Immunoassay or molecular diagnostic? Context is everything

Immunoassays and molecular diagnostic tests are invaluable tools in the fields of oncology and infectious disease diagnostics. However, commercial and marketing leads must still make the most appropriate choice of testing technology and decide which platforms they will be developed on to ensure their testing technology has the broadest reach. Several factors should be considered before selecting the final diagnostic approach beginning with the intended clinical use and test performance (sensitivity, specificity, precision), alongside the relative importance of speed, cost, resource availability and throughput. When choosing between developing a molecular-based assay or an immunoassay, the clinical context of the test is paramount – as is consideration of world market trends.

Whatever your diagnostic test, SEKISUI Diagnostics is ideally placed to support your R&D needs. Our products are optimized to support both a wide range of immunoassay applications as well as the development of molecular diagnostic tests.

Diagnostic test performance in both the infectious disease and oncology settings would ideally prioritize sensitivity and specificity. However, these may come at the cost of speed and throughput, and in the context of a pandemic or any highly communicable disease, the need for speed is often paramount. These seemingly conflicting demands can be reconciled by the appropriate choice of a molecular- or immune-based assay. In general, molecular assays offer the sensitivity and specificity needed for accurate and reliable mass testing at the expense of speed. However, point-of-care (POC) molecular assays can offer higher sensitivity and specificity without compromising on speed, generating results in 10-30 minutes. Meanwhile, POC immunoassays typically offer speed with reduced sensitivity, as demonstrated by recent rapid antigen tests; however, those compatible with high throughput autoanalyzer platforms can be as sensitive as molecular assays. The two approaches are often compensatory in the infectious disease setting. With the focus on speed, immunoassays are the intuitive option for diagnostic purposes – yet there remains a space for molecular diagnostics and it is important to note that significant advances are being made in POC molecular tests to provide faster result times.

Identifying infectious diseases: putting assay choice into context

Immunoassays are the traditional gold standard in infectious disease diagnostics, detecting microbial antigens or pathogen-specific antibodies in crude samples such as blood, saliva, or nasal swabs, which saves significant time compared to culturing approaches, a critical bottleneck for treating patients with severe disease.

Infectious disease encompasses the largest application served by molecular assays, with total procedural volume advancing 5.8% per year and projected to reach 436.6 million in 2025

The most commonly used immunoassays for infectious disease serological testing include enzyme-linked immunosorbent assays (ELISAs), lateral flow immunoassays (LFIAs), and chemiluminescent immunoassays (CLIAs). LIFAs are a particularly attractive option, as they require less operator skill and have potential use in point-of-care settings. Indeed, the recent role of LIFAs have become pronounced in the diagnosis of upper respiratory infections and sexually transmitted infections where accuracy and immediacy of the result for subsequent prescribing is of the essence. For screening programs (e.g. fecal occult blood) where higher throughput is required and the lack of an immediate result doesn’t pose a significant risk to patient wellbeing, central lab tests are typically employed – with a 3–4 day turnaround. Further benefits to centralized testing include accuracy, as highly-trained staff run tests in a controlled environment where the performance of distribution analysis and repeatability can be ensured – alongside processing significant sample quantities with great efficiency and with a high level of automation.

On the other side of the coin, molecular diagnostics is instrumental in scenarios where time is of the essence; a requirement emphasized by the ongoing pandemic. Infectious disease encompasses the largest application served by molecular assays, with total procedural volume advancing 5.8% per year and projected to reach 436.6 million in 2025 – a rate fueled by efforts to reduce antibiotic overuse, increasing rates of healthcare-acquired infections (HAIs), continuing emergence of disease-resistant microbial strains and the periodic emergence of new pathogenic threats. HAIs alone will see a 6.3% per year increase in the number of total procedures conducted, reaching 99.1 million in 2025, and a projected 92.6 million molecular assay procedures conducted worldwide in 2025 will involve the diagnosis and measurement of hepatitis, mostly involving the hepatitis B virus (HBV) and hepatitis C virus (HCV).

As the main counterpart to immunoassays, polymerase chain reaction (PCR) tests detect single or multiple (multiplex) target amplicons. In the molecular assay space, 80% of commercially-produced molecular assays are based on PCR with real-time PCR expanding molecular testing capabilities. Providing quantitative results through amplification-generated fluorescence or other optically detectable changes in real-time accelerates the testing process, while minimizing procedural time and enhancing sensitivity. Other variations of PCR succeed in minimizing time to result – and possess an environmental benefit, eliminating energy-intensive thermocycling. These encompass isothermal amplification methods, e.g. helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), nicking enzyme amplification reaction (NEAR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification and transcription-mediated amplification (TMA). Together with speed and low-energy advantages, simpler workflows and applicability to sample-to-answer platforms make them well-suited to decentralized and POC molecular testing – a salient requirement of the COVID-19 pandemic where adequate access to timely laboratory services created substantial barriers to adequate management. Meanwhile, a molecular POC test for Strep A comes with the advantage of not requiring additional confirmatory testing following a negative result – unlike immunoassays. Within the diagnostics of respiratory disease, molecular diagnostics gain the upper hand; viruses associated with influenza including pneumonia, and bacterial infections including tuberculosis and streptococcus cannot be differentiated by immunoassays. Therefore, molecular diagnostics is confirmatory – and its role expected to grow, reaching 36.7 million in volume by 2025 globally.

Representing the future of molecular diagnostics, next-generation sequencing (NGS) is a recent, ground-breaking innovation that combines large-scale parallel sequencing, high throughput and economy of operation. The approach uses miniaturized platforms in parallel to process up to 600 million short reads of DNA per 10-hour run. NGS assays relying on amplicon sequencing are instrumental in the management of several viral infections, including identifying drug-resistant mutations in human immunodeficiency virus (HIV) and cytomegalovirus (CMV). Studies have also revealed a potential role in the identification of suspected viral pathogens not detected by conventional diagnostic methods.

Relative to their immunoassay counterparts, these sequence-based approaches can report on additional clinical parameters beyond pathogen presence. For example, nucleic-acid tests against several viruses (HIV, HBV and HCV) can provide quantitative information on pathogen burden and treatment efficacy, while tests targeting specific regions of the Mycobacterium tuberculosis genome can simultaneously report on tuberculosis (TB) infection and the presence of key drug resistance mutations. The growing availability of new antiviral therapies directed at various pathogenic genotypes has proven to be 90% effective in curing the disease – and this has seen a concurrent rapid growth in the use of molecular testing in HCV detection, which are instrumental in reducing its threat of mortality.

In this setting, however, PCR has a fundamental flaw – the inability to differentiate between DNA signals from pathogenic and non-pathogenic viruses, live bacteria, dead bacteria or extracellular DNA. Analysis based on DNA signals alone results in an increased number of false-positive results or an overestimation of bacterial numbers. Outside of diagnostic purposes, PCR may complicate disease management; failing to detect the impact of antimicrobial agents on viral or bacterial populations.

When deliberating between these two methods, it is important to have a comprehensive understanding of the advantages, disadvantages, and precautions for interpretation (Table 1):

Molecular diagnostics in oncology; is there a role for immunoassays?

While immunoassays and molecular diagnostics are frequently compensatory in the identification of infectious diseases, the reliance on speed in oncology places molecular profiling as the standard practice for most patients with advanced disease. This technique replaces the historical treatment paradigm of prescribing chemotherapy based on the tumor’s organ of origin, histology and stage.

Excluding tissue-based histology assays, molecular cancer tests are projected to generate a total global procedural volume of 94.7 million in 2025, up 11.9% annually from 2020. This projected growth is a consequence of advances in liquid

biopsy and related technologies, alongside the ever-growing demand for the development of personalized medicine regimens for cancer patients. As molecular profiling evolves, so too has the focus ­– from single, predictive, disease-specific tests to broader panel testing that measure genomic changes. These serve as biomarkers of both response prediction and patient prognosis, and can be used for:

• Cancer screening in high-risk populations
• Early diagnosis of cancer and monitoring therapeutic response
• Prediction of metastasis and drug resistance following therapy
• Detection of minimal residual disease
• Detection of new driver mutations
• Assessment of tumor heterogeneity to guide personalized treatment decisions

The preferred target of molecular approaches are liquid biopsy targets including cell-free DNA and circulating tumor DNA (ctDNA). These blood-based liquid biopsies, which have been on the market for several years, have recently carved out a centralized role in cancer detection, characterization and staging. At present, there are several commercial tests based on markers for hematological cancer or cancer-related genomic DNA markers. These include JAK2, BCR-ABL and PML-RARA gene mutation and fusion detection kits and quantitative PCR kits for transcript-based disease monitoring. In the next several years, the liquid biopsy market is expected to be driven primarily by tests that detect mutations significant to targeted therapy selection. This possibility is driven by the utility of ctDNA, a molecule that can be used beyond the detection of point mutations and disease monitoring – encompassing the study of copy number aberrations, chromosomal rearrangements, fragmentation, methylation, and gene expression – all expanding our understanding of an individual’s cancer. This includes the potential for earlier detection through identification of early-stage biomarkers, the selection of, and resistance to, treatment, and the detection of minimal residual disease. Overall, a paradigm shift from direct tumor tissue biopsy to the liquid variety is underway, and it involves a range of PCR, quantitative PCR (qPCR), microarray, sequence-based techniques; in 2020/21 several products launched following FDA approval, or received a CE-mark.

The rapid uptake of NGS is expected to have a significant influence on cancer diagnostics and cancer management, but it is plagued by a major limitation; determining the threshold for ctDNA quantities between early-onset cancer requiring intervention and non-lethal cancers or malignant cells that can be managed by the patient’s immune system. Another significant sticking point is the poor sensitivity of ctDNA detection – this requires significant improvement before the widespread use of NGS is possible. Because of these limitations, present uptake is incremental; integration of EGFR mutation and BRCA1/2 assays took several years. This has resulted in a subsequent delay in clinical validation of molecular cancer diagnostics methods.

Molecular cancer tests are projected to generate a total global procedural volume of 94.7 million in 2025, up 11.9% annually from 2020.

So, does the continued rise of molecular diagnostics spell the end for immunoassays in the diagnosis and analysis of cancer? Perhaps not; immunoassays have been developed that overcome limitations in sensitivity and robustness to assess a series of tumor biomarkers that help diagnose, as well as predict and monitor treatment response in a similar manner to molecular-based methodologies. Projected future estimates suggest that they will account for nearly $24.6 billion in 2025, an increase in 5.1% per year from 2020 – and over the past several years, analytes for markers have been adapted to high throughput in vitro diagnostic (IVD) systems. In the cancer setting, this includes HE4 cancer markers and procalcitonin. Serum and tissue biomarkers can be detected by various applied immunoassay techniques including ELISA, electrochemiluminescence (ECL) and CLIA. These methods can help us understand a patient’s progress on therapy, while strides in proteomics technology have facilitated an explosion in biomarker research, uncovering new therapeutic targets for cancer. One such development is ELISA-based bulk sequencing proteomics toolkits, that enable the detection of autoantibodies against tumor proteins in serum that can represent novel biomarkers.

SEKISUI Diagnostics has developed Smartbond Streptavidin, a specialized streptavidin for the enhancement of signal generation, and offers Smartbond Streptavidin Magnetic Beads, which feature our core Smartbond Streptavidin covalently coupled to superparamagnetic beads via proprietary linkers. These are surface-modified, optimized, and validated for use with high-throughput random access magnetic platform analyzers in the application of immunodiagnostics.