Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus

Abstract

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.

A novel conserved B-cell epitope in pB602L of African swine fever virus

“…As the major ASFV envelope protein, p72 is one of the first viral proteins to induce the antibody production after viral infection and is an important marker in ASFV diagnosis and vaccine studies (Nan Wang et al 2019 ; Heimerman et al 2018 ). p30 is abundantly expressed in early cell stages, has good antigenicity, and is also an important target for early virus diagnosis (Wang et al 2022 ). Anti-p54 antibodies appear as early as 10 days after ASFV infection, but p54 is prone to false negative results due to amino acid sequence variations, and is not typically used as an ASF detection antigen (Alonso et al 2001 ; Gao et al 2022 ).…”

Identification of Two Novel Linear B Cell Epitopes on the CD2v Protein of African Swine Fever Virus Using Monoclonal Antibodies

“…The recombinant CD2v extracellular domain (CD2v-ex, GenBank: MK128995.1, 1–588 bp) was prepared in our previous study [ 34 ], and the recombinant ASFV p30 protein with His-tag was maintained in our laboratory [ 35 ]. The SP2/0 myeloma cell line (ATCC, Manassas, VA, USA) was cultured in RPMI 1640 (Solarbio, Beijing, China) with 10% ( v / v ) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA).…”

Identification of a novel linear B-cell epitope on the p30 protein of African swine fever virus using monoclonal antibodies

“…The development of diagnostic agents and vaccines against the p30 protein has been a hot research topic. The preparation of p30 protein McAb will be of great significance to the in-depth study of the pathogenic mechanism of ASFV ( Wang et al., 2022 ).…”