designed and wrote the manuscript. Peer review Peer review information thanks David (T) Harris, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. using finger-prick dried blood spots displays 91C97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood Rabbit Polyclonal to Thyroid Hormone Receptor alpha spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern. Subject terms: Immunological techniques, PCR-based techniques, Contamination, Antibodies, Assay systems Neutralizing antibodies are critical for conferring immunity against SARS-CoV-2. Here, Dahn et al. report a PCR assay termed SONIA (Split-Oligonucleotide Neighboring Inhibition Assay) for measuring neutralizing antibodies against multiple SARS-CoV-2 strains in fingerprick dried blood spot samples. Introduction The current epidemic of COVID-19 (novel coronavirus disease-2019) caused by SARS-CoV-2 has propagated globally at unprecedented velocity1C5. It has led to more than 522 million confirmed infections worldwide and over 6.2 million deaths1C5. SARS-CoV-2 virus enters human cells via binding between the viral surface spike protein and the human ACE2 receptor5. Neutralizing antibodies (Nab) are capable of disrupting this conversation and have been shown to result in enhanced disease survival and reduced viral loads in swab specimens3,4. NAb can be found in patient specimens after natural contamination, vaccination and/or receipt of convalescent plasma treatment. Monitoring of Nab after these events can provide useful information to both predict disease progression and confirm vaccination or treatment efficacy. The virus plaque reduction neutralization test (PRNT) is the current gold standard assay for NAb6. However, PRNTs reliance on infectious SARS-CoV-2 virions limits the use of this potentially hazardous and time-consuming assay to relatively few well-resourced institutions equipped with biosafety level 3 (BSL3) laboratories. Modifications to the PRNT such as pseudovirus neutralization assays insert sections of the virus in question into benign viral targets to allow for a safer approximation of PRNT, but are still reliant on time consuming cell-based methods6 and give results that do not always match those of live-virus PRNT Thrombin Receptor Activator for Peptide 5 (TRAP-5) Thrombin Receptor Activator for Peptide 5 (TRAP-5) assays7. ELISA and microbead-based Thrombin Receptor Activator for Peptide 5 (TRAP-5) methods have been reported, but they are either not multiplexable or may not be applicable to challenging sample types such as dried blood spots8,9. In this study we develop and validate an assay, termed SONIA (Fig.?1), to measure NAb using several cohorts of well-characterized specimens. This assay is usually inspired by our previous work of an ultrasensitive and highly specific assay method termed antibody detection by agglutination PCR (ADAP). The ADAP platform has been successfully applied to a wide variety of infections and autoimmune diseases10C14. Notably, we also present data on a multiplex version of the cell-free PCR assay to measure NAb against the alpha and delta SARS-CoV-2 variants in finger-prick dried blood spot specimens. Open in a separate window Fig. 1 Theory of SONIA neutralization Thrombin Receptor Activator for Peptide 5 (TRAP-5) PCR test.a Viral entry of SARS-CoV-2 is mediated by the binding of the spike protein to the human receptor angiotensin-converting enzyme 2 (ACE2). Disruption of this conversation forms the basis of neutralization by antibodies (NAb). b SONIA Neutralization PCR test reconstructs this conversation using a combination of S1 subunits of the spike protein- and ACE2-DNA conjugates. In the absence of NAb, S1 and ACE2 engage with strong affinity, thereby positioning the two DNA barcodes in proximity for subsequent ligation and PCR-amplification. On the other hand, binding of NAb blocks S1 subunit from binding ACE2, leaving the two DNA barcodes separated. Since each barcode has only one PCR primer binding site, they cannot be separately amplified. Therefore, the quantities of NAb are correlated with the decrease of PCR amplicon formation. Results Selection of antigens for the SONIA neutralization PCR assay to measure Nab The successful development of the NAb assay relies heavily on the proper choice of the antigens used. To that end, we first evaluated assay performance using the S1 portion of the spike Thrombin Receptor Activator for Peptide 5 (TRAP-5) protein versus the receptor binding domain name (RBD) fragments of the S1 protein. We assayed two convalescent COVID-19 patient samples and four control specimens from healthy blood donors collected prior to the outbreak (Fig.?2). The COVID-19 samples had been analyzed using a cell-based pseudovirus neutralization assay15,16 and confirmed to contain high titers of NAb. For both antigens, we observed no competition signals from the unfavorable control specimens, and strong competition signals from the COVID-19 samples, indicating effective competition and neutralization of the S1-ACE2 conversation. Given the observation of much stronger signals in the S1 protein-based neutralization assay (Fig.?2), we chose to proceed with the S1.
