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Unmasking Multiplexing Complexities: Overcoming Challenges for Respiratory Diagnostics
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In today's world, shaped by the aftermath of the COVID-19 pandemic, there is no doubt that lower respiratory tract infections (LRTIs) pose a substantial global health and economic threat. They are a significant cause of morbidity and mortality worldwide, particularly during the winter months due to seasonal respiratory epidemics. With the changing genetic landscape of virus strains post-COVID 1 , the critical role of diagnostics in managing patient outcomes and optimizing healthcare resources is amplified. Traditionally, the diagnostic process has revolved around identifying single targets. The newer multiplexing approach, in contrast, allows for simultaneous identification of multiple pathogens, enabling healthcare providers rapid insight and a comprehensive diagnostic profile. Overall, multiplexing has proven to accelerate diagnoses, create cost efficiencies, optimize resources, and enhance patient care.
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Benefits to Multiplex Testing: Molecular & Immunoassay-Based Techniques
Multiplex testing and syndromic panels save significant time and resources by testing for multiple diseases using a single sample. Flu, COVID-19, RSV and other respiratory diseases cause similar symptoms and differential diagnosis is not possible without a laboratory test. Combination testing provides physicians the unique opportunity to quickly and accurately diagnose a patient and provide the right treatment. This is important for respiratory infections such as COVID-19, RSV and flu, which require different treatments and put different patient groups, such as babies or the elderly, at risk from serious complications if mistreated or left untreated.
Several assay techniques are used for multiplex testing including immunoassay-based rapid antigen detection and nucleic acid amplification using molecular methods. Throughout the COVID-19 pandemic, molecular testing remained the predominant test used for diagnosis due to its high sensitivity and specificity. However, molecular tests require a laboratory to perform, involve complex logistics for sample transportation and are vulnerable to bottlenecks in machine capacities and reagent supplies, all impacting the turn-around-times which can be up to several days. In contrast, rapid antigen assays are highly
scalable, can be used at the point of care (such as a school or airport) and can provide a result in 10-15 minutes. Yet their sensitivity is lower (>80% as compared to RT-PCR) (CDC 2021) or more, they are prone to cross-reactivity, and they have a higher incidence of false positive and false negative results compared to molecular tests. Depending on the available resources, accuracy required and level of urgency, one testing method may prove to be more useful than another for a given situation.
Figure 1. Targets for Viral Detection Methods
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Multiplex Molecular Testing
Molecular multiplex assays have the ability to significantly lower the cost of testing multiple targets at one time. Compared to singleplex reactions, multiplex requires less reagents, less time, and less labor to achieve the same result. However, the performance of a molecular assay depends on its ability to accurately detect low levels of a pathogen, and in multiplex analysis, this challenge grows substantially. The overall variability in the levels of the targets can result in preferential amplification of one target over another, as well as PCR drift—stochastic variation caused by low template concentration. Variability in the physicochemical characteristics of the amplified sequences, its length, GC content, flanking regions and secondary structures also may add to the imbalance of the reaction and impact on Ct values. 1
To overcome these challenges, it is important to use a master mix optimized for multiplexing to ensure that Ct value remains the same and the assay retains its high sensitivity for each of its targets. Meridian’s Fast 1-Step RT-qPCR Mix (Catalog MDX032) is designed for multiplex RT-qPCR diagnostic tests and for high-throughput, automated platforms. The amount of polymerase, dNTP, and Mg 2+ has been optimized and the mix’s buffering capacity (to stop the pH changing) has been enhanced to provide optimal performance. Another major concern for multiplexing assays, especially ones on high-complexity platforms that involve the manipulation of amplified PCR products, is the potential for false-negative results from PCR inhibition or false-positive results from PCR amplicon carryover. Most assay protocols require DNA or RNA extraction prior to testing in order to remove PCR inhibitors found in clinical samples such as nasopharyngeal or sputum specimens. However, these purification methods are problematic, can cause sample loss, and are not completely effective at removing all inhibitors. In addition, during the height of the first wave of the COVID-19 pandemic, the huge demand for molecular testing created an RNA extraction reagent shortage. Faced with the challenge of overcoming this supply shortage, assay manufacturers looked for alternative methods that rely on direct detection, entirely avoiding the need to purify the DNA or RNA. To address these issues, Meridian focused on creating inhibitor-tolerant molecular master mixes that allow clinical crude specimens to be run directly on a PCR machine, without performing purification or extraction first. Meridian’s first direct detection mix, Inhibitor- Tolerant RT-qPCR Mix (Catalog MDX016) was designed for qualitative multiplex assays using crude lysates or inhibitor-rich samples from different sources. The mixes
can be used for direct amplification to substantially reduce steps in the assay workflow and improve assay turn-around times.
Figure 2. Schematic of direct detection from clinical sample
Compared to singleplex reactions, multiplex requires less reagents, less time, and less labor to
achieve the same result.
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POCT Molecular Assays & Multiplexing
Molecular testing methods require skilled technicians, sophisticated lab equipment, and cold shipment and storage to maintain the shelf-life of the product. Although these challenges are manageable in a centralized large laboratory, they are not suitable in the field where point of care (POCT) assays are needed.
Meridian has developed two reagent POCT solutions for molecular assays. The first is a group of lyophilization compatible mixes (Lyo-Ready ™ ) that are glycerol-free and fully optimized with a specialized blend of lyo-excipients and which only require the addition of primers and probes to complete the assay. The formulation can be used as a liquid mix for an instant multiplex RT-qPCR diagnostic test or lyophilized into cakes or beads to create an ambient temperature stable assay. The Lyo-Ready ™ 1-Step RT-qPCR Mix (Catalog MDX024) and Lyo-Ready ™ 1-Step RT-qPCR Virus Mix (Catalog MDX062) are widely used formulations in regulated assays around the world. The virus mix (Catalog MDX062) is specifically optimized for amplification of RNA or DNA viruses with a high secondary structure (reverse transcriptase remains active at 55-60°C, refer to Figure 3 for performance data).
mixes have been designed to overcome the inhibitors present in saliva/ sputum, blood, urine and stool. The new Air-Dryable ™ Direct RNA/DNA qPCR Saliva (Catalog MDX131) is a unique alternative for multiplexing flu/COVID/ RSV assays and is optimized for the sensitive detection of RNA targets from crudely processed saliva or sputum samples and only requires the addition of primers and probes to complete the assay (refer to Figure 4). The mix can be used in a liquid format or oven-dried to create a highly sensitive, ambient temperature stable, multiplex assay. Air-Dryable ™ Direct RNA/DNA qPCR Saliva (Catalog MDX131) can tolerate complex inhibitors in crude saliva such as sputum, mucin, UTM swabs. Syndromic respiratory panels have adopted this master mix to detect COVID-19, Flu, RSV, M. pneumoniae , etc), because of its sensitivity to less than 10 copies and its compatibility with liquid or dry assay formats.
MERS-CoV
Influenza A
RSV
The second POCT solution is Meridian’s new Air- Dryable ™ Direct RNA/DNA product line which combines the benefit of inhibitor tolerance with the ability to air-dry the liquid mix to create an ambient-temperature stable assay. Unlike lyophilization, air-drying is relatively quick, inexpensive, and can be performed in-house with an oven. Novel sample-specific solutions of Air-Dryable ™ Respiratory Syncytial Virus (RSV) (green), Rotavirus A (purple) and Dengue Type 2 Virus (DENV-2) (red) were amplified in a single multiplexed RT-qPCR assay using inactivated crude viral lysates and lyophilized Lyo-Ready ™ 1-Step RT-qPCR Virus Mix (MDX062). The result illustrates that Lyo-Ready ™ 1-Step RT-qPCR Virus Mix can be used in a multiplex RT-qPCR assay to detect several low-copy number RNA targets simultaneously from a single sample. Figure 3. Multiplex Analysis of DNA and RNA Viral Targets Using Lyo-Ready ™ 1-Step RT-qPCR Virus Mix
Figure 4. Respiratory Multiplex Testing Using Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131) Three respiratory pathogens, Influenza A, Middle East Respiratory syndrome coronavirus (MERS-CoV) and Respiratory Syncytial Virus (RSV) were amplified in a triplex qPCR assay in the presence of 35% Universal Transport Media (UTM) with 50% artificial sputum swab. The results illustrate that a higher performance was achieved with Air-Dryable ™ Direct RNA/DNA qPCR Saliva (blue) compared to an inhibitor-tolerant RT-qPCR mix (TaqPath ™ 1-step Multiplex Mix) from supplier T (red) and supplier Q (grey) (Ultra-Plex ™ 1-Step Tough Mix).
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Rapid Lateral Flow Assays Rapid lateral flow tests were adopted at an unprecedented scale during the COVID-19 pandemic, enabling access to testing beyond healthcare settings 3 . SARS-CoV-2 antigen rapid tests were developed within months and in 2022, the WHO ‘strongly endorsed’ COVID-19 self-testing with rapid tests 4 , putting the public at the heart of the public health response. By late 2022, more than 3 billion tests for SARS-CoV-2 had been conducted worldwide 5 .
Figure 5. Timeline of key advances in lateral flow testing.
Retrieved from: Budd, J., Miller, B.S., Weckman, N.E. et al. (2023) Lateral flow test engineering and lessons learned from COVID-19. Nat Rev Bioeng 1, 13–31. https://doi.org/10.1038/s44222-022-00007-3
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Rapid antigen assays are highly scalable, can be stored at room temperature, require minimal training, and can provide a result in less than 10-15 minutes. However, immunoassays have challenges with multiplex testing due to the overall variability in the levels of targets present in the patient sample. Compared to singleplex immunoassays, multiplex versions must have a wider dynamic range in order to detect targets that are present at radically different concentrations. Although multiplex ELISA assays are reported to maintain linearity better than singleplex ELISA assays (over three or even five orders of magnitude), a key component to an assay’s performance in the selection of antibodies. In order to detect both low and high-abundance targets, antibodies selected for a multiplex assay must be highly sensitive and they must be very specific so that the antibodies do not cross-react with each other or with other proteins in the assay mixture or patient sample. In addition, the antibody binding affinity is also a critical factor, especially in lateral flow assays where the association (k on ) and dissociation rate (k off ) can directly influence the sensitivity, specificity, and overall performance of an assay. A high antibody on-rate is desirable as the analyte has only a limited amount of time for interacting with the immobilized capture antibody as the sample diffuses along the test device. A fast on-rate and a slow off-rate will enhance the analyte binding to antibody which correlates to enhanced performance in assay sensitivity, specificity, signal intensity, dynamic range and limit of detection. 5
Flu A mAb Pairs – Nucleoprotein (NP) Capture Detection BN1072 BN1071 BN1073 BN1071 BN1074 BN1071 C01736M C01731M C01760M C01731M Antigens BN1119 – Recombinant Ag to NP Flu B mAb Pairs – Nucleoprotein (NP) Capture Detection BN1075 BN1076 C01744M C01747M C01744M C01897M C01746M C01747M C01746M C01897M C01897M C01747M Antigens BN1120 – Recombinant Ag to NP RSV mAb Pairs Capture Detection C01770M C01773M C65063M C65065M C01769M C01773M pAb – Goat anti RSV B65860G SARS-CoV-2 mAb to SARS-CoV-2 Nucleocapsid Capture Detection 9547 9548 ^ BN1060 BN1061 * mAb to SARS-CoV-2 Nucleocapsid Capture Detection C01769M C01773M C65063M C65065M Antigens BN1006 | BN1011 ^Pair detects Omicron down to 1.31 x 10 2 PFU/mL, and 1.88 x 10 2 PFU/mL for other variants. *New Product Meridian Top Performing Pairs.
Antibody
K D (M)
K on (M
-1 s -1 )
k
off (s
-1 )
X 2
R 2
MAb to Flu A (BN1069)
2.60x10 -11
4.80x10 5
1.25x10 -5
0.0110 0.9992
MAb to Flu A (BN1070)
6.10x10 -11
6.90x10 5
4.21x10 -5
0.0074
0.9979
MAb to Flu B (BN1224)
<1.00x10 -12
2.83x10 5
<1.00x10 -5
0.0133
0.9993
MAb to Flu B (BN1222)
<1.00x10 -12
2.15x10 5
<1.00x10 -5
0.0146
0.9996
MAb to Flu B (BN1223)
2.23x10 -11
3.29x10 5
7.34x10 -6
0.0176
0.9992
Figure 6. Binding kinetics of interactions between soluble Influenza A/B and immobilized Influenza antibodies from Meridian, as measured by bio-layer interferometry. Binding kinetics and affinity for the interactions of Influenza antibodies with Influenza A/B antigens were measured using an Octet QK384 instrument (ForteBio). All kinetic experiments were conducted in 69-well black plates (Greiner, 655209). Each purified Influenza antibody was diluted to 10 μ g/mL in buffer [1X PBS, containing 0.09% Tween 20] and directly immobilized onto AR2G sensors (ForteBio). The association of the antigen was measured for 600 s, followed by a dissociation step for 500 s. The association (k on ) and dissociation rate (k off ) constants as well as the equilibrium dissociation constant (KD) were determined by fitting to sensorgrams via the 1:1 binding model with a correlation coefficient (R2) value greater than 0.99.
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Given many respiratory disease symptoms overlap, it is not possible to reliably differentiate an infection with one of these viruses on clinical grounds alone.
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Rapid Test Targets for COVID-19, Flu A/B and RSV
Rapid tests can be designed to target different analytes, but generally target viral antigens and antibodies (IgG or IgM). They can detect analytes in blood, urine, saliva or vaginal swabs, with sampling protocols (sample collection, buffers, incubation time) varying by disease, sample matrix and analyte.
SARS-CoV-2 The nucleocapsid (N protein) is the main protein targeted in SARS-CoV-2 for rapid antigen testing as it is also the most abundant protein present on the surface of the virus and can easily be detected at low viral loads. Meridian’s highly sensitive antibody pair to SARS-CoV-2 Nucleocapsid Protein (Catalog 9548 and 9547) is proven to detect all of the WHO Variants of Concern (Alpha B.1.1.7, Beta B.1.351, Gamma P.1/P.2, Delta B.1.617.2 and Omicron B.1.1.529) and is used in FDA and CE-marked commercial rapid antigen tests around the world. The antibodies detect a conserved region N protein and they do not exhibit any cross-reactivity to influenza A/B, RSV or seasonal coronavirus strains. Influenza A & B For influenza, immunoassay testing typically detects the main circulating seasonal strains of both influenza type A and B. Influenza viruses are RNA viruses that are prone to antigenic drift and reassortment, which enables new strains of the virus to emerge each year. Rapid assays for influenza typically detect the nucleoprotein (NP) which is one of the more conserved proteins in the influenza virus and subsequently less likely to undergo mutations that lead to antigenic drift (which in turn can cause the functional components of an assay to not recognize a current influenza strain) 7 . Meridian offers several high performing antibody pairs for both influenza A and B that are ideal for rapid diagnostic testing and that do not cross-react with RSV or SARS-CoV-2 antigens.
SARS-COV-2 virus structure
Influenza virus structure
Subtype/ lineage
Titer (TCID 50 /mL)
PCR ct value*
Antibody
Strains
H1N1pdm A/Mexico/4108/09
1.51x10 3
28.1002
RSV virus structure
MAb to Flu A (BN1069-BN1070)
H3N2
A/Brisbane/10/07
7.44x10 2
31.4854
Yamagata lineage
B/Florida/04/06
3.56x10 3
22.7323
MAb to Flu B (BN1224 - BN1223)
Victoria lineage
B/Malaysia/2506/04 9.08x10 2
24.1907
Figure. 8 Limit of detection of RDTs using Meridian antibodies. *PCR: cobas 6800/8000 SARS-Cov-2/Flu 384T detection kit.
Figure 7. Virus structures and their major immunogenic proteins
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Rapid Test Targets for COVID-19, Flu A/B and RSV continued.
RSV is recognized as one of the most common causes of childhood illness and can lead to serious illnesses such as bronchiolitis and pneumonia in infants and older adults. In fact, almost two out of every one hundred children younger than six months of age with an RSV infection are at risk for hospitalization 8 . Structurally, RSV virus consists of three main proteins and the fusion protein (which is responsible for fusion to the host membrane) is the current leading target for diagnostic assays and the majority of vaccines and immunotherapies under development. This is due to the protein’s unique aspects in that it is only one of only two antigens that induce an RSV-neutralizing antibody response, it has a high degree of sequence conservation among RSV strains (>90%) 9 and it is highly immunogenic. Meridian offers several antibody pairs targeting the fusion protein which do not cross-react with SARS-CoV-2 or influenza and are for rapid antigen testing solutions. While immunoassay multiplexing offers numerous efficiency advantages, it also introduces several technical challenges that make assay design more complicated. One of the biggest challenges is controlling for interference between the various antibodies and proteins in the assay. Sample dilution can help with limiting interference caused by proteins and other substances present in complex sample types. However, in order to remove potentially interfering particles including endogenous antibodies such as heterophilic antibodies (HA) (e.g. HAMA) and rheumatoid factor (RF), it is important to incorporate immunoassay blockers into the assay design. Double
mouse monoclonal assays such as those for rapid respiratory antigen tests, are specifically prone to HAMA and RF interference and require a specialized blocker to ensure the assay’s accuracy. Meridian manufactures multikilogram scales of passive blockers such as Mouse IgG and animal serums as well a proprietary active blocker, TRU Block ™ , which contains specific binders directed against all types of heterophilic interference including HAMA and RF. Once bound to the interfering antibodies, TRU Block ™ prevents further binding of HA to other assay components through steric hindrance. Active blockers can typically be used in lower concentrations than passive blocking reagents, which minimizes the reduction in assay signal commonly associated with passive blockers. Overall, immunoassay multiplexing calls for carefully chosen reagents that allow the antibodies to work together to produce an accurate, meaningful result. Interfering factors must be minimized to prevent a false positive or a false negative error.
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Conclusion
Multiplex assays that combine the detection of several targets at once have several advantages including their high-throughput potential, ability to provide more results per sample, and lower reagent consumption (i.e., miniaturization). All of these benefits translate to a lower price-per-data point compared to traditional singleplex assays. Given many respiratory disease symptoms overlap, it is not possible to reliably differentiate an infection with one of these viruses on clinical grounds alone. Multiplex testing addresses the higher demand for screening between infections due to SARS-CoV-2, influenza A/B and RSV during what is expected to be higher than normal flu season. Molecular multiplex testing offers high sensitivity and specificity, detecting infected individuals early in the course of the disease and
enabling adequate time for the appropriate treatment. However, molecular tests require sophisticated equipment, experienced technicians and generally have a turn-around time of 1-5 days. In contrast, immunoassay rapid antigen assays are easy to use and can provide a result in 10-15 minutes, but they are considered to have a lower performance and are less sensitive. Each testing type, molecular or immunoassay, has advantages and disadvantages and they can be used independently from one another or in a testing algorithm that leverages the advantages of both. By employing multiplexing assays for the flu, COVID and RSV, patients will be able to be treated faster and more effectively, regardless of the testing type employed.
References 1 Jong-Yoon Jong. “Multiplex molecular diagnostics: shifting the paradigm.” Medica Laboratory Observer. Feb 17, 2013. Accessed Sept 12, 2023: https://www.mlo-online.com/home/article/13005002/multiplex- molecular-diagnostics-shifting-the-paradigm 2 The University of Sydney. “How COVID-19 created dramatic changes in a ‘winter virus’”. May 31, 2022. Accessed Sept 12, 2023: https://www.sydney.edu.au/news-opinion/news/2022/05/31/how-covid19- created-dramatic-changes--in-a-winter-virus.html 3 Budd, J., Miller, B.S., Weckman, N.E. et al. (2023) Lateral flow test engineering and lessons learned from COVID-19. Nat Rev Bioeng 1, 13–31. https://doi.org/10.1038/s44222-022-00007-3 4 Use of SARS-CoV-2 antigen-detection rapid diagnostic tests for COVID-19 self-testing.4 ), WHO https:// www.who.int/publications-detail-redirect/WHO-2019-nCoV-Ag-RDTs-Self_testing-2022.1 (2022). World Health Organization interim guidance on recommending COVID-19 self-testing using SARS-CoV-2 antigen tests; the web annexes include useful information on implementation 5 World Health Organization. “The ACT-Accelerator: two years of impact”. April 26, 2022. Accessed Sept 12, 2023: https://www.who.int/publications/m/item/the-act-accelerator--two-years-of-impact (2022). 6 Gasperino, D. et al. (2018) Improving Lateral Flow Assay Performance Using Computational Modeling. Annu. Rev. Anal. Chem. 11:219–44. https://doi.org/10.1146/annurev-anchem-061417-125737 7 Centers for Disease Control and Prevention. “Rapid Diagnostic Testing for Influenza: Information for Clinical Laboratory Directors”. https://www.cdc.gov/flu/professionals/diagnosis/rapidlab.htm. Accessed October 26, 2021 8 Centers for Disease Control and Prevention. “RSV in Infants and Young Children”. https://www.cdc.gov/ rsv/high-risk/infants-young-children.html. Accessed October 26, 2021. 9 Meng, J., Stobart, C. C., Hotard, A. L. & Moore, M. L. (2014). An overview of respiratory syncytial virus. PLoS Pathog. 24;10(4):e1004016. doi: 10.1371/journal.ppat.1004016.
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About Meridian
Meridian is a fully integrated life science company that develops, manufactures, markets, and distributes a broad range of innovative diagnostic products and critical raw materials. We are dedicated to developing and delivering better solutions that give answers with speed, accuracy, and simplicity that redefine the possibilities of life from discovery to diagnosis. As the Life Science division of Meridian, our focus is on supporting immunological and molecular test manufacturers with original raw materials for human, animal, plant, and environmental applications. The large portfolio of antigens, antibodies, blockers, molecular enzymes, nucleotides, and optimized mixes for qPCR and isothermal amplification applications are designed to simplify assay design and enable accurate test results. We strive to provide our customers with solutions they need when they need them – from novel antigens and antibodies to major disease outbreaks such as Zika and SARS-CoV-2 to pioneering the market with our innovative air-dried qPCR/RT-qPCR mixes. We take pride in providing our customers with unparalleled support, customer service, and quality.
Authors
Valerie Midgley, Ph.D. Senior Manager, Commercial Marketing LIFE SCIENCE
Steve Hawkins, Ph.D. Product Manager, Molecular LIFE SCIENCE
Ryan Hughes, Ph.D., MBA Product Manager, Immunology LIFE SCIENCE
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