Development of a chemiluminescence immunoassay to accurately detect African swine fever virus antibodies in serum
Introduction
African Swine Fever (ASF) is an acute, febrile, and highly contagious animal infectious disease of pigs caused by African Swine Fever Virus (ASFV) (Galindo and Alonso, 2017). Its clinical symptoms mainly include skin congestion and cyanosis, hämorrhages, high morbidity, high mortality, and a short disease course (Costard et al., 2009). The virus is transmitted through contact with infected animals and contaminated feed or pollutants, and may also be transmitted by soft ticks; it affects both domestic and wild pigs (Costard et al., 2009; Frant et al., 2017). The ASFV belongs to the Asfarviridae and is a double-stranded linear DNA virus with a icosahedral structure, a full-length genome of 170∼190 kb, 151 open reading frames, and an envelope (Gaudreault et al., 2020). In addition, there is a variable region at each end of the ASFV genome; its genome has 24 genotypes, which encodes 150–200 proteins (Gaudreault et al., 2020). The ASFV p54 protein, encoded by the E183 L gene, is an important structural protein and is located in the inner envelope of the virus particle; its molecular weight is approximately 29 kDa (Alonso et al., 2001; Rodriguez et al., 1996). The ASFV p54 protein plays an important role in the process of viral infection, especially in the conversion of viral proteins into viral envelope precursors through the endoplasmic reticulum membrane (Hernaez et al., 2004). The ASFV p54 protein is a late viral protein and thus, transcription of the ASFV p54 gene occurs in the late stage of virus infection (Alonso et al., 2001; Rodriguez et al., 2004).P54 is strongly conserved and is a good immunogen, suggesting it may be used as a detection antigen of an ASFV antibody (Tesfagaber et al., 2021). Duet to the lack of an available vaccine, ASFV-specific antibodies are always the result of current or prior ASFV infection (or are maternally-derived). ASFV-specific antibodies in convalescent animals can persist for months or years (Gaudreault et al., 2020; Lu et al., 2020). Therefore, the detection of ASFV antibody is very important.
The chemiluminescence immunoassay (CLIA) was proposed by Halman et al. in 1977 (Halmann et al., 1977). CLIA, which combines a highly sensitive chemiluminescence reaction system with a highly specific immune response system, is currently the most widely used analytical technique (Holec-Gasior et al., 2018). The basic principle of CLIA is labeling antigens or antibodies with tracers, such as luminescent agents or catalysts of a chemiluminescent reaction (Wang et al., 2009). The labeled antigens or antibodies react with the detected substances in a series of immune reactions, and the detected substances are quantified by measuring the luminescence intensity (Long et al., 2009). CLIA is divided into three types: chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence labeling immunoassay (CLL), and electrochemiluminescence immunoassay (ECL) (Mahler et al., 2016). CLIA has a high sensitivity, wide detection range, fast reaction speed, short analysis time, and simple operation (Mahler et al., 2016; Welch et al., 2016). CLIA is a combination of the chemiluminescence and immunoassay methods (Li et al., 2020). With its high sensitivity and specificity, CLIA has continuously contributed to a new understanding of scientific technology and research fields (Li et al., 2020; Shim et al., 2017). With the rapid development of CLIA in recent years, CLIA has been widely used in the fields of life science research, drug analysis, medical clinical research, food analysis and detection, and animal medicine and environmental management; many important research results have been achieved using CLIA (Shim et al., 2017). For example, Liu et al. have established a CLIA method for proteins based on recombinant non-structural epitopes that accurately distinguishes cattle infected and inoculated with foot-and-mouth disease virus with high sensitivity (Liu et al., 2018). D. Chen et al. have compared CLIA, enzyme-linked immunosorbent assay (ELISA), and the passive agglutination method by detecting Mycoplasma pneumoniae infection. They find that CLIA and ELISA have higher sensitivity than that of the passive agglutination method, and the CLIA results are highly consistent with the ELISA results (Chen et al., 2018). Although it has been indicated that CLIA may be used for disease detection with a high sensitivity and specificity, CLIA has relatively few clinical applications in veterinary medicine. In this study CLIA was used to detect ASFV, specifically to establish an assay which was highly sensitive, so as to facilitate clinical detection of ASFV.
Section snippets
Material sources
ASFV was preserved in our laboratory. Positive sera against pathogenic pseudorabies virus (PRV), Porcine parvovirus (PPV), Porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis (JE), and classical swine fever virus (CSFV) was prepared in our laboratory. ASFV standard immunized sera (n = 1) and infected sera (n = 33) were obtained from the Laboratory of Animal Health and Epidemiology Center in China. Clinical samples (n = 159) from pig farms in China. The ASFV ELISA
Generation of mAbs against ASFV p54
The obtained recombinant proteins p54-HIS and p54-GST were subjected to SDS-PAGE, and the two purified proteins were 37 kDa (Fig. 1a) and 43 kDa (Fig. 1b), respectively. Western blotting was performed using an anti-HIS tag mAb, anti-GST tag mAb and ASFV positive serum. P54-HIS showed specific reaction bands with the anti-HIS tag mAb and ASFV positive serum, but not the anti-GST tag mAb (Fig. 2). In contrast, p54-GST showed specific reaction bands with the anti-GST tag mAb and ASFV positive
Discussion
ASFV was first reported in Kenya, Africa in 1921, and was introduced to Portugal, a European country, in 1957. From the 1970s to the 1980s, ASFV was identified in many countries in Europe and America (Gaudreault et al., 2020; Sanchez-Vizcaino et al., 2013). China reported African swine plague for the first time in 2018, and then the disease spread rapidly, causing huge economic losses to the pig industry and the national economy (Lu et al., 2020; Cwynar et al., 2019). ELISA is the gold standard
Author statement
The authors declare that the article is my original work, hasn’t received prior publication and isn’t under consideration for publication elsewhere.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by The Major Projects of Technological Innovation in Hubei Province (2019ABA089) and the Key R&D Program of Hunan Province (2019NK2171).
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