key: cord-0686290-ew78trl9
authors: Conzelmann, Carina; Gilg, Andrea; Groß, Rüdiger; Schütz, Desirée; 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: 2020-06-15
journal: bioRxiv
DOI: 10.1101/2020.06.14.150862
sha: 4b0e3fa6b73af1edc2cfc96997d03deb88a05e55
doc_id: 686290
cord_uid: ew78trl9

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 TCID50 of virus stocks, antiviral efficiencies (IC50 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. Highlights 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)

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 directly 35

quantifies the amount of de novo synthesized viral spike protein within fixed and permeabilized cells. This 36 in-cell ELISA enables a rapid and quantitative detection of SARS-CoV-2 infection in microtiter format, 37 regardless of the virus isolate or target cell culture. It follows the established method of performing ELISA 38 assays and does not require expensive instrumentation. Utilization of the in-cell ELISA allows to e.g. 39

determine TCID 50 of virus stocks, antiviral efficiencies (IC 50 values) of drugs or neutralizing activity of sera. 40

Thus, the in-cell spike ELISA represents a promising alternative to study SARS-CoV-2 infection and 41

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 2019 47

(COVID-19) that if symptomatic manifests as fever, cough, and shortness of breath, and can progress to 48 pneumonia, acute respiratory distress syndrome resulting in septic shock, multi-organ failure and death. As 49 of end of May 2020, more than 376,000 deaths worldwide occurred upon SARS-CoV-2 infection which 50

forced governments to implement strict measures of social distancing to limit the spread of the virus but 51 greatly impacted individual freedom and economy. Due to its high transmissibility, without such harsh 52

interventions its pandemic spread is unlikely to be stopped without the cost of a substantial death toll. 53

Therefore, the development of prophylactics or therapeutics against SARS-CoV-2 is imperative. 54 SARS-CoV-2 is a positive-sense single-stranded RNA virus with diameters of 60-140 nanometers (Zhu et 55 al., 2020) . Like other coronaviruses, SARS-CoV-2 has four structural proteins, the S (spike), E (envelope), 56 M (membrane), and N (nucleocapsid) proteins. The S protein is responsible for allowing the virus to attach 57

to and fuse with the membrane of a host cell. It is primed by the transmembrane serine protease 2 58

(TMPRSS2) resulting in interactions of the S1 subunit with the angiotensin converting enzyme 2 (ACE2) 59

and rearrangements in S2 to form a six-helix bundle structure that triggers fusion of the viral with the cellular 60 membrane ( CoV-2 isolates tested and can be easily performed in any format including 96-well plates. It can be used to 87 measure the TCID 50 , to screen for antivirals, and to determine antiviral potencies of drugs (as inhibitory 88

concentration 50), neutralizing sera or antibodies in a timely and cost-effective manner, within only two 89 days. 90 fresh medium was added and the cells were inoculated with 18 µl of a 10-fold titration series of SARS-135

CoV-2. One to three days later, SARS-CoV-2 S protein staining was assessed using an anti-SARS-CoV-2 136 S protein antibody. To this end, cells were fixed by adding 180 µl 8% PFA and 30 min of room temperature 137

incubation. Medium was then discarded and the cells permeabilized for 5 min at room temperature by adding 138 100 µl of 0.1% Triton in PBS. Cells were then washed with PBS and stained with 1:1,000, 1:5,000 or 139 1:10,000 diluted mouse anti-SARS-CoV-2 S protein antibody 1A9 (Biozol GTX-GTX632604) in antibody 140 buffer (PBS containing 10% (v/v) FCS and 0.3% (v/v) Tween 20) at 37°C. After one hour, the cells were 141

washed three times with washing buffer (0.3% (v/v) Tween 20 in PBS) before a secondary anti-mouse or 142

anti-rabbit antibody conjugated with HRP was added (1:10,000, 1:15,000, 1:20,000 or 1:30,000) and 143

incubated for 1 h at 37°C. Following four times of washing, the 3,3',5,5'-tetramethylbenzidine (TMB) 144

peroxidase substrate (Medac #52-00-04) was added. After 5 min light-protected incubation at room 145 temperature, reaction was stopped using 0.5 M H 2 SO 4 . The optical density (OD) was recorded at 450 nm 146 and baseline corrected for 620 nm using the Asys Expert 96 UV microplate reader (Biochrom). 147 seeded in 96 well plates in 100 µl and the next day 62 µl fresh medium was added. The sera were heat-174 inactivated (30 min at 56°C), titrated 2-fold starting with a 5-fold dilution, and mixed 1:1 with SARS-CoV-175

2 France/IDF0372/2020. After 90 min incubation at room temperature, the mix was used to infect the cells 176

with 18 µl in triplicates at a MOI of 0.01. Two days later, SARS-CoV-2 S protein expression was quantified 177 as described above. 178 TCID50 determination by in-cell SARS-CoV-2 ELISA. Vero E6 cells were inoculated as described above 179

for the TCID 50 endpoint titration. Cells were then incubated and CPE development observed by microscopy. 180

At day 4 cells were then fixed (8% PFA), permeabilized (0.1% Triton), stained (1:5,000 1A9; 1:15,000 anti-181 mouse-HRP), visualized (TMB) and detected in a microplate reader as described above. Infected wells were 182 defined as having a higher signal than the uninfected control plus three times the standard deviation.

TCID 50 /ml was calculated as described. 184

Cell viability assay. The effect of investigated compounds on the metabolic activity of the cells was 185 analyzed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega #G7571). Metabolic 186 activity was examined under conditions corresponding to the respective infection assays. The CellTiter-187

Glo® assay was performed according to the manufacturer's instructions. Briefly, medium was removed 188 from the culture after 2 days of incubation and 50% substrate reagent in PBS was added. After 10 min, the 189 supernatant was transferred into white microtiter plates and luminescence measured in an Orion II 190

Microplate Luminometer (Titertek Berthold). Untreated controls were set to 100% viability. diisopropylethylamine. The coupling reaction was performed with microwaves in a few minutes followed 197 by a DMF wash. Once the synthesis was completed, the peptide was cleaved in 95% trifluoroacetic acid, 198

2.5% triisopropylsilane, and 2.5% H2O for one hour. The peptide residue was precipitated and washed with 199 cold diethyl ether and allowed to dry under vacuum to remove residual ether. The peptide was purified using 200 reversed phase preparative high-performance liquid chromatography (HPLC; Waters) in an 201

acetonitrile/water gradient under acidic conditions on a Phenomenex C18 Luna column (5 mm pore size, 202

100 Å particle size, 250 -21.2 mm). Following purification, the peptide was lyophilized on a freeze dryer 203

(Labconco) for storage prior to use. The purified peptide mass was verified by liquid chromatography mass 204 spectroscopy (LCMS; Waters). and Caco-2 (heterogeneous human epithelial colorectal adenocarcinoma cells), were seeded in 96-well 219

plates and inoculated with increasing multiplicities of infection (MOIs) of a SARS-CoV-2 isolate from 220

France (BetaCoV/France/IDF0372/2020). After 2, 24, 48 or 72 hours, cells were fixed, permeabilized, and 221 stained with 1:1,000, 1:5,000, or 1:10,000 dilutions of the anti-SARS-CoV-2 S protein antibody 1A9 for 1 222

hour. After washing, a 1:20,000 dilution of a secondary HRP-coupled anti-mouse antibody was added, cells 223

were incubated for 1 hour, washed again before TMB peroxidase substrate was added. After 5 min, reaction 224 was stopped using H2SO4 and optical density (OD) recorded at 450 nm and baseline corrected for 620 nm 225 using a microplate reader (Biochrom). 226

Already at day 1, we observed a significant increase in ODs upon infection with the highest MOI in Vero 227 E6 (Fig. 1a) and Caco-2 (Fig. 1b) with a MOI ≥ 0.05 (Fig. 1c) , in Caco-2 cells already highly significant with a MOI of ≥ 0.008 (Fig. 1d) and 239

in Calu-3 cells at a MOI of 0.014 (Fig. 1e) . Thus, under these experimental conditions, Caco-2 and Calu-3 240 cells allow a more sensitive detection of SARS-CoV-2 infection and replication as Vero E6 cells. 241

To optimize assay sensitivity, i.e. the signal-to-noise (S/N) ratio, we evaluated different secondary antibody 242

dilutions. For this, Caco-2 cells were inoculated with SARS-CoV-2 (MOIs of 0.0002 to 0.05), fixed at day 243 2, and stained with the anti-S protein antibody. Thereafter, four different dilutions of the HRP-coupled 244 secondary antibody were added. OD measurements revealed that highest ODs were obtained with 10,000-245 fold diluted secondary antibody (Fig. 1f) . However, when calculating the S/N ratios (OD of infected wells 246 divided by OD of uninfected cells), also the 15,000-fold dilution revealed a similar assay sensitivity with 247 maximum S/N values of 7.9 as compared to 7.5 for the 1:10,000 dilution (Fig. 1g) . Thus, all subsequent 248 experiments were performed in Caco-2 cells that were seeded at a density of 30,000 cells per well to increase 249

ODs, and stained with 5,000-fold diluted anti-S and 15,000-fold diluted secondary antibodies. 250 Netherlands/01 isolate as well as two isolates from Ulm, Southern Germany. Intracellular S protein 270 expression was determined 2 days later by in-cell ELISA. As shown in Fig. 2 

detectable even upon infection with very low MOIs, suggesting that the ELISA may be applied to all SARS-272

CoV-2 isolates. 273 

Results shown in Fig. 2 indicate that the assay allows to detect infected wells even after inoculation with 279

very low viral MOIs, e.g. a MOI of 0.0003 of the Ulm/01/2020 isolate resulted in a significantly increased 280

OD as compared to uninfected controls. We were wondering whether this high sensitivity and ease of 281 quantitation may also allow to determine the TCID 50 of virus stocks, that is usually done on Vero E6 cells 282 by manually counting infected wells using a microscope. To test this, we titrated virus, inoculated Vero E6 283 cells and incubated them for 4 days. We identified infected wells by eye (Fig. 3a) , but also performed the 284 in-cell ELISA and set a threshold of three times the standard deviation above the uninfected control to 285 determine the number of infected wells per virus dilution (Fig. 3b) . The subsequent calculation of TCID 50 /ml 286 by Reed and Muench revealed exactly the same viral titer for the in-cell ELISA (Fig. 3b ) as for microscopic 287 evaluation (Fig. 3a) showing that the established ELISA is suitable for determination of viral titers. 288 with SARS-CoV-2. In-cell ELISAs performed 2 days later demonstrated a concentration-dependent 314 antiviral activity of the tested compounds reflecting typical dose-response curves of antiviral agents 315 (Fig. 5a-d) . This also allowed the calculation of the inhibitory concentration 50 (IC 50 ) values, i.e. 23.9 µM 316

for chloroquine (Fig. 5a) , 21.0 µM for lopinavir (Fig. 5b) , 32.4 nM for remdesivir (Fig. 5c) , and 303.5 nM 317

for EK1 (Fig. 5d) . demonstrate that the in-cell S protein ELISA can be easily adapted to determine antiviral activities of 321 candidate drugs. Cytotoxicity assays that were performed simultaneously in the absence of virus revealed 322 no effects on cell viability by antivirally active concentrations of lopinavir, remdesivir and EK1 (Fig. 5b-d) .

However, reduced cellular viability rates were observed in the presence of chloroquine concentrations >1 324 µM (Fig. 5a) 

Finally, we evaluated whether the assay determines the neutralization activity of serum from SARS-CoV-2 335 convalescent individuals. For this, sera that were tested positive or negative for anti-SARS-CoV-2 336

immunoglobulins, were serially titrated and incubated with SARS-CoV-2 for 90 minutes at room 337 temperature before inoculation of Caco-2 cells. Two days later, we performed the in-cell ELISA as 338

described. As shown in Fig. 6 , the two control sera, that were obtained before the COVID-19 outbreak or 339

shown to contain no SARS-CoV-2 immunoglobulins, did not affect infection. In contrast, both COVID-19 340 sera neutralized SARS-CoV-2 infection (Fig. 6) . Serum 1 resulted in a more than 50% inhibition at a titer 341 of 640 and Serum 2 already neutralized SARS-CoV-2 at the 1,280-fold dilution. This confirms that the in-342

cell ELISA is suitable to detect neutralizing sera. Furthermore, analogous to the IC50, we calculated the 343 "inhibitory titers 50" using nonlinear regression, and determined titers of 654 and 1,076 respectively. These 344 titers corresponded well to the presence of immunoglobulins which suggests that the here established 345 method can be used to detect and quantify the neutralizing capacities of sera from COVID-19 patients. 346 in-cell ELISA is easy to perform and follows standard ELISA readouts using HRP-mediated TMB substrate 368

conversion and OD measurements after acidification with no need for expensive equipment. 369

Notably, the assay has been developed to be carried out in microtiter plates and should allow a convenient 370 medium-to-high throughput testing of antivirals, antibodies, or antisera with timely availability of results, 371

which is in the fast development of antivirals and in diagnostics. Due to targeting a highly conserved region 372 and the relatively high sequence homology of global SARS-CoV-2 isolates, it is also applicable to other 373

isolates as those that were tested herein. Furthermore, conservation in between related viruses suggest that, Vero E6 and Caco-2 cells and confirmed using Calu-3 cells, but principally all other cell lines or primary 377 cells supporting productive SARS-CoV-2 infection may also be used. In addition, SARS-CoV-2 is a BSL-378 3 pathogen which requires high safety requirements, which are usually at the expense of throughput. One 379 additional advantage of the in-cell ELISA is that treatment of cells with paraformaldehyde results in the 380 fixation and inactivation of virions, allowing a downstream processing of the plates outside a BSL-3 facility. 381

Another application of the in-cell S protein ELISA is to reliably determine infectious viral titres in virus 382 stocks, cell culture supernatants or from patient swabs. Viral titers are usually quantified by limiting dilution 383

analysis and microscopic determination of infected wells or staining of SARS-CoV-2 induced plaques or 384

foci with crystal violet, neutral red or specific antibodies for SARS-CoV-2 antigens. We found that the in-385

cell ELISA allows to i) discriminate infected from uninfected wells, and ii) even after infection with very 386

low MOIs (as low as 0.000005, which corresponds to one virion per three wells) at 4 days post infection 387 (Fig. 3) , representing an alternative for non-biased determining the TCID50 without the need of counting 388

infected wells or plaques. 389

Conclusively, the S protein specific in-cell ELISA quantifies SARS-CoV-2 infection rates of different cell 390

lines and allows to rapidly screen for and determine the potency of antiviral compounds. Thus, it represents 391 a promising, rapid, readily available and easy to implement alternative to the current repertoire of laboratory 392

techniques studying SARS-CoV-2 and will facilitate future research and drug development on COVID-19. 

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