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Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus.

Okda F, Liu X, Singrey A, Clement T, Nelson J, Christopher-Hennings J, Nelson EA, Lawson S - BMC Vet. Res. (2015)

Bottom Line: The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively.Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies.In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests.

View Article: PubMed Central - PubMed

Affiliation: Veterinary & Biomedical Sciences Department, South Dakota State University, Brookings, SD, USA. faten.okda@sdstate.edu.

ABSTRACT

Background: Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies.

Results: A recombinant North American nucleoprotein (NP) based iELISA was developed and validated along with a bELISA using newly developed PEDV-NP specific biotinylated monoclonal antibodies (mAbs) and an FMIA using magnetic beads coupled with expressed NA PEDV-NP. Receiver operating characteristic (ROC) analysis was performed using swine serum samples (iELISA n = 1486, bELISA n = 1186, FMIA n = 1420). The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. The iELISA and bELISA showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. Inter-rater (kappa) agreement was calculated to be 0.941 between iELISA and IFA, 0.945 between bELISA and IFA and 0.932 between FMIA and IFA. Similar comparative kappa values were observed between the iELISA, bELISA and FMIA, which demonstrated a significant level of testing agreement among the three assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted with these assays. All three assays detected seroconversion of naïve animals within 6-9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples.

Conclusion: Well-validated iELISA, bELISA and FMIA assays for the detection of PEDV antibodies were developed and showed good correlation with IFA and each other. Each assay format has advantages that dictate how they will be used in the field. Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies. In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. Measurement of neutralizing antibody responses using the FFN assay may provide a valuable tool for assessment of vaccine candidates or protective immunity.

No MeSH data available.


Related in: MedlinePlus

Antigen/antibody specificity. Western blot analysis showing detection of recombinant expressed NA PEDV-NP protein and specificity of the monoclonal antibody used in the bELISA. L- Molecular weight ladder. A- anti-PEDV-NP mAb 6–29. B- anti-polyhistidine mAb. C- anti PEDV-NP convalescent swine serum
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Fig2: Antigen/antibody specificity. Western blot analysis showing detection of recombinant expressed NA PEDV-NP protein and specificity of the monoclonal antibody used in the bELISA. L- Molecular weight ladder. A- anti-PEDV-NP mAb 6–29. B- anti-polyhistidine mAb. C- anti PEDV-NP convalescent swine serum

Mentions: The development and validation of the iELISA and bELISA made use of a recombinantly expressed full length NA PEDV-NP. The NP open reading frame (ORF) of PEDV was amplified from RNA extracted directly from intestinal contents by RT-PCR from a case submitted to the South Dakota ADRDL. It was subsequently directionally cloned into the E. coli, pET 28a(+), plasmid expression vector (Novagen, Madison, WI), then transformed into BL21-Codon Plus (DE3)-RP competent cells (Stratagene, La Jolla, CA) for protein expression. Primers used for the amplification of the full length (1323 bp) nucleoprotein were: PEDV-NP-fwd (5′-CGCGGATCCATGGCTTCTGTCAGTTTTCAG-3′); PEDV-NP-rev (5′- CACACTCGAGATTTCCTGTGTCGAAGATCTC-3′). Next, 20 μl of transformed cells were plated onto Luria-Bertani agar plates containing 50 μg of kanamycin/ml and incubated overnight. The following morning, colonies from the agar plates were added to 1 L of pre-warmed 2X yeast extract tryptone (YT) culture medium containing 50 μg kanamycin/ml and allowed to grow to an OD600 of 0.5 at 37 °C. PEDV-NP expression was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1.0 mM to induce transcription of the Lac operon, and the E. coli was allowed to incubate for an additional 8 h at 37 °C with shaking at 200 RPM. The agar was strained out and bacteria pelleted by centrifugation at 12,000 g for 10 min at 4 °C. The pellet was resuspended in 40 ml of lysis buffer solution (B-PER, Pierce, Rockford, IL), incubated for 15 min at 20–22 °C, then centrifuged at 12,000 g to separate the soluble from insoluble proteins. The PEDV-NP recombinant protein was expressed as insoluble periplasmic inclusion bodies. The resulting 441 amino acid recombinant protein was denatured using 8 M urea, subsequently purified three times using nickel-NTA affinity column chromatography and refolded back to its native conformational state. Individual affinity column elutions were collected, pooled and confirmed by SDS-PAGE, then aliquoted/frozen at −80 °C. The correct nucleotide sequence was confirmed by sequence and restriction endonuclease analysis. The average protein yield produced by the pET28a-PEDV-NP plasmid construct was calculated to be 11 mg PEDV-NP per liter of 2XYT under the aforementioned conditions. The recombinant protein was detected and a predicted molecular weight of 51 kDa was confirmed via Western Blotting using convalescent sera, a 6X histidine-specific mAb (Novagen, Madison, WI) and a PEDV-NP specific mAb (Figs. 1 and 2).Fig. 1


Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus.

Okda F, Liu X, Singrey A, Clement T, Nelson J, Christopher-Hennings J, Nelson EA, Lawson S - BMC Vet. Res. (2015)

Antigen/antibody specificity. Western blot analysis showing detection of recombinant expressed NA PEDV-NP protein and specificity of the monoclonal antibody used in the bELISA. L- Molecular weight ladder. A- anti-PEDV-NP mAb 6–29. B- anti-polyhistidine mAb. C- anti PEDV-NP convalescent swine serum
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4522128&req=5

Fig2: Antigen/antibody specificity. Western blot analysis showing detection of recombinant expressed NA PEDV-NP protein and specificity of the monoclonal antibody used in the bELISA. L- Molecular weight ladder. A- anti-PEDV-NP mAb 6–29. B- anti-polyhistidine mAb. C- anti PEDV-NP convalescent swine serum
Mentions: The development and validation of the iELISA and bELISA made use of a recombinantly expressed full length NA PEDV-NP. The NP open reading frame (ORF) of PEDV was amplified from RNA extracted directly from intestinal contents by RT-PCR from a case submitted to the South Dakota ADRDL. It was subsequently directionally cloned into the E. coli, pET 28a(+), plasmid expression vector (Novagen, Madison, WI), then transformed into BL21-Codon Plus (DE3)-RP competent cells (Stratagene, La Jolla, CA) for protein expression. Primers used for the amplification of the full length (1323 bp) nucleoprotein were: PEDV-NP-fwd (5′-CGCGGATCCATGGCTTCTGTCAGTTTTCAG-3′); PEDV-NP-rev (5′- CACACTCGAGATTTCCTGTGTCGAAGATCTC-3′). Next, 20 μl of transformed cells were plated onto Luria-Bertani agar plates containing 50 μg of kanamycin/ml and incubated overnight. The following morning, colonies from the agar plates were added to 1 L of pre-warmed 2X yeast extract tryptone (YT) culture medium containing 50 μg kanamycin/ml and allowed to grow to an OD600 of 0.5 at 37 °C. PEDV-NP expression was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1.0 mM to induce transcription of the Lac operon, and the E. coli was allowed to incubate for an additional 8 h at 37 °C with shaking at 200 RPM. The agar was strained out and bacteria pelleted by centrifugation at 12,000 g for 10 min at 4 °C. The pellet was resuspended in 40 ml of lysis buffer solution (B-PER, Pierce, Rockford, IL), incubated for 15 min at 20–22 °C, then centrifuged at 12,000 g to separate the soluble from insoluble proteins. The PEDV-NP recombinant protein was expressed as insoluble periplasmic inclusion bodies. The resulting 441 amino acid recombinant protein was denatured using 8 M urea, subsequently purified three times using nickel-NTA affinity column chromatography and refolded back to its native conformational state. Individual affinity column elutions were collected, pooled and confirmed by SDS-PAGE, then aliquoted/frozen at −80 °C. The correct nucleotide sequence was confirmed by sequence and restriction endonuclease analysis. The average protein yield produced by the pET28a-PEDV-NP plasmid construct was calculated to be 11 mg PEDV-NP per liter of 2XYT under the aforementioned conditions. The recombinant protein was detected and a predicted molecular weight of 51 kDa was confirmed via Western Blotting using convalescent sera, a 6X histidine-specific mAb (Novagen, Madison, WI) and a PEDV-NP specific mAb (Figs. 1 and 2).Fig. 1

Bottom Line: The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively.Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies.In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests.

View Article: PubMed Central - PubMed

Affiliation: Veterinary & Biomedical Sciences Department, South Dakota State University, Brookings, SD, USA. faten.okda@sdstate.edu.

ABSTRACT

Background: Recent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies.

Results: A recombinant North American nucleoprotein (NP) based iELISA was developed and validated along with a bELISA using newly developed PEDV-NP specific biotinylated monoclonal antibodies (mAbs) and an FMIA using magnetic beads coupled with expressed NA PEDV-NP. Receiver operating characteristic (ROC) analysis was performed using swine serum samples (iELISA n = 1486, bELISA n = 1186, FMIA n = 1420). The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. The iELISA and bELISA showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. Inter-rater (kappa) agreement was calculated to be 0.941 between iELISA and IFA, 0.945 between bELISA and IFA and 0.932 between FMIA and IFA. Similar comparative kappa values were observed between the iELISA, bELISA and FMIA, which demonstrated a significant level of testing agreement among the three assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted with these assays. All three assays detected seroconversion of naïve animals within 6-9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples.

Conclusion: Well-validated iELISA, bELISA and FMIA assays for the detection of PEDV antibodies were developed and showed good correlation with IFA and each other. Each assay format has advantages that dictate how they will be used in the field. Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies. In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. Measurement of neutralizing antibody responses using the FFN assay may provide a valuable tool for assessment of vaccine candidates or protective immunity.

No MeSH data available.


Related in: MedlinePlus