scholarly journals Tergitol-15-S-9 inactivates SARS-CoV-2 and boosts immunoassay signals

BioTechniques ◽  
2021 ◽  
Author(s):  
Michael G Berg ◽  
Eitan Israeli ◽  
Erin Quaco ◽  
Gavin A Cloherty ◽  
Philip M Hemken
Keyword(s):  
Nucleocapsid Protein ◽  
Full Length ◽  
Heat Inactivation ◽  
Diagnostic Assay ◽  
Assay Performance ◽  
Nonionic Detergents

Inactivation of SARS-CoV-2 virus is necessary to mitigate risk but may interfere with diagnostic assay performance. We examined the effect of heat inactivation on a prototype SARS-CoV-2 antigen immunoassay run on the ARCHITECT automated analyzer. Recombinant full-length SARS-CoV-2 nucleocapsid protein and virus lysate detection was reduced by 66 and 31%, respectively. Several nonionic detergents were assessed as inactivation alternatives based on infectivity in cultured Vero CCL81 cells. Incubation of SARS-CoV-2 in 0.1% Tergitol 15-S-9 for 10 min significantly reduced infectivity and increased the immunoassay signal for cultured lysate and patient specimens. Tergitol 15-S-9 can inactivate SARS-CoV-2 while preserving epitopes on the nucleocapsid protein for enhanced detection by immunoassay antibodies.

scholarly journals Several Recombinant Capsid Proteins of Equine Rhinitis A Virus Show Potential as Diagnostic Antigens

2005 ◽  
Vol 12 (6) ◽  
pp. 778-785 ◽  
Author(s):  
Fan Li ◽  
Rachel A. Stevenson ◽  
Brendan S. Crabb ◽  
Michael J. Studdert ◽  
Carol A. Hartley
Keyword(s):  
Recombinant Proteins ◽  
Foot And Mouth Disease ◽  
Terminal Region ◽  
Full Length ◽  
Capsid Proteins ◽  
Diagnostic Assay ◽  
B Cell Epitopes ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Native Antigen

ABSTRACT Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

scholarly journals Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

2020 ◽  
Author(s):  
Corinne A. Lutomski ◽  
Tarick J. El-Baba ◽  
Jani R. Bolla ◽  
Carol V. Robinson
Keyword(s):  
Antibody Response ◽  
Nucleocapsid Protein ◽  
Electron Transfer Dissociation ◽  
Rna Binding ◽  
Vaccine Development ◽  
Cyclophilin A ◽  
Proteolytic Processing ◽  
Full Length ◽  
Oligomeric State ◽  
N Protein

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.

scholarly journals Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding

2007 ◽  
Vol 35 (6) ◽  
pp. 2026-2034 ◽  
Author(s):  
Anwer Mujeeb ◽  
Nikolai B. Ulyanov ◽  
Stefanos Georgantis ◽  
Ivan Smirnov ◽  
Janet Chung ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Full Length ◽  
Initiation Complex ◽  
Sequence Effects ◽  
Hiv 1

scholarly journals Detection of Nucleocapsid Antibody to SARS-CoV-2 is More Sensitive than Antibody to Spike Protein in COVID-19 Patients

2020 ◽  
Author(s):  
Peter D. Burbelo ◽  
Francis X. Riedo ◽  
Chihiro Morishima ◽  
Stephen Rawlings ◽  
Davey Smith ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Spike Protein ◽  
Heat Inactivation ◽  
Cross Sectional ◽  
Immunoprecipitation Assay ◽  
Specific Antibodies ◽  
Quantitative Measurements ◽  
Initial Symptoms ◽  
Antibody Levels ◽  
Immunocompetent Patients

ABSTRACTBackgroundSARS-CoV-2, the cause of coronavirus disease 2019 (COVID-19), is associated with respiratory-related morbidity and mortality. Assays to detect virus-specific antibodies are important to understand the prevalence of infection and the course of the immune response.MethodologyQuantitative measurements of plasma or serum antibodies by luciferase immunoprecipitation assay systems (LIPS) to the nucleocapsid and spike proteins were analyzed in 100 cross-sectional or longitudinal samples from SARS-CoV-2-infected patients. A subset of samples was tested with and without heat inactivation.ResultsFifteen or more days after symptom onset, antibodies against SARS-CoV-2 nucleocapsid protein showed 100% sensitivity and 100% specificity, while antibodies to spike protein were detected with 91% sensitivity and 100% specificity. Neither antibody levels nor the rate of seropositivity were significantly reduced by heat inactivation of samples. Analysis of daily samples from six patients with COVID-19 showed anti-nucleocapsid and spike antibodies appearing between day 8 to day 14 after initial symptoms. Immunocompromised patients generally had a delayed antibody response to SARS-CoV-2 compared to immunocompetent patients.ConclusionsAntibody to the nucleocapsid protein of SARS-CoV-2 is more sensitive than spike protein antibody for detecting early infection. Analyzing heat-inactivated samples by LIPS is a safe and sensitive method for detecting SARS-CoV-2 antibodies.

scholarly journals Activation of NF-kappaB by the Full-length Nucleocapsid Protein of the SARS Coronavirus

2005 ◽  
Vol 37 (9) ◽  
pp. 607-612 ◽  
Author(s):  
Qing-Jiao LIAO ◽  
Lin-Bai YE ◽  
Khalid Amine TIMANI ◽  
Ying-Chun ZENG ◽  
Ying-Long SHE ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Full Length ◽  
Sars Coronavirus ◽  
Nf Kappab

scholarly journals Sensitivity in Detection of Antibodies to Nucleocapsid and Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Coronavirus Disease 2019

2020 ◽  
Vol 222 (2) ◽  
pp. 206-213 ◽  
Author(s):  
Peter D Burbelo ◽  
Francis X Riedo ◽  
Chihiro Morishima ◽  
Stephen Rawlings ◽  
Davey Smith ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nucleocapsid Protein ◽  
Spike Protein ◽  
Heat Inactivation ◽  
Cross Sectional ◽  
Quantitative Measurements ◽  
Initial Symptoms ◽  
Antibody Levels ◽  
Immunocompetent Patients ◽  
Spike Proteins

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is associated with respiratory-related disease and death. Assays to detect virus-specific antibodies are important to understand the prevalence of infection and the course of the immune response. Methods Quantitative measurements of plasma or serum antibodies to the nucleocapsid and spike proteins were analyzed using luciferase immunoprecipitation system assays in 100 cross-sectional or longitudinal samples from patients with SARS-CoV-2 infection. A subset of samples was tested both with and without heat inactivation. Results At >14 days after symptom onset, antibodies against SARS-CoV-2 nucleocapsid protein showed 100% sensitivity and 100% specificity, whereas antibodies to spike protein were detected with 91% sensitivity and 100% specificity. Neither antibody levels nor the rate of seropositivity were significantly reduced by heat inactivation of samples. Analysis of daily samples from 6 patients with COVID-19 showed anti-nucleocapsid and spike protein antibodies appearing between days 8 and 14 after initial symptoms. Immunocompromised patients generally had a delayed antibody response to SARS-CoV-2, compared with immunocompetent patients. Conclusions Antibody to the nucleocapsid protein of SARS-CoV-2 is more sensitive than spike protein antibody for detecting early infection. Analyzing heat-inactivated samples with a luciferase immunoprecipitation system assay is a safe and sensitive method for detecting SARS-CoV-2 antibodies.

Baculovirus expression and purification of peste-des-petits-ruminants virus nucleocapsid protein and its application in diagnostic assay

Biologicals ◽  
2018 ◽  
Vol 55 ◽  
pp. 38-42 ◽  
Author(s):  
Suresh H. Basagoudanavar ◽  
Madhusudan Hosamani ◽  
D. Muthuchelvan ◽  
R.P. Singh ◽  
R. Santhamani ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Peste Des Petits Ruminants ◽  
Diagnostic Assay ◽  
Expression And Purification ◽  
Baculovirus Expression

scholarly journals Antigenicity Analysis of Different Regions of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

2004 ◽  
Vol 50 (6) ◽  
pp. 988-995 ◽  
Author(s):  
Zeliang Chen ◽  
Decui Pei ◽  
Lingxiao Jiang ◽  
Yajun Song ◽  
Jin Wang ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nucleocapsid Protein ◽  
Vaccine Candidate ◽  
Protein Microarray ◽  
Full Length ◽  
Protein Fragments ◽  
N Protein ◽  
Conformational Epitopes ◽  
Glass Slides ◽  
Linear Epitopes

Abstract Background: The widespread threat of severe acute respiratory syndrome (SARS) to human health has made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use. For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein. Methods: The cDNA for full-length N protein and its various regions from the SARS-CoV was cloned and expressed in Escherichia coli. After purification, all of the protein fragments were printed on glass slides to fabricate a protein microarray and then probed with the sera from SARS patients to determine the reactivity of these protein fragments. Results: The full-length protein and two other fragments reacted with all 52 sera tested. Four important regions with possible epitopes were identified and named as EP1 (amino acids 51–71), EP2 (134–208), EP3 (249–273), and EP4 (349–422), respectively. EP2 and EP4 possessed linear epitopes, whereas EP1 and EP2 were able to form conformational epitopes that could react with most (>80%) of the tested sera. EP3 and EP4 also formed conformational epitopes, and antibodies against these epitopes existed in all 52 of the sera tested. Conclusion: The N protein is a highly immunogenic protein of the SARS-CoV. Conformational epitopes are important for this protein, and antigenicity of the COOH terminus is higher than that of the NH2 terminus. The N protein is a potential diagnostic antigen and vaccine candidate for SARS-CoV.

scholarly journals Deciphering the Role of Humoral and Cellular Immune Responses in Different COVID-19 Vaccines—A Comparison of Vaccine Candidate Genes in Roborovski Dwarf Hamsters

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2290
Author(s):  
Jakob Trimpert ◽  
Susanne Herwig ◽  
Julia Stein ◽  
Daria Vladimirova ◽  
Julia M. Adler ◽  
...  
Keyword(s):  
Immune Responses ◽  
Nucleocapsid Protein ◽  
Neutralizing Antibodies ◽  
Specific Antigen ◽  
T Cell Response ◽  
Full Length ◽  
Spike Protein ◽  
Protective Effects ◽  
Cellular Immune Responses ◽  
Cellular Immune

With the exception of inactivated vaccines, all SARS-CoV-2 vaccines currently used for clinical application focus on the spike envelope glycoprotein as a virus-specific antigen. Compared to other SARS-CoV-2 genes, mutations in the spike protein gene are more rapidly selected and spread within the population, which carries the risk of impairing the efficacy of spike-based vaccines. It is unclear to what extent the loss of neutralizing antibody epitopes can be compensated by cellular immune responses, and whether the use of other SARS-CoV-2 antigens might cause a more diverse immune response and better long-term protection, particularly in light of the continued evolution towards new SARS-CoV-2 variants. To address this question, we explored immunogenicity and protective effects of adenoviral vectors encoding either the full-length spike protein (S), the nucleocapsid protein (N), the receptor binding domain (RBD) or a hybrid construct of RBD and the membrane protein (M) in a highly susceptible COVID-19 hamster model. All adenoviral vaccines provided life-saving protection against SARS-CoV-2-infection. The most efficient protection was achieved after exposure to full-length spike. However, the nucleocapsid protein, which triggered a robust T-cell response but did not facilitate the formation of neutralizing antibodies, controlled early virus replication efficiently and prevented severe pneumonia. Although the full-length spike protein is an excellent target for vaccines, it does not appear to be the only option for future vaccine design.

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