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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...

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Autores principales: Shukla, Esha, Choudhury, Lipsa, Rastogi, Saurabh, Chawla, Arshmeet, Bhattacharya, Sanghati, Kaushik, Umesh, Mittal, Manan, Rathore, Anurag Singh, Pandey, Gaurav
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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.
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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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