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.
| العنوان: | 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. |
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| المؤلفون: | Mladenović Stokanić, Maja, Simović, Ana, Jovanović, Vesna, Radomirović, Mirjana, Udovički, Božidar, Krstić Ristivojević, Maja, Djukić, Teodora, Vasović, Tamara, Aćimović, Jelena, Sabljić, Ljiljana, Lukić, Ivana, Kovačević, Ana, Cujic, Danica, Gnjatović, Marija, Smiljanić, Katarina, Stojadinović, Marija, Radosavljević, Jelena, Stanić-Vučinić, Dragana, Stojanović, Marijana, Rajković, Andreja, Ćirkovic Veličković, Tanja |
| المصدر: | International Journal of Molecular Sciences |
| بيانات النشر: | MDPI |
| سنة النشر: | 2024 |
| مصطلحات موضوعية: | antigen test, COVID-19 diagnosis, ELISA, nucleocapsid protein, polyclonal antibodies |
| الوصف: | 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 ... |
| نوع الوثيقة: | dataset |
| اللغة: | English |
| تدمد: | 1422-0067 |
| Relation: | info:eu-repo/grantAgreement/MESTD/inst-2020/200168/RS//; info:eu-repo/grantAgreement/MESTD/inst-2020/200007/RS//; info:eu-repo/grantAgreement/MESTD/inst-2020/200177/RS//; info:eu-repo/grantAgreement/ScienceFundRS/Fond_2020_COVID19/7542203/RS//; Serbian Academy of Sciences and Arts GA No. F-26; Ghent University Global Campus and Belgian Special Research Fund BOF StG No. 01N01718; https://doi.org/10.3390/ijms25010333; https://intor.torlakinstitut.com/handle/123456789/858; http://intor.torlakinstitut.com/handle/123456789/859; http://intor.torlakinstitut.com/bitstream/id/2041/ijms-2756843-supplementary.pdf; https://hdl.handle.net/21.15107/rcub_intor_859 |
| الاتاحة: | http://intor.torlakinstitut.com/handle/123456789/859 http://intor.torlakinstitut.com/bitstream/id/2041/ijms-2756843-supplementary.pdf https://hdl.handle.net/21.15107/rcub_intor_859 |
| Rights: | openAccess ; https://creativecommons.org/licenses/by/4.0/ ; BY |
| رقم الانضمام: | edsbas.42BC0229 |
| قاعدة البيانات: | BASE |
Full Text Finder | تدمد: | 14220067 |
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