Ongoing global efforts are working to communicate and facilitate new diagnostic assay development and worldwide test kit delivery. It is likely that the COVID-19 diagnostic market will continue to thrive well into the future.
Diagnostic technology is rapidly evolving, and over the last decade, substantial progress has been made even for the identification of antibodies, increasingly approaching this type of diagnostic to that of automated clinical chemistry laboratory.
Numerous analytical methods have been proposed for autoantibody detection. Of these, immunoassay has undergone important and radical changes in recent years owing to continuous technological development, which has been spurred on by an increased demand for services analogous to those already occurring in other sectors of modern laboratory diagnostics. Fundamentally, immunoassay evolution corresponds to the evolution of immunoassay labelling technology. Radioimmunoassay (RIA) was the first immunoassay to be developed, and could be considered the forefather of the modern immunoassay.
COVID-19 and immunoassays
A number of immunoassays have received approval for diagnostic use for COVID-19, utilizing formats like ELISA and lateral flow.
ELISA. The enzyme-linked immunosorbent assay (ELISA) uses enzyme-substrate reactions to produce measurable signals proportional to the concentration of the target analyte. A defining feature of ELISA is the immobilization of the target antigen upon a solid surface, usually in microplate wells. This can occur by coating the surface with specific antibodies. If the targets are immunoglobulins, the surface is coated with recombinant proteins instead. The general procedure follows in a set of sequential steps: sample application and analyte binding, addition of enzymes conjugated to specific antibodies (usually alkaline phosphatase or horseradish peroxidase), incubation with the chemical substrate, and detection on a microplate reader. ELISA kits are sensitive, quantitative tests that can be used to measure a variety of analyte types within complex samples.
Lateral flow immunoassays. In this chromatography type of assay, capture antibodies are immobilized onto the surface of a support membrane (in antibody tests, recombinant proteins are immobilized). When the sample is applied, its various components will travel across the membrane. The target analytes, however, will become trapped in the designated region, producing a color observable in plain sight or with a detection instrument. Compared to ELISA, lateral-flow assays are generally qualitative, rather than quantitative. However, the relative simplicity of the procedure has led to the widespread development of various point-of-care and direct-to-consumer devices.
Immunoassay tools. Many tools are available that make it possible to run immunoassay experiments or even develop new, customized tests. At the heart of the assay are specific SARS-CoV-2 antibodies for targeting various antigens. Recombinant SARS-CoV-2 proteins can be used to develop controls or targets or to serve as antigens for immunoglobulins. Plate readers, essential in the analysis of microplate-based assays, come in many models with an assortment of features.
While the past few months have witnessed rapid progress in diagnostic kit development for COVID-19, the race continues to develop even more efficient laboratory techniques and cost-effective, point-of-care test kits that can be deployed in mass quantities.
Contrasting roles in diagnostics
There are several fundamental differences between molecular tests and immunoassays. Molecular assays are sensitive to viral nucleic acids, while immunoassays detect proteins and antigens. The experimental workflows also differ. Molecular tests generally require more steps and instruments, thus taking more time to complete. PCR amplification, which involves repetitive temperature cycling, is a rate-limiting step that takes roughly 1–2 hours to complete. Including sample prep, RNA extraction, and data analysis, a full manual procedure can take several hours.
In contrast, immunoassay procedures are relatively simpler and require shorter incubations. ELISAs can take a few hours while some rapid disposable tests can produce results within the hour. Notably, both ELISAs and real-time RT-PCR assays in multi-well formats are scalable, and can undergo automated processes, greatly increasing throughput.
As for diagnostic roles, only molecular tests are currently intended to diagnose acute infection, as these recognize viral components directly from respiratory samples. On the other hand, antibody tests inform whether the body has responded to an infection but not when the infection has taken place. While these tests cannot diagnose infected individuals, they can be used to conduct serosurveys – studies to determine which people have developed antibodies against SARS-CoV-2. Serosurveys are important in that they can be used to determine sources of convalescent plasma as well as to track the spread of the disease within a population. The growing development, centered around these two contrasting test methods, presents a hopeful avenue in mitigating the COVID-19 pandemic.
The Indian immunochemistry instruments and reagents market, in 2019, is estimated at `1912 crore, with reagents dominating the market, with a 90-percent share at `1721 crore. Immunochemistry has witnessed extraordinary advancement in every aspect of basic and clinical research, and this is owing to technological advances, which enable quantitatively and comprehensively measuring immune response to genetic or environmental perturbations. Immunochemistry analyzers are becoming more and more popular these days as they have proved to be very effective tools to diagnose cancer, hepatitis, illegal drugs, fertility problems, infectious diseases, and many chronic diseases.
Nowadays, chemiluminescence immunoassay (CLIA) systems are gaining momentum; this in turn results in a decline in ELISA tests. RIA and Fluorescence Immunoassay (FIA) are age-old procedures, which over time have been replaced by ELISA. Rapid tests can reveal the test results in less than 30 minutes; hence these are preferred for screening. ELISA, on the other hand, continues to be trusted as a gold standard, owing to extremely high sensitivity, specificity, precision, and throughput.
The last two decades have seen tremendous automation in ELISA technology. From a compact bench-top micro-strip processor, ELISA processors are now available with six plates, allowing reporting of over 500 test results at one go. Automated processors are equipped with robotic probes and dedicated workstations, which drastically improve ELISA workflows.
These fully automated processors are complete walkaway systems that minimize manual errors, while improving the accuracy and turnaround time for critical tests. Usually being open systems, the limitation of ELISA kits from a single source can also be ruled out.
The diagnostic landscape in India is largely varied, with the chain labs concentrated in the big metros, and the rural areas often devoid of even the most basic facilities for routine testing. Moreover, public hospitals in metros are often overloaded. In either of these scenarios, ELISA is often the preferred choice for being affordable and reliable.
Immunochemistry plays an important role in development of biomarkers too as growth in incidence and prevalence of infectious diseases, cancer, gastrointestinal and cardiovascular diseases, endocrine diseases, and many other chronic diseases.
The evolution of diagnostic and prognostic markers is significantly changing the clinical practice, and this can be demonstrated by the use of immunochemistry. The demand for immunochemistry has increased owing to overall increase in healthcare expenditure, decreasing mortality, increased awareness, and lifestyle-related diseases.
Immunochemistry is the backbone of diagnosis and prognosis of diseases in healthcare system, and is thus the need of today for accurate and precise measurement of the sensitive parameters, and for their rapid diagnosis.
An extensive menu of immunoassay tests required
With more and more requests pouring in from clinicians, particularly endocrinologists, medical diagnostic laboratories have to equip themselves to provide extensive menu of immunoassay tests. This also poses a challenge on how to select an immunoassay equipment.
Test menu. It is important to understand the test menu the lab has to offer. Although majority of the tests are available with all leading manufacturers, it is important to check that the lab’s requirement is fulfilled. Even if one test is not available, the lab may have to procure another equipment to carry out the missing test.
Hence, checking test menu is very important. One also has to check additional tests available on the equipment so that the facility is available for future needs too.
Throughput. Due to large variation in assay timings for different tests, throughput of immunoassay depends on the combination of assays the equipment has to perform at any given time. It is never a fixed value and is always stated as up to. Hence, it is advised to choose equipment that has throughput higher than the actual laboratory requirement. This also takes care of the future increase in workload.
Proficiency testing performance. There is tremendous variation in test results amongst different immunoassay equipment. What is reported normal by one equipment may be reported as hypo or hyper by other equipment. Since there are challenges of harmonization of results across all immunoassay platforms, it is advised to study proficiency testing performance of various equipment for different analytes from reputed PT providers.
This gives an idea on total number of labs using similar equipment and their overall performance. Also, PT providers rank top 8–10 equipment’s performance for each analyte. Based on this study, one can find out the most popular equipment, the equipment giving consistent performance, and equipment-wise accuracy for every single analyte. This exercise can be done by studying at least past one-year data of popular PT providers.
Software for prenatal screening. If the lab is planning to provide prenatal screening tests, then it is important to know which software is available for reporting. Many referring obstetricians are inclined toward a particular software. They are comfortable with a specific software report format. Choosing any other software increases the risk of losing business of prenatal screening tests.
Others. Other points like initial and recurring cost, cost per reportable test, service back-up, long-term equipment performance, support in terms of consumables, reagents, user-friendliness, etc., remain the same as that of any other laboratory equipment, and needs to be looked into before finalizing the equipment.
CLIA technology has just begun to evolve
Regardless of its current optimal analytical performance, CLIA technology is destined for further development. The new flow-injection chemiluminescent immunoassay (FI-CLIA) technology, which is based on the fast injection of micro-bubbles into the reaction system with the aim of ensuring a more efficient reagent mixture and of reducing incubation times and increasing temperature control, is able to improve the immunoreaction kinetics and, therefore, significantly reduce analysis time.
Current chemiluminescent immunoassay consists of discrete tests, i.e., measures one autoantibody at a time. However, the need is emerging for multiparametric tests that can identify all the components of a complex immunological picture in a single analytical step, efficiently and at reasonable cost. Use of the two-dimensional resolution for CL multiplex immunoassay could open doors for the setting up of multiparametric CLIA tests. The technique is based on a multichannel sampling strategy, in combination with the use of various enzyme labels.
After a brief period of batch incubation, the free conjugated enzymes separate from those bound to immunocomplexes and captured by magnetic microparticles; at the same time, activation of the magnet leads to the arrest of the immunological reaction. Subsequently, application of the channel-resolved approach makes it possible to record signals coming from various parallel measuring channels, and thus enables the sequential determination of multiple analytes.
Multianalyte assay has also been realized with the zone-resolved strategy, a method that implements the array – different antigens are immobilized in different positions on the solid phase in such a way that the individual immunoreactions can occur simultaneously and be distinctly individuated by array detectors. Use of specific CCD (charge-coupled device) equipment has accelerated the development of chemiluminescent imaging techniques, and is making CLIA array more convenient compared to those employing alternative labels.
Finally, applying ultrasensitive chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) technology further increases the analytic sensitivity of the CLIA method. In this non-competitive and direct-type immunoassay, where the solid phase is made up of magnetic beads, gold nanoparticles with double labelling are used.
One label is a monoclonal antibody specific for the analyte and the other label is horseradish peroxidase, thus amplifying the luminescent signal derived from the immunoreaction and the associated enzymatic reaction in an exponential manner.
In conclusion, the evolutionary process of the CLIA method is, in all likelihood, merely beginning. In the coming years, new and more efficient analytical methods based on the principle of chemiluminescence will be introduced into autoimmune diagnostics, at steadily reduced cost.
This transformation will align antibody diagnostics with already consolidated biochemistry and immunoassay methods, with noticeable advantages in terms of both diagnostic accuracy and expediency, to great clinical benefit.
COVID-19 finds immunoassays valuable
The urgent need for accurate and rapid diagnosis of SARS-CoV-2 infection remains critical as global healthcare systems continue to operate during the course of the COVID-19 pandemic. In particular, serological and immunological testing of infected asymptomatic and symptomatic individuals, and their close contacts, is expected to be in high demand.
In addition to its role, complementary to molecular genetic testing to confirm suspected cases, this type of testing would provide valuable information about the course and degree of immune response as well as the durability of immunity in both infected individuals and participants in vaccine clinical trials. The results from these tests may assist epidemiological assessment, and can be used to manage the return to normal activities.
However, many questions regarding serological tests remain to be addressed, including their degree of sensitivity and specificity. Finally, it remains to be confirmed that the presence of antibodies against SARS-CoV-2 indeed correlates with immunity to the virus.
In summary, significant progress has been made in the development of diagnostic tests despite all the remaining questions and challenges. Ongoing global efforts are working to communicate and facilitate new diagnostic assay development and worldwide test kit delivery. To promote more accurate and faster diagnostic solutions, a number of organizations are supporting these efforts by inviting assay developers to submit their test products for independent evaluation or by providing huge investments for greater collaboration.
As similar initiatives and knowledge sharing become available, including collaborative technological advancements, it is likely that the COVID-19 diagnostic market will continue to thrive well into the future.