Expression of the porcine circovirus type 2 capsid protein subunits and application to an indirect ELISA

Wu, Pei-Ching; Chien, Maw-Sheng; Tseng, Yu-Yao; Lin, Jeng-Feng; Lin, Weili; Yang, Cheng‐Yao; Huang, Chi‐Hsiang

doi:10.1016/j.jbiotec.2007.09.015

Cited by 31 publications

(26 citation statements)

References 21 publications

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“…Several indirect ELISAs using the recombinant Cap protein expressed in insect cells or E. coli as a coating antigen have been described (4,20,25,28,34,44). These studies indicated that the recombinant PCV2 Cap protein expressed in either prokaryotes or eukaryotes showed good immunoreactivity and that these proteins were effective diagnostic antigens for establishment of an ELISA method for PCV2 epidemiological investigation and clinical diagnosis.…”

Section: Discussionmentioning

confidence: 99%

“…Fortunately, it has been reported that this NLS has poor reactivity with PCV2-positive swine sera and that deletion of NLS has little effect on the immunoreactivity of the Cap protein (18,44). Therefore, the 41-amino-acid signal peptide sequence at the 5= end of the ORF2 gene was deleted, and the resulting ORF2 gene was inserted into the pET28-a(ϩ) and pET32-a(ϩ) expression vectors to construct recombinant plasmids pET28a-ORF2⌬123 and pET32a-ORF2⌬123.…”

Section: Discussionmentioning

confidence: 99%

“…The amino acid sequence identity of Cap between PCV1 and PCV2 is only 65% (27); consequently, although there is some antigenic relationship, only PCV2 Cap protein is recognized by PCV2 polyclonal antibodies, which is the basis for the establishment of methods for specific serological detection of PCV2 (24). Thus, besides indirect immunofluorescence (IIF) (1), immunoperoxidase monolayer assay (IPMA) (8), and some enzyme-linked immunosorbent assays (ELISAs) based on the PCV2 virion (38,40), more and more ELISAs, mostly indirect, have been developed using recombinant PCV2 Cap protein (4,20,25,28,34,44). Here, we report a double-antigen sandwich ELISA, which has certain advantages over current methods for detection of antibodies to PCV2.…”

mentioning

confidence: 99%

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Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

Luo

Jiang

et al. 2012

Clin Vaccine Immunol

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ABSTRACTA double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

Luo

Jiang

et al. 2012

Clin Vaccine Immunol

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“…11 No statistical analysis was performed in that trial. In addition, other studies have been published that compared newly developed serologic tests with either an IFAT 12,16,17,20 or an IPMA. 10 In a 2008 study in which 2 commercially available ELISAs and 1 inhouse ELISA were compared, it was found that the assays were able to detect both PCV-2a and PCV-2b, and that all assays had high diagnostic accuracies based on receiver operating characteristic (ROC) analysis.…”

Section: Introductionmentioning

confidence: 99%

Interlaboratory Comparison of Porcine Circovirus-2 Indirect Immunofluorescent Antibody Test and Enzyme-Linked Immunosorbent Assay Results on Experimentally Infected Pigs

et al. 2011

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Abstract. A blinded interlaboratory assessment of the diagnostic agreement and accuracy of serologic tests for routine detection of antibodies against Porcine circovirus-2 (PCV-2), including indirect fluorescent antibody tests (IFATs) and enzyme-linked immunosorbent assays (ELISAs) was conducted in 7 North American laboratories. Serum samples were collected weekly, on trial days 0, 7, 14, 21, 28, 35, 42, and 49, from the following groups of animals: 1) negative controls (n 5 7), 2) PCV-2a (n 5 8), 3) PCV-2b (n 5 8), 4) PCV-1 (n 5 8), 5) PCV-2 vaccine A (n 5 8; IngelvacH CircoFLEX TM ), 6) PCV-2 vaccine B (n 5 8; CircumventH PCV2), and 7) PCV-2 vaccine C (n 5 8; SuvaxynH PCV2 One Dose). Results from each laboratory were analyzed by kappa and receiver operating characteristic (ROC) analysis. Kappa analysis indicated that, by trial day 49, IFATs had almost perfect agreement, in-house ELISAs had fair to almost perfect agreement, and commercially available anti-PCV-2 immunoglobulin G ELISAs (I or S) had moderate to substantial agreement. From trial days 14-49, the area under the ROC curve for the 2 laboratories that offered IFATs, the 4 laboratories that offered in-house ELISAs, and the 3 laboratories that used commercially available ELISAs ranged from 0.94 to 1.00, 0.72 to 1.00, and 0.95 to 1.00, respectively. However, test sensitivities varied based on laboratory-specific cutoffs that were used to dichotomize test results.

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“…One method of assessing when and if PCV-2 is circulating in a herd is to conduct a cross-sectional serological survey to determine the prevalence and levels of anti-PCV-2 antibodies over time using 1 or more of the serological tests for PCV-2 including various enzymelinked immunosorbent assays (ELISAs). 3,16,20,25,29,30 Most ELISA tests use the ORF2 region as antigen since it is specific and immunogenic for PCV-2. 28 Although most assays are based on PCV-2 ORF2, the methods of antigen production differ among assays.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Three Enzyme-Linked Immunosorbent Assays to Detect Porcine Circovirus-2 (PCV-2)—Specific Antibodies after Vaccination or Inoculation of Pigs with Distinct PCV-1 or PCV-2 Isolates

et al. 2008

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Abstract. Porcine circovirus-2 (PCV-2) serology is frequently used to determine PCV-2 status and optimal timing of PCV-2 vaccination in the field. The objectives of the current study are to determine the diagnostic accuracy of 3 currently available commercial anti-immunoglobulin G (IgG) PCV-2 enzyme-linked immunosorbent assays (ELISAs) and to compare the ability of the 3 assays to detect and differentiate between anti-PCV-2a and anti-PCV-2b antibodies, as well as anti-PCV-2 and anti-PCV-1 antibodies. Fiftyfive 3-week-old, conventional pigs were randomly allocated to 7 groups: 1) negative controls (n 5 7), 2) PCV2a (n 5 8; inoculated with PCV-2 ISU-40895), 3) PCV-2b (n 5 8; inoculated with PCV-2 NC-16845), 4) PCV-1 (n 5 8), 5) vaccine A (n 5 8; IngelvacH CircoFLEX TM ), 6) vaccine B (n 5 8; CircumventH PCV2), and 7) vaccine C (n 5 8; SuvaxynH PCV2 One Dose). Blood samples were collected weekly, and all sera were tested by 3 different anti-PCV-2 IgG ELISAs. The results indicated that all ELISAs had area under the receiver operating curve values greater than 0.94, detected both anti-PCV-2a and -2b antibodies with no differentiation, and did not detect anti-PCV-1 antibodies in infected animals. One of the ELISAs was able to distinguish pigs vaccinated with vaccine B from pigs inoculated with either PCV-2a or PCV-2b.

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Expression of the porcine circovirus type 2 capsid protein subunits and application to an indirect ELISA

Cited by 31 publications

References 21 publications

Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

Development and Application of a Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Porcine Circovirus 2

Interlaboratory Comparison of Porcine Circovirus-2 Indirect Immunofluorescent Antibody Test and Enzyme-Linked Immunosorbent Assay Results on Experimentally Infected Pigs

Comparison of Three Enzyme-Linked Immunosorbent Assays to Detect Porcine Circovirus-2 (PCV-2)—Specific Antibodies after Vaccination or Inoculation of Pigs with Distinct PCV-1 or PCV-2 Isolates

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