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1.
Viruses ; 16(5)2024 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-38793544

RESUMO

The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.


Assuntos
Antígenos Virais , COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Fosfoproteínas , SARS-CoV-2 , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/virologia , Antígenos Virais/imunologia , Antígenos Virais/genética , Fosfoproteínas/imunologia , Fosfoproteínas/genética , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Teste Sorológico para COVID-19/métodos , Teste Sorológico para COVID-19/normas , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Biologia Computacional/métodos , Mutação , Animais
2.
Viruses ; 14(12)2022 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-36560827

RESUMO

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2 , COVID-19/diagnóstico , Anticorpos Antivirais , Anticorpos Neutralizantes , Glicoproteína da Espícula de Coronavírus
3.
Data Brief ; 38: 107278, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34401413

RESUMO

We present supplementary data for the published article, "Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer" [1]. The assay described is designed to be performed at home or in a clinic without expensive instrumentation or professional training. SARS-CoV-2 is detected by an aptamer-based assay that targets the Nucleocapsid (N) or Spike (S) antigens. Binding of the N or S protein to their respective aptamer results in the competitive release of a complementary antisense-invertase enzyme complex. The released enzyme then catalyzes the conversion of sucrose to glucose that is measured by an off-the-shelf glucometer. The data presented here describe the optimization of the assay parameters and their contribution to developing this aptamer-based assay to detect SARS-CoV-2. The assay performance was checked in a standard buffer, contrived samples, and patient samples validated with well-established scientific methods. The resulting dataset can be used to further develop glucometer-based assays for diagnosing other communicable and non-communicable diseases.

4.
Biosens Bioelectron ; 180: 113111, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33743492

RESUMO

Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of widely available antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally, making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones, providing an opportunity to integrate with contact tracing apps, medical providers, and electronic health records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within 1 h, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach provides an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale.


Assuntos
Antígenos Virais/análise , Técnicas Biossensoriais/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Testes Imediatos , SARS-CoV-2/imunologia , Saliva/virologia , Adulto , Teste para COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus/análise , Feminino , Humanos , Masculino , Fosfoproteínas/análise , SARS-CoV-2/isolamento & purificação , Técnica de Seleção de Aptâmeros , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/análise
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