key: cord-308093-m40czdsr
authors: Matthews, M. M.; Kim, T. G.; Shibata, S.; Shibata, N.; Butcher, C.; Hyun, J.; Kim, K. Y.; Robb, T.; Jheng, S. S.; Narita, M.; Mori, T.; Collins, M.; Wolf, M.
title: COVID-19 serological survey using micro blood sampling
date: 2020-10-13
journal: nan
DOI: 10.1101/2020.10.09.20209858
sha: 
doc_id: 308093
cord_uid: m40czdsr

During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol developed by the Krammer Lab in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.

with at-home self-collection of blood samples using a custom low-cost finger prick-based 23 capillary blood collection kit. Although our survey did not find any COVID-19 seropositive 24 individuals among the OIST cohort, it reliably detected all positive control samples obtained 25 from a local hospital and excluded all negatives controls. Among our controls, we found 26 strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in 27 the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with 28 patient-based capillary blood collection using as little as one drop of blood can reliably assess 29 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint 48 Serological surveys which detect the presence of antibodies against SARS-CoV-2 49 antigens can provide important information to the government for issuing health care 50 guidelines. At the beginning of April 2020, we obtained plasmids for coronavirus surface 51 antigens from the Krammer Lab at the Icahn School of Medicine (NY, NY, USA). We established 52 protein expression and purification in a mammalian cell line and set up an ELISA following the 53 2-step assay developed by the same group (1). Their assay has received emergency use 54 authorization by the U.S. Food & Drug Administration (FDA) (2). We secured PCR-confirmed 55 human sera from COVID-19 positive patients at the local hospital as well as negative controls 56 from serum collected before December 2019. Once the assay itself was validated, we set up 57 partially automated sample handling on a robotic liquid handler, established a website with 58 a barcoding system for anonymous sample tracking, and conducted a serological survey of 59 staff and students at our institution. 60 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint

In emergency situations such as during COVID-19, the health care system is under 61 stress; it cannot be expected that trained clinical personnel are available to draw patient 62 blood by venous puncture. Furthermore, non-essential human traffic in hospitals and other 63 health care institutions should ideally be limited to protect vital health care workers from risk 64 of exposure to potential carriers of the virus. To overcome this limitation, we distributed easy-65 to-use, self-administered micro blood sampling kits to participants. The kit uses a single-use 66 safety lancet to collect a few drops of capillary blood from the participant's finger. We show 67 that antibody titers obtained by micro blood sampling are equivalent to serum antibody titers 68 from blood drawn by conventional venous puncture. This low-cost, easily deployable self-69 sampling method in combination with a highly sensitive and specific ELISA in a centralized 70 testing lab provides a scalable solution that can enable serological surveys of larger by Ni-NTA affinity and size-exclusion chromatography as described previously (1). The yield 81 was 15 mg/L and 4 mg/L for RBD and the trimeric SARS-CoV-2 S, respectively. 500 µL aliquots 82 of purified protein at a concentration of 10 µg/mL were frozen in liquid nitrogen and stored 83 at -80 °C until use. We expressed and purified both the SΔcs (furin cleavage site deletion RRAR 84 to A) and SΔcspp construct (cleavage site deletion and stabilizing mutations K986P and V987P) 85

(3), but only the SΔcs protein was used in this study. Protein purity was verified by SDS-PAGE 86 and confirmed by Western blot with MonoRab™ Anti-His Tag (C-term) Antibody (Nr. 25B6E11, 87

GenScript, USA). 88 89

The quality and folding of two proteins, the RBD and SARS-CoV-2 S, were verified by 91 biochemical methods and correct folding and assembly of the trimeric spike protein was 92 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint confirmed using cryo-electron microscopy ( Figure 1 ). Sample solution was applied to a carbon 93 coated copper grid and stained with 1 % uranyl acetate. The protein particles were visualized 94 with a Talos L120 transmission electron microscope (Thermo Fisher Scientific, USA) operating 95 at 120 kV acceleration voltage. 96

For cryo-EM, 3 µL of purified trimeric spike sample solution was applied to UltrAuFoil 97 R1.2/1.3 grids pre-treated with a Solarus advanced plasma system (Model 950, Gatan, USA) 98

for 60 seconds at 25 °C in a 23 % H2, 77 % O2 gas mix. The grids were blotted and vitrified in a 99

Vitrobot Mark IV (Thermo Fisher Scientific, USA) using a liquid ethane-propane mixture. Cryo-100 EM grids and particle density were optimized by screening based on protein concentration in 101 the range of 2-3 mg/mL. Particles Images of trimeric spike protein in amorphous ice were 102 collected with a Titan Krios transmission electron microscope (Thermo Fisher Scientific, US) 103 operating at 300 kV on a Falcon-3EC camera (Thermo Fisher Scientific, US) in counting mode, 104 at a pixel size of 1.08 Å (at the specimen level). Frames from 1,479 movies were aligned, dose-105 weighted and summed using motioncor2 (4). Contrast transfer function (CTF) estimation, 106 particle picking and 2D classification were performed with RELION 3.1 (5). A total of 1,119,504 107 particles were picked semi-automatically, of which 297,144 particles were retained after 2D 108 classification. 109 110

The micro blood sampling kits were assembled in 8x12 cm sealable plastic bags, including BD 112

Microtainer Ⓡ contact-activated lancet (Becton Dickison, NJ, USA), blood collection tube 113 containing a coagulant and a separator gel (Greiner Bio-one MiniCollect Ⓡ TUBE 0.5/0.8 CAT 114

Serum Separation Clot Activator gold cap), packaged alcohol wipes, adhesive bandage, and 115 peelable sticker with a linear tube barcode (Code 128) and a matching QR code encoding a 116 link to our website. After finger prick with the safety lancet, users collected 1-5 drops (approx. 117 30-150 µL) of capillary blood into the serum separation tube and attached the linear barcode. 118

Serum separation tubes were centrifuged at 5,000╳g for 5 min at 4 ºC under biosafety level 119 2 (BSL2) conditions, then inactivated in a water bath at 56 ºC for 1 hr. Heat-inactivated 120 samples were stored at 4 ºC until use for ELISA. After scanning tube barcodes for each plate 121 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint into a CSV file with a hand-held scanner, separated serum was transferred into barcoded 96-122 well plates using manual pipetting. 123 124

We followed the 2-step ELISA protocol developed by the Krammer lab (1). ELISA plate formats 126 were modified as depicted in Supplemental Figure calculate threshold values for each plate and output a "positive" or "negative" result for each 151 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. The threshold for each step 1 plate was defined as the average of the negative controls plus 170 3 standards deviations of the negative controls as described previously (1). The average and 171 median threshold for all step 1 plates was 0.161 AU and 0.166 AU, respectively. Initially, the 172 threshold for the step 2 plate was calculated in the same manner as step 1. However, the 173 threshold calculated with only three negative controls was below the recommended range 174 sample IDs were composed of six characters generated randomly from the set of 211 alphanumeric characters (a-zA-Z0-9), excluding letters O, I, l, Z, Q and numbers 0, 1, 2, 9 to 212 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint minimize human read errors. Out of approximately 10 10 combinations, 1000 unique IDs were 213 randomly selected. Some additional security features, such as request rate limiting, were 214 implemented to restrict the use of automated tools to retrieve results for all possible sample 215

IDs. Basic functions were developed to allow batch import and export of test results in a 216 delimited text format, allowing easy transfer of data between the testing system and the web 217 application database. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint Samples were typically collected, serum separated, and heat-inactivated at the end of 278 each day. Nonetheless, most blood samples can be stored at room temperature in the serum 279 separation tube for several days, in some cases with larger blood volume for up to 1 week. 280 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint Longer than 1 week is not recommended because the blood begins to dry up. Antibody titers 281 after serum separation have been reported as stable for up to 6 weeks when stored at 4 °C 282 (8). Some participants had difficulty collecting their blood by themselves. In such cases, we 283 encouraged participants to visit the nurses in our institute's Health Center for assistance. 284

The results for RBD ELISA step 1 are summarized in Figure 3A perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint costs low. Serum can be separated from whole blood up to 1 week after collection. Provided 349 that temperatures are kept close to ambient temperature, this would allow for shipping of 350 samples from collection site to the testing site by mail. The main bottleneck is serum transfer 351 from collection tube to ELISA plate. This step may be accelerated with a sophisticated 352 integrated liquid handling system. We showed that antibody titers from capillary blood serum 353 are equivalent to titers measured from venous blood (Figure 2) . The Infectious Diseases 354

Society of America Guidelines on the Diagnosis of COVID-19 currently make no 355 recommendations for or against the use of capillary blood in serological assays due to a 356 knowledge gap on the subject (10). We hope that the success of our study begins to fill this 357 knowledge gap, paving the way for further proof-of-concept studies using capillary blood. Our 358 survey revealed that micro blood sampling of capillary blood is a practical, cost-efficient 359 method using as little as one drop of blood (approximately 30 µL) per sample. Such a small 360 sample volume would even allow testing of newborns using neonatal heel prick. 361 362 Anonymization and web-based reporting 363

The web-based platform served as an efficient means to conduct this study anonymously 364 while providing instructions and communicating results to participants. Anonymization was 365 achieved by randomly barcoded samples without the need to enter personal data. From the 366 1,154 staff and students at OIST (as of May 2020), 675 samples were received, of which only 367 41 samples (6.1%) could not be tested due to lack of serum or improper sample collection. 368

The success of our practical web-based method indicates that this method may be used for 369 larger epidemiological studies. 370 371

The serological survey of students and employees carried out at OIST on Okinawa, Japan, in 373

August 2020 revealed no seroconversion among this small population. The test has reliably 374 identified all previously confirmed PCR-positive individuals and controls, with reactivities as 375 high as 7 times threshold. Our results assert strong confidence in this 2-step assay, which has 376 received FDA-emergency approval (2). 377

Okinawa has, thus far, enjoyed moderately low numbers of COVID-19 cases among its 378 population. Other recent serological surveys in Japan, have found seroprevalence as low as 379 0.43 % (among 44,066 employees and business partners of the company Softbank and 380

Accelerated emergency use authorization (EUA) summary COVID-19 ELISA IGG 447 antibody test

A serological assay to detect SARS-CoV-2 seroconversion in humans

MotionCor2: anisotropic correction of beam-induced motion for 452 improved cryo-electron microscopy

New tools for automated high-resolution cryo-EM structure 454 determination in RELION-3

Comparative assessment of multiple COVID-19 serological 456 technologies supports continued evaluation of point-of-care lateral flow assays in 457 hospital and community healthcare settings

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Anti-SARS-CoV -2 Spike Antibodies are Stable in Convalescent 461

Plasma when Stored at 4° Celsius for at Least 6 Weeks

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Angiotensin-converting enzyme 2 is a functional receptor for the SARS 475 coronavirus

Structural biology: Structure of SARS coronavirus 477 spike receptor-binding domain complexed with receptor. Science

Coronavirus membrane 480 fusion mechanism offers a potential target for antiviral development

Dipeptidyl peptidase 4 is a functional receptor for the emerging human 483 coronavirus-EMC

Structure of MERS-CoV spike receptor-binding domain complexed 485 with human receptor DPP4

Cross-reactive Antibody Response between SARS-CoV-2 and SARS-CoV 487

Convalescent plasma anti-SARS-CoV-2 spike protein ectodomain and 489 receptor binding domain IgG correlate with virus neutralization

A neutralizing human antibody binds to the N-terminal domain of the 492

Spike protein of SARS-CoV-2. Science (80-. )

A human SARS-CoV neutralizing antibody against epitope on S2 protein

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint healthcare workers across Japan) (11), 0.1 % (1,971 citizens of Tokyo), 0.03 % (3,009 citizens 381 in Miyagi Prefecture) and 0.17 % (2,970 citizens of Osaka prefecture) (12). At such low 382 seroprevalence, high assay specificity is critical to achieving a high predictive value (13, 14) . 383Although accurate estimation of the actual seroprevalence among OIST staff and students 384 (including the 45 % of employees that did not participate in the survey) is not possible, 385 assuming a seroprevalence between 0.03 % (Tokyo) and 0.43 % (Softbank), with a known 386 assay specificity of close to 100 % and a sensitivity of 92.5 % for the Mt. Sinai Antibody Test 387 used here (9), we estimate the negative predictivity value of our assay to be greater than 388 99.9%. 389 SARS-CoV, SARS-CoV-2 and other human coronaviruses such as NL63 use aceE2 as 390 receptor (15, 16) mediated by the class-1 fusion protein S before entry through the plasma 391 membrane, or via a clathrin-dependent endosomal pathway (17). MERS-CoV utilizes a similar 392 mechanism but uses the dipeptidyl peptidase 4 receptor (dpp4) instead (18). Dpp4 does not 393 share sequence and structural similarity to previously identified human coronavirus receptors 394 such as ACE2 or APN (15, 16, 19) . Convalescent plasma samples from SARS-CoV-infected 395 patients have moderate cross-reactivity with SARS-CoV-2 spike protein, but no cross-396 neutralization (20). The assay used here has been shown to have no cross-reactivity with the 397 seasonal human coronavirus NL63 (3). Interestingly, when we tested human convalescent 398 serum from MERS patients with our ELISA, we found explicit cross-reactivity between MERS 399 serum and SARS-CoV-2 spike with an antibody titer similar to that of SARS-CoV-2 plasma. Our found to be ≥80 % (21). Our data shows strong MERS cross-reactivity with SARS-CoV-2 spike 408 at titers close to 1:1350. Although most effective neutralizing antibodies against 409 coronaviruses target the RBD, neutralizing antibodies against SARS-CoV-2 S1-N-terminal 410 domain (22) and SARS-CoV S2 domain (23) have also been identified. Our data suggests that 411 MERS convalescent serum may also contain such neutralizing antibodies against SARS-CoV-2. 412 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. Plates are modified versions of those described previously (1). 505 506 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209858 doi: medRxiv preprint