key: cord-0846549-m6tkvyl6
authors: Liang, Yuanhao; Yan, Huanchang; Huang, Liping; Zhao, Jianhui; Wang, Haiying; Kang, Min; Wan, Zhengwei; Shui, Jingwei; Tang, Shixing
title: A luciferase immunosorbent assay for quantitative detection of IgG antibodies against SARS-CoV-2 nucleoprotein
date: 2021-03-19
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114141
sha: b995774280f23bbfa82401112e72c07ba82d64b6
doc_id: 846549
cord_uid: m6tkvyl6

In this study, we developed and evaluated a luciferase immunosorbent assay (LISA) for quantitative detection of IgG antibody against SARS-CoV-2 nucleoprotein (NP). Anti-SARS-CoV-2 NP antibody in serum or plasma samples was captured by protein G-coated microtiter plate and detected using the crude cell lysates expressing Nanoluc luciferase (Nluc) enzyme fused with SARS-CoV-2 NP. After the addition of furimazine substrate, the levels of anti-SARS-CoV-2 NP IgG antibody were quantitatively measured as luciferase light units. As expected, SARS-CoV-2 NP showed cross-reactivity with the monoclonal antibodies against SARS-CoV NP, but not MERS-CoV NP-specific monoclonal antibodies or the monoclonal antibodies against SARS-CoV Spike protein. LISA for detecting murine monoclonal antibody against SARS-CoV NP showed a low limit of detection of 0.4 pg/μl and linear detection range from 0.4 pg/μl to 75 pg/μl. Furthermore, LISA had a sensitivity of 71 % when testing COVID-19 patients at the second week post onset and a specificity of 100 % when testing healthy blood donors.

. To control the epidemics of emerging infectious diseases, high-throughput serological assays are important for screening population infection and evaluating population immunity, and tracing the source and animal hosts of infection. In this study, we evaluated a luciferase immunosorbent assay (LISA), which is easily and quickly developed, semi-quantitative and is suitable for detecting the specific antibody in a wide range of species including human and animals.

The gene fragment of nucleoprotein (aa 1-419) of SARS-CoV-2 (NCBI accession number MN908947) was amplified by RT-PCR using the following primers: 5′-GCGATCGCTTCCGAATTCATGTCTGATAATGGAC-3′ and 5′-CGGTTGAGCTCTGAATTCTTAGGCCTGAGTTGAG-3′. The PCR products were purified and subcloned into the pNLF1-N vector (Promega, Madison, USA) downstream of the Nluc luciferase gene with a linker of 30 base pairs. DNA sequencing was performed to confirm the integrity of the Nluc-SARS-CoV-2 NP fusion constructs. (Burbelo et al., 2009) . The lysis buffer composed of 50mM Tris, pH7.5, 100mM NaCl, 5mM MgCl2, 1% Triton X-100, 50% glycerol and protease inhibitors (Roche, Mannheim, Germany). The cell lysates containing Nluc-fusion antigen were harvested after centrifugation at 12,000 × rpm for Briefly, 96-well Costar flat-bottomed luminometry plates (Corning, New York, USA) were coated with 50 µl of 5 μg/mL protein G (Genscript, Nanjing, China) in carbonate buffer (pH 9.6) overnight at 4 °C. The best concentration of protein G for coating plate was experimentally determined to be 5 ug/mL and 250ng per well in our assay. After three washes with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-T), the plates were incubated with blocking solution (PBS containing 5% non-fat milk)

for 1 h at 37 °C. The wells were then washed, and incubated with 50 μL of diluted sera (Burbelo et al., 2020) . However, LIPS needs pre-incubation of Ruc-antigen and samples and a plate washer with vacuum to capture the antigen-antibody-beads complex (Burbelo et al., 2009 ). Our LISA is simplified by using protein G coated 96well microtiter plate to capture the total IgG antibody from a wide range of species and followed by the addition of Nluc-fusion antigen and luciferase specific reagent.

Therefore, it is a simple and universal testing system. More importantly, LISA is more suitable for rapid development of serological assays for detecting and diagnosing emerging infectious diseases in the light of no need for purification process when In this study, a total of 4 monoclonal antibodies (mAb) against SARS-CoV used, in which the two mAb (N-14A3 and N-1E8) were produced by immunizing BALB/c mice with the recombinant NP protein of SARS-CoV so that they are specifically target the SARS-CoV nucleoprotein (Che et al., 2003) . In addition, the other two mAb (E26A15 and E14A9) were obtained by immunizing Balb/c mice with inactivated SARS-CoV isolates. Aforementioned four monoclonal antibodies were generously provided by the Central Laboratory, Zhujiang Hospital, Southern Medical University, Guangzhou,

China, but the epitope mapping and biological function analysis of mAb E26A15 and E14A9 remain to be done. A dose-dependent reactivity was observed among the 2 mAb J o u r n a l P r e -p r o o f against SARS-CoV NP, i.e. N-14A3 ( Fig. 2A) , N-1E8 (Fig.2B) , and the 2 mAb against SARS-CoV, i.e. E26A15 (Fig. 2C) and E14A9 (Fig. 2D) , but not the mAb against MERS-CoV NP (Fig. 2E ). LISA showed a linear range of detection from 0.4 pg/µl to 75 pg/µl (Fig. 2F) . We would like to emphasize that the quantitation range of LISA was determined based on murine monoclonal antibody against SARS-CoV NP, not the human antibody against SARS-CoV-2 due to the lack of anti-SARS-CoV-2 NP antibody.

However, it has been reported that the affinity of protein G for human IgG is higher than that of murine IgG, therefore, in theory, a wider quantitation range of LISA would be achieved for detecting human IgG antibodies. Then the wells were washed, and 50 µl of luciferase substrate were added to each well to determine the luciferase light units. Each sample was tested in triplicate. The cross reactivity between the mAb of anti-SARS-CoV NP and SARS-CoV-2 NP was inhibited by about 75% using 1:100 diluted serum from CVOID-19 patient 20SF15 (Fig. 3A ) and

J o u r n a l P r e -p r o o f 94% using 1:100 diluted serum from CVOID-19 patient 20SF35 (Fig. 3B) with a dosedependent response. However, the sera from the two COVID-19 patients could not inhibit the cross reactivity between the mAb of anti-MERS-CoV NP and SARS-CoV-2 NP ( Fig. 3C and 3D ). These results indicate the specificity of SARS-CoV-2 NP-based LISA for detecting antibodies against SARS-CoV NP and SARS-CoV-2 NP due to the 94% homogeneity of these two NP (Chan et al., 2020a) .

There are several limitations in our study. First, in this study, no serum samples from patients infected with other acute respiratory infections were tested. Therefore, the potential cross-reactivity of the LISA assay with other respiratory pathogens could not be evaluated although no false positive results were observed in the healthy blood donors collected before the COVID-19 pandemic. Second, the high degree of similarity between SARS-CoV and SARS-CoV-2 NP sequences and the cross-reactivity between the specific monoclonal antibodies against SARC-CoV NP and serum samples from SARS-CoV-2 infected patients could overestimate the seroprevalence of SARS-CoV-2 infection among the population ever experienced SARS-CoV infection; however, it has been reported that SARS-CoV specific antibodies could not be detected in about 90%

(21/23) serum samples of SARS patients 6 years after infection (Tang et al., 2011) .

Therefore, false-positives results caused by the cross-reactivity of SARS-CoV specific antibodies presented in the population would be rare because SARS-CoV infection has not been documented since 2003. Third, in the current study, we have only detected human serum samples, and did not evaluate the performance of this SARS-CoV-2 J o u r n a l P r e -p r o o f specific serological assay in testing samples of various animal species due to the lack of these samples. However, in our previous study, we evaluated and found that the LISA assay achieved very good performance in detecting MERS-CoV specific IgG antibody in various animal samples including camel, marmoset, rhesus and mice .

In conclusion, a LISA assay for detection of antibody against SARS-CoV-2 nucleoprotein was developed and evaluated, and demonstrated superior performance in discriminating COVID-19 patients and uninfected subjects. Additionally, attributed to the high homogeneity between SARS-CoV NP and SARS-CoV-2 NP, SARS-CoV-2 NP showed cross-reactivity with SARS-CoV NP-specific monoclonal antibodies.

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