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Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering
The COVID-19 pandemic has had a significant impact on human health management. A rapid diagnosis of SARS-CoV2 at the point-of-care (POC) is critical to prevent disease spread. As a POC device for remote settings, a LFIA should not require cold-chain maintenance and should be kept at normal temperatu...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10604654/ https://www.ncbi.nlm.nih.gov/pubmed/37892206 http://dx.doi.org/10.3390/biom13101524 |
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author | Shukla, Esha Choudhury, Lipsa Rastogi, Saurabh Chawla, Arshmeet Bhattacharya, Sanghati Kaushik, Umesh Mittal, Manan Rathore, Anurag Singh Pandey, Gaurav |
author_facet | Shukla, Esha Choudhury, Lipsa Rastogi, Saurabh Chawla, Arshmeet Bhattacharya, Sanghati Kaushik, Umesh Mittal, Manan Rathore, Anurag Singh Pandey, Gaurav |
author_sort | Shukla, Esha |
collection | PubMed |
description | The COVID-19 pandemic has had a significant impact on human health management. A rapid diagnosis of SARS-CoV2 at the point-of-care (POC) is critical to prevent disease spread. As a POC device for remote settings, a LFIA should not require cold-chain maintenance and should be kept at normal temperatures. Antigen stability can be enhanced by addressing instability issues when dealing with fragile components, such as proteinaceous capture antigens. This study used immunologically guided protein engineering to enhance the capture nucleocapsid (NP) antigen stability of SARS-CoV2. A search of the IEDB database revealed that antibodies detecting epitopes are almost uniformly distributed over NP(1-419). In contrast, N-terminal stretches of NP(1-419) are theoretically more unstable than C-terminal stretches. We identified NP(250-365) as a NP stretch with a low instability index and B-cell epitopes. Apart from NP(1-419), two other variants (NP(121-419) and NP(250-365)) were cloned, expressed, and purified. The degradation pattern of the proteins was observed on SDS-PAGE after three days of stability studies at −20 °C, 4 °C, and 37 °C. NP(1-419) was the most degraded while NP(250-365) exhibited the least degradation. Also, NP(1-419), NP(250-365), and NP(121-419) reacted with purified antibodies from COVID-19 patient serum. Our results suggest that NP(250-365) may be used as a stable capture antigen in LFIA devices to detect COVID-19. |
format | Online Article Text |
id | pubmed-10604654 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-106046542023-10-28 Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering Shukla, Esha Choudhury, Lipsa Rastogi, Saurabh Chawla, Arshmeet Bhattacharya, Sanghati Kaushik, Umesh Mittal, Manan Rathore, Anurag Singh Pandey, Gaurav Biomolecules Article The COVID-19 pandemic has had a significant impact on human health management. A rapid diagnosis of SARS-CoV2 at the point-of-care (POC) is critical to prevent disease spread. As a POC device for remote settings, a LFIA should not require cold-chain maintenance and should be kept at normal temperatures. Antigen stability can be enhanced by addressing instability issues when dealing with fragile components, such as proteinaceous capture antigens. This study used immunologically guided protein engineering to enhance the capture nucleocapsid (NP) antigen stability of SARS-CoV2. A search of the IEDB database revealed that antibodies detecting epitopes are almost uniformly distributed over NP(1-419). In contrast, N-terminal stretches of NP(1-419) are theoretically more unstable than C-terminal stretches. We identified NP(250-365) as a NP stretch with a low instability index and B-cell epitopes. Apart from NP(1-419), two other variants (NP(121-419) and NP(250-365)) were cloned, expressed, and purified. The degradation pattern of the proteins was observed on SDS-PAGE after three days of stability studies at −20 °C, 4 °C, and 37 °C. NP(1-419) was the most degraded while NP(250-365) exhibited the least degradation. Also, NP(1-419), NP(250-365), and NP(121-419) reacted with purified antibodies from COVID-19 patient serum. Our results suggest that NP(250-365) may be used as a stable capture antigen in LFIA devices to detect COVID-19. MDPI 2023-10-14 /pmc/articles/PMC10604654/ /pubmed/37892206 http://dx.doi.org/10.3390/biom13101524 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Shukla, Esha Choudhury, Lipsa Rastogi, Saurabh Chawla, Arshmeet Bhattacharya, Sanghati Kaushik, Umesh Mittal, Manan Rathore, Anurag Singh Pandey, Gaurav Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering |
title | Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering |
title_full | Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering |
title_fullStr | Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering |
title_full_unstemmed | Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering |
title_short | Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering |
title_sort | improved stability and manufacturability of nucleocapsid antigens for sars-cov2 diagnostics through protein engineering |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10604654/ https://www.ncbi.nlm.nih.gov/pubmed/37892206 http://dx.doi.org/10.3390/biom13101524 |
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