Developing an Indirect ELISA for the Detection of African Swine Fever Virus Antibodies Using a Tag-Free p15 Protein Antigen

Abstract

African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in Escherichia coli (E. coli) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential.

Establishment of an ELISA Based on a Recombinant Antigenic Protein Containing Multiple Prominent Epitopes for Detection of African Swine Fever Virus Antibodies

“…Moreover, the sensitivity test of the developed ELISA was evaluated in the present assessment and the result showed that the current ELISA kit was capable of detecting a commercially positive ASFV serum at a high dilution of 1:32, which is consistent with the results of a previous study [17]. In addition, the agreement rate between the k3-iELISA and INGENASA's commercial ASFV antibody detection kit was 99.00% [14,16]. These results emphasize the significant clinical potential of the k3-iELISA.…”

“…Additionally, the samples were tested using a commercial ASFV antibody detection kit (Ingenasa, Madrid, Spain) provided by Qingdao RealVet Bio-Technology Co., Ltd., Qingdao, China. (ASF.K001/5) based on the p72 protein for comparison with the established ELISA method as previously described [1,16]. This comparison aimed to determine the effectiveness and coincidence rate, evaluating the consistency between the established ELISA and the commercial kit based on the test results of each sample.…”

“…For the method comparison with the commercial Kit, relative sensitivity, relative specificity, and coincidence rates were calculated as described previously [16].…”

“…In response to the challenges posed by ASFV infection and the significant role played by various proteins at distinct infection stages, our study explores a novel approach in indirect ELISA design. Rather than focusing on a single protein target as seen in previous studies [16][17][18], we propose the use of multiple prominent B-cell epitopes named k3 derived from 11 ASFV antigenic proteins. This innovative approach aims to achieve heightened sensitivity and specificity compared to currently available commercial assays.…”

Overview of Modern Commercial Kits for Laboratory Diagnosis of African Swine Fever and Swine Influenza A Viruses

“…Some studies include p30 and p54 proteins as potential indicators [ 87 ]. The last studies using new antigens include core–shell protein p15 [ 88 ], transmembrane protein pI329L [ 83 ] or K205R protein [ 89 ]…”