A novel rapid detection for SARS-CoV-2 spike 1 antigens using human angiotensin converting enzyme 2 (ACE2)

Abstract

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 × 10⁵ 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.

Keywords: COVID-19; Human ACE2; Lateral flow immunoassay; SARS-CoV-2; Spike antigens.

Fig. 1

Cellular receptor (ACE2)-based LFIA. a) Schematic of ACE2 receptor recognition by SARS-CoV-2. ACE2, a type 1 membrane protein expressed in the lung, heart, kidney, and intestine, is the cellular receptor for SARS-CoV-2. b) Schematic of an ACE2-based LFIA consisting of a sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. The test line placed on the nitrocellulose membrane contains ACE2 for detection of the SARS-CoV-2 spike antigen. Anti-IgG antibody is used in the control line. The proposed LFIA can achieve sensitive and selective detection of SARS-CoV-2 spike antigen within 20 min.

Fig. 2

Indirect ELISA results from spike antigens of three different coronaviruses (SARS-CoV S1, SARS-CoV-2 S1, and MERS S1). a) The interactions between these S1 antigens and ACE2 were examined with serially diluted samples (concentration range: 200 to 0.05 ng/mL). In addition, three different antibodies, CR3022 (black) (b); F26G19 (red) (c); and S1-mAb (orange) (d), were tested for their interaction with spike antigens, using the same concentration range. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Biolayer interferometry (BLI) results of ACE2, CR3022, F26G19, and S1-mAb against the SARS-CoV-2 S1 antigen. Dotted lines represent the response curves of BLI measurement, and solid lines represent the fitting curves based on a 1:1 binding model. Binding kinetics were measured for four different concentrations of the S1 antigen.

Fig. 4

Identification of the sandwich pair for detection of SARS-CoV-2 spike antigen. a) Schematic diagram of LFIA using ACE2 as the capture probe and sandwich analysis results obtained from paired antibodies (CR3022, F26G19, and S1mAb). SARS-CoV-2 S1 antigen (50 ng) was used as a positive control, and buffer containing no S1 antigen was used as a negative control. After 20 min, the strips were captured by a smartphone, and their peak intensities were analyzed. b) Schematic diagram of LFIA, using antibodies as the capture probe, and their sandwich analysis results. c) Peak intensities of capture probe (P_C)–detection probe (P_D) pairs. A total of 12 pairs of positive controls (50 ng S1 antigen) were tested, and their intensities were analyzed. Peak intensity was calculated by subtracting the background intensity of the strip from the average intensities of the dots.

Fig. 5

Sensitivity and specificity of the ACE2-based LFA a) Results of ACE2-based LFA for the detection sensitivity of SARS-CoV-2 S1 antigen. Serially diluted antigen concentrates (concentration range: 500 ng/mL to 5 ng/mL) were tested by ACE2-based LFA. After 20 min, the LFA strips were photographed with a smartphone. Moreover, the intensity of the test and control lines was converted to a peak histogram by an image analyzer. b) Results of the comparative analysis for the detection selectivity: positive control – SARS-CoV-2 S1, negative control – SARS-CoV S1, MERS S1, and buffer solution. Using three different concentrates (1 μg/mL, 200 ng/mL, and 50 ng/mL) of the antigen sample, the detection performance of ACE2-based LFA was demonstrated. c) Bar graph of peak intensities for test lines. After 20 min for the sample flow, the intensity of the test lines was measured by a portable line analyzer. Inset) the detection intensity for the 5 ng antigen per reaction of each control. Limit of detection (LOD) was determined by the mean value of negative controls plus three times the standard deviation. P-values: ns > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Fig. 6

Laboratory confirmation of ACE2-based LFIA using clinical samples a) Schematic diagram of COVID-19 test using ACE2-based LFIA. A nasopharyngeal swab from the COVID-19 patient is placed into the UTM. 50 μL of UTM containing the SARS-CoV-2 is mixed with running buffer in a 1:1 (v/v) ratio, and 100 μL of mixed solution is loaded into the LFIA device. After 20 min, the line intensity of the LFIA strip is semi-quantified by the portable analyzer. b) Results of ACE2-based LFA for the detection sensitivity of cultured SARS-CoV-2. Serially diluted virus concentrates (concentration range: 1.07 × 10⁸ copies/mL to 5.35 × 10⁶ copies/mL) were tested. After 20 min, the LFIA strip was taken with a smartphone and scanned with an image analyzer. The line intensities of the test and control lines were converted to peak histograms. Also, the intensity of the test lines was measured by a portable line analyzer (I_L: line intensity of test line). Furthermore, human coronavirus (OC43) was tested as a negative control. c) Bar graph of intensities for test lines measured by the portable analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (0 copies/mL of SARS-CoV-2) plus three times the standard deviation. d) Laboratory confirmation of ACE2-based LFIA compared to the RT-qPCR using clinical samples. i) Nasopharyngeal swab samples of COVID-19 patients (n = 4) and healthy subjects (n = 4) were tested both ACE2-based LFIA and RT-qPCR. Sensitivity was determined by the number of true positive samples divided by the number of positive samples tested. Moreover, specificity was determined by the number of true negative samples divide by the number of negative samples tested. ii) RT-qPCR results for the detection of the SARS-CoV-2 specific gene (Env gene). C_t value and their correspondent viral load in the clinical samples were evaluated. e) Results of ACE2-based LFIA on laboratory confirmation using clinical COVID-19 patient samples. Twenty minutes after sample loading, the test line intensities of the LFIA strips were measured with a portable line analyzer. The limit of detection (LOD) was determined by the mean value of negative controls (healthy control) plus three times the standard deviation.