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. 2002 Nov 13;20(2):111–122. doi: 10.1016/0378-1135(89)90034-5

Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: Establishment of an efficient ELISA for antigen detection in feces

C-P Czerny 1, W Eichhorn 1
PMCID: PMC7117518  PMID: 2549679

Abstract

Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40 000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera.

Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay.

The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.

References

  1. Almeida J.D., Waterson A.P. The morphology of virus-antibody interaction. Adv. Virus Res. 1969;15:307–338. doi: 10.1016/S0065-3527(08)60878-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Avrameas S., Ternynck T. Peroxidase-labelled antibody and fab conjugates with enhanced intracellular penetration. Immunochemistry. 1971;8:1175–1179. doi: 10.1016/0019-2791(71)90395-8. [DOI] [PubMed] [Google Scholar]
  3. Bade H., Stegemann H. Rapid method of extraction of antibodies from hen egg yolk. J. Immunol. Methods. 1984;72:421–426. doi: 10.1016/0022-1759(84)90010-3. [DOI] [PubMed] [Google Scholar]
  4. Baljer G., Bachmann P.A. Nachweis enteropathogener Escherichia coli-Stämme und Rotaviren in Kotproben von Kälbern mit Diarrhoe. Zentralbl. Veterinaermed. Reihe B. 1980;27:606–615. [PubMed] [Google Scholar]
  5. Baljer G., Eichhorn W., Göbel E., Wolf M., Bachmann P.A. Vorkommen und Verbreitung wichtiger Durchfallerreger bei neugeborenen Källbern in Süddeutschland im Zeitraum 1984 bis 1986. Tieräerztl. Umsch. 1987;42:56–65. [Google Scholar]
  6. Crouch C.F., Raybould T.J.G., Acres S.D. Monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus. J. Clin. Microbiol. 1984;19:388–393. doi: 10.1128/jcm.19.3.388-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deregt D., Sabara M., Babiuk L.A. Structural proteins of bovine coronavirus and their intracellular processing. J. Gen. Virol. 1987;68:2863–2877. doi: 10.1099/0022-1317-68-11-2863. [DOI] [PubMed] [Google Scholar]
  8. Ellens D.J., Balken J.A.M.V., Leeuw P.D. Proceedings of the 2nd International Symposium on Neonatal Diarrhoea. 1978. Diagnosis of bovine coronavirus infections with haemadsorption-elution-haemagglutination assay (HEHA) and with enzyme linked immunosorbent assay (ELISA) pp. 321–330. [Google Scholar]
  9. Fazekas de St. Groth S., Scheidegger D. Production of monoclonal antibodies: Strategy and tactics. J. Immunol. Methods. 1980;35:1–21. doi: 10.1016/0022-1759(80)90146-5. [DOI] [PubMed] [Google Scholar]
  10. Hogue B.G., King B., Brian D.A. Antigenic relationships among proteins of coronavirus human respiratory coronavirus OC43 and mouse hepatitis coronavirus A59. J. Virol. 1984;51:384–388. doi: 10.1128/jvi.51.2.384-388.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lannèr M., Berquist R., Carlsson L., Joklik W. Purification of enzyme-labelled conjugate by affinity chromatography. In: Hoffman-Ostenhof O., editor. Affinity Chromatography. Pergamon Press; Oxford: 1978. pp. 237–241. [Google Scholar]
  12. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacterophage T4. Nature, (London) 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Mayr A., Bachmann P.A., Bibrack B., Wittmann G. VEB Gustav Fischer Verlag; Jena: 1974. Virologische Arbeitsmethoden. Bd. 2: Serologie. [Google Scholar]
  14. Mebus C.A., Stair E.L., Rhodes M.B., Twiehaus M.J. Neonatal calf diarrhea: Propagation, attenuation and characteristics of coronavirus-like agent. Am. J. Vet. Res. 1973;34:145–150. [PubMed] [Google Scholar]
  15. Merill C.R., Goldman D., Sedman S.A., Ebert M.H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981;211:1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  16. Peters T.H., Baumgarten H., Schulze M. Springer Verlag; Berlin: 1985. Monoklonale Antikörper, Herstellung und Charakterisierung. [Google Scholar]
  17. Reynolds D.J., Chassey D., Scott A.C., Bridger J.C. Evaluation of ELISA and electro microscopy for the detection of coronavirus and rotavirus in bovine faeces. Vet. Rec. 1984;114:397–401. doi: 10.1136/vr.114.16.397. [DOI] [PubMed] [Google Scholar]
  18. Reynolds D.J., Morgan J.H., Chanter N., Jones P.W., Bridger J.C., Debney T.G., Bunch K.J. Microbiology of calf diarrhea in southern Britain. Vet. Rec. 1986;119:34–38. doi: 10.1136/vr.119.2.34. [DOI] [PubMed] [Google Scholar]
  19. Sato K., Inaba Y., Tokuhisa S., Miura Y., Kaneko N., Matumoto M. Detection of bovine coronavirus in feces by reversed passive hemagglutination. Arch. Virol. 1984;80:20–31. doi: 10.1007/BF01315291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sharpee R.L., Mebus C.A., Bass E.P. Characterization of a calf diarrheal coronavirus. Am. J. Vet. Res. 1976;37:1031–1041. [PubMed] [Google Scholar]
  21. Snodgrass D.R., Terzolo H.R., Sherwood D., Campbell J., Menzies J.D., Synge B.A. Aetiology of diarrhea in young calves. Vet. Rec. 1986;119:31–39. doi: 10.1136/vr.119.2.31. [DOI] [PubMed] [Google Scholar]
  22. Stair E.L., Rhodes M.B., White R.G., Mebus C.A. Purification and electron microscopy of a coronavirus-like agent. Am. J. Vet. Res. 1972;33:1147–1155. [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Vol. 76. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications; pp. 4350–4354. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vautherot J.F., Laporte J. Utilization of monoclonal antibodies for antigenic characterization of coronaviruses. Ann. Rech. Vet. 1983;14:437–444. [PubMed] [Google Scholar]
  25. Woode G.N., Bridger S.C., Meyling A. Significance of bovine coronavirus infection. Vet. Rec. 1978;102:15–16. doi: 10.1136/vr.102.1.15. [DOI] [PubMed] [Google Scholar]
  26. Yolken R.H. Enzyme immunoassays for the detection of infectious antigens in body fluids: current limitations and future prospects. Rev. Infect. Dis. 1982;4:35–67. doi: 10.1093/clinids/4.1.35. [DOI] [PubMed] [Google Scholar]

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