23. Weng X, Gaur G, Neethirajan S. Rapid detection of food allergens by microfluidics ELISA-based optical sensor. Biosensors. 2016;6(2):24. doi: 10.3390/bios6020024 24. Uddin MJ, Bhuiyan NH, Shim JS. Fully integrated rapid microfluidic device translated from conventional 96-well ELISA Listicle CURRENT AND EMERGING APPLICATIONS OF ELISAS 5 Listicle kit. Sci Rep. 2021;11(1):1986. doi: 10.1038/s41598-021-81433-y 25. Wu L, Li G, Xu X, Zhu L, Huang R, Chen X. Application of nano-ELISA in food analysis: Recent advances and challenges. Trends Analyt Chem. 2019;113:140-156. doi: 10.1016/j.trac.2019.02.002 26. Zhan S, Hu J, Li Y, Huang X, Xiong Y. Direct competitive ELISA enhanced by dynamic light scattering for the ultrasensitive detection of aflatoxin B1 in corn samples. Food Chem. 2021;342:128327. doi: 10.1016/j.foodchem.2020.128327 27. Wang W, Liu L, Song S, Xu L, Zhu J, Kuang H. Gold nanoparticle-based paper sensor for multiple detection of 12 Listeria spp. by P60-mediated monoclonal antibody. Food Agr Immunol. 2017;28(2):274-287. doi: 10.1080/09540105.2016.1263986 28. Song M, Xiao Z, Xue Y, Zhang X, Ding S, Li J. Development of an indirect competitive ELISA based on immunomagnetic beads’ clean-up for detection of maduramicin in three chicken tissues. Food Agr Immunol. 2018;29(1):590-599. doi: 10.1080/09540105.2017.1418842 29. Agricultural consumption of pesticides worldwide from 1990 to 2021. Statista. https://www.statista.com/statistics/ 1263077/global-pesticide-agricultural-use/. Published July 2023. Accessed April 02, 2024. 30. Aga DS, Thurman EM. Environmental immunoassays: Alternative techniques for soil and water analysis. In: Aga DS, Thurman EM, eds. Immunochemical Technology for Environmental Applications. ACS Sym Ser;1997:1-20. doi: 10.1021/bk- 1997-0657.ch001 31. Jaria G, Calisto V, Otero M, Esteves VI. Monitoring pharmaceuticals in the aquatic environment using enzyme-linked immunosorbent assay (ELISA)—a practical overview. Anal Bioanal Chem. 2020;412(17):3983-4008. doi: 10.1007/s00216- 020-02509-8 32. Khan AH, Barros R. Pharmaceuticals in water: Risks to aquatic life and remediation strategies. Hydrobiology. 2023;2(2):395-409. doi: 10.3390/hydrobiology2020026 About the author: Kate Harrison is a senior science writer and is responsible for the creation of custom-written projects. She holds a PhD in virology from the University of Edinburgh. Before working at Technology Networks, she was involved in developing vaccines for neglected tropical diseases and held a lectureship position teaching immunology.