key: cord-266903-lxtxqdst
authors: Lee, Jong-Hwan; Choi, Minsuk; Jung, Yujin; Lee, Sung Kyun; Lee, Chang-Seop; Kim, Jung; Kim, Jongwoo; Kim, Nam Hoon; Kim, Bum-Tae; Kim, Hong Gi
title: A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)
date: 2020-10-15
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2020.112715
sha: 
doc_id: 266903
cord_uid: lxtxqdst

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a newly emerging human infectious disease. Because no specific antiviral drugs or vaccines are available to treat COVID-19, early diagnostics, isolation, and prevention are crucial for containing the outbreak. Molecular diagnostics using reverse transcription polymerase chain reaction (RT-PCR) are the current gold standard for detection. However, viral RNAs are much less stable during transport and storage than proteins such as antigens and antibodies. Consequently, false-negative RT-PCR results can occur due to inadequate collection of clinical specimens or poor handling of a specimen during testing. Although antigen immunoassays are stable diagnostics for detection of past infection, infection progress, and transmission dynamics, no matched antibody pair for immunoassay of SARS-CoV-2 antigens has yet been reported. In this study, we designed and developed a novel rapid detection method for SARS-CoV-2 spike 1 (S1) protein using the SARS-CoV-2 receptor ACE2, which can form matched pairs with commercially available antibodies. ACE2 and S1-mAb were paired with each other for capture and detection in a lateral flow immunoassay (LFIA) that did not cross-react with SARS-CoV Spike 1 or MERS-CoV Spike 1 protein. The SARS-CoV-2 S1 (<5 ng of recombinant proteins/reaction) was detected by the ACE2-based LFIA. The limit of detection of our ACE2-LFIA was 1.86 × 10(5) copies/mL in the clinical specimen of COVID-19 Patients without no cross-reactivity for nasal swabs from healthy subjects. This is the first study to detect SARS-CoV-2 S1 antigen using an LFIA with matched pair consisting of ACE2 and antibody. Our findings will be helpful to detect the S1 antigen of SARS-CoV-2 from COVID-19 patients.

1 2.1 Enzyme-linked immunosorbent assay 2 MaxiSorp immunoplates (ThermoFisher SCIENTIFIC, MA, USA) were coated overnight at 4°C with 3 varying amounts (200, 50, 12.5, 3.13, 0.78, 0.2, 0 .05, and 0 ng/mL) of SARS-CoV-2 S1 protein (S1 4 subunit, Sino Biological, Beijing, China) in 100 µL coating buffer per well.

The immunoplates were blocked for 1 hour with blocking buffer (Cat. No. DS98200; Invitrogen, CA, 6 USA), and then hACE2, CR3022, F26G19, or S1-mAb in 100 µL blocking buffer was added to each Fc-tag tagged human ACE2 (ACE2, Cat. No. 10108-H05H), SARS-CoV-2 spike monoclonal antibody 18 (S1-mAb, Cat. No. 40150-R007), SARS-CoV-2 spike protein (S1 subunit, Cat. No. 40591-V08H), 19 and SARS-CoV-2 RBD were purchased from Sino Biological. CR3022 and 20 F26G19 were purchased from Abclon (Seoul, Korea). Plasmids encoding heavy and light chains of 21 each antibody at a 1:6 ratio were transiently cotransfected into 293-F cells using PEI reagent 22 (PolyScience, PA, USA). Six days after transfection, the supernatant was collected, and CR3022 and 23 F26G19 were purified on Protein A columns (GE Healthcare, IL, USA). Binding affinities between conjugated CNB was approximately 0.1%. The exact concentration of the CNB was calculated by 1 measuring the maximum absorbance at 554 nm by UV-vis spectrophotometry (Synergy H1; BioTek). amino-2-methyl-1-propanol (pH 9.0), 0.5% BSA, 0.5% β-Lactose, 0.05% Triton X-100, and 0.05% 10 sodium azide) was sprayed on the conjugate pad, followed by incubation for 1 hour at 37°C in a 11 vacuum oven (FDU-1200, EYELA, Tokyo, Japan; JSVO-30T, JSR, Gongju, Korea). A test line 12 containing the capture probe and a control line were dispensed onto the nitrocellulose membrane 13 using a line dispenser (BTM Inc., Uiwang, Korea) under the following conditions: dispensing speed, 14 50 mm/s; dispensing rate, 1 μL/cm. ACE2 (1 mg/mL) and 0.5 mg/mL anti-IgG antibody mixture hour at 3°C. To decrease the non-specific interaction between capture probes in test lines and 20 detection probes, the nitrocellulose membrane was treated with the blocking solution (10 mM 2-21 amino-2-methyl-1-propanol (pH 9.0), 0.5% BSA, 0.5% β-Lactose, 0.05% Triton X-100, 0.05% 22 sodium azide) for 1 hour in a vacuum oven (37°C). Each component of the LFIA strip was precisely 23 assembled and cut to a 38 mm width, followed by integration into a housing for a single LFIA test. To discover the optimum pair for detection of the SARS-CoV-2 spike antigen, a total of 12 capture 2 probe-detection probe pairs (Supplementary Table 1 ) were tested. The affinity of the four different 3 antibodies for SARS-CoV-2 S1 and RBD was confirmed through ELISA, western blot, and BLI.

Capture probe (1 mg/mL) and 0.5 mg/mL anti-IgG antibody mixture [1:1:1 (v/v/v) anti-human IgG 5 antibody/anti-rabbit IgG antibody/anti-mouse IgG antibody] were used to form test dots and control 6 dots, respectively. Dots were immobilized onto a nitrocellulose membrane (Advanced Microdevices) 7 by loading 0.5 μL of the test or control solution. The nitrocellulose membrane was incubated with 8 blocking solution (10 mM 2-amino-2-methyl-1-propanol (pH 9.0), 0.5% BSA, 0.5 % β-lactose, 0.05% 9 Triton X-100, 0.05% sodium azide) in a vacuum oven (1 hour, 37°C). The LFIA strip for the dot-blot 10 assay was constructed as described in the previous section. For comparative analysis between the 12 11 pairs, 50 ng target antigen (SARS-CoV-2 Spike S1) was incubated with each detection probe in optimized. Finally, 0.05 % of CNB for the detection probe, 1 mg/mL of ACE2 for the immobilized capture probes, and running buffer [10 mM AMP, (pH 9.0), 5 mM EDTA, 200 mM urea, 1% Triton X-1 100, 0.5% Tween 20, 500 mM NaCl, 1% PEG (MW 200)] were selected. The SARS-CoV-2 S1 and 2 SARS-CoV-2 RBD antigens were serially diluted in sample diluent buffer (Cat. No. ab154873; 3 Abcam), and diluted samples were mixed with running buffer at a ratio of 1:9 (v/v). The final 4 concentrations of the diluted samples ranged from 500 to 5 ng/mL. One hundred microliters of 5 running buffer containing each antigen concentrate were added to the inlet of the LFIA device. In this 6 system, the sample flows along with the LFIA strip by capillary force and first encounters the 7 antibody (S1-mAb)-conjugated CNB. The antigen in the sample is captured by the S1-mAb 8 conjugated CNB, and this antigen-probe complex is detected by a pre-immobilized capture reporter 9 (ACE2) on the nitrocellulose membrane. After 20 minutes of sample loading, the appearance of red 10 color in the test and control lines is confirmed and analyzed on a Sapphire Biomolecular Imager.

For specificity testing, two different corona-related spike antigens (i.e., SARS-CoV S1, and 12 MERS S1 antigens) were prepared at three different concentrations (100, 20, and 5 ng/mL). Three between target antigen and antibodies (or receptor) is a prerequisite for development of not only 7 therapeutics and vaccines, but also sensitive and accurate diagnostic platforms for antigen detection. 8 We measured the interaction between SARS-CoV-2 S1 protein and ACE2 by western blot analysis, 9 indirect ELISA, and BLI to detect SARS-CoV-2 S protein..

The interaction between receptor (or antibodies) and SARS-CoV-2 S1 protein was 11 characterized by western blot (Supplementary Figure 4) . The results revealed that anti-SARS-CoV-2 12 antibodies (CR3022, F26G19, S1-mAb) and human Fc tagged ACE2 receptor (ACE2-Fc) detect 13 SARS-CoV-2 S1 protein. These interactions were also confirmed by ELISA showing that ACE2 14 receptor bound the SARS-CoV-2 S1 protein more strongly than the SARS-CoV S1 protein, but it did 15 not bind the MERS-CoV S1 protein (Figure 2a ). By contrast, the commercial anti-SARS-CoV-2 16 antibodies bound to the SARS-CoV S1 protein and SARS-CoV-2 S1 protein with similar affinities 17 (Figure 2b -d) . In the ELISA, the detection limits of the ACE2 receptor, CR3022, F26G19, and S1-18 mAb against SARS-CoV-2 S1 protein were approximately 125, 3.13, 3.13, and 0.78 ng/mL, 19 respectively. In addition, the detection limits of the ACE2 receptor, CR3022, F26G19, and S1-mAb 20 against SARS-CoV-2 RBD were approximately 3.13, 125, 0.05, and 0.05 ng/mL (Supplementary 21 Figure 5 ). To confirm the detailed kinetics of binding between ACE2 and the SARS-CoV-2 S1 protein, 22 we performed BLI to measure the affinity of ACE2 for two different variants of the SARS-CoV-2 23 spike (S1 and RBD) protein. In addition, we used three different commercial antibodies (CR3022, 24 F26G19, and S1-mAb) that bind SARS-CoV-2 S1 in the BLI experiments. BLI is a label-free technology for measuring biomolecular interactions based on changes in the interference pattern 1 before and after binding events. The end of the BLI biosensor tip was coated with Protein A, which 2 enables the efficient capture of the target antibody. Three commercial antibodies (CR3022, F26G19, 3 and S1-mAb) and ACE2-Fc were immobilized onto the surface of the BLI biosensor through the 4 specific interaction with protein A. We then examined the interactions of these antibodies with SARS-5 CoV-2 S1 and SARS-CoV-2 RBD. Representative real-time binding sensorgrams (dotted lines) and Table 2 ). The K D values of ACE2 for S1 and RBD were 319.7 10 and 13.18 nM, respectively. The RBD of S1 is mainly in charge of engagement with a host cell protein of SARS-CoV-2 by proprotein convertase furin has had a higher binding affinity to ACE2 14 through its RBD (Shang et al. 2020). Therefore, we assume that the K D value of ACE2 is lower for 15 RBD binding than for S1 binding because RBD can access ACE2 more efficiently than S1. This also 16 implies that targeting monomeric RBD of SARS-CoV-2 S1 antigens to detect antigens using ACE2 17 and antibody pair may be more sensitive than targeting S1 protein in the present study although we 18 have not checked out the detection of RBD itself in complex biological sample yet.

purpose, anti-IgG Ab was used to capture all antibodies that were already conjugated with the CNBs.

The test and control lines were formed on the nitrocellulose membrane using a line dispenser. As the 2 detection of the target antigen on the test lines of LFIA, the red signal from the CNBs makes it 3 possible to visually confirm whether the sample contains the target antigen or not. To avoid non-4 specific interaction between capture probes in test lines and detection probes, the nitrocellulose 5 membrane was adequately treated with blocking solution. Unbound detection probes pass through the 6 nitrocellulose membrane and ultimately reach the absorbent pad located at the end of the strip, which 7 serves to maintain the capillary force that drives sample flow.

To demonstrate the detection capability of the ACE2-based LFIA (ACE2-LFIA), we 9 performed the sensitivity analysis using serially diluted samples of SARS-CoV-2 specific antigen (S1 10 and RBD, concentration range: 5-500 ng/mL) as shown in Figure 5a and Supplementary Figure 7 and 11 8. Twenty minutes after sample loading, the window of the LFIA device was photographed using a 12 smartphone, and the intensity of the test and control lines was analyzed with an image scanner and 13 analyzer that converts the line color intensity to signal peaks, as shown in Figure 5a . In the case of S1 14 detection, the detection signal gradually decreased as the dilution factor increased, but even if only 5 15 ng antigen was present in the sample, the signal was still present. In addition, we confirmed that the 16 detection signal was clearly higher for RBD than for S1 for all diluted samples, and even 1 ng RBD 17 present in the sample could be successfully detected by our ACE2-LFIA. In addition, we observed no 1 coronaviruses (SARS-CoV and MERS-CoV). Three different concentrations (100, 20, and 5 2 ng/reaction) of each S1 antigen were introduced into the LFIA device, and the intensity of test lines 3 was measured after 20 minutes using a portable line analyzer. It took less than 10 seconds to measure Figure 8 ). The sensitivity difference between SARS-CoV-2 S1 and RBD using ACE2-9 based LFIA might be associated with the K D value of ACE2 and S1-mAb were lower for RBD 10 binding than for S1 binding (Supplementary Table 2 ). Meanwhile, the MERS-CoV S1 antigen was not 11 detected even at relatively high concentrations (100 ng antigen); however, 100 ng antigen of SARS-12 CoV S1 was slightly detected, as shown in Figures 5b and 5c , and Supplementary Table 3 . This means 13 that more than 100 ng antigen of SARS-CoV S1 might not be distinguished using the ACE2-LFIA. 14 The intensity slightly increased at a high concentration of SARS-CoV S1; however, the proposed 15 ACE2-LFIA has the potential to distinguish SARS-CoV-2 from other coronaviruses due to the 16 significant difference in intensity. The inset graph in Figure 5c shows the detection intensity for the S1 17 antigens from three different coronaviruses at an antigen concentration of 5 ng. The detection signal 18 of SARS-CoV-2 S1 was clearly higher than the limit of detection (LOD, mean value of negative 19 controls + 3 × standard deviation), whereas those of SARS-CoV S1 and MERS-CoV S1 were similar 20 to that of the negative control. Thus, the proposed ACE2-LFIA has the ability to detect the SARS-

CoV-2 antigen with high sensitivity and without significant cross-reactivity from other coronaviruses. ACE2, a type 1 membrane protein expressed in the lung, heart, kidney, and intestine, is the cellular receptor for 3 SARS-CoV-2. b) Schematic of an ACE2-based LFIA consisting of a sample pad, conjugate pad, nitrocellulose

-The human ACE2 and commercial antibody were paired with each other as capture and detection probes in a lateral flow immunoassay that was not cross-reactive with SARS-CoV S1 and MERS-CoV S1 proteins.

-The SARS-CoV-2 S1 (< 5 ng of recombinant proteins/reaction) was detected by the ACE2-based LFIA. The limit of detection of our ACE2-LFIA was 1.86x105 copies/mL in the clinical specimen of COVID-19 Patients without no cross-reactivity for nasal swabs from healthy subjects.

-This is the first study to detect SARS-CoV-2 S1 antigen using an LFIA with matched pair consisting of ACE2 and antibody. Our findings will be helpful to detect the S1 antigen of SARS-CoV-2 from COVID-19 patients.

Schematic illustration of human ACE2-based later flow immunoassay.

J o u r n a l P r e -p r o o f

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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