Goals:

COVID-19 emerged and spread rapidly around the world. Testing strategies that focus on COVID-19 patients require high throughput, low risk of infection, and small sample volumes assays. Antigen surveillance can be used to identify exposure to pathogens and measure acute infections.

Methods:

A total of 914 serum samples, collected from 309 currently infected COVID-19 patients, 48 ​​recovered, and 410 non-COVID-19 patients, were used to measure N protein antigen levels by chemiluminescence immunoassay. Diagnostic results were analyzed at different periods after initiation.

Results:

There was a high level of protein N antigen in COVID-19 patients (0.56 COI), compared to recovered patients (0.12 COI) and controls (0.19 COI). In receptor performance characteristic curve analysis, the area under the curve for serum protein N antigen was 0.911 in the first week after initiation. In this period, the sensitivity and specificity of the serological tests for N protein antigens were 76.27 and 98.78%. The performance of the diagnosis of specific antibodies improved from the third week after initiation. Subgroup analysis suggested that severely ill patients had higher antigen levels than mild patients.

Conclusions:

The high serum antigen level suggested early infection and severe disease. The serum protein N antigen test by chemiluminescence immunoassay is considered a viable assay that is used to improve the diagnostic sensitivity of current patients.

Materials and methods

1. Patients, samples and data collection

Consecutive COVID-19 patients who were presented or admitted to Wuhan University Zhongnan Hospital from January 28 to March 10, 2020, were enrolled to test for SARS-COV-2 protein N antigen and antibody specific in serum. The patients with COVID-19 and those recovered included in this study were diagnosed according to the diagnostic guide published by the National Health Commission (16). Healthy volunteers and other virus-infected patients who were examined or admitted to Wuhan University Zhongnan Hospital from November 18, 2020, to December 6, 2020, were enrolled in the control group.

Exclusion criteria for controls were as follows: (a) positive SARS-COV-2 RNA in throat swab, (b) patients deficient in basic clinical data. All throat swabs and venous blood samples were collected and processed at Wuhan University Zhongnan Hospital. The remaining sera were collected and stored at -80 ° C for the SARS-CoV-2 specific antibody and protein N antigen test. Clinical features, laboratory findings, and results were obtained from electronic medical records. . The study was reviewed and approved by the Ethics Committee of Wuhan University Zhongnan Hospital. All study objectives have signed informed consent.

2. Real-time RT-PCR assay for SARS-CoV-2 RNA

Throat swabs were collected from COVID-19 patients for SARS-CoV-2 RNA testing. First, total RNAs were extracted from a swab within 3 h using a respiratory sample RNA isolation kit (Zhongzhi, Wuhan, China). Briefly, 40 µL of cell lysis solution was transferred to a collection tube consisting of the swab followed by vortexing for 30 s. After incubation at room temperature for 15 min, the collection tube was centrifuged at 1,000 rpm/min for 5 min. The suspension was used as a template for amplification using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay kits (Daan Gene, Guangzhou, China). Two target genes, the nucleocapsid protein (N) and the SARS-CoV-2 open reading frame 1ab (ORF1ab), were simultaneously amplified and detected during the real-time RT-PCR assay.

The volume of the real-time RT-PCR reaction system was 25 μL, including 2 μL of template, 3 μL of pure water, 17 μL of Mix A, and 3 μL of Mix B. Each amplification was performed in an Eppendorf tube. with ABI prism 7500 (Thermo Fisher Scientific, Waltham, MA, USA). The reaction conditions were as follows: transcription at 50 ° C for 15 min and pre-denaturation at 95 ° C for 15 min, followed by 45 cycles of denaturation at 94 ° C for 15 s and extension at 55 ° C for 45 s. Fluorescence was collected at regular intervals during each extension phase.

3. Chemiluminescence Immunoassay to Test for SARS-CoV-2 Specific Antibody

Serum IgM antibody against SARS-CoV-2 N and peak protein were determined using the iFlash immunoassay analyzer (Shenzhen Yhlo Biotech Co., Ltd, Shenzhen, China) and the iFlash-SARS IgM Detection Kit- CoV-2 (Shenzhen Yhlo Biotech Co., Ltd, Shenzhen, China) approved by the China Food and Drug Administration (FDA). It was a two-step indirect immunoassay. First, the SARS-CoV-2-specific IgM in the serum binds to the SARS-CoV-2 N and the spike protein coating the paramagnetic microparticles to form a complex, after washing the unbound materials in the IgM.

Acridinium-labelled magnetic anti-human. The conjugate was added so that the reaction continued to form a new complex, then it was washed again and the Pre-Trigger and Trigger solutions were added to the reaction mixture. Finally, the serum levels of SARS-CoV-2 specific IgM were calculated based on the relative light units (RLU) resulting from the reaction mixture using a 2-point calibration curve, and a value> 10 AU / ml.

SARS-CoV-2 N serum IgG antibody and peak protein were determined using the Administration approved iFlash-SARS-CoV-2 IgG Detection Kit (Shenzhen Yhlo Biotech Co., Ltd, Shenzhen, China) Food and Drug Administration of China (FDA). ). The principle and procedure for IgG were similar to IgM. The cut-off value given by the manufacturer is 10 AU / ml.

4. Chemiluminescence Immunoassay for SARS-CoV-2 N Serum Antigen

Serum SARS-CoV-2 N protein antigen was determined using a double-antibody sandwich chemiluminescence immunoassay by iFlash immunoassay analyzer (Shenzhen Yhlo Biotech Co., Ltd, Shenzhen, China). Paramagnetic carboxylated microparticles (Thermo Scientific) were coated with one of the 10 candidate-specific antibodies (Shenzhen YHLO Biotech Co., Ltd, Shenzhen, China) by crosslinking with N-ethyl-N ‘- (3-dimethyl aminopropyl) carbodiimide (Thermo Scientific) for N protein antigen-capture as described above. Another antibody was conjugated to NSP-DMAE-NHS (Maxchemtech) for antigen detection.

Recombinant nucleocapsid protein SARS-CoV-2 (Shenzhen YHLO Biotech) dissolved in healthy human serum was used as calibrators. Tests can be run after calibration. In the test, the capture antibody coated paramagnetic carboxylated microparticles collected protein N antigens. After washing off unbound material, the antibody compounds captured by the antibody-protein N-antigen reacted with the acridinium-labelled antibody. The mixture was held in a tube under the magnetic field. And then the pre-activation and activation solution were added to calculate the protein N antigen based on the resulting relative light units (RLU) through a 2-point calibration curve.