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. 2024 Jun;39(3):478-489.
doi: 10.1016/j.virs.2024.04.002. Epub 2024 Apr 6.

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies

Jiahong Zhu  1 , Qingyuan Liu  1 , Liuya Li  1 , Runyu Zhang  1 , Yueting Chang  1 , Jiakai Zhao  1 , Siyu Liu  1 , Xinyu Zhao  1 , Xu Chen  1 , Yani Sun  2 , Qin Zhao  3
Affiliations

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies

Jiahong Zhu et al. Virol Sin. 2024 Jun.

Abstract

African swine fever virus (ASFV) poses a significant threat to the global swine industry. Currently, there are no effective vaccines or treatments available to combat ASFV infection in pigs. The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig. Recently, a lower virulent ASFV isolate with a deleted EP402R gene (CD2v-deleted) has been reported in China, which further complicates the control of ASFV infection in pig farms. Furthermore, an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain. Therefore, it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections. In this study, two recombinant ASFV-p72 and -CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels. Subsequently, eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened. Following the production of these nanobodies with horse radish peroxidase (HRP) fusion proteins, the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs (cELISAs) to detect anti-ASFV antibodies. The two cELISAs exhibited high sensitivity, good specificity, repeatability, and stability. The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6% and 97.6%, respectively. Collectively, the two cELISA for detecting antibodies against ASFV demonstrated ease of operation, a low cost, and a simple production process. The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV, and could play an important role in monitoring ASFV infections in pig farms.

Keywords: ASFV-CD2v; ASFV-p72; African swine fever virus (ASFV); Competitive ELISA; Nanobody-HRP.

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Conflict of interest statement

Conflict of interest The authors report no conflicts of interest in this work.

Figures

Fig. 1
Fig. 1
Screening and identification of nanobodies against the ASFV-p72 and -CD2v proteins. A Titration of the sera from the immunized camel against the recombinant ASFV-p72 protein. Data are presented as means ​± ​SD. B Titration of the sera from the immunized camel against the recombinant ASFV-CD2v protein. C Indirect ELISA using periplasmic extracts from the 96 clones to identify specific binding to ASFV-p72. A total of 56 clones were positive. D Indirect ELISA using periplasmic extracts from the 96 clones to identify specific binding to ASFV-CD2v. All the 96 clones were positive. NDV-NP was used as the negative control and expressed using the same system as the two proteins. E Amino acid alignment of the eight nanobodies against the ASFV-p72 protein. F Amino acid alignment of the ten nanobodies against the ASFV-CD2v protein. The hydrophilic amino acids residues at positions 37, 44, 45, and 47 are indicated by red arrows.
Fig. 2
Fig. 2
Expression of the nanobody-HRP fusion proteins in HEK293T cells. A Identification of the eight nanobody-HRP fusion proteins against the ASFV-p72 protein expressed in HEK293T cells using indirect immunofluorescence assay (IFA). B Identification of the ten nanobody-HRP fusion proteins against the ASFV-CD2v protein expressed in HEK293T cells using IFA. C Direct ELISA to identify the specific reactions between the eight nanobody-HRP fusion proteins with ASFV-p72. D Direct ELISA to identify the specific reactions between the ten nanobody-HRP fusion proteins with ASFV-CD2v. Data are presented as means ​± ​SD. E Identification of the eight nanobody-HRP fusion proteins against the ASFV-p72 protein binding to the ASFV-infected PAM cells by IFA. A representative image of ASFV-p72-Nb3-HRP is shown here, which is the same as the other three nanobody-HRP fusion proteins that do not target the ASFV-p72 protein. F Identification of the 10 nanobody-HRP fusion proteins against ASFV-CD2v protein binding to the ASFV-infected PAM cells by IFA. A representative image of ASFV-CD2v-Nb88-HRP is shown here, which is the same as the other seven nanobody-HRP fusion proteins that do not target the ASFV-CD2v protein. Scalar bar, 100 ​μm.
Fig. 3
Fig. 3
Optimization of the best nanobody-HRP fusion proteins for separately developing p72-cELISA and CD2v-cELISA. A Analysis of the negative and positive pig sera for anti-ASFV antibody blocking the reaction between ASFV-p72 and the eight nanobody-HRP fusion proteins. B Analysis of the negative and positive pig sera blocking the reaction between ASFV-CD2v and the 10 nanobody-HRP fusion proteins. Data are presented as means ​± ​SD. Statistical analyses were performed using a t-test with GraphPad Prism software. ∗P ​< ​0.05; ∗∗P ​< ​0.01; ∗∗∗P ​< ​0.001; ∗∗∗∗P ​< ​0.0001; ns, not significant.
Fig. 4
Fig. 4
Sensitivity and specificity analysis of the two developed cELISAs. A Determination of the limit of detection of the p72-cELISA. Data are presented as means ​± ​SD. B Determination of the limit of detection of the CD2v-cELISA. C Analysis of positive samples for antibodies against other pig pathogens by p72-cELISA. D Analysis of the positive samples for antibodies against other pathogens by CD2v-cELISA. E Detection of the 23 positive sera for anti-wild-type ASFV (red) antibodies and 36 positive sera for anti-CD2v-deleted ASFV (blue) antibodies by p72-cELISA. F Detection of the 23 positive sera for anti-wild-type ASFV (red) antibodies and 36 positive sera for anti-CD2v-deleted ASFV (blue) antibodies by CD2v-cELISA.
Fig. 5
Fig. 5
Stability analysis of the two developed cELISAs stored at 4 ​°C for 180 days. A Binding analysis of the nanobody-HRP fusion proteins to ASFV-p72 or ASFV-CD2v by direct ELISAs. B Analysis of the positive pig sera blocking the nanobody-HRP fusion protein to react with the ASFV-p72 and ASFV-CD2v proteins by cELISAs. The nanobody fusion proteins and coated plates were stored at 4 ​°C for 180 days.
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