Development of a Dual Fluorescent Microsphere Immunological Assay for Detection of Pseudorabies Virus gE and gB IgG Antibodies
Chihai Ji,
Yingfang Wei,
Jingyu Wang,
Yuchen Zeng,
Haoming Pan,
Guan Liang,
Jun Ma,
Lang Gong,
Wei Zhang,
Guihong Zhang,
Heng Wang
Affiliations
Chihai Ji
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Yingfang Wei
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Jingyu Wang
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Yuchen Zeng
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Haoming Pan
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Guan Liang
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Jun Ma
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Lang Gong
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Wei Zhang
Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, Guangzhou 510642, China
Guihong Zhang
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Heng Wang
Guangdong Provincial Key Laboratory of Prevention and Control for Severe Clinical Animal Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Pseudorabies, also known as Aujezsky’s disease, is an acute viral infection caused by pseudorabies virus (PRV). Swine are one of the natural hosts of pseudorabies and the disease causes huge economic losses in the pig industry. The establishment of a differential diagnosis technique that can distinguish between wild-type infection and vaccinated responses and monitor vaccine-induced immunoglobulin G(IgG) is crucial for the eventual eradication of pseudorabies. The aim of this study was to develop a rapid dual detection method for PRV gE and gB protein IgG antibodies with high specificity and sensitivity. PRV gE codons at amino acid residues (aa) 52–238 and gB codons at aa 539–741 were expressed to obtain recombinant PRV gE and gB proteins via a pMAL-c5x vector. After purification with Qiagen Ni–nitrilotriacetic acid (NTA) agarose affinity chromatography, the two proteins were analyzed via SDS-PAGE and immunoblotting assays. Two single fluorescent-microsphere immunoassays (FMIAs) were established by coupling two recombinant proteins (gE and gB) to magnetic microbeads, and an effective dual FMIA was developed by integrating the two single assays. Optimal serum dilution for each assay, correlation with other common swine virus-positive sera, and comparison with ELISA for two PRV antigens were tested for validation. Compared with ELISA, the specificity and sensitivity were 99.26% and 92.3% for gE IgG antibody detection, and 95.74% and 96.3% for the gB IgG antibody detection via dual FMIA. We provide a new method for monitoring PRV protective antibodies in vaccinated pigs and differentiating wild-type PRV infection from vaccinated responses simultaneously.
