Abstract
The p30 protein is abundantly expressed in the early stage of African swine fever virus (ASFV) infection. Thus, it is an ideal antigen candidate for serodiagnosis with the use of an immunoassay. In this study, a chemiluminescent magnetic microparticle immunoassay (CMIA) was developed for the detection of antibodies (Abs) against ASFV p30 protein in porcine serum. Purified p30 protein was coupled to magnetic beads, and the experimental conditions including concentration, temperature, incubation time, dilution ratio, buffers, and other relevant variables were evaluated and optimized. To evaluate the performance of the assay, a total of 178 pig serum samples (117 negative and 61 positive samples) were tested. According to receiver operator characteristic curve analysis, the cut-off value of the CMIA was 104,315 (area under the curve, 0.998; Youden’s index, 0.974; 95% confidence interval: 99.45 to 100%). Sensitivity results showed that the dilution ratio of p30 Abs in ASFV-positive sera detected by the CMIA is much higher when compared to commercial blocking ELISA kit. Specificity testing showed that no cross-reactivity was observed with sera positive for other porcine disease viruses. The intraassay coefficient of variation (CV) was < 5%, and the interassay CV was < 10%. The p30-magnetic beads could be stored at 4 °C for more than 15 months without loss of activity. The kappa coefficient between CMIA and INGENASA blocking ELISA kit was 0.946, showing strong agreement. In conclusion, our method showed superiority with high sensitivity, specificity, reproducibility, and stability and potentialized its application in the development of a diagnostic kit for the detection of ASF in clinical samples.
Key points
• ASFV tag-free p30 was successfully purified.
• High sensitivity, specificity, relatively simple, and time-saving to detect antibody against ASFV were developed.
• The development of CMIA will help the clinical diagnosis of ASFV and will be useful for large-scale serological test.
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Data availability
The data that support the results of this study are available from the corresponding author upon reasonable request.
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Funding
This work was supported by the National Key R&D Program of China (2021YFD1801300), Gansu Province Animal Biological Products Innovation Consortium Project (22ZD6NA012), the earmarked fund for CARS-35 (CARS-35), open competition program of top ten critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2022SDZG02).
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Y. H., H. T., and H. Z. designed the study. Z. S., L. C., J. L., and Q. Z. performed the experiments. G. Z. contributed new reagents. X. L. provided the serum samples. Z. S. and L. C. analyzed the data. L. C. wrote the manuscript. All authors discussed the results.
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Shi, Z., Cao, L., Luo, J. et al. A chemiluminescent magnetic microparticle immunoassay for the detection of antibody against African swine fever virus. Appl Microbiol Biotechnol 107, 3779–3788 (2023). https://doi.org/10.1007/s00253-023-12518-z
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DOI: https://doi.org/10.1007/s00253-023-12518-z