Ferritin-displayed antigen nanoparticles and nanobody-horseradish peroxidase fusions based-competitive ELISA for the rapid and sensitive detection of antibody against African swine fever virus

Abstract

African swine fever (ASF) is a highly contagious and lethal hemorrhagic disease, which has brought great distress and economic losses to the world pig industry. For lack of approved vaccines and effective treatment, accurate early detection, both ASF virus (ASFV) antigen and antibody detection in the field, is one of the most important aspects for preventing the outbreak and spread of ASFV. Here we screened the specific nanobodies against the ASFV-P30 protein from the library of immunized Bactrian camel and fused them with horseradish peroxidase (HRP) for expression in HEK293T cells. Moreover, the P30 protein was designed and displayed on the surface of ferritin, the fusion nanocages. Then we developed a novel competitive ELISA (cELISA) based on ferritin-displayed P30 nanoparticles protein (P30-Fn) and nanobody-horseradish peroxidase fusions (P30–Nb71-vHRP) for rapid detection of ASFV antibody in serum sample with high sensitivity and specificity. The cELISA can detect as far as 1:200 diluted clinically positive serums and show no cross-reactivity with positive serums against others porcine viruses, such as PRRSV, PPV, CSFV, TGEV, PEDV, PCV and SIV. The agreement rate is 98.1% with the test results of the commercial ELISA kit for the detection of a total of 267 clinical samples, and the kappa value is 0.96. Furthermore, the cELISA exhibits a good repeatability and the intra- and inter-assay coefficient of variation are less than 10%. In summary, the newly developed cELISA is a simple, rapid, sensitive and low-cost immunoassay, which has a great potential for ASFV antibody detection in clinical samples.