Supplementary information for the article: Mladenovic Stokanic, M.; Simovic, A.; Jovanovic, V.; Radomirovic, M.; Udovicki, B.; Krstic Ristivojevic, M.; Djukic, T.; Vasovic, T.; Acimovic, J.; Sabljic, L.; Lukic, I.; Kovacevic, A.; Cujic, D.; Gnjatovic, M.; Smiljanic, K.; Stojadinovic, M.; Radosavljevic, J.; Stanic-Vucinic, D.; Stojanovic, M.; Rajkovic, A.; Cirkovic Velickovic, T. Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species. International Journal of Molecular Sciences 2024, 25 (1), 333. https://doi.org/10.3390/ijms25010333.

Abstract

S1.1. Checking of N protein purity Recombinant N protein purity was checked after imidazole removal and buffer exchange by SDS PAGE (Figure 6.). For comparison, commercial high-purity HSA was also analyzed. S1.2. Identification of N protein Tandem mass spectrometry identification of proteins in an in-gel digested band of N protein (Figure S1, lane 3), confirmed the identity of N protein with high scores and peptide coverage (Fig. S2.). S2. Purification of polyclonal antibodies from mice and rabbit sera For the development of an ELISA test specific for the detection of SARS-CoV-2 N protein, recombinantly produced N protein was used for the immunization of mice and rabbits. Sera obtained from rabbits and mice were then tested for titer and specificity (Figure S3 and Figure 1). To determine the titer of polyclonal sera required to detect N protein in samples, we use wells coated with N protein and serial dilution of sera pools from different animals. After multiple washing steps, we detected the binding of rabbit and mice antibodies using secondary biotinylated antibodies and streptavidin-alkaline phosphatase chimaera or secondary antibodies with previously coupled alkaline phosphatase, where the amount of enzymes’ substrate converted to the product was measured as an increase in absorbance at 405 nm. As shown in Figure S3A, unpurified sera pools from both animals showed very high titers and expected logarithmic decrease of signal with dilution. Based on the obtained data titer for unpurified sera was determined to be X. The same trend was observed for pools purified using AS precipitation and rabbit sera purified using protein A affinity chromatography (Figure S3B and S3C). As shown in Figure S3D, clear bands from antibodies could be observed in both full and purified samples. Western blot analysis showed only one protein band on mass around 40 kDa, a Accession number / Protein Name Score Coverage (%) Unique peptides P0DTC9|NCAP_SARS2 Nucleoprotein OS=Severe acute respiratory syndrome coronavirus 2, 46 kDa 504.9 74.22 183 mass of purified N protein suggesting that the obtained sera is highly specific for N protein (Figure 2). Section S3 Diagnostic validationS3.1. Stabilization of capture antibodies Pre-coated ELISA plates were prepared for usage in clinical practice. To ensure the preservation of the biofunctionality of the surface-bound capture antibodies, the commonly used stabilizing excipient, 3% sucrose with 10% glycerol in MilliQ water was used. The plates were incubated with 300 μL per well of a stabilizing agent for 1 hour at room temperature. After an hour of incubation, the solution was carefully aspirated from each well. The plate was then blotted against clear paper towels to remove any remaining liquid, and the plates were allowed to air dry for 3 hours at RT. Dried plates were wrapped in parafilm and stored at 4 °C for later use. To remove the stabilizing agent coating, wells were washed with slightly acidic distilled water (pH of 6) three times, leaving the plate prepared for subsequent assay steps. Section S4. Characterization of N protein by HRMS S4.1. SDS PAGE and in-gel digestion Characterization of the produced recombinant N protein was done by HRMS after its in-gel digestion. A total of 10 μg of purified protein(s) were loaded in a 0.5 cm wide well and after SDSPAGE gel was stained with Coomassie Brilliant Blue R-250 (CBB). Protein gel bands were washed, reduced with dithiothreitol, and alkylated with iodoacetamide, followed by in-gel trypsin digestion1 (Shevchenko et al. 2006) with some minor modifications. The amount of trypsin was leveled to a trypsin/sample ratio of 1:30 (w/w). The final concentration of MS-grade trypsin (diluted in 25 mM ammonium bicarbonate buffer) was 1 ng/μL. Sample clean-up was performed using zip tips HyperSep C18 (Thermo Fisher Scientific Inc., Bremen, Germany). S5.1 Immunization of rabbits and mice Mice immunization Swiss Webster mice (n=10) were immunized subcutaneously with N protein formulated with Complete Freund`s adjuvant (CFA; 1st dose, 100 μg N protein / dose) or Incomplete Freund`s adjuvant (IFA; 2nd and 3rd doses, 50 μg N protein / dose) in three-week intervals. Mice were housed in small groups of up to six animals and had access to commercial mice food and water ad libitum. N protein solution (500ug/ml in PBS) was sterilized by filtering through 0.22 um filters. Sterile N protein solution was mixed with CFA (Sigma, Cat. No. F5881) at ratio 1:1 (v/v) under aseptic conditions. In total 400 ul of N protein-CFA emulsion (N protein final concentration 250ug/ml) was applied per immunization per mouse. Initial immunization was done by injection of N protein in CFA given subcutaneously (SC) in four sites (thigh pocket, base of tail, and mediastinum) with a 100 ul using 23-25 gauge needle. In total 100 ug of N protein was applied per mouse (25 ug per site). Subsequent immunizations with booster doses were done in the same way, but using IFA (Sigma, Cat. No. F5506) instead of CFA and N protein final concentration was 125 ug/ml. . In total 50 ug of N protein was applied per mouse (12.5 ug per site). Immunizations were done every three weeks. Mice immunization scheme: 1. day 0 – N protein in PBS: CFA = 1:1 (v/v); N protein final concentration was 250 μg/mL; 400 μL per mice (4x100 μL), e.g. 100 μg per mice 2. day 21 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μL per mice (4x100 μL), e.g. 50 μg per mice 3. day 42 - N protein in PBS: IFA = 1:1 (v/v); N protein final concentration was 125 μg/mL; 400 μl per mice (4x100 μL), e.g. 50 μg per mice First bleeding was performed two weeks after the 3rd dose, and then in intervals not shorter than two weeks. The sera obtained after the first bleeding was tested for the production of specific anti-N protein antibodies.