Reagents
Reagents were obtained from following manufacturers: Advansta Corporation (San Jose, USA): WesternBright Sirius®; Carl Roth GmbH & Co. KG (Karlsruhe, Germany): Carbenicillin disodium salt, lysozyme (≥ 45 000 FIP U/mg), ROTI®Fair Carbonate Bicarbonate Buffer pH 9.6, ROTI®Stock 10 × PBS, ROTI®Stock 10 × PBS-T, sodium chloride (≥ 99.5%), sodium dodecyl sulfate (SDS, ≥ 99.5%), sulfuric acid, Terrific-Broth- (TB-) Medium and urea (> 99.5%); GenScript Biotech BV (Leiden, Netherlands): SARS-CoV-2 Nucleocapsid protein (His-tag), SARS-CoV-2 Nucleocapsid Protein mAbs; MORPHISTO GmbH: Acetate buffer pH 5.0; Promega GmbH (Mannheim, Germany): Peroxidase-conjugated anti-human IgG antibody; Roche Deutschland Holding GmbH (Mannheim, Germany): cOmplete™ Mini EDTA-free protease inhibitor cocktail (from bovine pancreas); Seramun Diagnostika GmbH (Heidesee, Germany): TMB substrate solution; SERVA Electrophoresis GmbH (Heidelberg, Germany): Acrylamide/bis(acrylamide) (30% T, 2.67% C), BlueBlock PF 10x, Coomassie Brilliant Blue G-250, TEMED and trypsin (sequencing grade, MS approved); Sigma Aldrich Chemie GmbH (Taufkirchen, Germany): 2-mercaptoethanol (BioUltra), Antifoam Y-30 emulsion and imidazole (≥ 99.5%); Surmodics IVD, Inc. (Eden Prairie, USA): StabilZyme™ SELECT; Thermo Fisher Scientific (Waltham, Massachusetts, USA): AcroMetrix™ Inhibition Panel, SuperBlock® (PBS).
Protein expression and purification
The coding sequence of SARS-CoV-2 N-protein (NCBI accession # YP_009724397.2, downloaded from https://www.ncbi.nlm.nih.gov/protein) containing a N-terminal His-tag was codon-optimized for Escherichia coli, synthesized, cloned into a pET45b(+) vector (GenScript Biotech BV), and expressed in E. coli BL21(DE3). Briefly, an overnight culture from TB-Medium containing Carbenicillin (0.5 g/L) and Antifoam Y-30 emulsion (0.0075%, v/v) was inoculated with a single colony of E. coli BL21(DE3) pET45b(+)-N-protein and incubated on a horizontal shaker (180 rpm, Certomat® BS-T, B. Braun Biotech GmbH, Melsungen, Germany) at 30 °C for 17 h. Cells were harvested, resuspended in lysis buffer (PBS (337 mmol/L NaCl, 2.7 mmol/L potassium phosphate, 10 mmol/L disodium hydrogen phosphate, 2 mmol/L potassium dihydrogen phosphate) containing 50 µmol/L lysozyme and 1 tablet/10 mL protease-inhibitor-mix), and disrupted on a French® Press (Thermo Fisher Scientific, Waltham USA). For purification by immobilized metal affinity chromatography the protein concentration determined by NanoPhotometer NP80® (Implen GmbH, München, Germany) was adjusted with purification buffer (PBS, pH 7.4) to 10 g/L and imidazole added to obtain a final concentration of 25 mmol/L. The sample was loaded and proteins were eluted using a linear 25-min gradient from 25 mmol/L–0.5 mol/L imidazole in purification buffer. Fractions were collected in 1.5-min intervals and analyzed by SDS-PAGE. Fractions containing mostly the N-protein were combined, dialyzed against storage buffer (PBS), and stored at -20 °C. The purity of the N-protein was verified by SDS-PAGE. The most intense band expected to contain the N-protein was excised from the gel and the protein digested with trypsin (in-gel digest). The extracted tryptic peptides were analyzed by LC–MS. Additionally, proteins in the gel were semi-dry electroblotted onto a PVDF membrane (Bio-Rad Laboratories GmbH, Feldkirchen, Germany), blocked with BlueBlock solution at room temperature for 1 h and incubated with an anti-SARS-CoV-2 Nucleocapsid protein antibody (GenScript Biotech BV) diluted to 10,000-fold in BlueBlock solution at room temperature. After 1 h, peroxidase-conjugated anti-Human IgG-HRP (Promega GmbH, 1: 30,000 in BlueBlock) was added at room temperature. After 1 h, the bands were visualized with WesternBright™ Sirius substrate solution and the chemiluminescence was recorded on ChemiDoc MP (Bio-Rad Laboratories GmbH).
Serum collection
Serum samples from PCR-confirmed SARS-CoV-2 positive patients were obtained from two individual donors and hospitalized patients (Krankenhaus Nordwest, Frankfurt, Germany, Klinikum Chemnitz gGmbH, Chemnitz, Germany, Institut für Transfusionsmedizin, Universitätsklinkum Leipzig, and Hospital St. Georg gGmbH, Leipzig, Germany) (Additional file 1: Tables S1–S4). These investigations represent parts of the analyzes in the COVID genetics cohort Leipzig-Chemnitz, which was approved by the Institutional Review Board of Leipzig University (reference numbers 195/20-ek and EK-allg-37/10–1). Control serum samples collected from 2009 to 2014 were enriched for older people, higher BMI, and a history of chronic obstructive pulmonary disease (COPD) to specifically test the assay specificity in populations at higher risk for severe COVID-19 conditions and with presumably a higher incidence of previous viral infections including other coronaviruses, although this information was not available (Additional file 1: Table S5). These 1500 serum samples obtained from the population-based LIFE-Adult study of the Leipzig Research Center for Civilization Disease (LIFE) [10]. All samples have been processed and stored by the team of the Leipzig Medical Biobank. Probands were collected all over the year and thus should well represent different antibody titers in response to seasonal bacterial and viral infections, e.g., influenza, coronaviruses, and rhinoviruses, as well as allergies to achieve a robust performance in epidemiological studies. Furthermore, serum samples from persons with confirmed antibody titers against human immunodeficiency viruses (HIV) 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were used for cross-reactivity studies (INSTAND, Düsseldorf, Germany).
Enzyme-linked immunosorbent assay (ELISA)
Medium binding microplates (Greiner Bio-One, Frickhausen, Germany; 12xF8, PS, F-bottom) were coated with 150 ng SARS-CoV-2 N-protein per well in PBS at 4 °C overnight. All following steps were performed at room temperature. Wells were washed three times with PBS-T (300 µL) using a Hydro Flex ELISA washer (Tecan Group AG, Männedorf, Switzerland) and blocked with superblock (200 µL) for 60 min. Human serum (off-the-clot, sterile filtered; PAN-Biotech GmbH, Aidenbach, Germany) was used as negative control. The positive control consisted of an anti-SARS-CoV-2 N-protein antibody (1 g/L, GenScript Biotech BV) 100-fold diluted in the negative control human serum. Both controls and the serum samples were diluted 100-fold in sample buffer (Indical Bioscience, Leipzig, Germany) and incubated for 45 min. Wells were washed with PBS-T (300 µL) using the Hydro Flex ELISA washer before conjugate solution (100 µL/well) was added (anti-Human IgG-HRP, 1:30,000 in Stabilzyme Select). After 30 min, wells were washed three times as described above and TMB substrate solution added (100 µL/well). The reaction was stopped after 10 min by the addition of sulfuric acid (0.3 mol/L; 100 µL/well) and the absorbance recorded at 450 nm using a SUNRISE microplate reader (Tecan Group AG, Männedorf, Switzerland).
Additionally, serum samples were tested with a commercial anti-SARS-CoV-2 ELISA (IgG) kit (SARS-CoV-2 N-protein based ELISA, EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) according to the manufacturer’s instruction.
To test potential interference, a positive serum sample was 128-fold diluted with plasma containing different interfering substances: hemoglobin (up to 20 g/L), bilirubin (up to 0.3 g/L), and triglycerides (up to 15 g/L).
ELISA validation and statistical analysis
The absorbance values of serum samples were converted to sample-to-positive (S/P-) ratios using the absorbances recorded at 450 nm (OD450) of the positive (PC) and negative controls (NC), using the following equation:
$${\text{S/P-ratio}}\;(\%) = 100 \times ({\text{OD}}_{{{45}0}} {\text{(sample)}} - {\text{OD}}_{{{45}0}} ({\text{NC}})){\text{/(OD}}_{{{45}0}} {\text{(PC)}} - {\text{OD}}_{{{45}0}} ({\text{NC}}){)}$$
The cut-off value was determined by Receiver Operating Characteristics (ROC) to obtain the best sensitivity and specificity. ROC analysis was performed by GraphPad Prism 9.0.2 (Graph Pad Software, La Jolla, CA, USA).
Western Blot analysis of serum samples
Control sera collected before 2015 but unexpectedly tested positive in the N-protein ELISA, were probed by an immunoblot. Commercial and in-house expressed N-proteins were separated by SDS-PAGE (0.5 µg per lane) using 12% gels. Gels were stained with brilliant Coomassie Blue G250 or semi-dry electroblotted onto a PVDF membrane (Bio-Rad Laboratories GmbH) [7]. Briefly, the membrane was blocked with BlueBlock solution at 4 °C overnight and incubated with SARS-CoV-2 positive sera, negative sera or potentially false-positive sera (1:10,000) at room temperature for one hour. After three washing steps with PBS-T for 5 min, the membrane was incubated with peroxidase-conjugated anti-human IgG-HRP (Promega GmbH, 1: 20,000 in BlueBlock) at room temperature for one hour. After washing, bands were detected with WesternBright™ Sirius substrate solution and the chemiluminescence was recorded on ChemiDoc MP (Bio-Rad Laboratories GmbH).