key: cord-0690111-nq3lsle6
authors: MacMullan, M. A.; Chellamuthu, P.; Mades, A.; Das, S.; Turner, F.; Slepnev, V. I.; Ibrayeva, A.
title: Rapid detection of SARS-CoV-2 antibodies in oral fluids
date: 2020-10-14
journal: nan
DOI: 10.1101/2020.10.12.20210609
sha: 2c4d309c58913bf4b1e1cc97592b44043a406691
doc_id: 690111
cord_uid: nq3lsle6

Current commercially available methods for reliably detecting antibodies against SARS-CoV-2 remain expensive and inaccessible due to the need for whole blood collection by highly trained phlebotomists using personal protective equipment (PPE). We evaluated an antibody detection approach utilizing the OraSure Technologies' Oral Antibody Collection Device (OACD) and their proprietary SARS-CoV-2 total antibody detection enzyme-linked immunosorbent assay (ELISA). We found that the OraSure test for total antibody detection in oral fluid had comparable sensitivity and specificity to serum-based ELISAs while presenting a more affordable and accessible system with the potential for self-collection.

Introduction 36

The COVID-19 pandemic has had a severe impact on populations and economies 37 worldwide (1). While the relationship between antibodies and their protective role against 38 reinfection of SARS-CoV-2 is under investigation, it has been shown that antibodies 39 consistently develop as part of the immune response against the virus in some, if not 40 most, infected individuals (2). Neutralizing antibodies were effective and sustained for at 41 least two and potentially up to twelve years against SARS-CoV, the first SARS 42 coronavirus, strongly suggesting the potential for significant immunity conferred by 43 antibodies against SARS-CoV-2 and prompting extensive studies to identify neutralizing 44 antibodies for therapeutic development (3, 4) . Further, although residues of the receptor 45 binding domains (RBD) of SARS-CoV and SARS-CoV-2 differ by about 55% (18/33), it 46 has been shown that neutralizing antibodies against SARS-CoV are reactive against 47 reason to believe that the monitoring of antibodies is crucial to understanding global 57 disease spread, immunity against re-infection by the virus, and the effectiveness of 58 potential vaccine candidates. 59

The industry standard for antibody detection relies on the collection and analysis 60 of patient serum, requiring trained phlebotomists to draw the whole blood from a patient 61

following suspected viral infection (7). That process is time-consuming, costly, and 62 potentially hazardous to the phlebotomist. There is also a substantial need to protect 63

To evaluate this, we selected sixteen out of our original sixty-nine positive participants 103 and collected their paired oral fluid and serum specimens at least 60 days after the original 104 collection date. We found that the oral fluid-based total antibody ELISA from OraSure® 105

Technologies performed with statistical equivelance in comparison to the serum-based 106

IgG antibody ELISA from EuroImmun, with all positive specimens passing the antibody 107 detection cutoff after both 21+ days and 60+ days (Fig 3. ) 108

To evaluate the potential time and cost savings of using the OACD for antibody 109 detection instead of traditional serum-based methods, we estimated that a trained 110 phlebotomist operating at a full-capacity drive-thru center can only collect and process 111 blood serum from ~40 patients in a 5-hour period, resulting in collection of one patient 112 specimen per 7.5 minutes. Because oral fluid can potentially be self-collected using the 113 OACD, face-to-face specimen collection time can be eliminated. This dramatically 114 reduces the amount of time needed for each specimen to be collected, leading us to 115 estimate that we can collect devices from ~1,000 patients in a similar drive-thru setting 116 over the same 5-hour period, resulting in collection of one specimen in approximately 0.3 117 minutes. Note that this time does not include the actual time for collection of the sample, 118 which we are assuming individuals would perform while they are waiting in the drive-thru 119 line, thus highlighting the dramatic benefit of self-collected oral samples. This represents 120 approximately a 25-fold increase in sample collection throughput versus traditional blood 121 serum sample collection in the same time frame (Fig. 4) . 122

To increase testing capacity and decrease turnaround time for antibody results, 123

we have automated the process for running the OraSure® Anti-SARS-CoV-2 total 124 antibody ELISA and will continue evaluating the efficiency and reliability of the test. 125 . CC-BY 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 October 14, 2020 . . https://doi.org/10.1101 Discussion 126

While antibody testing has previously been limited by the requirement of trained 127 personnel for specimen collection, we have evaluated here a technology for non-invasive 128 self-collection which we believe will be able to be scaled up and mass-produced for 129 widespread testing. Based on the test efficacy and time-savings afforded by this method 130 of antibody detection, OraSure® Technologies has developed a more accessible and 131 affordable testing option for patients interested in understanding whether they have been 132 exposed to and developed antibodies against SARS-CoV-2. The simplicity of the OACD 133 allows us to speculate that this will enable specimens to be theoretically self-collected 134 and eliminate the need for a trained medical associate to administer the collection. The OACD, when used with the Anti-SARS-CoV-2 total antibody ELISA kit 143 developed by OraSure® Technologies, has achieved sensitivity and specificity which are 144 equivalent to commercially available serum-based ELISAs(9). This represents a unique 145 and highly successful advancement in antibody detection technology. As we move closer 146 to the development and implementation of a successful vaccine against SARS-CoV-2, 147

we need reliable methods for monitoring antibody production on a wide scale and over 148 . CC-BY 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 October 14, 2020. . https://doi.org/10. 1101 time. This antibody detection system allows us to overcome the limitations presented by 149 currently administered serum antibody tests, increasing accessibility to antibody testing 150 and enabling a better understanding of the trends of immune response against this virus 151 over time. 152 . CC-BY 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 October 14, 2020. CC-BY 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 October 14, 2020. (10,15). 184

The pad was brushed briefly on the lower and upper gums and then held between the 185 lower gum and the cheek for 2-5 minutes. The pad was then placed into the storage tube, 186 with the provided storage solution. Specimens were kept on ice until they reached the 187 lab. The specimens were processed as recommended by the manufacturer before being 188 aliquoted and stored at -80°C until use (16). three times with a provided wash buffer (10X dilution with ddH2O, 0.35ml per well), before 197 . CC-BY 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 October 14, 2020. Data were then analyzed as recommended by the manufacturer and results reported as 204 a ratio (Equation 1). Specimens whose ratio exceeded or was equal to 1.1 were 205 considered positive, while specimens with a ratio greater than 0.8 and less than 1.1 were 206 considered equivocal and specimens with a ratio less than or equal to 0.8 were 207 considered negative. Specimens with ratios in the equivocal range were recommended 208 to be rerun, but this did not apply to any specimens in this study. antigens S1 and S2 was run manually according to the manufacturer provided 218 protocol(11). 25uL of provided sample diluent buffer was added to the desired wells of 219 the plate followed by 100uL of each specimen. Specimens were incubated at ambient 220 . CC-BY 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 October 14, 2020. . https://doi.org/10.1101/2020.10.12.20210609 doi: medRxiv preprint temperature for 1 hour. Sample wells were washed six times with a provided wash buffer 221 (20X dilution with ddH2O, 0.35ml per well), before the provided conjugate solution was 222 added (0.1ml per well) and incubated at ambient temperature for 1 hour. After a second 223 wash step, the provided substrate solution was added (0.1ml per well) and incubated at 224 ambient temperature for 30 minutes. The provided stop solution was then added (0.1ml 225 per well) and absorbance of sample wells measured immediately at 450 nm and 630 nm, 226

with output reports generated with O.D. at 630nm subtracted from O.D. at 450nm. 227

Data were analyzed as recommended by the manufacturer and results reported as a 228 specimen to cutoff (S/CO) ratio (Equation 2). Specimens whose ratio was equal to or 229 exceeding 1.0 were considered positive, while specimens with a ratio greater than 0.8 230 and less than 1.0 were considered equivocal and specimens with a ratio less than or 231 equal to 0.8 were considered negative. Specimens with ratios in the equivocal range were 232 recommended to be rerun, but this did not apply to any specimens in this study. 

Using the automated Dynex DSX 5 plate ELISA Processing System, we developed a 240 quick two-step automated method for SARS-CoV-2 antibody detection involving: 1) 241 centrifugation of the oral fluid collection devices in a secondary tube to transfer oral fluid 242 . CC-BY 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 October 14, 2020. . https://doi.org/10.1101/2020.10.12.20210609 doi: medRxiv preprint in the device and 2) processing of the collected saliva specimens on OraSure® 243

Technologies SARS-CoV-2 Total Antibody ELISA plates. Diego, USA), with a 95% confidence interval. Area under the curves was also calculated. 248

Correlation between serum and oral fluid specimen IgG values were calculated using a 249

Pearson correlation computing R between the two datasets, with a 95% confidence 250 interval. 251 252

The data that support the findings of this study are available from the authors upon 254 reasonable request. 255 . CC-BY 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 October 14, 2020. 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 October 14, 2020. 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 October 14, 2020. 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 October 14, 2020. . https://doi.org/10.1101/2020.10.12.20210609 doi: medRxiv preprint Step 3. After securing the vial cap, place the vial into a plastic biohazard collection bag and label with patient name and date. This bag will be transported or collected for processing.

. CC-BY 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 October 14, 2020 . . https://doi.org/10.1101 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 October 14, 2020 . . https://doi.org/10.1101 

. CC-BY 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 October 14, 2020. . IgG Antibody S/CO Ratio in Serum . CC-BY 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 October 14, 2020. . https://doi.org/10.1101/2020.10.12.20210609 doi: medRxiv preprint 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 October 14, 2020. . https://doi.org/10. 1101 

CEPAR)), Fernando R 269 (Australian NU and the C of E in PAR (CEPAR)). The economic impact of COVID-270 19

Antibody responses 273 to SARS-CoV-2 in patients with COVID-19

The trinity of COVID-19: 275 immunity, inflammation and intervention

IgG Antibodies in SARS-CoV Infected Healthcare Workers

Characterization of the 280 receptor-binding domain (RBD) of 2019 novel coronavirus: implication for 281 development of RBD protein as a viral attachment inhibitor and vaccine

The authors would like to thank Joseph Kapcia III, Cedie Bagos, Aaron Angel, Marilisa 257Santa-Cruz and Matthew Geluz from Curative, Inc. as well as Kerry Phillips, Toral Zaveri,