A triple protein-based ELISA for differential detection of ASFV antibodies

Abstract

African swine fever (ASF) caused by the ASF virus (ASFV) is a severe and highly contagious viral disease that poses a significant threat to the global pig industry. As no vaccines or effective drugs are available to aid prevention and control, early detection is crucial. The emergence of the low-virulence ASFV strain not expressing CD2v/MGFs (ASFVΔCD2v/ΔMGFs) has been identified domestically and internationally and has even become an epidemic in China, resulting in a complex epidemic. The commercialized ASFV ELISA kits available can detect the presence of ASFV infection in pigs, but they are unable to distinguish wild-type ASFV from gene-deleted strains. The current published ELISA assays can distinguish between the wild-type and CD2v gene-deleted ASFV but cannot differentiate wild-type and MGF505 gene-deleted ASFV or CD2v and MGF505 double-gene deleted ASFV infection, posing new challenges for an effective prevention and control of ASFV. In this study, the ASFV-p30, ASFV-CD2v, and ASFV-MGF505 proteins were expressed using a prokaryotic expression system, and a triple protein-based ELISA antibody detection method based on these proteins was successfully established to effectively differentiate between wild-type ASFV and ASFVΔCD2v and/or ASFVΔMGF505 virus infection. This triple protein-based ELISA showed good analytical specificity without cross-reactivity with antibodies against PRRSV, CSFV, PRV, and PCV2. Moreover, it demonstrates remarkable analytical sensitivity by allowing the identification of clinical samples even at dilutions as high as 1:800. The coefficient of variation the intra-assay and inter-assay were below 5%, indicating strong repeatability and reproducibility. To evaluate the performance of the triple protein-based ELISA, a total of 59 clinical serum samples were detected using the triple protein-based ELISA. The results showed that 22 samples were positive for ASFV, of which 19 were ASFV wild-type, one was ASFVΔCD2v, and two were ASFVΔMGF505. Compared with the commercialized triplex qPCR kit, the triple protein-based ELISA exhibited high diagnostic sensitivity and diagnostic specificity. The test accuracy with the commercialized triplex qPCR kit was 98.31% (58/59), and the test accuracy with the commercialized ELISA kit was 96.61% (57/59). These results indicated that the developed triple protein-based ELISA performs well in detection and differentiation. Therefore, it will be useful for the ASFV serological differential diagnosis and epidemiology study.

Keywords: African swine fever virus; differential diagnosis; gene-deleted strain; indirect ELISA; wild-type strain.

Figure 1

SDS-PAGE and Western blot analysis of purified recombinant proteins ASFV-p30, ASFV-CD2v, and ASFV-MGF505. SDS-PAGE analysis of purified recombinant proteins ASFV-p30, ASFV-CD2v, and ASFV-MGF505 (A–C). The expression and purification of the recombinant proteins ASFV-p30 (39.6 kDa) (A), ASFV-CD2v (46.8 kDa) (B), and ASFV-MGF505 (42.3 kDa) (C) were analyzed by SDS-PAGE. Lane M: protein marker; Lane 1: pET-32a; Lane 2: unpurified protein; Lane 3: purified protein. Analysis of immunogenic identification of the ASFV-p30, ASFV-CD2v, and ASFV-MGF505 proteins by Western blot assay (D–G). ASFV-p30, ASFV-CD2v, and ASFV-MGF505 proteins could react with ASFV⁺ serum (D). ASFV-p30 and ASFV-CD2v proteins could react with ASFV⁺ΔMGF505 serum (E). ASFV-p30 and ASFV-MGF505 proteins could react with ASFV⁺ΔCD2v serum (F). ASFV-p30, ASFV-CD2v, and ASFV-MGF505 proteins could not react with ASFV⁻ serum (G). Lane M: protein marker; Lane 1: ASFV-p30; Lane 2: ASFV-CD2v; Lane 3: ASFV-MGF505.

Figure 2

Optimization of experimental conditions for the triple protein-based ELISA. Determination of optimal protein coating concentration. P/N ratios of an ASFV⁺ and ASFV⁻ serum using different concentrations of the ASFV-p30, ASFV-CD2v, and ASFV-MGF505 proteins (A). Determination of optimal serum dilution. P/N ratios of an ASFV⁺ and ASFV⁻ serum with various dilutions of serum samples (B). Determination of optimal antigen coating conditions. P/N ratios of an ASFV⁺ and ASFV⁻ serum with various coating times and temperatures of each antigen (C). Determination of the best blocking buffer. P/N ratios of an ASFV⁺ and ASFV⁻ serum with various blocking buffer (D). Determination of the optimal dilution of the HRP labeled Rabbit Anti-Pig IgG (H + L). P/N ratios of an ASFV⁺ and ASFV⁻ serum with various dilutions of HRP labeled Rabbit Anti-Pig IgG (H + L) (E). P/N ratio data represent mean ± SD.

Figure 3

Confirmation of the cut-off value for the triple protein-based ELISA. ASFV⁻ serum samples (n = 90) were tested by the triple protein-based ELISA to determine the cut-off value under the optimal test conditions. The cut-off value of each ELISA was established at the mean OD_{450 nm} value (⁻x) + 3 × the standard deviation (SD).

Figure 4

Determination of the analytical specificity and analytical sensitivity of the triple protein-based ELISA. The triple protein-based ELISA cannot detect ASFV⁻, PRRSV⁺, PRV⁺, CSFV⁺, and PCV2⁺ serum samples, but can detect standard ASFV⁺, ASFV⁺ΔCD2v, and ASFV⁺ΔMGF505 serum samples at 1:100 dilution (A). Standard serum ASFV⁺ was diluted to test the detection limit of triple protein-based ELISA. According to the cut-off value, the ELISA based on the ASFV-p30 and ASFV-CD2v proteins could detect a 1,600-fold dilution of standard serum ASFV⁺, and the ELISA based on the ASFV-MGF505 protein could detect a 800-fold dilution of standard serum ASFV⁺. The dashed lines indicate the cut-off value (B).

Figure 5

Clinical serum samples (n = 59) detection results of the triple protein-based ELISA.