key: cord-0949111-4bai2wg7
authors: Conzelmann, Carina; Gilg, Andrea; Groß, Rüdiger; Schütz, Desiree; Preising, Nico; Ständker, Ludger; Jahrsdörfer, Bernd; Schrezenmeier, Hubert; Sparrer, Konstantin M.J.; Stamminger, Thomas; Stenger, Steffen; Münch, Jan; Müller, Janis A.
title: An enzyme-based immunodetection assay to quantify SARS-CoV-2 infection
date: 2020-07-29
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2020.104882
sha: 14775fc7745ca44ea6c00d0ed8dcb8a0e4d7ff63
doc_id: 949111
cord_uid: 4bai2wg7

SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These methods are laborious, time-consuming and expensive and therefore not suitable for a high sample throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. determine TCID(50) of virus stocks, antiviral efficiencies (IC(50) values) of drugs or neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and inhibition and may facilitate future research.

spike protein in bulk cell cultures 23

• Targeting a highly conserved region in the S2 subunit of the S protein allows broad detection of 24 several SARS-CoV-2 isolates in different cell lines 25

• Screening of antivirals in microtiter format and determining the antiviral activity as inhibitory 26

concentrations 50 (IC 50 ) 27 28

Abstract: 29 SARS-CoV-2 is a novel pandemic coronavirus that caused a global health and economic crisis. The 30 development of efficient drugs and vaccines against COVID-19 requires detailed knowledge about SARS-31

CoV-2 biology. Several techniques to detect SARS-CoV-2 infection have been established, mainly based 32 on counting infected cells by staining plaques or foci, or by quantifying the viral genome by PCR. These 33 methods are laborious, time-consuming and expensive and therefore not suitable for a high sample 34 throughput or rapid diagnostics. We here report a novel enzyme-based immunodetection assay that 35

directly quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized 36

cells. This in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter 37

format, regardless of the virus isolate or target cell culture. It follows the established method of 38

performing ELISA assays and does not require expensive instrumentation. Utilization of the in-cell 39 ELISA allows to e.g. determine TCID 50 of virus stocks, antiviral efficiencies (IC 50 values) of drugs or 40 neutralizing activity of sera. Thus, the in-cell spike ELISA represents a promising alternative to study 41 SARS-CoV-2 infection and inhibition and may facilitate future research. 42 43 44

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel human pathogen 46

at the end of 2019 and spread around the globe within three months. It causes the coronavirus disease 47 2019 (COVID-19) that if symptomatic manifests as fever, cough, and shortness of breath, and can 48 progress to pneumonia, acute respiratory distress syndrome resulting in septic shock, multi-organ failure 49

and death. As of end of June 2020, more than 500,000 deaths worldwide occurred upon SARS-CoV-2 50

infection which forced governments to implement strict measures of social distancing to limit the spread 51 of the virus but greatly impacted individual freedom and economy. Due to its high transmissibility, 52 without such harsh interventions its pandemic spread is unlikely to be stopped without the cost of a 53 substantial death toll. Therefore, the development of prophylactics or therapeutics against SARS-CoV-2 is 54

imperative. 55 SARS-CoV-2 is a positive-sense single-stranded RNA virus with diameters of 60-140 nanometers (Zhu et 56 al., 2020) . Like other coronaviruses, SARS-CoV-2 has four structural proteins, the S (spike), E (envelope), 57 M (membrane), and N (nucleocapsid) proteins. The S protein is responsible for allowing the virus to 58

attach to and fuse with the membrane of a host cell. It is primed by the transmembrane serine protease 2 59

(TMPRSS2) resulting in interactions of the S1 subunit with the angiotensin converting enzyme 2 (ACE2) 60

and rearrangements in S2 to form a six-helix bundle structure that triggers fusion of the viral with the 61 cellular membrane (Hoffmann et cells were seeded in 24-well plates in 500 µl medium incubated over night at 37°C. The next day, medium 111 was replaced by 400 µl of 2.5 µg/ml amphotericin B containing medium. Then, 100 µl of throat swabs that 112

were tested positive for SARS-CoV-2 by qRT-PCR were titrated 5-fold on the cells and incubated for 3 to 113 5 days. Upon visible CPE, supernatant was taken and virus expanded by inoculation of Vero E6 cell in 75 114 cm² flasks and propagated as above described, resulting in the two viral isolates 115

BetaCoV/Germany/Ulm/01/2020 and BetaCoV/Germany/Ulm/02/2020. 116 Plaque assay. To determine plaque forming units (PFU), SARS-CoV-2 stocks were serially diluted 10-117

fold and used to inoculate Vero E6 cells. To this end, 800,000 Vero E6 cells were seeded per 12 well in 1 118 ml medium and cultured overnight to result in a 100% confluent cell monolayer. Medium was removed, 119 cells were washed once with PBS and 400 µl PBS were added. Cells were then inoculated with 100 µl of 120 titrated SARS-CoV-2 and incubated for 1 to 3 h at 37°C with shaking every 15 to 30 min. Next, cells were 121 overlayed with 1.5 ml of 0.8% Avicel RC-581 (FMC Corporation) in medium and incubated for 3 days. 122

Cells were fixed by adding 1 ml 8% paraformaldehyde (PFA) and incubation at room temperature for 45 123 min. Supernatant was discarded, cells were washed with PBS once, and 0.5 ml of staining solution (0.5% 124 crystal violet and 0.1% Triton in water) was added. After 20 min incubation at room temperature, the 125 staining solution was washed off with water, virus-induced plaques were counted, and PFU per ml 126 calculated. Based on the applied PFU per cell the MOIs were calculated. 127 TCID 50 endpoint titration. To determine the tissue culture infectious dose 50 (TCID 50 ), SARS-CoV-2 128 stocks were serially diluted 10-fold and used to inoculate Vero E6 or Caco-2 cells. To this end, 6,000 Vero 129 E6 or 10,000 Caco-2 cells were seeded per well in 96 flat bottom well plates in 100 µl medium and 130 incubated over night before 62 µl fresh medium was added. Next, 18 µl of titrated SARS-CoV-2 of each 131 dilution was used for inoculation, resulting in final SARS-CoV-2 dilutions of 1:10 1 to 1:10 9 on the cells in 132

sextuplicates. Cells were then incubated for 5 days and monitored for CPE. TCID 50 /ml was calculated 133

according to Reed and Muench. 134

6,000 Vero E6 or 10,000 Caco-2 target cells were seeded in 96 well plates in 100 µl. The next day, 62 µl 136 fresh medium was added and the cells were inoculated with 18 µl of a 10-fold titration series of SARS-137

CoV-2. One to three days later, SARS-CoV-2 S protein staining was assessed using an anti-SARS-CoV-2 138 S protein antibody. To this end, cells were fixed by adding 180 µl 8% PFA and 30 min of room 139 temperature incubation. Medium was then discarded and the cells permeabilized for 5 min at room 140 temperature by adding 100 µl of 0.1% Triton in PBS. Cells were then washed with PBS and stained with 141 1:1,000, 1:5,000 or 1:10,000 diluted mouse anti-SARS-CoV-2 S protein antibody 1A9 (Biozol GTX-142 GTX632604) in antibody buffer (PBS containing 10% (v/v) FCS and 0.3% (v/v) Tween 20) at 37°C. After 143 one hour, the cells were washed three times with washing buffer (0.3% (v/v) Tween 20 in PBS) before a 144 secondary anti-mouse or anti-rabbit antibody conjugated with HRP was added (1:10,000, 1:15,000, 145

1:20,000 or 1:30,000) and incubated for 1 h at 37°C. Following four times of washing, the 3,3',5,5'-146 tetramethylbenzidine (TMB) peroxidase substrate (Medac #52-00-04) was added. After 5 min light-147

protected incubation at room temperature, reaction was stopped using 0.5 M H 2 SO 4 . The optical density 148

(OD) was recorded at 450 nm and baseline corrected for 620 nm using the Asys Expert 96 UV microplate 149 reader (Biochrom). 150

applied to analyze SARS-CoV-2 infection and inhibition. See Table I for reagents. To determine SARS-152

CoV-2 infection, 12,000 Vero E6 or 30,000 Caco-2 target cells were seeded in 96 well plates in 100 µl. 153

The next day, fresh medium and the respective compound of interest (chloroquine (Sigma-Aldrich 154 #C6628); lopinavir (Selleck Chemicals #S1380); EK1 (Core Facility Functional Peptidomics, Ulm); 155 remdesivir (Selleck Chemicals #S8932)) was added and the cells inoculated with the desired multiplicity (v/v) Tween 20 in PBS) before a secondary anti-mouse antibody conjugated with HRP (Thermo Fisher 164 #A16066) was added (1:15,000) and incubated for 1 h at 37°C. Following four times of washing, the TMB 165 peroxidase substrate (Medac #52-00-04) was added. After 5 min light-protected incubation at room 166 temperature, reaction was stopped using 0.5 M H 2 SO 4 . The optical density (OD) was recorded at 450 nm 167 and baseline corrected for 620 nm using the Asys Expert 96 UV microplate reader (Biochrom). Values 168

were corrected for the background signal derived from uninfected cells and untreated controls were set to 169 100% infection. seeded in 96 well plates in 100 µl and the next day 62 µl fresh medium was added. The sera were heat-180 inactivated (30 min at 56°C), titrated 2-fold starting with a 5-fold dilution, and mixed 1:1 with SARS-181

CoV-2 France/IDF0372/2020. After 90 min incubation at room temperature, the mix was used to infect 182 the cells with 18 µl in triplicates at a MOI of 0.01. Two days later, SARS-CoV-2 S protein expression was 183 quantified as described above. 184 TCID 50 determination by in-cell SARS-CoV-2 ELISA. Vero E6 cells were inoculated as described 185

above for the TCID 50 endpoint titration. Cells were then incubated and CPE development observed by 186 microscopy. At day 4 cells were then fixed (8% PFA), permeabilized (0.1% Triton), stained (1:5,000 1A9; 187 1:15,000 anti-mouse-HRP), visualized (TMB) and detected in a microplate reader as described above.

Infected wells were defined as having a higher signal than the uninfected control plus three times the 189 standard deviation. TCID 50 /ml was calculated as described. Glo® assay was performed according to the manufacturer's instructions. Briefly, medium was removed 194 from the culture after 2 days of incubation and 50% substrate reagent in PBS was added. After 10 min, the 195 supernatant was transferred into white microtiter plates and luminescence measured in an Orion II 196

Microplate Luminometer (Titertek Berthold). Untreated controls were set to 100% viability. (BetaCoV/France/IDF0372/2020). After 2, 24, 48 or 72 hours, cells were fixed, permeabilized, and stained 252 with 1:1,000, 1:5,000, or 1:10,000 dilutions of the anti-SARS-CoV-2 S protein antibody 1A9 for 1 hour. 253

After washing, a 1:20,000 dilution of a secondary HRP-coupled anti-mouse antibody was added, cells 254

were incubated for 1 hour, washed again before TMB peroxidase substrate was added. After 5 min, 255 reaction was stopped using H 2 SO 4 and optical density (OD) recorded at 450 nm and baseline corrected for 256 620 nm using a microplate reader (Biochrom) (see listed reagents in table I) . 257

Already at day 1, we observed a significant increase in ODs upon infection with the highest MOI in Vero 258 E6 (Fig. 1a) and Caco-2 (Fig. 1b) after infection with a MOI ≥ 0.05 (Fig. 1c) , in Caco-2 cells already highly significant with a MOI of ≥ 270 0.008 (Fig. 1d ) and in Calu-3 cells at a MOI of 0.014 (Fig. 1e) . Thus, under these experimental conditions, 271

Caco-2 and Calu-3 cells allow a more sensitive detection of SARS-CoV-2 infection and replication as 272

Vero E6 cells. 273

To optimize assay sensitivity, i.e. the signal-to-noise (S/N) ratio, we evaluated different secondary 274 antibody dilutions. For this, Caco-2 cells were inoculated with SARS-CoV-2 (MOIs of 0.0002 to 0.05), 275

fixed at day 2, and stained with the anti-S protein antibody. Thereafter, four different dilutions of the 276 HRP-coupled secondary antibody were added. OD measurements revealed that highest ODs were obtained 277 with 10,000-fold diluted secondary antibody (Fig. 1f) . However, when calculating the S/N ratios (OD of 278 infected wells divided by OD of uninfected cells), also the 15,000-fold dilution revealed a similar assay 279 sensitivity with maximum S/N values of 7.9 as compared to 7.5 for the 1:10,000 dilution (Fig. 1g) . Thus, 280

all subsequent experiments were performed in Caco-2 cells that were seeded at a density of 30,000 cells 281

per well to increase ODs, and stained with 5,000-fold diluted anti-S and 15,000-fold diluted secondary 282 antibodies. A schematic illustration of the final protocol is shown in Fig. 2 . 

(1:20,000) or 1.7 ng/well (1:30,000). Caco-2 cells infected with indicated MOIs of SARS-CoV-2 and stained 2 days 292 later with anti-S protein antibody were treated with four dilutions of the HRP-coupled secondary antibody before OD 293 was determined. g) Corresponding maximum signal-to-noise (S/N) ratios observed in Fig. 1f . All values show in 294 panels a-e are means of raw data obtained from technical triplicates ± sd. ns not significant, * P < 0.01, ** P < 0.001, 295 *** P < 0.0001 (by one-way ANOVA with Bonferroni's post-test). 

Having demonstrated that the in-cell ELISA quantifies infection by a French SARS-CoV-2 isolate, we 306

wanted to validate that isolates from other geographic areas are also detected. The French isolate clusters 307

with the reference Wuhan-Hu-1/2019 isolate whereas the Netherlands/01 strain can be grouped to clade 308

A2a (www.nextstrain.org (Hadfield et al., 2018) ). The antibody-targeted S2 domain is generally conserved 2018)) which allows detection of the S protein of the France/IDF0372/2020 as well as the Netherlands/01 311 isolate and two isolates from Ulm, Southern Germany (Fig. S1 ). To test the performance of the in-cell 312 ELISA, Caco-2 cells were inoculated with increasing MOIs of the isolates and intracellular S protein 313 expression was determined 2 days later. As shown in Fig. 3 , virus infection was readily detectable even 314 upon infection with very low MOIs, suggesting that the ELISA may be applied to all SARS-CoV-2 315

isolates. 316 317 Fig. 3 . The in-cell S protein ELISA detects SARS-CoV-2 isolates from different geographic regions. Caco-2 318 cells were infected with increasing MOIs of three SARS-CoV-2 isolates and intracellular S protein expression was 319 quantified 2 days later by in-cell ELISA. Data shown represent means of raw data obtained from technical triplicates 320 ± sd. ns not significant, ** P < 0.001, *** P < 0.0001 (by one-way ANOVA with Bonferroni's post-test).

Results shown in Fig. 3 indicate that the assay allows to detect infected wells even after inoculation with 322

very low viral MOIs, e.g. a MOI of 0.0003 of the Ulm/01/2020 isolate resulted in a significantly increased 323

OD as compared to uninfected controls. We were wondering whether this high sensitivity and ease of 324 quantitation may also allow to determine the TCID 50 of virus stocks, that is usually done on Vero E6 cells 325 by manually counting infected wells using a microscope. To test this, we titrated virus, inoculated Vero E6 326 cells and incubated them for 4 days. We identified infected wells by eye (Fig. 4a) , but also performed the 327 in-cell ELISA and set a threshold of three times the standard deviation above the uninfected control to 328 determine the number of infected wells per virus dilution (Fig. 4b) . The subsequent calculation of 329 TCID 50 /ml by Reed and Muench revealed exactly the same viral titer for the in-cell ELISA (Fig. 4b) as for 330 microscopic evaluation (Fig. 4a) showing that the established ELISA is suitable for determination of viral 331

titers. 332 dependent antiviral activity of the tested compounds reflecting typical dose-response curves of antiviral 345 agents (Fig. 5a-d) . This also allowed the calculation of the inhibitory concentration 50 (IC 50 ) values, i.e. 346

23.9 µM for chloroquine (Fig. 5a) , 21.0 µM for lopinavir (Fig. 5b) , 32.4 nM for remdesivir (Fig. 5c) , and 347 303.5 nM for EK1 (Fig. 5d) . These values are in the same range as previously reported for Vero E6 cells 348

( al., 2020a)), and demonstrate that the in-cell S protein ELISA can be easily adapted to determine antiviral 351 activities of candidate drugs. Cytotoxicity assays that were performed simultaneously in the absence of 352 virus revealed no effects on cell viability by antivirally active concentrations of lopinavir, remdesivir and 353 EK1 (Fig. 5b-d) . However, reduced cellular viability rates were observed in the presence of chloroquine 354

concentrations >1 µM (Fig. 5a) , which is in line with the fact that part of the anti-SARS-CoV-2 activity of 355 this anti-malaria drug is attributed to its interference with cell organelle function 

Finally, we evaluated whether the assay determines the neutralization activity of serum from SARS-CoV-366 2 convalescent individuals. For this, sera that were tested positive or negative for anti-SARS-CoV-2 367

immunoglobulins, were serially titrated and incubated with SARS-CoV-2 for 90 minutes at room 368 temperature before inoculation of Caco-2 cells. Two days later, we performed the in-cell ELISA as 369

described. As shown in Fig. 6 , the two control sera, that were obtained before the COVID-19 outbreak or 370

shown to contain no SARS-CoV-2 immunoglobulins, did not affect infection. In contrast, both COVID-19 371 sera neutralized SARS-CoV-2 infection (Fig. 6 ). Serum 1 resulted in a more than 50% inhibition at a titer 372 of 640 and Serum 2 already neutralized SARS-CoV-2 at the 1,280-fold dilution. This confirms that the in-373

cell ELISA is suitable to detect neutralizing sera. Furthermore, analogous to the IC 50 , we calculated the 374 "inhibitory titers 50" using nonlinear regression, and determined titers of 654 and 1,076 respectively. 375

These titers corresponded well to the presence of immunoglobulins which suggests that the here 376 established method can be used to detect and quantify the neutralizing capacities of sera from COVID-19 377 patients. 378 

We here describe a novel assay that allows quantification of SARS-CoV-2 infection by measuring 389 intracellular levels of the viral S protein in bulk cell cultures. The assay is based on the detection of de 390 novo synthesized S protein by a S2-targeting antibody, and quantification via a corresponding secondary 391

horseradish peroxidase (HRP) -linked antibody. This more sensitively detects nuances of viral replication 392 than counting infected cells and is faster than determining titers of progeny virus. At high viral input (e.g.

MOI 3), infection can already be detected after 24 hours, and at low viral input (e. To exclude such misconceptions, we in parallel performed viability assays under the same conditions but 411

in the absence of virus. 412

Notably, the assay has been developed to be carried out in microtiter plates and should allow a convenient 413 medium-to-high throughput testing of antivirals, antibodies, or antisera with timely availability of results, 414

which is in the fast development of antivirals and in diagnostics. We chose the SARS-CoV-2 S protein 415 antibody as it specifically detects this viral antigen in western blotting (Fig. S1) , was verified by 416 fluorescence microscopy (Zheng et al., 2020) , and gave low background signals in the in-cell ELISA. Due 417 to targeting a highly conserved region and the relatively high sequence homology of global SARS-CoV-2 418

isolates, it is also applicable to other isolates as those that were tested herein. Furthermore, conservation in 419 between related viruses suggest that, also SARS-CoV, and related civet SARS-CoV and bat SARS-like 420 coronavirus infection can be detected with this assay (Ng et al., 2014; Walls et al., 2020) . The in-cell 421

ELISA was established using permissive Vero E6 and Caco-2 cells and confirmed using Calu-3 cells, but 422 principally all other cell lines or primary cells supporting productive SARS-CoV-2 infection may also be 423

used. In addition, SARS-CoV-2 is a BSL-3 pathogen which requires high safety requirements, which are 424

usually at the expense of throughput. One additional advantage of the in-cell ELISA is that treatment of 425 cells with paraformaldehyde results in the fixation and inactivation of virions, allowing a downstream 426 processing of the plates outside a BSL-3 facility. 427

Another application of the in-cell S protein ELISA is to reliably determine infectious viral titres in virus 428 stocks, cell culture supernatants or from patient swabs. Viral titers are usually quantified by limiting 429 dilution analysis and microscopic determination of infected wells or staining of SARS-CoV-2 induced 430 plaques or foci with crystal violet, neutral red or specific antibodies for SARS-CoV-2 antigens. We found 431

that the in-cell ELISA allows to i) discriminate infected from uninfected wells, and ii) even after infection 432

with very low MOIs (as low as 0.000005, which corresponds to one virion per three wells) at 4 days post 433 infection (Fig. 4) , representing an alternative for non-biased determining the TCID 50 without the need of 434 counting infected wells or plaques. 435

Conclusively, the S protein specific in-cell ELISA quantifies SARS-CoV-2 infection rates of different cell 436

lines and allows to rapidly screen for and determine the potency of antiviral compounds. Thus, it 437

represents a promising, rapid, readily available and easy to implement alternative to the current repertoire 438 of laboratory techniques studying SARS-CoV-2 and will facilitate future research and drug development 439

on COVID-19. 440 441

Raw data is available upon request. 443 444

This project has received funding from the European Union's Horizon 447 2020 research and innovation programme under grant agreement No 101003555 (Fight-nCoV) to J.M

is indebted to the Baden-Württemberg Stiftung for the financial support of this research 450 project by the Eliteprogramme for Postdocs. C

Inactivation of Zika virus in plasma with 457 amotosalen and ultraviolet A illumination

Stability of SARS-CoV-2 in different environmental conditions

Comparative tropism, replication kinetics, and cell damage profiling of SARS-465

CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory 466 studies of COVID-19: an observational study

Storage-Dependent Generation of Potent Anti-ZIKV Activity in Human Breast Milk

NextStrain: Real-time tracking of pathogen evolution

Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease 477 Inhibitor

Identification of antiviral 479 drug candidates against SARS-CoV-2 from FDA-approved drugs

Inactivation of severe acute 482 respiratory syndrome coronavirus 2 in plasma and platelet products using a riboflavin and ultraviolet 483 light-based photochemical treatment

Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 486 infection in vitro

Potential therapeutic effects of dipyridamole in the 490 severely ill patients with COVID-19

Liu Shen capsule shows antiviral and anti-inflammatory abilities 494 against novel coronavirus SARS-CoV-2 via suppression of NF-κB signaling pathway

Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live 498 virus micro-neutralization assay in human serum samples

Chloroquine inhibits autophagic flux by decreasing autophagosome-502 lysosome fusion

Inhibition of SARS-CoV-2 Infections in 506 Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2

Semen inhibits Zika virus infection of cells and tissues from the anogenital region

Development of a high-throughput 515 colorimetric Zika virus infection assay

Substitution at 518 aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-519 targeting neutralizing monoclonal antibody

Characterization of spike glycoprotein 523 of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

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Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus 533 (SARS-CoV-2)

Structure, Function, 535 and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

A human monoclonal antibody blocking 539 SARS-CoV-2 infection

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) 542 in vitro

Structural and Functional Basis of SARS-CoV-2 Entry by Using 544 Human ACE2

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection 547 by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high 548 capacity to mediate membrane fusion

550 Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein

Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome 555 Coronavirus 2 (SARS-CoV-2)

Monoclonal antibodies for the 558 S2 subunit of spike of SARS-CoV cross-react with the newly-emerged SARS-CoV-2

A Novel Coronavirus from 562 Patients with Pneumonia in China

Western blot of SARS-CoV-2 infected cell lysates. Caco-2 cells were mock-infected or infected with four 568

SARS-CoV-2 isolates (MOI 0.1) and 2 days later cell lysates were harvested. Lysates were used for western blot 569 analysis for detection of SARS-CoV-2 S protein (S2 subunit ~77 kDa

SARS-CoV-2 S protein antibody 1A9 and the housekeeping protein β-actin using a mouse anti-β-actin antibody

445

• Determination of SARS-CoV-2 infection by enzymatically quantifying the expression of viral spike protein in bulk cell cultures • Targeting a highly conserved region in the S2 subunit of the S protein allows broad detection of several SARS-CoV-2 isolates in different cell lines • Screening of antivirals in microtiter format and determining the antiviral activity as inhibitory concentrations 50 (IC50)