HRP-conjugated-nanobody-based cELISA for rapid and sensitive clinical detection of ASFV antibodies

Abstract

African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and wild boars and brings huge economic losses to world swine industry. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, and cost-effective detection and strict control and elimination strategies. Traditional serological testing methods are generally associated with high testing costs, complex operations, and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity, although the system is dependent on the immunization of Bactrian camels to obtain the specific VHH library of the target protein. The application of Nbs in the detection of ASFV antibodies has not yet been reported yet. Using a phage display technology, one Nb against the ASFV p54 protein that exhibited high specificity and affinity, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. There was no cross-reaction with healthy pig serum, porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and classical swine fever virus (CSFV) positive sera. The optimal cut-off value for the cELISA by ROC analysis was 52.5%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The results showed that the relative specificity of the cELISA was 98.97%, and the relative sensitivity of the cELISA was 93.3%, with the percent agreement between the two ELISA methods being 98.56%. In conclusion, a specific, sensitive, and repeatable cELISA was successfully developed based on the Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum. Key points • We successfully screened a specific, high affinity nanobody against ASFV p54 protein. • We establish a method for continuous and stable expression of Nb-HRP fusion protein using a lentiviral packaging system. • We establish a nanobody cELISA detection method that can monitor an ASF infection. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-11981-4.

K205R specific nanobody-horseradish peroxidase fusions as reagents of competitive ELISA to detect African swine fever virus serum antibodies

“…These provide a strong guarantee for establishing rapid and timely detection of ASFV. In addition, some studies have also reported that ASFV proteins such as p54, p30 and p72 were used for ASFV serum antibody detection [ 13 , 36 , 37 ]. The difference of sensitivity and specificity between using K205R protein as antigen and other antigens are still unclear.…”

Identification and characterization of nanobodies specifically against African swine fever virus major capsid protein p72

“…Owing to their intrinsic and unique properties, nanobodies have been widely used in the detection of specific causative agents in laboratory tests ( Salvador et al, 2019 ). More recently, several nanobodies against the p54 and K205R protein of ASFV have been isolated from the camelid immune library, they were subsequently applied in the development of nanobody-based cELISA for detection of ASFV antibodies ( Zhang et al, 2022 ; Zhao et al, 2022 ). In the present study, the nanobodies specific for native p72 protein were selected by a sandwich ELISA, and the Nb25 can specifically recognize the virus particles captured by polyclonal antibody against ASFV.…”

Development of a Competitive Enzyme-Linked Immunosorbent Assay Targeting the-p30 Protein for Detection of Antibodies against African Swine Fever Virus

“…It can also illustrate epidemiological features of outbreaks such as the incubation period of the viral infection and the infections caused by the low pathogenic strains of ASFV [ 11 ]. The routine diagnostic method for ASF approved by the World Organization of Animal Health (WOAH) is the ELISA after preliminary screening, followed by Western blotting [ 12 , 13 ]. Either in endemic or ASF-free countries, the ELISA is commonly used to diagnose viral diseases and to assess serosurveys.…”

“…It will make it easier to clarify viral pathogenesis and the immune response of the host. Furthermore, in the serological diagnosis of viral diseases, mAbs are the most widely used antibodies [ 13 ]. In this study, we generated four monoclonal antibodies targeting p30, and mapped the epitope of one mAb which showed good ability to distinguish the positive and negative serum of ASF.…”