Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein as antigen

Dea, S.; Wilson, Louise E.; Therrien, Dominic; Cornaglia, E.

doi:10.1016/s0166-0934(00)00158-0

Cited by 36 publications

(29 citation statements)

References 30 publications

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“…The VN tests were performed in triplicates with 100 TCID50 of the virus/well as previously described [22]. To monitor humoral immune response following challenging of vaccinated pigs, an IIF using PRRSV infected MARC-145 cells as well as a competitive ELISA for detection of antibodies to PRRSV using recombinant E. coli-expressed N protein as antigen and a commercial indirect ELISA (IDEXX) for detection of anti-PRRSV antibodies were used as previously described [23].…”

Section: Virus Neutralization and Serological Testsmentioning

confidence: 99%

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP5 glycoprotein and improves immune response in challenged pigs

Kheyar

Jabrane

Zhu

et al. 2005

Vaccine

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Pigs exposed to GP5 protein of PRRSV by means of DNA immunization develop specific neutralizing and protecting antibodies. Herein, we report on the consequences of codon bias, and on the favorable outcome of the systematic replacement of native codons of PRRSV ORF5 gene with codons chosen to reflect more closely the codon preference of highly expressed mammalian genes. Therefore, a synthetic PRRSV ORF5 gene (synORF5) was constructed in which 134 nucleotide substitutions were made in comparison to wild-type gene (wtORF5), such that 59% (119) of wild-type codons were replaced with known preferable codons in mammalian cells. In vitro expression in mammalian cells of synORF5 was considerably increased comparatively to wtORF5, following infection with tetracycline inducible replication-defective human adenoviral vectors (hAdVs). After challenge inoculation, SPF pigs vaccinated twice with recombinant hAdV/synORF5 developed earlier and higher antibody titers, including virus neutralizing antibodies to GP5 than pigs vaccinated with hAdV/wtORF5. Data obtained from animal inoculation studies suggest direct correlation between expression levels of immunogenic structural viral proteins and immune response

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Section: Virus Neutralization and Serological Testsmentioning

confidence: 99%

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP5 glycoprotein and improves immune response in challenged pigs

Kheyar

Jabrane

Zhu

et al. 2005

Vaccine

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“…The primary structural proteins of PRRSV are the nucleocapsid (N) protein, the membrane/matrix (M) protein and the primary envelope glycoprotein GP5 (Plagemann et al 2006). Early antibodies mounted by PRRSV infection are mainly directed to the N-protein and, to a lesser extent, to the M-protein and are non-neutralizing (Nelson et al 1994, Gonin et al 1999, Dea et al 2000. Neutralizing antibodies are mainly directed against GP5, and the primary neutralization epitope of PRRSV strain VR-2332 is located in the middle of the GP5 ectodomain (amino acids 36-52) (Pirzadeh and Dea 1997, Gonin, et al 1999, Plagemann et al 2002, Ferrin et al 2004.…”

Section: Introductionmentioning

confidence: 99%

“…The immunoperoxidase monolayer assay (IPMA) (Wensvoort et al 1991) and the indirect immunofluorescence assay (IFA) (Yoon et al 1992) are based on the specific binding of antibodies to viral antigens cultivated in porcine alveolar macrophages or MARC-145 cells. Yet both tests are laborious and not suitable for routine testing on a large scale with the disadvantages of the need of a replicating virus (Dea et al 2000). Indirect or competitive enzyme-linked immunosorbent assays (ELISAs) have been reported by several groups of investigators (Albina et al 1993, Cho et al 1996, Dea et al 2000, Nowicka et al 2014.…”

Section: Introductionmentioning

confidence: 99%

“…Yet both tests are laborious and not suitable for routine testing on a large scale with the disadvantages of the need of a replicating virus (Dea et al 2000). Indirect or competitive enzyme-linked immunosorbent assays (ELISAs) have been reported by several groups of investigators (Albina et al 1993, Cho et al 1996, Dea et al 2000, Nowicka et al 2014. These ELISA procedures are simple, quick, sensitive, and specific.…”

Section: Introductionmentioning

confidence: 99%

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GP5 Protein-based ELISA for the Detection of PRRSV Antibodies

Wang¹,

Guo²,

Qiao³

et al. 2016

Polish Journal of Veterinary Sciences

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Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen, causing huge economic losses each year worldwide. Immunization with vaccines containing the glycoprotein 5 (GP5) of PRRSV is the main measure to induce neutralizing antibodies and control the disease. Here, we developed a GP5 protein-based ELISA for detecting antibodies against PRRSV. The overall yield of purified GP5 in E. coli flask culture was more than 45 mg/L cell culture. Western blot and IFA indicated that the GP5 protein was highly immunogenic. After optimization and validation with IDEXX PRRS using 566 clinical sera, the DSN, DSP, and accuracy of GP5-ELISA were 81.39%, 75.96%, and 80.39%, respectively. Besides, GP5-ELISA is highly specific, showing no cross-reactions with sera against other important swine pathogens. Hence, GP5 is a good diagnostic antigen and the GP5 protein-based ELISA has the potential to be used in the field.

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“…Since the majority of antibodies produced during PRRSV infection in pigs are specific for the N protein, for which major antigenic determinants are highly conserved, the N protein has been targeted as a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. Indeed, recombinant N protein expressed either in insect cells (9,16) or in Escherichia coli (8) has been used as an antigen in the development of indirect and competitive enzyme-linked immunosorbent assays, respectively, for detection of serum antibodies against PRRSV. These methods are relatively inexpensive, sensitive, and easy to perform and therefore represent a feasible economic alternative to the present methods that rely upon whole-virus antigen (14).…”

mentioning

confidence: 99%

Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

Wootton

Koljesar

Yoon

et al. 2001

Clin Diagn Lab Immunol

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Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.

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Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein as antigen

Cited by 36 publications

References 30 publications

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP5 glycoprotein and improves immune response in challenged pigs

Alternative codon usage of PRRS virus ORF5 gene increases eucaryotic expression of GP5 glycoprotein and improves immune response in challenged pigs

GP5 Protein-based ELISA for the Detection of PRRSV Antibodies

Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

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