Development of a Ready‐To‐Use Bioluminescence Immunosensor for the One‐Step Sensitive Detection of Antibodies Against African Swine Fever Virus

Figures

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  FIGURE 1. Schematic showing the development of the immunosensor for detecting ASFV antibodies via split‐NanoLuc complementation technology. The ASFV p30/SmBiT and protein G/LgBiT fusion probes of the immunosensor bind to the Fab and Fc regions of IgG antibodies against ASFV p30. They subsequently form a ternary immunocomplex and reconstitute functional luciferase, which can generate a detectable signal by catalysing the substrate furimazine.

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  FIGURE 2. Selection of sensor pairs. Four sensor combinations (LN‐C2 + SN‐p30, LN‐C2 + SC‐p30, LC‐C2 + SN‐p30, and LC‐C2 + SC‐p30) were investigated for their ability to reconstitute functional luciferase by binding with anti‐ASFV antibodies. All sensor pairs produced detectable luminescent signals that differentiated ASFV‐positive sera from negative controls, and the sensor pair LN‐C2 and SN‐p30 achieved the highest signal‐to‐noise (S/N) ratio and was identified as optimal. Data are presented as the means of duplicate tests, and error bars indicate the standard deviations of duplicate tests.

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  FIGURE 3. Determination of the optimal doses of the fusion sensors. The maximum luminescence signal and less background were observed at 50 ng of LN‐C2 and 6.25 ng of SN‐p30, yielding the highest signal‐to‐noise (S/N) ratio. The data are presented as the means of duplicate samples.

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  FIGURE 4. Determination of the optimal reaction conditions. (a) The optimal amount of serum. (b) The optimal incubation time for sample detection. Data is presented as the means of duplicate tests, and error bars indicate the standard deviations of duplicate tests.

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  FIGURE 5. Specificity and analytical performance of the immunosensor. (a) Specificity analysis of the proposed assay by testing sera positive for PCV2, CSFV, PRV, FMDV‐O, PRRSV, APP, and HPS. Apart from ASFV, the signal‐to‐noise (S/N) values of those serum samples were lower than the defined cutoff value of the assay. Data are presented as the means of duplicate tests, and error bars indicate the standard deviations of duplicate tests. (b) Comparison of analytical sensitivity between the proposed assay and ELISA with dilutions of ASFV‐positive samples.