A bioluminescence immunosensor using engineered antibody for quantitative detection of African swine fever virus

Abstract African swine fever (ASF), caused by ASF virus (ASFV), poses a significant threat to global swine industries due to its high mortality and the lack of an effective vaccine. Consequently, it is critical to develop a rapid and quantitative detection method for timely monitoring of swine populations and accurate confirmation of ASF infection, thereby enhancing ASF prevention and control measures. Here, we developed an ultrasensitive bioluminescence immunosensor for quantitative detection of ASFV p30 (p30-UBI) by genetically fusing HiBiT (an 11-amino-acid peptide that binds the NanoLuc fragment LgBiT with high affinity, reconstituting bright bioluminescence) to the light chain constant region of monoclonal antibody (mAb) 16-5E7E8 as the detection antibody and using mAb 2-1B3G3 as the capture antibody. The p30-UBI exhibited broad linear ranges of 46 pg/mL to 250 ng/mL for recombinant p30 protein (a combined quantitative range covered by two calibration equations) with detection limits of 15.3 pg/mL. The practical utility of the p30-UBI was evaluated using tissue, serum, and swab samples from ASFV-challenged pigs. P30 was readily detectable in tissue and serum samples, whereas lower concentrations were measured in swabs, and p30 levels showed broadly similar patterns to ASFV p72 gene copy numbers determined by quantitative PCR (qPCR). Notably, in an exploratory analysis in which pigs from different vaccine groups and dose groups were combined, lower serum p30 levels at 7 dpc were significantly associated with longer survival after ASFV challenge (Spearman ρ = − 0.62, P < 0.0001, n = 53). These findings indicate that the p30-UBI is a promising quantitative analytical method evaluated in samples from experimentally infected pigs. Key points • A HiBiT-tagged antibody-based bioluminescence immunosensor was developed for ASFV p30 detection • The assay achieves detection limits of 15.3 pg/mL for p30 within 70 min • Lower serum p30 levels at 7 dpc were associated with longer survival in an exploratory analysis