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. 2022 Aug 20;18(1):321.
doi: 10.1186/s12917-022-03423-0.

K205R specific nanobody-horseradish peroxidase fusions as reagents of competitive ELISA to detect African swine fever virus serum antibodies

Angke Zhang #  1   2 , Shuya Wu #  1   2 , Xiaohong Duan  3 , Huijun Zhao  1   2 , Haoxin Dong  1   2 , Jiahui Ren  1   2 , Mingfang Zhang  1 , Jiaji Li  1 , Hong Duan  4 , Gaiping Zhang  5   6   7
Affiliations

K205R specific nanobody-horseradish peroxidase fusions as reagents of competitive ELISA to detect African swine fever virus serum antibodies

Angke Zhang et al. BMC Vet Res. .

Abstract

Background: African swine fever virus (ASFV) is a highly contagious hemorrhagic disease and often lethal, which has significant economic consequences for the swine industry. Due to lacking of commercial vaccine, the prevention and control of ASF largely depend on early large-scale detection and screening. So far, the commercial ELISA kits have a long operation time and are expensive, making it difficult to achieve large-scale clinical applications. Nanobodies are single-domain antibodies produced by camelid animals, and have unique advantages such as smaller molecular weight, easy genetic engineering modification and low-costing of mass production, thus exhibiting good application prospects.

Results: The present study developed a new method for detection of ASFV specific antibodies using nanobody-horseradish peroxidase (Nb-HRP) fusion proteins as probe. By using camel immunization, phage library construction and phage display technology, five nanobodies against K205R protein were screened. Then, Nb-HRP fusion proteins were produced using genetic modification technology. Based on the Nb-HRP fusion protein as specific antibodies against K205R protein, a new type of cELISA was established to detect ASFV antibodies in pig serum. The cut-off value of the cELISA was 34.8%, and its sensitivity, specificity, and reproducibility were good. Furthermore, the developed cELISA exhibited 99.3% agreement rate with the commercial available ELISA kit (kappa value = 0.98).

Conclusions: The developed cELISA method has the advantages of simple operation, rapid and low-costing, and can be used for monitoring of ASFV infection in pigs, thus providing a new method for the prevention and control of ASF.

Keywords: ASFV; Antibody; K205R; Nanobody-HRP; cELISA.

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Conflict of interest statement

All the authors approved the final manuscript and they have no competing interests to declare.

Figures

Fig. 1
Fig. 1
Expression and purification of K205R-His protein. A The K205R-His protein was analyzed by SDS-PAGE. B and C Western blotting and iELISA analysis of the antigenic property of the K205R protein using inactivated ASFV antibody-positive serum as the primary antibody. Lane 1: pET30a empty vector control; Lane 2: IPTG, 0.5 mM; Lane 3: Supernatant soluble K205R protein; Lane 4: Inclusion body of K205R protein; Lane 5: Purified K205R protein
Fig. 2
Fig. 2
Screening of K205R specific nanobody. A iELISA analysis of serum antibody titer against K205R protein of the immunized camel. B ELISA analysis the enrichment of specific phage. C iELISA analysis of periplasmic extracts from 96 clones that specifically bind to K205R protein. D Five K205R specific nanobodies were screened according to amino acid sequence alignment. The red line indicates the hallmark residues at 37, 44, 45, and 47
Fig. 3
Fig. 3
Identification of the nanobodies against the K205R protein. A iELISA identification of nanobodies specifically recognized K205R protein. ASFV DP96R protein was used as a negative control with the same expression system as K205R. B Affinity analysis of nanobodies in cytoplasmic extracts for K205R protein
Fig. 4
Fig. 4
Secretory expression and functional analysis of Nbs-HRP in HEK-293 T cell culture supernatants. A Schematic representation of Nbs-HRP fusion protein construction. B IFA identification of Nbs-HRP expression in HEK-293 T cells using anti-HA monoclonal antibody. C Detection of secretory expression of Nbs-HRP fusion protein in HEK-293 T cell culture supernatants. D Direct ELISA analysis of Nbs-HRP specifically reacted with K205R protein. ASFV DP96R was used as a control. E Titration of the Nbs-HRP binding with the K205R protein. F cELISA comparative analysis of Nb1, Nb35 and Nb82 blocking capability of the binding of ASFV antibody-positive serum to K205R protein
Fig. 5
Fig. 5
Determination of optimal reaction conditions of the cELISA. A cELISA analysis of the optimal dilution ratio of pig serum. B Determination of optimal competition time for K205R protein binding between ASFV antibody-positive serum and Nb1-HRP fusion protein. C Optimal colorimetric reaction time of the developed cELISA
Fig. 6
Fig. 6
Sensitivity analysis of the developed cELISA. A Determination of the maximum dilution of inactivated ASFV antibody-positive pig serum. B Distribution of the PI values by testing the positive clinical sera for anti-ASFV antibodies using cELISA
Fig. 7
Fig. 7
Specificity analysis of the developed cELISA. A Specificity analysis of the developed cELISA by detection of PRRSV-, PEDV-, PRV-, CSFV-, and PCV2-antibody positive serum. B ASFV antibody-negative serum samples were tested using the cELISA to analyze distribution of the PI values
Fig. 8
Fig. 8
Evaluation of the discrepancy of test results between the cELISA and commercial ELISA kit. Western blotting of the 2 swine serum samples with a discrepant result between the developed cELISA and commercial ELISA kit. A negative serum and a inactivated standard ASFV antibody-positive serum were used simultaneously as the control. PC: positive serum control; NC: negative serum control
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References

    1. Sanchez-Vizcaino JM, Mur L, Martinez-Lopez B. African swine fever: an epidemiological update. Transbound Emerg Dis. 2012;59(Suppl 1):27–35. doi: 10.1111/j.1865-1682.2011.01293.x. - DOI - PubMed
    1. Gallardo C, Reis AL, Kalema-Zikusoka G, Malta J, Soler A, Blanco E, Parkhouse RM, Leitao A. Recombinant antigen targets for serodiagnosis of African swine fever. Clin Vaccine Immunol. 2009;16:1012–1020. doi: 10.1128/CVI.00408-08. - DOI - PMC - PubMed
    1. Zhao D, Liu R, Zhang X, Li F, Wang J, Zhang J, Liu X, Wang L, Zhang J, Wu X, Guan Y, Chen W, Wang X, He X, Bu Z. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg Microbes Infect. 2019;8:438–447. doi: 10.1080/22221751.2019.1590128. - DOI - PMC - PubMed
    1. Zhou X, Li N, Luo Y, Liu Y, Miao F, Chen T, Zhang S, Cao P, Li X, Tian K, Qiu HJ, Hu R. Emergence of African Swine Fever in China, 2018. Transbound Emerg Dis. 2018;65:1482–1484. doi: 10.1111/tbed.12989. - DOI - PubMed
    1. Bellini S, Rutili D, Guberti V. Preventive measures aimed at minimizing the risk of African swine fever virus spread in pig farming systems. Acta Vet Scand. 2016;58:82. doi: 10.1186/s13028-016-0264-x. - DOI - PMC - PubMed

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