Development of New 30 Minute COVID-19 Diagnostic Testing

 A new SARS-CoV-2-specific serology assay, using a rapid gel agglutination assay, has been designed and analysis shows that the tests can provide serological results for SARS-CoV-2 infection within 30 min, using an approach consistent with blood typing assays used routinely in hospital labs around the world.

While large-scale efforts are underway to develop vaccines and antiviral therapies,  the rapid development and deployment of diagnostic tests is of key importance. As we learn more about the immune response to SARS-CoV-2, recent reports suggest that IgG and IgM antibodies are produced either sequentially or simultaneously, with concentrations reaching a plateau 6 days after seroconversion, and that SARS-CoV-2 elicit highly specific antibody responses not present in naiv̈e individuals, including those previously infected with other coronaviruses. Several approaches for serology testing are already being distributed around the world and point-of-care paper-based tests for antibodies are under evaluation and available in some countries, though they cannot be used for high-throughput screening (15−30 min/sample), and specificity/sensitivity is not expected to meet the standard of laboratory-based tests. The current standard for serology methods is laboratory- based indirect enzyme-linked immunosorbent assay (ELISA), in which antibodies from patient serum are captured onto a protein-coated microwell plate followed by enzymatic detection using an anti-Ig secondary antibody. These assays can be performed manually or using automated systems; however, they are still multistep processes requiring multiple antibodies and reagents. A new SARS-CoV-2-specific serology assay, using a rapid gel agglutination assay, has been designed and analysis shows that the tests can provide serological results for SARS-CoV-2 infection within 30 min, using an approach consistent with blood typing assays used routinely in hospital labs around the world.

This rapid gel agglutination assay approach is designed for the widespread availability of the simple CAT technology commonly employed for blood typing because it meets the ASSURED (afford- able, sensitive, specific, user-friendly, rapid/robust, equipment- free, deliverable to end-users) criteria and be rapidly scalable and customizable.

Blood typing and antibody screening are performed in hospital laboratories all over the world, using the robust column agglutination test (CAT) technology. Detection of antibodies in patient plasma or serum involves pipetting a mixture of reagent red blood cells (RRBCs) and antibody- containing serum/plasma onto a gel card containing separation media, incubating the card for 5−15 min and using a centrifuge to separate agglutinated cells from free cells, resulting in strong red lines on top of the gel column in the case of a “positive” test. A wide range of RRBCs expressing different surface antigens are available for assay development, along with corresponding antibodies of varying affinity and avidity.

In this study, the scientists developed a serology test to detect SARS- CoV-2 antibodies from human plasma using gel card agglutination tests. The CAT technology was selected for rapid and high throughput testing and comprehensive serology mapping, for two reasons. First, CAT is currently available in the blood/analytical laboratory of all major hospitals throughout the world as an automated and high throughput platform (>100 tests/h), with equipment and trained person- nel already in place. Second, many companies are currently manufacturing the gel cards widely used for blood typing analysis. Production of SARS-CoV-2 gel card diagnostics only requires the substitution of the current RRBCs with bioconjugated cells, using the current processing and technology. They have found that by producing bioconjugates of anti-D-IgG and peptides from SARS-CoV-2 spike protein, and immobilizing these to RRBCs, they observe selective agglutination assays in gel cards in the presence of plasma collected from patients recently infected with SARS-CoV-2 in comparison to healthy plasma and negative controls.

Antibody−peptide bioconjugates were designed to bind SARS-CoV-2-specific antibodies raised in response to viral infection. First, their research suggested that operating the assays under conditions of bioconjugate saturation is required for successful agglutination and retention of aggregated cells above the gel column. Then, they progressed to optimize gel card agglutination assays to distinguish between SARS-CoV-2-positive and SARS-CoV- 2-negative patient samples. Reactions involving mixed bioconjugates (mixtures prepared after cell incubation) were also tested, showing similar results to those for single-bioconjugate reactions, which is important, as they anticipate that multiple immunodominant peptides will be required to minimize false positives in large cohorts. Importantly, during the optimization phase of assay development, they occasionally observed false-positive results(a red line appearing above the gel column after incubation of bioconjugate-coated RRBCs and SARS-CoV-2-negative plasma.) However, upon microscopy, it was revealed that these cells were not agglutinated; this was mainly related to the presence of glycerol carried over from the bioconjugate stock solution.

Following optimization of the gel card assays to distinguish between SARS-CoV-2-positive samples and negative controls, they tested 10 clinical samples in both gel cards and indirect IgG ELISA. The ELISA was designed to capture and detect IgG antibodies from plasma which bound to SARS-CoV-2 proteins coated onto the plates. This assay cannot detect IgM antibodies, which are also likely to be present in many samples; however, they expect that IgM levels are likely to recede over time in at least some individuals, whereas IgG levels are likely to remain high over time and hence are appropriate to confirm immune response to infection.

The lack of positive results in ELISA or agglutination tests for these negative samples is particularly encouraging, because in a recent study, four out of five SARS-CoV-2-negative blood samples showed high levels of antibodies against seasonal coronaviruses but no cross-reactive antibodies that bind SARS-CoV-2. This is consistent with earlier estimates that nearly 100% of adolescents have antibodies against seasonal coronaviruses. This analysis shows that the gel card agglutination tests can provide serological results for SARS-CoV-2 infection within 30 min, using an approach consistent with blood typing assays used routinely in hospital labs around the world.

This study utilized widely used blood typing tests and converted them into SARS-CoV-2 serology tests. Given the rapid turnaround time, high throughput, and level of clinical acceptance, they suggest that with further testing in large sample cohorts to accurately characterize false-positive/- negative rates, CAT assays could provide an alternative to ELISAs. Currently, key limitations include the lack of knowledge on which SARS-CoV-2 peptides are immunodominant, whether or not predicted B-cell epitopes will efficiently bind antibodies (IgG, IgM, etc.) in patient blood, and what is the level of cross-reactivity between antibodies raised against previous corona-virus infections in large sample cohorts.

This study confirms that pursuing an approach involving multiple bioconjugates is likely required to minimize false-negative results in large sample cohorts. Noting, an alternative approach would be to create bioconjugates using whole proteins instead of peptide epitopes; the advantage would be that protein sequences are often much faster to determine in comparison to identifying a minimal list of immunodominant peptides, which is important in terms of pandemic response. However, bioconjugation reactions involving whole (and potentially novel) proteins are likely to be less efficient due to size/charge comparisons, requiring individual tailoring of reaction conditions, whereas any peptide bioconjugates can be prepared simultaneously.


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