scholarly journals Expression of the Nucleoprotein of the Puumala Virus from the Recombinant Semliki Forest Virus Replicon: Characterization and Use as a Potential Diagnostic Tool

2003 ◽  
Vol 10 (4) ◽  
pp. 658-663 ◽  
Author(s):  
A. Billecocq ◽  
D. Coudrier ◽  
F. Boué ◽  
B. Combes ◽  
H. Zeller ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Semliki Forest Virus ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Puumala Virus ◽  
Wild Rodents ◽  
Serological Tests ◽  
N Protein ◽  
Antigenic Properties ◽  
Native Antigen

ABSTRACT Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), μ-capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.

scholarly journals Expression and novel alternative purification of the recombinant nucleocapsid (N) protein of SARS-CoV-2 in Escherichia coli for the serodiagnosis of COVID-19

2021 ◽  
Author(s):  
Jose David Rosales ◽  
William Quintero ◽  
Jhon Cruz ◽  
Balbino Perdomo ◽  
Militza Quintero ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein S ◽  
Exchange Chromatography ◽  
Diagnostic Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Metal Exchange ◽  
Serological Tests ◽  
N Protein ◽  
Global Pandemic ◽  
Novel Strategy

The SARS-CoV-2 coronavirus causes severe acute respiratory syndrome and has caused a global pandemic by causing the COVID-19 disease. To monitor and control it, diagnostic methods such as molecular and serological tests are necessary. The serological approach use SARS-CoV-2 antigens to detect the antibodies present in patients using quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or qualitative rapid tests such as lateral flow chromatography (RDT's). The main antigens used are the spike protein (S) and the nucleocapsid protein (N). Both proteins are obtained in different expression systems, in eukaryotic cells, their production is expensive, so in this work we chose a simpler and cheaper system such as prokaryotic to express and purify the N protein. Thereore, the nucleotide sequence had to being optimized to be expressed in Escherichia coli. The protein N is sensitive to E.coli proteases and also has the ability to self-proteolyze under native conditions, degrading into different fragments. However, under denaturing conditions, using urea and at pH 5.3 it is stable and efficiently purified using metal exchange chromatography (IMAC). In our purification strategy, we surprisingly found that by not using a sonicator, a homogeneous and time-stable preparation of the recombinant antigen is obtained. An approximate yield of 200 mg / L was obtained. It was then tested with healthy sera and sera from COVID-19 convalescent patients in Wester-blot tests that were able to recognize it. Our work provides a novel strategy to produce the SARS-CoV-2 protein N so that it can be used as an input in the development and innovation of serological tests in the diagnosis of COVID-19.

scholarly journals Comparative analysis of antigen-specific anti-SARS-CoV-2 antibody isotypes in COVID-19 patients

2020 ◽  
Author(s):  
Hidetsugu Fujigaki ◽  
Masato Inaba ◽  
Michiko Osawa ◽  
Saya Moriyama ◽  
Yoshimasa Takahashi ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Serological Test ◽  
Polymerase Chain Reaction Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
S Protein ◽  
Neutralizing Activity ◽  
N Protein ◽  
Specificity And Sensitivity ◽  
Antibody Isotypes

AbstractSerological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect antibodies against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various antigen-specific antibody isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via polymerase chain reaction test. We developed IgG, IgM and IgA measurement assays for each antigen, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full length S protein, S trimer and nucleocapsid (N) domain, based on enzyme-linked immunosorbent assay. The assays of the S protein for all isotypes showed high specificity, while the assays for all isotypes against N protein showed lower specificity. The sensitivity of all antigen-specific antibody isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 days post-symptom onset. The best correlation with virus neutralizing activity was found for IgG against RBD (RBD-IgG), and levels of RBD-IgG in sera from four severe COVID-19 patients increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

scholarly journals Development of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay To Differentiate Human Flavivirus Infections Occurring in Australia

2005 ◽  
Vol 12 (3) ◽  
pp. 371-374 ◽  
Author(s):  
Carmel Taylor ◽  
Russell Simmons ◽  
Ina Smith
Keyword(s):  
Immunosorbent Assay ◽  
Specific Antibody ◽  
Hemagglutination Inhibition ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Original Diagnosis ◽  
Specific Diagnosis

ABSTRACT We report the development of a flavivirus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MAC-ELISA) which improves the determination of an infecting flavivirus serotype over that by current serological methods. A panel of 165 IgM-positive sera from flavivirus patients with specific diagnostic results was tested by the flavivirus MAC-ELISA using a panel of 10 antigens. For 134 of these sera (81.2%), the highest reactivity was demonstrated against the infecting virus, which was consistent with the original diagnostic result. Specific antibody reactions inconsistent with the original diagnosis were found for six sera (3.6%). In our experience, the flavivirus-serotyping ELISA provides a rapid and accurate alternative to other serological tests, such as hemagglutination inhibition, for the specific diagnosis of flavivirus infections.

scholarly journals Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19

2021 ◽  
Author(s):  
Wajihul Hasan Khan ◽  
Nida Khan ◽  
Avinash Mishra ◽  
Surbhi Gupta ◽  
Vikrant Bansode ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Accessible Surface Area ◽  
Solvent Accessible Surface Area ◽  
Enzyme Linked Immunosorbent Assay ◽  
Monomeric Form ◽  
N Protein ◽  
Diagnostic Assays ◽  
Antigenic Properties ◽  
Dimeric Form ◽  
Accessible Surface

Diagnostics has played a significant role in effective management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays. Thus far, limited knowledge exists about the antigenic properties of the N protein. In this paper, we demonstrate the significant impact of dimerization of SARS-CoV-2 nucleocapsid protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics of COVID-19. The expressed purified protein from E.coli consists of two forms, dimeric and monomeric forms, which have been further characterized by biophysical and immunological means. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form of the protein. These findings have also been confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. It is evident that use of the dimeric form will increase the sensitivity of the current nucleocapsid dependent ELISA for rapid COVID-19 diagnostic. Further, the results indicate that monitoring and maintaining of the monomer-dimer composition is critical for accurate and robust diagnostics.

scholarly journals Obtaining Multiprotein Antigens Of Brucella And Study Of Their Immunoreactivity

2021 ◽  
Author(s):  
B.K. Ingirbai ◽  
A. Syzdykova ◽  
A.K. Kurmasheva ◽  
A.K. Bulashev
Keyword(s):  
Complement Fixation Test ◽  
Recombinant Dna ◽  
Outer Membrane Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Producer Strain ◽  
Recombinant Dna Technology ◽  
Serological Tests ◽  
Highly Sensitive ◽  
Antigenic Properties ◽  
Low Sensitivity

Rose-Bengal test, complement fixation test and the agglutination test are mainly used for the diagnosis of animal brucellosis. These tests are characterized by low sensitivity and specificity, which is one of the main reasons for the low effectiveness of measures aimed at eradicating brucellosis. The use of modern highly sensitive serological tests requires the availability of antigens specific to Brucella spp. The aim of the study was to obtain multiproteins of the pathogen by recombinant DNA technology and to study their antigenic properties. In the course of the study, three types of multiproteins were obtained, constructed from diagnostically important peptides that form B.abortus and B.melitensis outer membrane proteins. All target products were synthesized by the producer strain in a form that is authentic to natural proteins, and showed immunogenicity in mouse model. Antibodies produced against the multiproteins were specific for the single proteins of the pathogen's cell wall. The data obtained indicate the need to continue studies to determine the possibility of using multiproteins as an antigen in an enzyme-linked immunosorbent assay to detect anti-Brucella specific antibodies.

scholarly journals Evaluation of the Indirect Hemagglutination Assay for Diagnosis of Acute Leptospirosis

1998 ◽  
Vol 36 (1) ◽  
pp. 11-14 ◽  
Author(s):  
Paul N. Levett ◽  
Carol U. Whittington
Keyword(s):  
Predictive Value ◽  
Venereal Disease ◽  
Agglutination Test ◽  
Immunoglobulin M ◽  
Microscopic Agglutination Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hemagglutination Assay ◽  
Serological Tests ◽  
Indirect Hemagglutination ◽  
Human Sera

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources and expertise to perform the microscopic agglutination test. There is a need for rapid and simple serological tests which facilitate the early diagnosis of leptospirosis, while antibiotic therapy may be most effective. A commercially available indirect hemagglutination assay (IHA; MRL Diagnostics, Cypress, Calif.) was evaluated with multiple serum specimens from 107 patients being investigated for leptospirosis. By using a combination of enzyme-linked immunosorbent assay (ELISA) methods for immunoglobulin M (IgM) and IgG antibodies and the microscopic agglutination test, 54 patients were found to have leptospirosis and 53 were found not to have leptospirosis. The sensitivity of IHA for the detection of acute leptospirosis was 100%, the specificity was 94%, the positive predictive value was 95%, and the negative predictive value was 100%. IHA was negative when 13 antinuclear antibody-positive sera, 24 serum specimens from patients with syphilis, and 16 serum specimens false positive by the Venereal Disease Research Laboratory test were tested. IHA was shown to detect both IgM and IgG classes of antibodies in human sera. Serum specimens from 27 dogs investigated for leptospirosis were studied: 3 samples gave nonspecific hemagglutination, but for all remaining samples, the results of IHA and an IgM ELISA were concordant. Performance of IHA was simple, and IHA requires no specialized equipment. It represents a useful assay for laboratories which require a leptospiral diagnostic capability but lack the expertise to perform specialist investigations.

Evaluation of crude hydatid cyst fluid antigens for the serological diagnosis of hydatidosis in cattle

2010 ◽  
Vol 85 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Lemu Golassa ◽  
Tamrat Abebe ◽  
Asrat Hailu
Keyword(s):  
Hydatid Cyst ◽  
Addis Ababa ◽  
Cyst Fluid ◽  
Epidemiological Surveillance ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Efficiency ◽  
Infected Cattle ◽  
Serological Tests ◽  
Antigenic Properties ◽  
Hydatid Cyst Fluid

AbstractEchinococcosis is a zoonotic infection caused by adult or larval (metacestode) stages of cestodes belonging to the genusEchinococcus. The purpose of this study was to evaluate the antigenic ability of hydatid cyst fluid antigen for the diagnosis of hydatidosis in cattle using enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination test (IHA). The source of the antigens for the serological tests was fertile crude cyst fluids collected from naturally infected sheep at the Addis Ababa abattoir. A total of 502 sera were collected from 329 uninfected cattle and 173 hydatid-infected cattle which were confirmed by post-mortem examination. Most cysts were sterile and multiple organ infection predominated. Of 173 infected cattle, 166 (96.0%; confidence interval (CI) 91.8–98.4) were positive using ELISA while 7 (4.0%) were negative. Of 329 sera from uninfected cattle, 274 (83.3%; CI 78.8–87.2) were found to be negative and the remaining 55 (16.7%) were positive by ELISA. Of 173 infected cattle, 151 (87.3%; CI 81.4–91.9) were positive and 22 (12.7%) were negative by IHA. Of 329 negative sera tested using IHA, 266 (80.9%; CI 76.2–85.0) were negative and the remaining 63 (19.1%) were positive. The false positive and negative values of ELISA were 4.0 and 16.7%, respectively, and the corresponding values of IHA were 12.7 and 19.1%. The sensitivity and diagnostic efficiency of IHA were 87.2 and 83.6%, respectively. Crude hydatid cyst fluid antigen seems to have reasonable antigenic properties and hence could be employed for epidemiological surveillance of cattle hydatidosis.

scholarly journals The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests

2021 ◽  
Vol 12 ◽  
Author(s):  
Laura Williams ◽  
Silvia Jurado ◽  
Francisco Llorente ◽  
Alberto Romualdo ◽  
Sara González ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Cost Effective ◽  
City Council ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Serological Tests ◽  
K Value ◽  
N Protein ◽  
Effective Manner

BackgroundThe fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms.MethodsWe expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test.ResultsThe performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05–98.44) and diagnostic specificity of 96.37 (95% CI: 93.05–98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892–0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA).ConclusionThis study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.

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