Limits...
Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies.

Nainys J, Lasickiene R, Petraityte-Burneikiene R, Dabrisius J, Lelesius R, Sereika V, Zvirbliene A, Sasnauskas K, Gedvilaite A - BMC Biotechnol. (2014)

Bottom Line: Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells.The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania. juozas.nainys@gmail.com.

ABSTRACT

Background: Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.

Results: In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.

Conclusions: We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

Show MeSH

Related in: MedlinePlus

Titration curves of swine serum samples. Either positive or negative for PCV2 antibodies swine serum samples were titrated by the newly developed indirect IgG PCV2 Cap VLP-based ELISA using recombinant PCV2 Cap VLPs at a concentration of 0.5 μg/ml (solid lines) and control antigen (HaPyV-VP1) at a concentration of 2 μg/ml (dotted line). Sera were tested in serial duplicate twofold dilutions ranging from 1:100 to 1:3200. The results are expressed as the OD450 and correspond to the average of the values obtained in at least two different analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4265424&req=5

Fig3: Titration curves of swine serum samples. Either positive or negative for PCV2 antibodies swine serum samples were titrated by the newly developed indirect IgG PCV2 Cap VLP-based ELISA using recombinant PCV2 Cap VLPs at a concentration of 0.5 μg/ml (solid lines) and control antigen (HaPyV-VP1) at a concentration of 2 μg/ml (dotted line). Sera were tested in serial duplicate twofold dilutions ranging from 1:100 to 1:3200. The results are expressed as the OD450 and correspond to the average of the values obtained in at least two different analyses.

Mentions: As a first step in the development of PCV2-Cap VLP-based ELISA, checkerboard titrations were performed to determine the optimal concentration of the Cap antigen and the test serum. To optimize plate coating, the recombinant PCV2-Cap 622 protein was immobilized on ELISA plates at five different concentrations: 2; 1; 0.5; 0.25; 0.125 μg/mL. The optimal antigen concentration for plate coating as determined by a checkerboard titration was 0.5 μg/mL. To determine the optimal serum sample dilution, serum samples identified by the commercial SERELISA test (Synbiotics, Lyon, France) as high positive, low positive and negative were diluted in serial two fold dilutions ranging from 1:100 to 1:3200. The OD450 values obtained in an indirect ELISA were plotted against the dilutions (Figure 3). High OD readings with positive control serum and low background signals with negative sera were obtained at serum dilution 1:800. The absorbance values declined at serum dilution 1:1600. As the commercial SERELISA test uses serum dilution 1:1000 and the differences between OD values at serum dilutions 1:800 and 1:1000 were insignificant in our newly developed ELISA, for convenience the appropriate dilution (1:1000) was selected for the indirect IgG PCV2 Cap-based ELISA. Control antigen – yeast expressed Hamster polyomavirus (HaPyV) VP1 protein VLPs [28] was included in the test to determine the specificity of the assay. The positive samples revealed significantly higher OD values (0.9–2.5) with PCV2 Cap protein as compared to the control HaPyV-VP1 antigen (0.05–0.1) (Figure 4, Dotted lines).Figure 3


Generation in yeast of recombinant virus-like particles of porcine circovirus type 2 capsid protein and their use for a serologic assay and development of monoclonal antibodies.

Nainys J, Lasickiene R, Petraityte-Burneikiene R, Dabrisius J, Lelesius R, Sereika V, Zvirbliene A, Sasnauskas K, Gedvilaite A - BMC Biotechnol. (2014)

Titration curves of swine serum samples. Either positive or negative for PCV2 antibodies swine serum samples were titrated by the newly developed indirect IgG PCV2 Cap VLP-based ELISA using recombinant PCV2 Cap VLPs at a concentration of 0.5 μg/ml (solid lines) and control antigen (HaPyV-VP1) at a concentration of 2 μg/ml (dotted line). Sera were tested in serial duplicate twofold dilutions ranging from 1:100 to 1:3200. The results are expressed as the OD450 and correspond to the average of the values obtained in at least two different analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265424&req=5

Fig3: Titration curves of swine serum samples. Either positive or negative for PCV2 antibodies swine serum samples were titrated by the newly developed indirect IgG PCV2 Cap VLP-based ELISA using recombinant PCV2 Cap VLPs at a concentration of 0.5 μg/ml (solid lines) and control antigen (HaPyV-VP1) at a concentration of 2 μg/ml (dotted line). Sera were tested in serial duplicate twofold dilutions ranging from 1:100 to 1:3200. The results are expressed as the OD450 and correspond to the average of the values obtained in at least two different analyses.
Mentions: As a first step in the development of PCV2-Cap VLP-based ELISA, checkerboard titrations were performed to determine the optimal concentration of the Cap antigen and the test serum. To optimize plate coating, the recombinant PCV2-Cap 622 protein was immobilized on ELISA plates at five different concentrations: 2; 1; 0.5; 0.25; 0.125 μg/mL. The optimal antigen concentration for plate coating as determined by a checkerboard titration was 0.5 μg/mL. To determine the optimal serum sample dilution, serum samples identified by the commercial SERELISA test (Synbiotics, Lyon, France) as high positive, low positive and negative were diluted in serial two fold dilutions ranging from 1:100 to 1:3200. The OD450 values obtained in an indirect ELISA were plotted against the dilutions (Figure 3). High OD readings with positive control serum and low background signals with negative sera were obtained at serum dilution 1:800. The absorbance values declined at serum dilution 1:1600. As the commercial SERELISA test uses serum dilution 1:1000 and the differences between OD values at serum dilutions 1:800 and 1:1000 were insignificant in our newly developed ELISA, for convenience the appropriate dilution (1:1000) was selected for the indirect IgG PCV2 Cap-based ELISA. Control antigen – yeast expressed Hamster polyomavirus (HaPyV) VP1 protein VLPs [28] was included in the test to determine the specificity of the assay. The positive samples revealed significantly higher OD values (0.9–2.5) with PCV2 Cap protein as compared to the control HaPyV-VP1 antigen (0.05–0.1) (Figure 4, Dotted lines).Figure 3

Bottom Line: Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells.The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania. juozas.nainys@gmail.com.

ABSTRACT

Background: Porcine circovirus type 2 (PCV2) is considered to be an important emerging pathogen associated with a number of different syndromes and diseases in pigs known as PCV2-associated diseases. It has been responsible for significant mortality among pigs and remains a serious economic problem to the swine industry worldwide leading to significant negative impacts on profitability of pork production.

Results: In this study we have demonstrated that PCV2 capsid (Cap) protein based virus-like particles (VLPs) were efficiently produced in yeast S. cerevisiae and induced production of monoclonal antibodies (MAbs) reactive with virus-infected cells. Moreover, PCV2 Cap VLPs served as a highly specific recombinant antigen for the development of an indirect IgG PCV2 Cap VLP-based ELISA for the detection of virus-specific IgG antibodies in swine sera. Four hundred-nine serum samples collected from pigs in Lithuania were tested for PCV2-specific IgG to determine the sensitivity and specificity of the newly developed ELISA in parallel using a commercial SERELISA test as a gold standard. From 409 tested serum samples, 297 samples were positive by both assays. Thirty-nine sera from 112 serum samples were determined as negative by SERELISA but were found to be positive both in the newly developed indirect IgG PCV2 Cap VLP-based ELISA and the PCR test.

Conclusions: We have demonstrated that S. cerevisiae expression system is an alternative to insect/baculovirus expression system for production of homogenous in size and shape PCV2 Cap protein-based VLPs similar to native virions. Yeast expression system tolerated native virus genes encoding PCV2 Cap protein variants as well as the codon-optimized gene. Moreover, yeast-derived PCV2 Cap VLPs were capable to induce the generation of PCV2-specific MAbs that did not show any cross-reactivity with PCV1-infected cells. The high sensitivity and specificity of the indirect IgG PCV2 Cap VLP-based ELISA clearly suggested that this assay is potentially useful diagnostic tool for screening PCV2-suspected samples.

Show MeSH
Related in: MedlinePlus