Archives of Virology

, Volume 163, Issue 6, pp 1549–1557 | Cite as

Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1

  • Greice Japolla
  • Jair Pereira Cunha-Junior
  • Ana Claudia Arantes Marquez Pajuaba
  • Ernesto Akio Taketomi
  • Samira Bührer-Sékula
  • Luiz Artur Mendes Bataus
  • Guilherme Rocha Lino de Souza
Original Article


Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.



We thank Prof. Dr. Frédérick Jean Georges Frézard (Federal University of Minas Gerais) and Dr. Sydnei Magno da Silva (Federal University of Uberlandia) for the gold nanoparticle analysis.


This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant number 564204/2010-2) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Grant number 1378410).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

Supplementary material

705_2018_3763_MOESM1_ESM.pdf (89 kb)
Supplementary material 1 (PDF 89 kb)


  1. 1.
    Marin MS, Leunda MR, Verna AE, Morán PE, Odeón AC, Pérez SE (2016) Distribution of bovine herpesvirus type 1 in the nervous system of experimentally infected calves. Vet J 209:82–86. CrossRefPubMedGoogle Scholar
  2. 2.
    Rissi DR, Pierezan F, Sa e Silva M, Flores EF, de Barros CS (2008) Neurological disease in cattle in southern Brazil associated with Bovine herpesvirus infection. J Vet Diagn Investig 20(3):346–349. CrossRefGoogle Scholar
  3. 3.
    Engels M, Ackermann M (1996) Pathogenesis of ruminant herpesvirus infections. Vet Microbiol 53(1–2):3–15CrossRefPubMedGoogle Scholar
  4. 4.
    Fulton RW (2009) Bovine respiratory disease research (1983–2009). Anim Health Res Rev 10(2):131–139. CrossRefPubMedGoogle Scholar
  5. 5.
    Takiuchi E, Alfieri AF, Alfieri AA (2001) Bovine herpervirus type 1: infection and diagnosis methods. Semina: Ciências Agrarias 22(2):203–209Google Scholar
  6. 6.
    Mahajan V, Banga HS, Deka D, Filia G, Gupta A (2013) Comparison of diagnostic tests for diagnosis of infectious bovine rhinotracheitis in natural cases of bovine abortion. J Comp Pathol 149(4):391–401. CrossRefPubMedGoogle Scholar
  7. 7.
    van Drunen Littel-van den Hurk S (2006) Rationale and perspectives on the success of vaccination against bovine herpesvirus-1. Vet Microbiol 113(3–4):275–282. CrossRefPubMedGoogle Scholar
  8. 8.
    van Oirschot JT, Kaashoek MJ, Rijsewijk FA, Stegeman JA (1996) The use of marker vaccines in eradication of herpesviruses. J Biotechnol 44(1–3):75–81. CrossRefPubMedGoogle Scholar
  9. 9.
    Raaperi K, Orro T, Viltrop A (2015) Effect of vaccination against bovine herpesvirus 1 with inactivated gE-negative marker vaccines on the health of dairy cattle herds. Prev Vet Med 118(4):467–476. CrossRefPubMedGoogle Scholar
  10. 10.
    Tignon M, De Baere M, Hanon JB, Goolaerts A, Houtain JY, Delooz L, Cay AB (2017) Characterization of three commercial ELISA kits for detection of BOHV-1 gE specific antibodies in serum and milk samples and applicability of bulk milk for determination of herd status. J Virol Methods 245:66–72. CrossRefPubMedGoogle Scholar
  11. 11.
    Muratore E, Bertolotti L, Nogarol C, Caruso C, Lucchese L, Iotti B, Ariello D, Moresco A, Masoero L, Nardelli S, Rosati S (2017) Surveillance of infectious bovine rhinotracheitis in marker-vaccinated dairy herds: application of a recombinant gE ELISA on bulk milk samples. Vet Immunol Immunopathol 185:1–6. CrossRefPubMedGoogle Scholar
  12. 12.
    Walz PH, Givens MD, Rodning SP, Riddell KP, Brodersen BW, Scruggs D, Short T, Grotelueschen D (2017) Evaluation of reproductive protection against bovine viral diarrhea virus and bovine herpesvirus-1 afforded by annual revaccination with modified-live viral or combination modified-live/killed viral vaccines after primary vaccination with modified-live viral vaccine. Vaccine 35(7):1046–1054. CrossRefPubMedGoogle Scholar
  13. 13.
    Cortese VS, Woolums A, Hurley DJ, Berghaus R, Bernard JK, Short TH (2017) Comparison of interferon and bovine herpesvirus-1-specific IgA levels in nasal secretions of dairy cattle administered an intranasal modified live viral vaccine prior to calving or on the day of calving. Vet Immunol Immunopathol 187:35–41. CrossRefPubMedGoogle Scholar
  14. 14.
    Silva MS, Brum MCS, Loreto ELS, Weiblen R, Flores EF (2007) Molecular and antigenic characterization of Brazilian bovine herpesvirus type 1 isolates recovered from the brain of cattle with neurological disease. Virus Res 129(2):191–199. CrossRefPubMedGoogle Scholar
  15. 15.
    Marin MS, Leunda MR, Verna AE, Faverín C, Pérez SE, Odeón AC (2012) In vitro replication of bovine herpesvirus types 1 and 5. J Virol Methods 181(1):80–85. CrossRefPubMedGoogle Scholar
  16. 16.
    House JA, Baker JA (1971) Bovine herpesvirus IBR-IPV. The antibody virus neutralization reaction. Cornell Vet 61(2):320–335PubMedGoogle Scholar
  17. 17.
    Choudhary N, Roy A, Leon MG, Wei G, Nakhla MK, Levy L, Brlansky RH (2017) Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays. J Virol Methods 243:177–181. CrossRefPubMedGoogle Scholar
  18. 18.
    Hauck TS, Giri S, Gao Y, Chan WCW (2010) Nanotechnology diagnostics for infectious diseases prevalent in developing countries. Adv Drug Deliv Rev 62(4–5):438–448. CrossRefPubMedGoogle Scholar
  19. 19.
    Wang Y, Wang L, Zhang J, Wang G, Chen W, Chen L, Zhang X (2014) Preparation of colloidal gold immunochromatographic strip for detection of Paragonimiasis skrjabini. PLoS One 9(3):e92034. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ju Y, Hao H-J, Xiong G-H, Geng H-R, Zheng Y-L, Wang J, Cao Y, Yang Y-H, Cai X-H, Jiang Y-Q (2010) Development of colloidal gold-based immunochromatographic assay for rapid detection of Streptococcus suis serotype 2. Vet Immunol Immunopathol 133(2–4):207–211. CrossRefPubMedGoogle Scholar
  21. 21.
    Bastús NG, Comenge J, Puntes V (2011) Kinetically controlled seeded growth synthesis of citrate-stabilized gold nanoparticles of up to 200 nm: size focusing versus ostwald ripening. Langmuir 27(17):11098–11105. CrossRefPubMedGoogle Scholar
  22. 22.
    Tram DT, Wang H, Sugiarto S, Li T, Ang WH, Lee C, Pastorin G (2016) Advances in nanomaterials and their applications in point of care (POC) devices for the diagnosis of infectious diseases. Biotechnol Adv 34(8):1275–1288. CrossRefPubMedGoogle Scholar
  23. 23.
    Wen-de W, Min L, Ming C, Li-ping L, Rui W, Hai-lan C, Fu-Yan C, Qiang M, Wan-wen L, Han-zhong C (2017) Development of a colloidal gold immunochromatographic strip for rapid detection of Streptococcus agalactiae in tilapia. Biosens Bioelectron 91:66–69. CrossRefPubMedGoogle Scholar
  24. 24.
    Meng K, Sun W, Zhao P, Zhang L, Cai D, Cheng Z, Guo H, Liu J, Yang D, Wang S, Chai T (2014) Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma. Biosens Bioelectron 55:396–399. CrossRefPubMedGoogle Scholar
  25. 25.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  26. 26.
    Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat Phys Sci 241:20–22CrossRefGoogle Scholar
  27. 27.
    Bright RM, Musick MD, Natan MJ (1998) Preparation and characterization of Ag colloid monolayers. Langmuir 14(20):5695–5701. CrossRefGoogle Scholar
  28. 28.
    Santos VO, Pelegrini PB, Mulinari F, Moura RS, Cardoso LPV, Buhrer-Sekula S, Miller RNG, Pinto ERC, Grossi-de-Sa MF (2015) A novel immunochromatographic strip test for rapid detection of Cry1Ac and Cry8Ka5 proteins in genetically modified crops. Anal Methods 7(21):9331–9339. CrossRefGoogle Scholar
  29. 29.
    Greiner M (1996) Two-graph receiver operating characteristic (TG-ROC): update version supports optimisation of cut-off values that minimise overall misclassification costs. J Immunol Methods 191(1):93–94CrossRefPubMedGoogle Scholar
  30. 30.
    Akobeng AK (2007) Understanding diagnostic tests 1: sensitivity, specificity and predictive values. Acta Paediatr (Oslo, Norway: 1992) 96(3):338–341. CrossRefGoogle Scholar
  31. 31.
    van Stralen KJ, Stel VS, Reitsma JB, Dekker FW, Zoccali C, Jager KJ (2009) Diagnostic methods I: sensitivity, specificity, and other measures of accuracy. Kidney Int 75(12):1257–1263. CrossRefPubMedGoogle Scholar
  32. 32.
    Medeiros LS, Carvalho YK, Maciel RCG, Lilenbaum W (2016) Análise de custo-efetividade de protocolos no diagnóstico da tuberculose bovina em um rebanho naturalmente infectado. Pesquisa Veterinária Brasileira 36:485–491CrossRefGoogle Scholar
  33. 33.
    Komorowski M, Raffa J (2016) Markov models and cost effectiveness analysis: applications in medical research. In: Secondary analysis of electronic health records, edn. Springer International Publishing, Cham, Swizerland, pp. 351–367.
  34. 34.
    Raaperi K, Bougeard S, Aleksejev A, Orro T, Viltrop A (2012) Association of herd BRSV and BHV-1 seroprevalence with respiratory disease and reproductive performance in adult dairy cattle. Acta Vet Scand 54:4. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Statham JME, Randall LV, Archer SC (2015) Reduction in daily milk yield associated with subclinical bovine herpesvirus 1 infection. Vet Rec 177(13):339. CrossRefPubMedGoogle Scholar
  36. 36.
    Sayers RG, Byrne N, O’Doherty E, Arkins S (2015) Prevalence of exposure to bovine viral diarrhoea virus (BVDV) and bovine herpesvirus-1 (BoHV-1) in Irish dairy herds. Res Vet Sci 100:21–30. CrossRefPubMedGoogle Scholar
  37. 37.
    Rola J, Larska M, Socha W, Rola JG, Materniak M, Urban-Chmiel R, Thiry E, Żmudziński JF (2017) Seroprevalence of bovine herpesvirus 1 related alphaherpesvirus infections in free-living and captive cervids in Poland. Vet Microbiol 204:77–83. CrossRefPubMedGoogle Scholar
  38. 38.
    Raaperi K, Orro T, Viltrop A (2014) Epidemiology and control of bovine herpesvirus 1 infection in Europe. Vet J 201(3):249–256. CrossRefPubMedGoogle Scholar
  39. 39.
    Jones C, Geiser V, Henderson G, Jiang Y, Meyer F, Perez S, Zhang Y (2006) Functional analysis of bovine herpesvirus 1 (BHV-1) genes expressed during latency. Vet Microbiol 113(3–4):199–210. CrossRefPubMedGoogle Scholar
  40. 40.
    Pierotti S, Albano C, Milandri A, Callegari F, Poletti R, Rossini GP (2007) A slot blot procedure for the measurement of yessotoxins by a functional assay. Toxicon 49(1):36–45. CrossRefPubMedGoogle Scholar
  41. 41.
    Dias JA, Alfieri AA, Ferreira-Neto JS, Goncalves VS, Muller EE (2013) Seroprevalence and risk factors of bovine herpesvirus 1 infection in cattle herds in the state of Parana, Brazil. Transbound Emerg Dis 60(1):39–47. CrossRefPubMedGoogle Scholar
  42. 42.
    Zhu D, Saul AJ, Miles AP (2005) A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli. J Immunol Methods 306(1–2):40–50. CrossRefPubMedGoogle Scholar
  43. 43.
    Pawar SS, Meshram CD, Singh NK, Saini M, Mishra BP, Gupta PK (2014) Development of a SYBR Green I based duplex real-time PCR for detection of bovine herpesvirus-1 in semen. J Virol Methods 208:6–10. CrossRefPubMedGoogle Scholar
  44. 44.
    Marin MS, Quintana S, Leunda MR, Recavarren M, Pagnuco I, Späth E, Pérez S, Odeón A (2016) A new method for simultaneous detection and discrimination of bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis. J Virol Methods 227:14–22. CrossRefPubMedGoogle Scholar
  45. 45.
    Casarin E, Lucchese L, Grazioli S, Facchin S, Realdon N, Brocchi E, Morpurgo M, Nardelli S (2016) A new ELISA using the ANANAS technology showing high sensitivity to diagnose the bovine rhinotracheitis from individual sera to pooled milk. PLoS One 11(1):e0145912CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Al-Yousif Y, Anderson J, Chard-Bergstrom C, Kapil S (2002) Development, evaluation, and application of lateral-flow immunoassay (immunochromatography) for detection of rotavirus in bovine fecal samples. Clin Diagn Lab Immunol 9(3):723–724. PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Zhang H-C, Liu C-Y, Liu G-Y, Chen X-L, Ye Y-D, Chai C-Y, Wang Y-R (2012) A portable photoelectric sensor based on colloidal gold immunochromatographic strips for rapid determination of clenbuterol in pig urine. Chin J Anal Chem 40(6):852–856. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Greice Japolla
    • 1
  • Jair Pereira Cunha-Junior
    • 2
  • Ana Claudia Arantes Marquez Pajuaba
    • 2
  • Ernesto Akio Taketomi
    • 2
  • Samira Bührer-Sékula
    • 3
  • Luiz Artur Mendes Bataus
    • 4
  • Guilherme Rocha Lino de Souza
    • 1
    • 4
  1. 1.Programa de Pós-graduação em Ciência Animal, Escola de Veterinária e Zootecnia (PPGCA/EVZ)Universidade Federal de GoiásGoiâniaBrazil
  2. 2.Departamento de Imunologia, Instituto de Ciências BiomédicasUniversidade Federal de UberlândiaUberlândiaBrazil
  3. 3.Instituto de Patologia Tropical e Saúde Pública, Setor ImunologiaUniversidade Federal de GoiásGoiâniaBrazil
  4. 4.Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas (ICB)Universidade Federal de GoiásGoiâniaBrazil

Personalised recommendations