Development of a recombinant nucleocapsid protein‐based ELISA for the detection of IgM and IgG antibodies to SARS‐CoV‐2

Ranjbar, Maryam; Asadi, Mohammad; Nourigorji, Marjan; Sarkari, Bahador; Mostafavi‐Pour, Zohreh; Zomorodian, Kamiar; Shabaninejad, Zahra; Taheri‐Anganeh, Mortaza; Maleksabet, Amir; Moghadami, Mohsen; Savardashtaki, Amir

doi:10.1002/bab.2308

Cited by 7 publications

(3 citation statements)

References 38 publications

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“…Enzyme-linked immunosorbent assay (ELISA) is often used for the detection of viral antibodies and has been developed as a rapid diagnostic test kit due to its ease of operation and use of inexpensive equipment. In ELISA of SARS-CoV-2, different structural proteins could be used as Ag to detect the corresponding antibodies and researchers have developed ELISAs with good sensitivity and specificity [ 83 – 86 ]. Using microfluidic technology, González-González et al [ 87 ] developed an automated ELISA chip for detecting antibodies to SARS-CoV-2, enabling on-site testing that may only require a smartphone with a camera.…”

Section: Rapid Detection Methodsmentioning

confidence: 99%

Rapid assays of SARS-CoV-2 virus and noble biosensors by nanomaterials

Liu,

Li,

Hang

et al. 2024

Nano Convergence

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The COVID-19 outbreak caused by SARS-CoV-2 in late 2019 has spread rapidly across the world to form a global epidemic of respiratory infectious diseases. Increased investigations on diagnostic tools are currently implemented to assist rapid identification of the virus because mass and rapid diagnosis might be the best way to prevent the outbreak of the virus. This critical review discusses the detection principles, fabrication techniques, and applications on the rapid detection of SARS-CoV-2 with three categories: rapid nuclear acid augmentation test, rapid immunoassay test and biosensors. Special efforts were put on enhancement of nanomaterials on biosensors for rapid, sensitive, and low-cost diagnostics of SARS-CoV-2 virus. Future developments are suggested regarding potential candidates in hospitals, clinics and laboratories for control and prevention of large-scale epidemic. Graphical Abstract

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Section: Rapid Detection Methodsmentioning

confidence: 99%

Rapid assays of SARS-CoV-2 virus and noble biosensors by nanomaterials

Liu,

Li,

Hang

et al. 2024

Nano Convergence

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“…The recombinant construct (pET23a-SsIR-Ss1a) was transformed into competent E . coli DH5α cells (Novagen, USA), using the heat shock technique [ 28 ]. The related SsIR-Ss1a was sequenced (S1, Sequence of the cloned antigen (rSsIR-Ss1a)) and used to verify the cloning accuracy of the plasmid isolated from transformants (GenScript, USA).…”

Section: Methodsmentioning

confidence: 99%

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity

Omidian,

Mostafavi-Pour,

Asadi

et al. 2024

PLoS Negl Trop Dis

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Background The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. Methodology/Principal findings The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest immunogenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. Conclusions/Significance The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

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“…Recombinant Ncap protein has also been used to facilitate development of both ELISA [9][10][11][12] and lateral flow diagnostics systems [12] [13] [14,15]. The SARS-CoV-2 Ncap protein has been suggested as a possible vaccine candidate [16,17] and it has been demonstrated that immunization with recombinant Ncap produced in Escherichia coli induced an antibody response in the lungs of rats [18].…”

Section: Introductionmentioning

confidence: 99%

Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins inEscherichia coli

Brudenell,

Pohare,

Zafred

et al. 2024

Preprint

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The fundamental biology of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (Ncap), its use in diagnostic assays and its potential application as a vaccine component have received considerable attention since the outbreak of the Covid19 pandemic in late 2019. Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap inEscherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled bylacoperator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200 mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.

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Development of a recombinant nucleocapsid protein‐based ELISA for the detection of IgM and IgG antibodies to SARS‐CoV‐2

Cited by 7 publications

References 38 publications

Rapid assays of SARS-CoV-2 virus and noble biosensors by nanomaterials

Rapid assays of SARS-CoV-2 virus and noble biosensors by nanomaterials

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity

Efficient overexpression and purification of SARS-CoV-2 Nucleocapsid proteins inEscherichia coli

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