A broad spectrum monoclonal antibody against porcine circovirus type 2 for antigen and antibody detection

2019 ◽  
Vol 103 (8) ◽  
pp. 3453-3464 ◽  
Author(s):  
Liping Huang ◽  
Yanwu Wei ◽  
Deli Xia ◽  
Dan Liu ◽  
Hongzhen Zhu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Broad Spectrum ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Antibody Detection ◽  
Porcine Circovirus

scholarly journals Purification of Porcine Circovirus Type 2 Using an Affinity Chromatography Based on a Neutralizing Monoclonal Antibody against Viral Capsid Protein

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1564
Author(s):  
Haiqiao Bian ◽  
Chong Yu ◽  
Yanwu Wei ◽  
Li Feng ◽  
Changming Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Affinity Chromatography ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chromatography Method ◽  
Viral Adsorption

Porcine circovirus type 2 (PCV2) is a DNA virus without an envelope. The viral particle is icosahedral and has a diameter of approximately 17 nm. In order to obtain the purified virus, a broad-spectrum monoclonal antibody 3A5 against PCV2 was coupled to CNBr-activated SepharoseTM 4B, and an affinity chromatography was established for PCV2 purification. A total of 6.5 mg of purified PCV2a/LG with 97% purity was obtained from 120 mL of the viral culture medium, and only PCV2 was detected by electron microscopy. No significant changes in the antigenic characteristics of the purified virus were detected by a capture enzyme-linked immunosorbent assay (ELISA). Furthermore, the titer of the purified PCV2 was 100 times higher than that of the unpurified virus. This affinity chromatography method was also used to purify PCV2b/LN590516 and PCV2d/SD446F16, and the purified viruses were detected by electron microscopy, capture ELISA, and virus titration, respectively. The results showed that these two strains can be successfully purified, but the yield is lower than that of the PCV2a strain. In addition, the purified virus could be used to study the viral adsorption and invasion of PK15 cells using indirect immunofluorescence assays. A large number of PCV2 signals were detected to transfer from the cellular surface to the periphery of the nucleus of the PK15 cells after 30 min of adsorption of the PCV2 to the PK15 cells. The affinity chromatography is a simple and convenient tool to obtain PCV2 with high purity. It could be applied for virus structure analysis, antibody preparation, and viral adsorption and invasion research.

scholarly journals A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

2016 ◽  
Vol 36 (12) ◽  
pp. 1171-1177 ◽  
Author(s):  
Taís F. Cruz ◽  
Tatiana M. Kanashiro ◽  
Alessandra M.M.G. de Castro ◽  
Cintia M. Baldin ◽  
Leonardo J. Richtzenhain ◽  
...  
Keyword(s):  
Cell Culture ◽  
Natural Infection ◽  
Sandwich Elisa ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Antibody Detection ◽  
Porcine Circovirus ◽  
Sucrose Cushion ◽  
Double Antibody

ABSTRACT: Few studies have described enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against porcine circovirus type 2 (PCV2) based on antigens produced in cell culture. Furthermore, few articles have described viral purification techniques for members of the family Circoviridae. This occurs because circoviruses are difficult to isolate, noncytopathogenic, and produce low viral titres in cell culture. Thus, for overcoming these difficulties in the cultivation of PCV2, this study aimed to develop a double-antibody sandwich ELISA based on the cell culture antigen PCV2b for the quantification of anti-PCV2 antibodies. A 20% and 50% discontinuous sucrose cushion was used for viral purification, which enabled the separation of cell culture proteins in the 20% sucrose cushion and a greater viral concentration in the 50% sucrose cushion. Following isopycnic centrifugation, PCV2 was concentrated in the band with density values from 1.330 to 1.395g/cm3. Viral purification was assessed using SDS-PAGE, indirect ELISA and electron microscopy. The standardised ELISA revealed a strong linear correlation (r= 0.826, p<0.001) when compared with a commercial ELISA kit. The assay exhibited low variability (inter-assay coefficient of variation of 4.24% and intra-assay of 1.80%) and excellent analytical specificity conferred by the capture antibody produced in rabbit. Thus, this ELISA is a rapid, specific and convenient method for the detection of antibodies against PCV2 in studies of experimental and natural infection, and in monitoring the response to vaccination on commercial farms.

Porcine circovirus type 2 antibody detection in backyard pigs from Mexico City

2007 ◽  
Vol 83 (1) ◽  
pp. 130-132 ◽  
Author(s):  
H. Ramírez-Mendoza ◽  
C. Martínez ◽  
C. Mercado ◽  
H. Castillo-Juárez ◽  
J. Hernández ◽  
...  
Keyword(s):  
Mexico City ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Antibody Detection ◽  
Porcine Circovirus ◽  
Backyard Pigs

Immunoenhancement of Recombinant Neisseria meningitides PorB Protein on Porcine Circovirus Type 2 and Mycoplasma hyopneumoniae Genetically Engineered Vaccines

2019 ◽  
Vol 26 (10) ◽  
pp. 776-784
Author(s):  
Rui Yang ◽  
Yu Tao ◽  
Gaojian Li ◽  
Jian Chen ◽  
Jianhong Shu ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Mycoplasma Hyopneumoniae ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Genetically Engineered ◽  
Porcine Circovirus ◽  
Practical Applications ◽  
Neisseria Meningitides ◽  
Short Period

Background:Porcine circovirus and Mycoplasma hyopneumoniae can cause respiratory diseases in pigs, which cause serious economic loss in the worldwide pig industry. Currently, these infections are mainly prevented and controlled by vaccination. The new vaccines on the market are mainly composed of subunits and inactivated vaccines but usually have lower antigenicity than traditional live vaccines. Thus, there is an increasing need to develop new adjuvants that can cause rapid and long-lasting immunity to enhance the antigenic efficacy for vaccines. Studies have shown that meningococcal porin PorB can act as a ligand to combine with Toll-like receptors to activate the production of immunological projections and act as a vaccine immunological adjuvant.Objective:In this article, we expressed and purified the recombinant PorB protein and verified its immunogenicity against porcine circovirus type 2 and Mycoplasma hyopneumoniae genetically engineered vaccine.Methods:In this article, we used prokaryotic expression to express and purify recombinant PorB protein, four different concentrations of PorB protein, Freund's adjuvant with two genetically engineered vaccines were combined with subcutaneous immunization of mice.Results:Our study shows that the appropriate dose of the recombinant protein PorB can enhance the levels of humoral and cellular responses induced by two genetically engineered vaccines in a short period of time in mice. The PorB adjuvant group may cause statistically higher antibody titers for both genetically engineered vaccines compared to Freund's commercial adjuvant (P<0.001).Conclusion:The recombinant protein PorB may be a good candidate adjuvant for improving the protective effect of vaccines against porcine circovirus type 2 and Mycoplasma hyopneumoniae, and the protein can be used for future practical applications.

scholarly journals A Comparison of Virulence of Three Porcine Circovirus Type 2 (PCV2) Genotypes (a, b, and d) in Pigs Singularly Inoculated with PCV2 and Dually Inoculated with PCV2 and Porcine Reproductive and Respiratory Syndrome Virus

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 891
Author(s):  
Jeongmin Suh ◽  
Taehwan Oh ◽  
Keehwan Park ◽  
Siyeon Yang ◽  
Hyejean Cho ◽  
...  
Keyword(s):  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Daily Weight Gain ◽  
Porcine Circovirus ◽  
Respiratory Syndrome Virus ◽  
Average Daily Weight Gain ◽  
Significant Difference ◽  
Lesion Severity ◽  
Severe Lung

The aim of this study was to compare the virulence of porcine circovirus type 2 (PCV2) genotypes in dually inoculated pigs with both three genotypes (a, b, and d) of PCV2 and porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) versus pigs singularly inoculated with the same three PCV2 genotypes (a, b, and d). Differences in this comparison were found in PCV2 viremia levels, lung and lymphoid lesion severity, and the amount of PCV2 antigen within the lymphoid lesions. Regardless of PCV2 genotypes, pigs that were dually inoculated with PCV2/PRRSV had significantly higher clinical scores, less average daily weight gain, higher levels of PCV2 viremia, and more severe lug and lymphoid lesions compared to pigs singularly inoculated with PCV2. Among the dually infected pig groups, pigs infected with PCV2d/PRRSV-2 had significantly higher levels of PCV2 viremia, more severe lung and lymphoid lesions, and more PCV2-positive cells within lymphoid lesions compared to pigs dually inoculated with PCV2a/PRRSV-2 and PCV2b/PRRSV-2. The results of this study demonstrated significant differences in the virulence among dual inoculation of PCV2a/PRRSV-2, PCV2b/PRRSV-2, and PCV2d/PRRSV-2. A significant difference in the virulence among PCV2a, PCV2b, and PCV2d single-inoculated pig groups was not found with respect to the levels of PCV2 viremia and production of PCV2-associated lymphoid lesions.

scholarly journals Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ellen Kathrin Link ◽  
Matthias Eddicks ◽  
Liangliang Nan ◽  
Mathias Ritzmann ◽  
Gerd Sutter ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Diagnostic Sensitivity ◽  
Locked Nucleic Acid ◽  
Porcine Circovirus ◽  
Amplification Efficiency ◽  
Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

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