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

Figures

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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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