key: cord-320845-imxby1b1 authors: Morley, G. L.; Taylor, S.; Jossi, S.; Perez-Toledo, M.; Faustini, S. E.; Marcial-Juarez, E.; Shields, A. M.; Goodall, M.; Allen, J. D.; Watanabe, Y.; Newby, M. L.; Crispin, M.; Drayson, M. T.; Cunningham, A. F.; Richter, A. G.; O'Shea, M. K. title: Sensitive detection of SARS-CoV-2-specific-antibodies in dried blood spot samples date: 2020-07-02 journal: nan DOI: 10.1101/2020.07.01.20144295 sha: doc_id: 320845 cord_uid: imxby1b1 Abstract Importance: Population-wide serological testing is an essential component in understanding the COVID-19 pandemic. The logistical challenges of undertaking widespread serological testing could be eased through use of a reliable dried blood spot (DBS) sampling method. Objective: To validate the use of dried blood spot sampling for the detection of SARS-CoV-2-specific antibodies. Design, setting and participants: Eighty-seven matched DBS and serum samples were obtained from eighty individuals, including thirty-one who were previously PCR-positive for SARS-CoV-2. DBS eluates and sera were used in an ELISA to detect antibodies to the viral spike protein. Results: Specific anti-SARS-Cov-2 spike glycoprotein antibodies were detectable in both serum and DBS eluate and there was a significant correlation between the antibody levels detected in matched samples (r = 0.96, p<0.0001). Using serum as the gold standard in the assay, matched DBS samples achieved a Cohens kappa coefficient of 0.975 (near-perfect agreement), a sensitivity of 98.1% and specificity of 100%, for detecting anti-spike glycoprotein antibodies. Conclusions and relevance: Eluates from DBS samples are a reliable and reproducible source of antibodies to be used for the detection of SARS-CoV-2-specific antibodies. The use of DBS sampling could complement the use of venepuncture in the immunosurveillance of COVID-19 in both low and high income settings. Confirmation of a diagnosis for acute coronavirus disease 2019 (COVID-19) is dependent upon the detection of RNA from the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, while serology is less useful for diagnosing acute stages of infection in most individuals, it can help diagnose atypical presentations of SARS-CoV-2 and asymptomatic infection 1 . Furthermore, serology is valuable for determining prior viral exposure at a population level, allowing for a more comprehensive understanding of the epidemiology of SARS-CoV-2, particularly given the limitations of polymerase chain reaction testing 2 . Antibody assessments can also help to establish transmission patterns, assess longitudinal humoral responses and identify correlates of protection, all of which may have a significant impact on public health and social policies 3, 4 . Currently, antibody testing for SARS-CoV-2 uses serum or plasma collected by venepuncture. The use of such sampling in large-scale seroepidemiological studies is limited by logistical challenges, resources and costs, together with the risk of SARS-CoV-2 exposure from direct patient contact. In contrast, dried blood spot (DBS) sampling is simple, inexpensive 5 and can be self-collected then sent by postal services to laboratories for processing 6 . It is a well-established method for detecting antibodies against a variety of infections 7, 8 and antibodies collected on DBS cards are stable for prolonged periods 9 . Moreover, DBS sampling provides one solution to widening access to serological platforms in low-and middle-income countries (LMICs). Nevertheless, the potential role of the DBS to study SARS-CoV-2 seroprevalence has not been fully explored; and there is limited understanding of how to recover antibody from DBS. Here, we describe the validation of DBS samples . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 2, 2020. . https://doi.org/10.1101/2020.07.01.20144295 doi: medRxiv preprint against matched serum in a highly sensitive and specific SARS-CoV-2 Enzyme Linked Immunosorbent Assay (ELISA). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted July 2, 2020. For DBS collection, finger-prick capillary blood samples (approximately 50 µL per spot) were collected onto forensic 226 grade DBS collection cards (Ahlstrom Munksjo, Germany) provided as part of the TakeATestUK postal testing kits 6 (Saving Lives, UK), using standard methods 10 . DBS cards were stored at room temperature (RT) in individual sample bags with desiccant. Concomitantly, venous blood was collected from volunteers and serum was separated by centrifugation at 9,700 x g for 5 minutes, RT. Laboratory analysis was blinded to PCR status and SARS-CoV-2specific-antibody results were reported as positive, negative or equivocal. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 2, 2020. An ELISA to measure IgG, IgA and IgM against soluble, stabilised, trimeric SARS-CoV-2 spike (S) glycoprotein 11, 12 , was performed as previously described 1, 13 . Briefly, 96-well plates (Nunc, UK) were coated with 50 µL of 2 µg/mL spike (S) glycoprotein. Plates were blocked and sample diluted with 2% BSA PBS-T 0.1% (starting dilutions: . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 2, 2020. . https://doi.org/10.1101/2020.07.01.20144295 doi: medRxiv preprint Statistical analyses were performed using Prism (version 8, GraphPad, USA). Correlations between continuous data were assessed using Spearman's rank test and a p value <0.05 was considered statistically significant. DBS sample ELISA performance was assessed by calculating the sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively) with 95 % confidence intervals (CIs). The agreement between DBS and serum ELISA results was assessed by determining the Cohen's kappa coefficient and Bland-Altman mean difference. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 2, 2020. . https://doi.org/10.1101/2020.07.01.20144295 doi: medRxiv preprint Quantification of total immunoglobulin concentrations in serum and DBS eluate was performed. A 7-11-fold reduction in mean immunoglobulin concentration (IgG, IgA and IgM) was observed in DBS eluate compared to matched serum (Fig 1a) . To detect antibodies to SARS-CoV-2-anti-spike (S) glycoprotein, matched serum and DBS titration curves were generated. Specific anti-S antibodies were detectable in both serum and DBS eluate with higher responses observed in PCR-positive matched samples, whilst pre-August 2019 DBS samples were negative across all dilutions (Fig 1b and 1c) . The OD 450 detected by ELISA for matched DBS samples and sera, diluted 1:10 and 1:100 respectively, were plotted. There was a significant correlation between matched serum and DBS samples (r = 0.96 (95% CI: 0.93-0.97), p<0.0001) (Fig 1d) and minimal differences in results using either antibody source in the assay (Bland-Altman bias 0.11±0.20) (Fig 1e) . Discordance occurred between only one matched sample, giving a Cohen's kappa coefficient of 0.975. DBS samples achieved a sensitivity of 98.11% and specificity of 100% for detecting anti-S glycoprotein antibodies, when compared to serum (Fig 1f) , with 100% of the PCR-positive samples (n=31) also antibody-positive in DBS eluate. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 2, 2020. . https://doi.org/10.1101/2020.07.01.20144295 doi: medRxiv preprint We show that DBS samples can be used for the detection of SARS-CoV-2-specific antibodies with high levels of sensitivity and specificity and compared well with matched serum samples. Taken together, these results demonstrate that DBS sampling could complement venepuncture for serological assessments, such as seroprevalence studies, during the COVID-19 pandemic. A current limitation in antibody assays is the necessity for venepuncture by skilled phlebotomists. The use of DBS overcomes this limitation and introduces the opportunity for wider population level sampling and improved surveillance in those groups shielding from the infection. For example, DBS could be delivered using postal services 6 to patients with chronic conditions, the immunocompromised and the elderly; groups which have been disproportionately affected by COVID-19 14 . Furthermore, the DBS method is simple and inexpensive 5,6 which could enhance sampling in LMICs, amongst groups where venepuncture is culturally unacceptable or, in a geographically dispersed populous 15 . . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 2, 2020. . Serology confirms SARS-CoV-2 infection in PCR-negative children presenting with Paediatric Inflammatory Multi-System Syndrome. medRxiv Detection of SARS-CoV-2 in Different Types of Clinical Specimens SARS-CoV-2 seroconversion in health care workers. medRxiv Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into populationbased research Dried blood spot and mini-tube blood sample collection kits for postal HIV testing services: a comparative review of successes in a real-world setting Evaluation of dried blood spot samples for screening of hepatitis C and human immunodeficiency virus in a real-world setting Detection of immunoglobulin G to measles virus, rubella virus, and mumps virus in serum samples and in microquantities of whole blood dried on filter paper Stability of human immunodeficiency virus type 1 antibodies in whole blood dried on filter paper and stored under various tropical conditions in Kinshasa Dried blood spots--preparing and processing for use in immunoassays and in molecular techniques Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Site-specific glycan analysis of the SARS-CoV-2 spike Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection. medRxiv We would like to thank the University of Birmingham Clinical Immunology Service for their invaluable support in sample collection and processing; and thank Cynthia