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ELISA holds promising results in early disease diagnosis

To date, numerous ELISA formats have been demonstrated with considerable improvements in sensitivity, duration, procedure, cost-effectiveness, simplicity, and automation and the trend will continue owing to emerging advances in nano- and micromaterials.

As an integral part of healthcare, in vitro diagnostics (IVD) enables the healthcare professionals to screen, diagnose, treat, monitor, and manage diseases. The highly sensitive and specific detection of biomarkers at an early stage of a disease is an essential requirement in healthcare as it enables the healthcare professionals to precisely diagnose patient’s condition and start an effective treatment regimen at the earliest. The enzyme-linked immunosorbent assay (ELISA) has been one of the most widely used immunodiagnostic format in healthcare, industrial, and bioanalytical settings. As ELISAs determine most of the biomarkers, they form an indispensable part of clinical decision-making and monitoring the efficacy of the treatment regimen. Their deployment has contributed to considerable advances in healthcare in terms of sustainable and better health outcomes, detection of diseases at early stages, lower healthcare costs, and advanced analytical capabilities.

Cancer is one of the major causes of mortality in the world. The burden of cancer in India is also increasing day by day. According to projections of the Indian Council of Medical Research (ICMR), the total number of new cancer cases is expected to touch 17.3 lakh by 2020 and the number of deaths due to cancer is estimated to likely reach 8.8 lakh cases by 2020. Its diagnosis is advised in early stages, when useful treatment is possible, in order to achieve longer survival of cancer patients. This clearly indicates the need for greater awareness and early detection. To achieve early stage diagnosis, researchers have proposed the use of proteins and oligonucleotides (biomarkers) released in the body during the early stages of cancer, which are not present in the same concentrations in healthy individuals. It is desired to detect these biomarkers in a non-invasive or minimally invasive manner with high selectivity, sensitively, and free from false positives and false negatives. ELISA is one of the commonly employed methods of cancer detection and also utilizes biomarkers for analysis and their estimated levels are related to cancer stage and inform cancer therapy.

The last few decades have witnessed the development of manual and automated ELISA formats. Initially, the manual ELISA was the predominant format when the field of clinical diagnostics was less advanced. Nevertheless, ELISA is still deployed in resource-limited settings. The trend has now shifted toward automated ELISAs, which can be performed in central laboratories using clinical analyzers. Indeed, a wide range of analyzers have become available to suit the customized needs and requirements of various healthcare settings. To date, numerous ELISA formats have been demonstrated with considerable improvements in sensitivity, duration, procedure, cost-effectiveness, simplicity, and automation. Emerging nano- and micromaterials further increase the sensitivity of ELISA while new detection concepts result in an analytically superior ELISA for multiplex detection of biomarkers.

The recent trend is also strongly inclined toward point-of-care (PoC) testing using advanced lab-on-a-chip (LOC) formats, rapid ELISA strategies, and portable, low-cost readout devices, notably smartphone readers. The advent of microfluidic protocols and LOC platforms has contributed to rapid and fully automated PoC ELISAs for remote and decentralized settings. Similarly, several prospective low-cost formats, mainly paper-based ELISAs, have also been advocated in the developing nations with limited resources. The trend toward SP-based ELISAs using LOC platforms and SP readers are paving the way to prospective next-generation ELISA formats equipped with mobile healthcare, telemedicine, and personalized healthcare monitoring and management tools. The advances in complementary technologies, such as multiplex detection, microfabrication techniques, and clinical scoring, are further paving the way to low-cost and analytically superior ELISAs.

Global market
The global ELISA market was valued at USD 1664.2 million in 2018 and is expected to witness a robust CAGR of 5.1 percent from 2017 – 2025, according to Coherent Market Insights. Increasing incidence of infectious diseases coupled with increasing demand for cost-effective diagnostic tools are expected to drive growth of the ELISA market. The increasing incidence of cancer is one of the key trends that will gain traction in the market during the next few years. According to the Centers of Disease Control and Prevention (CDC), 2015, the number of visits to physician offices for infectious and parasitic diseases in the US was 16.8 million. Furthermore, vector borne diseases comprise a major percentage of infectious diseases that affects a wide population. According to the World Health Organization (WHO), 2017, annually vector borne diseases accounts for over 17 percent of all infectious diseases causing over 700,000 deaths worldwide. Dengue is most commonly occurring vector borne disease with around 96 million new cases diagnosed annually.<

Moreover, advancements in the technology of ELISA are also fueling the market growth. For instance, in August 2017, researchers at University of Illinois, Urbana Campaign, developed a portable spectral analyzer based on ELISA assay that enables a smartphone to perform lab grade medical diagnostic tests, which usually requires expensive instruments. This test includes detection and measurement of variety of proteins and antibodies in blood, urine, and saliva samples that is commonly used for wide range of clinical diagnostic tests. Apart from the conventional medical applications such as disease diagnosis and transplantation, ELISA can also be used in the field of toxicology for determination of certain classes of drugs that is expected to aid the market growth in near future. However, cross reactivity leading to false positive and false negative results exhibited by the tests leading to incorrect diagnosis can obstruct the market growth. For instance, according to study report published by American Medical Association, the preliminary health department data identified 32 pregnant women with HIV positive through ELISA assays. However, after a confirmatory test, 17 of these women were confirmed to be negative for HIV virus.

In terms of region, North America dominated the global market. This is attributed to development of innovative products by manufacturers in the region. In January 2018, Eagle Biosciences, Inc. introduced a novel intact FGF23 ELISA kit assay, which is a 96 well Sandwich ELISA assay. It measures the full length active form of Human Intact FGF23 used for the detection of mineral bone disorder, chronic kidney disease, tumor induced osteomalacia, and hyperphosphatemia. Moreover, various private and public sectors carrying out research and development in immunoassay-based clinical diagnostics are commercializing their research inventions by collaborating with existing market players to introduce new products in the market. For instance, in March 2018, BARD1 Life Sciences, US-based science research company commercialized its BARD1-Ovarian blood based ELISA test to identify ovarian cancer in early stages. Furthermore, Asia-Pacific is projected to be the fastest growing region in ELISA market, owing to geographical expansion of regional players along with introduction of novel enzyme-based test kits at affordable prices.

The key players are developing novel, innovative, and smart products to maintain leading position in the global market. In January 2017 Illumina, Inc. and Bio-Rad Laboratories, Inc. introduced the Illumina Bio-Rad Single-Cell Sequencing Solution at the J.P. Morgan Healthcare Conference, San Francisco. The comprehensive solution is the first next-generation sequencing (NGS) workflow for single-cell analysis, providing researchers the ability to investigate the coordinated contribution of individual cells in tissue function, disease progression, and therapeutic responses.

Technological advances
The ELISA turnaround time has significantly decreased from 24 h to a few hours. Indeed, most of the current ELISAs being used in clinical diagnostics have a sample-to-answer time of less than an hour. Of notice is the microfluidic LOC formats with immunoassay (IA) duration of less than 30 min. Furthermore, the costs of analysis and the need for highly skilled analysts have decreased significantly during the last few decades. The current ELISA formats employ a minimal number of IA steps, an easy-to-use procedure, reduced reagent volumes, and an automated operational procedure. The readout devices and instruments required for performing ELISA have witnessed an evolving trend over the years. Most initial ELISA formats were bulky, expensive, and semi-automated instruments, including microplate readers. However, a wide range of fully automated and portable clinical analyzers have been developed recently. Most of the fully automated PoC analyzers in clinical diagnostics are integrated with advanced technologies, features, and information technology tools, to provide a complete system for healthcare needs. The market has also witnessed the emergence of portable immunoanalytical devices that enable a fully automated ELISA on LOC platforms and integrated readers. The most recent development of smartphone readers for ELISA can read out detection signals generated in ELISAs such as colorimetric, spectroscopic, chemiluminescence, fluorescence, surface plasmon resonance, Mie scattering, and electrochemical.

Smartphone camera-based analysis of ELISA using artificial neural network – It is an inexpensive, portable, and easily accessible technology for the quantitative analysis of medical samples for the detection of disease in the ELISA. The technology follows a PoC diagnostic model and attends to the several challenges in healthcare system in rural settings. The technology will alleviate the inconveniences faced by the average citizen of a country with insufficient resources to implement an affordable healthcare administration for its entire population. A smartphone is used to procure images of an ELISA containing para-nitrophenol samples which is then fed into a machine learning algorithm, specifically artificial neural network. The introduction of two relatively new technologies in medical aid – the smartphone and machine learning not only reduces cost and time of detection, but also presents ample possibility for further development. The predictions result in highly accurate diagnostic labels.

High-throughput microarray-based ELISA – A new generation biochip is capable of supporting high-throughput, multiplexed ELISAs. These biochips consist of an optically flat, glass plate containing 96 wells formed by an enclosing hydrophobic Teflon mask. The footprint dimensions of each well and the plate precisely match those of a standard microplate. Arrays are formed by a custom, continuous flow, capillary-based print head attached to a precise, high-speed, robot. The array printing capacity of a single robot exceeds 20000 arrays per day. Arrays are quantitatively imaged using a custom, high-resolution, scanning charge-coupled device (CCD) detector with an imaging throughput of 96 arrays every 30s. Using this new technology, arrayed antigens are individually and collectively detected using standard ELISA techniques. Because of the open microarray architecture, the 96-well microarray format is compatible with automated robotic systems and supports a low-cost, highly parallel assay format. Future applications of this new high-throughput screening format include direct cellular protein expression profiling, multiplexed assays for detection of infectious agents and cancer diagnostics.

Electrochemical ELISA-based systems – To shorten the time required and to improve the response and characteristics of traditional optical ELISA, various researchers have proposed newer technologies via development of improved sensor surfaces and detection probes. Also, to reduce cost, easier testing, and shorter measurement time, sandwich-based electrochemical ELISAs have been proposed, which utilize the specificity of optical ELISA and advantages of electrochemical measurements to achieve better response and characteristics for desired analyte estimation. Though at present there are not many commercial electrochemical ELISA based immunosensors, the required instrumentation is available and as such, there is huge potential for such sensors and their commercialization.

As standard ELISA can reliably and routinely be used for diagnosis, technological advances have focused on simpler or more cost-effective approaches that can be used as PoC tests. As part of the movement to more broadly define health, recent technological developments have enabled the assessment of large numbers of antigen-specific antibodies; for example against viruses, or autoantibodies reactive to self-proteins. One such approach utilizes immunoprecipitation allied to massively parallel DNA sequencing of a bacteriophage library displaying proteome-wide peptides from all human viruses. This results in the screening of 108 antibody–peptide interactions, offering great potential for population monitoring of environmental or pathogenic exposures by providing a comprehensive view of previous viral infections. It is now also possible to simultaneously monitor multiple protein concentrations in biological fluids. Notably, multiplex approaches such as multi-analyte profiling, PCR-based proximity ligation and extension assays, and aptamer-based arrays are used to simultaneously measure hundreds of analytes, providing a reproducible approach to the profiling of immune responses at the protein level. With the introduction of high-dimensional biomarker assays come new challenges such as ensuring that discovery studies are statistically powered to extract biologically meaningful signals and providing clinical validation of the findings in disease settings.

Road ahead
Favored for its simplicity, specificity, robustness, and cost effectiveness, ELISA is a useful and common technique for many laboratories. ELISA kits for a wide range of analytes can be purchased commercially, although these tend to be expensive and money is often tight, particularly in a research setting. Despite the ease of use of the technique as a whole, there are still a number of factors to be considered in order to design robust and reliable assays. For instance, they are usually limited to detection in the ng/mL range and can sometimes show limited dynamic range, high background, or matrix effects. Although more sensitive fluorogenic and chemiluminescent substrates are available for peroxidase-based assays, these substrates require equipment that is less common to laboratories, and can be less robust due to bleaching or decay.

Over the years many changes to the basic format have resulted in assay improvements, but some of the most recent ones look set to take the traditional ELISA to new levels. These include many vendors now addressing the automation of ELISA assays. Some automated platforms include new or alternative ELISA formats typically with miniaturized or microfluidic components. It is envisioned that further advancements in nano- and bio-technology along with chemistry, material science, physics, and electronics will pave the way to solve the limitations and result in larger acceptance of these devises in clinical practice.

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