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. 2022 Dec;37(6):922-933.
doi: 10.1016/j.virs.2022.09.004. Epub 2022 Sep 8.

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

Jiakai Zhao  1 , Jiahong Zhu  1 , Ying Wang  1 , Mengting Yang  1 , Qiang Zhang  1 , Chong Zhang  2 , Yuchen Nan  1 , En-Min Zhou  1 , Yani Sun  3 , Qin Zhao  4
Affiliations

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

Jiakai Zhao et al. Virol Sin. 2022 Dec.

Abstract

African swine fever virus (ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds. Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase (nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA (cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7% by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.

Keywords: ASFV-p30; African swine fever virus (ASFV); Competitive ELISA; Nanobody; Nanobody-HRP fusion Protein.

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Figures

Fig. 1
Fig. 1
Expression and purification of the recombinant ASFV-p30. A SDS-PAGE analysis of the recombinant ASFV-p30 expressed with E coli. B Western blot assay of the recombinant ASFV-p30. Positive pig sera for anti-ASFV antibodies was used as primary antibodies. M: molecular weight markers; lane 1: pET21b vector control; lane 2: Induction with 0.1 ​mmol/L IPTG; lane 3: soluble protein in the supernatant after sonication; lane 4: inclusion body in precipitation after sonication; lane 5: purified ASFV-p30.
Fig. 2
Fig. 2
Screening and identification of nanobodies against the ASFV-p30. A Titers of antibodies against ASFV-p30 in the sera from the immunized camel. B Total 48 clones were randomly picked to estimate the correct insertion rate by PCR. C Identification of the periplasmic extracts from the 96 clones specifically binding to the ASFV-p30 with indirect ELISA. 94 clones were identified as positive. D Alignment of amino acid sequences of seven screened nanobodies. E Titration of the seven screened nanobodies binding with the ASFV-p30 in the periplasmic extracts.
Fig. 3
Fig. 3
Characterization of seven ASFV-p30-Nbs-HRP fusion proteins secretory expressed in the HEK293T cells. A Expression of seven ASFV-p30-Nbs-HRP fusion proteins in the HEK293T cells. Anti-His mAb was used as the primary antibody. B ASFV-p30-Nbs-HRP fusion proteins in the culture medium of HEK293T cells showed HRP activity reacting with the substrate of TMB. C Specific reactions between different concentrations of ASFV-p30-Nbs-HRP and ASFV-p30 using direct ELISA. D ASFV-p30-Nbs-HRP specifically binds to p30 in ASFV-infected PAM cells in IFA assay. The anti-His mAbs were used as the primary antibody reacting with His tag of the ASFV-p30-Nbs-HRP and the FITC-goat anti-mouse IgG antibodies was used as the second antibody.
Fig. 4
Fig. 4
Selection of the best nanobody-HRP fusion protein for cELISA. A Analysis of the seven screened nanobodies blocking the binding of ASFV-p30 and the ASFV pig serum. B Binding affinity analysis of ASFV-p30-Nb75-HRP to different amounts of ASFV-p30 proteins by direct ELISA.
Fig. 5
Fig. 5
Specificity of the developed cELISA for detecting anti-ASFV antibodies using the ASFV-p30-Nb75-HRP fusions as a probe. A Distribution of the PI values of the clinical negative pig sera for anti-ASFV antibodies in the cELISA. B Evaluation of the cELISA for detecting the antibodies against other pig disease viruses, including PRRSV, PCV2, PRV, PEDV, and SIV.
Fig. 6
Fig. 6
Sensitivity of the developed cELISA with the ASFV-p30-Nb75-HRP fusion protein. A Distribution of the PI values of the clinical positive pig sera for anti-ASFV antibodies in the cELISA. B Determination of the largest dilution of positive pig sera for anti-ASFV antibodies. C Detection of antibodies against ASFV in the 36 sequential sera at different days post-inoculation from six pigs challenged with ASFV using the cELISA.
Fig. 7
Fig. 7
Stability of the developed cELISA. A Binding analysis of the ASFV-p30-Nb75-HRP fusion protein to ASFV-p30 at different times using direct ELISA. B Analysis of the ASFV-p30-Nb75-HRP fusion protein blocked to react with ASFV-p30 by the positive pig sera for anti-ASFV antibodies using the cELISA.
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