Multiplex testing for COVID-19, Flu and RSV

overcome the inhibitors present in saliva/ sputum, blood, urine and stool. The new Air-Dryable ™ Direct RNA/DNA qPCR Saliva mix (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. 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, Mycoplasma pne, etc), because of its sensitivity to less than 10 copies and its compatibility with liquid or dry assay formats. Immunoassays Although molecular tests are considered the gold standard for viral respiratory detection, they have limitations in accessibility, scalability, and point-of-care applications. In contrast, lateral flow immunoassays are perfectly suited for field-based testing. Rapid antigen assays are highly scalable, can be stored at room temperate, 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. The antibodies selected for a multiplex assay must be highly sensitive in order to detect both low and high abundance targets, 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. Immunoassay Testing for COVID-19, Flu A/B and RSV For COVID-19, the nucleocapsid (N protein) is the main protein targeted for rapid antigen testing as it is also the most abundant protein present on the surface of the SARS-CoV-2 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. 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 news 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). 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. 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 hospitalizaton (CDC, 2021). 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%) (Meng, 2014) 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

Fig. 4 Respiratory Multiplex Testing Using Air-Dryable ™ Direct RNA/DNA qPCR Saliva (MDX131)

MERS-CoV

RSV

Influenza A

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).