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Alabalıklarda (Oncorhynchus Mykiss, Walbaum) Viral Hemorajik Septisemi (VHS) Hastalığının Teşhisi İçin Enzymelinked Immunosorbent Assay (ELISA) Yönteminin Geliştirilmesi*

Yıl 2017, Cilt 2, Sayı 1, 7 - 10, 03.04.2017
https://doi.org/10.35229/jaes.288116

Öz

VHS, enfekte balık dokusundaki virüsün, duyarlı doku kültürlerinde izolasyonundan sonra ELISA, virüs nötralizasyon, immunofloresans, immunoperoksidaz

ve komplement fiksasyon gibi serolojik testlerle teşhis edilmektedir. ELISA, tekrarlanabilir olması, kullanımının basit olması, çabuk yapılabilmesi ve

ucuz olmasıyla diğer serolojik testlere göre daha çok tercih edilmektedir.

Bu çalışmada VHS antijenini tespit edebilecek tavşandan elde edilen sadece bir antikor kullanıldı. Bu antikor biotin ile işaretlenerek testte konjugat

olarak kullanıldı. Üretilen antikorun (anti-VHSV F1 ɣ-globulin fraksiyonu) protein konsantrasyonu 28 mg/ml olarak ölçüldü. Mikroplak kuyucukları 10

mikrogram/mililitre (μg/ml) anti-VHSV ɣ-globulin fraksiyonu ile kaplandı. Daha sonra şüpheli örnek ilave edildi. Örnekteki VHSV biotinlenmiş anti-VHSV ɣglobulin

fraksiyonuna bağlandı. Bu aşamadan sonra ilave edilen horse radish peroksidase (HRP) enzimi ile işaretli avidin, konjugattaki biotin ile bağlandı. Son

olarak tetramethylbenzidine substrat (TMB) eklenmesiyle renk şekillendi ve ELISA okuyucuda 450 nonometrede (nm) okutturuldu. Bu çalışmada geliştirilen ELISA

yöntemi ile bilinen tüm VHSV alttipleri (07.71, HE, 23.75) tespit edilebilmektedir. Yine bu yöntem VHSV’ye oldukça spesifiktir ve İnfeksiyöz Hematopoetik

Nekrozis Virüsu (IHNV), Pike Fry rhabdovirus (PFR), Perch rhabdovirus (PR) ve bilinen tüm İnfeksiyöz Pankreatik Nekrozis Virüsu (IPNV) serotipleriyle çapraz

reaksiyon vermemektedir. Sonuç olarak geliştirilen bu ELISA yöntemi 80 nanogram (ng) virüs proteinini ve DKID50 değeri 104,75/0,1 ml olan virüsü tespit

edebilmektedir.

Kaynakça

  • Adams A., (1992). Techniques in Fish İmmunology: Sandwich Enzyme Linked Immunosorbent Assay (ELISA) to Detect and Quantify Bacterial Pathogens In Fish Tissue, SOS publications, 43 Denormandie Ave Fair Haven NJ.07704-3303 USA, 177- 182 p.
  • Anderson D.P. and Dixon O. W., (1989). Fish Biology Guide, U.S. Fish and Wildlife Service National Fish Health Research Laboratory, West Virginia, USA, 167p.
  • Anonim, (2006). Council Directive 2006/88/EC of 24 October 2006. Official Journal of the European Union L 328/14
  • Anonim, (2007). 3285 Sayılı Hayvan Sağlığı Ve Zabıtası Kanununun 4 Üncü Maddesine Göre Tespit Edilen İhbarı Mecburi Hastalıklar Hakkında Tebliğ (Tebliğ No: 2007/32). Resmî Gazete, Tarih:12 Temmuz 2007 Sayı: 26580.
  • Anonim, (2010). OIE Listed diseases: Updated: 04.01.2010, http://www.oie.int/en/animal-health-in-the-world/oie-listed-diseases-2017/. (25.01.20017)
  • Beard C.W., (1989). Serological procedure. 192-200. In: Purchase H.G., Lawrence, C., Arp, L.H., Domermuth, C.H., Pearson J.E. (Eds): A Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Iowa, USA.
  • Cagirgan H., (1992). The Development of New ELISA Assays for Detecting Serum Antibody Levels Against VHS and IPN in Rainbow Trout (Oncorhynhus mykiss), MSc Thesis, University of Plymouth, 92 p (unpublished).
  • Enzmann P.J. and Konrad M., (1985). Inapparent infections of Brown trout with VHS-virus. Bull. Eur. Assoc. Fish Pathol. 5, 81–83 pp
  • Guesdon J.L., Ternynck, T. and Avrameas S. (1979). The use of avidin-biotin interaction in immunoenzymatic techniques, The Journal Of Histochemistry and Cytochemistry, 27 (8), 1131-1139pp.
  • Harlow E., Lane D., (1988). Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 101p
  • Hsu, S. and Raine, L., (1981). Protein A, Avidin, and Biotin in Immunohistochemistry, The Journal of Histochemistry and Cytochemistry, 29 (11), 1349-1353pp
  • Hudson L. and Hay F.C., (1991). Pratical Immunology, Blackwell Scientific Publication, Oxford Great Britain, Third Edition, 4-6pp
  • Jensen M. H., (1965). Research on the virus of Egtved disease. Ann. N.Y. Acad. Sci. 126, 422-426 pp.
  • Kalaycı, G., Inacoglu S. and Ozkan B., (2006). First isolation of viral haemorrhagic septisemia (VHS) virus from turbot (Scophthalmus maximus) cultured in the Trabzon coastal area of the Black Sea in Turkey . Bull. Eur. Ass. Fish Pathol., 26(4), 157p.
  • Medina J.M., Chang P.W., Bradley T.M., Yeh M.T. and Sadasiv E.C., (1992). Diagnosis of infectious hematopoietic necrosis virus in Atlantic salmon ,Salmo salar by enzyme-linked immunusorbent assay, Dis. of Aquat. Org., 13 147-150pp.
  • Mourton C., Bearzotti M., Piechaczyk M., Paolucci F., Pau B., Bastide J.M. and de Kinkelin P., (1990). Antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I, J Virol Methods. 29(3):325-333.
  • Mourton C., Romestand B., de Kinkelin P., Jeffroy J., Le Gouvello R. and Pau B., (1992). Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies, Journal of Clinical Microbiology, 30 (9): 2338-2345 pp.
  • Nishizawa T., Savas H., Isidan H., Ustundag C., Iwamoto H. and Yoshimizu M., (2006). Genotyping and Pathogenicity of Viral Hemorrhagic Septicemia Virus from Free-Living Turbot (Psetta maxima) in a Turkish Coastal Area of the Black Sea, Appl. Environ. Microbiol. Vol. 72, no. 4, pp. 2373-2378.
  • Olesen N.J. and Jǿrgensen P. E. V., (1991). Rapid detection of viral haemorrhagic septicaemia virüs in fish by ELISA, J. Appl. Ichthyol. 7 183-186.
  • Olesen N. J., Lorenzen N. and Jørgensen P. E. V., (1993). Serological differences among isolates of viral haemorrhagic septicaemia virus detected by neutralizing monoclonal and polyclonal antibodies, Diseases of Aquatic Organisms,16: 163-170.
  • Olesen N. J., (1998). Sanitation of viral haemorrhagic septicaemia (VHS). J Appl Ichthyol 14, 173–177pp.
  • Schäperclaus W., (1938). Die Schädigungen der deutschen Fischerei durch Fischparasiten und Fischkrankheiten. Allg. Fischztg. 41:256–259, 267–270 pp. in Wolf K., (1988). Viral Hemorrhagic Septicemia, Fish Viruses and Fish Viral Diseases. Cornell University Pres/Ithaca and Landon 217-249 pp.
  • Savigny D. and Voller A., (1980). The communication of ELISA data from laboratory to clinician, Journal of Immunoassay,1 (1) 105-128pp.
  • Skall H.F., Olesen N.J. and Mellergaard S., (2005). Viral haemorrhagic septicaemia virus in marine fishand its implications for fish farming – a review, Journal of Fish Diseases, 28, 509–529pp.
  • Sümbüloğlu K., ve Sümbüloğlu V., (1997). Mann- Whitney U testi, Biyoistatistik, Hatiboğlu Yayınevi 7. Baskı, Ankara, 269 s.
  • Vestergard Jorgensen P. E., (1970). The survival of viral hemorrhagic septicemia (VHS) virus associated with trout eggs. Riv. Ital. Piscicol. Ittiopatol. 5:13-14
  • Way K., and Dixon P. E., (1988). Rapid detection of VHS and IHN viruses by the enzymelinked immunosorbent assay (ELISA). J. Appl. Ichthyol. 4:182- 189pp.
  • Wolf K., (1988). Viral Hemorrhagic Septicemia, Fish Viruses and Fish Viral Diseases. Cornell University Pres/Ithaca and Landon 217-249 pp.

The Development of Enzyme-Linked Immunosorbent Assay (ELISA) Method for Diagnosis of Viral Hemorrhagic Septicemia (VHS) Disease in Rainbow Trout (Oncorhynchus mykiss, Walbaum)

Yıl 2017, Cilt 2, Sayı 1, 7 - 10, 03.04.2017
https://doi.org/10.35229/jaes.288116

Öz

VHS can be diagnosed by the isolation of virus from infected fish in an appropriate cell line then identified by different serological

techniques such as ELISA, virus neutralization, immunofluorescence, immunoperoxidase and complement fixation tests. ELISA is over to the other serological

tests by the repeatability, simplicity, rapidity and cheapness.

In this research only one antibody which raised in rabbit was used for the detection of VHSV antigen. Furthermore, this antibody was labeled with

biotin and used as test conjugate. Estimated protein concentration of produced antibody (anti-VHSV F1 ɣ-globulin fraction) was 28 mg/ml. The microwells were

coated with 10 μg/ml anti-VHSV ɣ-globulin fraction. Then the suspected samples were reacted. If the samples were containing virus, biotinylated anti VHSV ɣglobulin

fraction binds to the antigen and biotinylated ɣ-globulin fraction react with HRP labeled avidin. The colour is developed and read by a reader fallowing

addition of TMB substrate. In the research, developed ELISA was detecting all known VHSV Subtypes (strain F1, HE, 23.75). Also this developed ELISA was

highly specifique to VHSV and was not giving the cross reaction with other fish viruses such as IHNV, PFR, PR and all known IPN serotypes. As a result,

sensitivity of that ELISA method is 104.75/0,1 ml DKID50 and method can detect 80 ng virus protein.

Kaynakça

  • Adams A., (1992). Techniques in Fish İmmunology: Sandwich Enzyme Linked Immunosorbent Assay (ELISA) to Detect and Quantify Bacterial Pathogens In Fish Tissue, SOS publications, 43 Denormandie Ave Fair Haven NJ.07704-3303 USA, 177- 182 p.
  • Anderson D.P. and Dixon O. W., (1989). Fish Biology Guide, U.S. Fish and Wildlife Service National Fish Health Research Laboratory, West Virginia, USA, 167p.
  • Anonim, (2006). Council Directive 2006/88/EC of 24 October 2006. Official Journal of the European Union L 328/14
  • Anonim, (2007). 3285 Sayılı Hayvan Sağlığı Ve Zabıtası Kanununun 4 Üncü Maddesine Göre Tespit Edilen İhbarı Mecburi Hastalıklar Hakkında Tebliğ (Tebliğ No: 2007/32). Resmî Gazete, Tarih:12 Temmuz 2007 Sayı: 26580.
  • Anonim, (2010). OIE Listed diseases: Updated: 04.01.2010, http://www.oie.int/en/animal-health-in-the-world/oie-listed-diseases-2017/. (25.01.20017)
  • Beard C.W., (1989). Serological procedure. 192-200. In: Purchase H.G., Lawrence, C., Arp, L.H., Domermuth, C.H., Pearson J.E. (Eds): A Laboratory Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Iowa, USA.
  • Cagirgan H., (1992). The Development of New ELISA Assays for Detecting Serum Antibody Levels Against VHS and IPN in Rainbow Trout (Oncorhynhus mykiss), MSc Thesis, University of Plymouth, 92 p (unpublished).
  • Enzmann P.J. and Konrad M., (1985). Inapparent infections of Brown trout with VHS-virus. Bull. Eur. Assoc. Fish Pathol. 5, 81–83 pp
  • Guesdon J.L., Ternynck, T. and Avrameas S. (1979). The use of avidin-biotin interaction in immunoenzymatic techniques, The Journal Of Histochemistry and Cytochemistry, 27 (8), 1131-1139pp.
  • Harlow E., Lane D., (1988). Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 101p
  • Hsu, S. and Raine, L., (1981). Protein A, Avidin, and Biotin in Immunohistochemistry, The Journal of Histochemistry and Cytochemistry, 29 (11), 1349-1353pp
  • Hudson L. and Hay F.C., (1991). Pratical Immunology, Blackwell Scientific Publication, Oxford Great Britain, Third Edition, 4-6pp
  • Jensen M. H., (1965). Research on the virus of Egtved disease. Ann. N.Y. Acad. Sci. 126, 422-426 pp.
  • Kalaycı, G., Inacoglu S. and Ozkan B., (2006). First isolation of viral haemorrhagic septisemia (VHS) virus from turbot (Scophthalmus maximus) cultured in the Trabzon coastal area of the Black Sea in Turkey . Bull. Eur. Ass. Fish Pathol., 26(4), 157p.
  • Medina J.M., Chang P.W., Bradley T.M., Yeh M.T. and Sadasiv E.C., (1992). Diagnosis of infectious hematopoietic necrosis virus in Atlantic salmon ,Salmo salar by enzyme-linked immunusorbent assay, Dis. of Aquat. Org., 13 147-150pp.
  • Mourton C., Bearzotti M., Piechaczyk M., Paolucci F., Pau B., Bastide J.M. and de Kinkelin P., (1990). Antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I, J Virol Methods. 29(3):325-333.
  • Mourton C., Romestand B., de Kinkelin P., Jeffroy J., Le Gouvello R. and Pau B., (1992). Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies, Journal of Clinical Microbiology, 30 (9): 2338-2345 pp.
  • Nishizawa T., Savas H., Isidan H., Ustundag C., Iwamoto H. and Yoshimizu M., (2006). Genotyping and Pathogenicity of Viral Hemorrhagic Septicemia Virus from Free-Living Turbot (Psetta maxima) in a Turkish Coastal Area of the Black Sea, Appl. Environ. Microbiol. Vol. 72, no. 4, pp. 2373-2378.
  • Olesen N.J. and Jǿrgensen P. E. V., (1991). Rapid detection of viral haemorrhagic septicaemia virüs in fish by ELISA, J. Appl. Ichthyol. 7 183-186.
  • Olesen N. J., Lorenzen N. and Jørgensen P. E. V., (1993). Serological differences among isolates of viral haemorrhagic septicaemia virus detected by neutralizing monoclonal and polyclonal antibodies, Diseases of Aquatic Organisms,16: 163-170.
  • Olesen N. J., (1998). Sanitation of viral haemorrhagic septicaemia (VHS). J Appl Ichthyol 14, 173–177pp.
  • Schäperclaus W., (1938). Die Schädigungen der deutschen Fischerei durch Fischparasiten und Fischkrankheiten. Allg. Fischztg. 41:256–259, 267–270 pp. in Wolf K., (1988). Viral Hemorrhagic Septicemia, Fish Viruses and Fish Viral Diseases. Cornell University Pres/Ithaca and Landon 217-249 pp.
  • Savigny D. and Voller A., (1980). The communication of ELISA data from laboratory to clinician, Journal of Immunoassay,1 (1) 105-128pp.
  • Skall H.F., Olesen N.J. and Mellergaard S., (2005). Viral haemorrhagic septicaemia virus in marine fishand its implications for fish farming – a review, Journal of Fish Diseases, 28, 509–529pp.
  • Sümbüloğlu K., ve Sümbüloğlu V., (1997). Mann- Whitney U testi, Biyoistatistik, Hatiboğlu Yayınevi 7. Baskı, Ankara, 269 s.
  • Vestergard Jorgensen P. E., (1970). The survival of viral hemorrhagic septicemia (VHS) virus associated with trout eggs. Riv. Ital. Piscicol. Ittiopatol. 5:13-14
  • Way K., and Dixon P. E., (1988). Rapid detection of VHS and IHN viruses by the enzymelinked immunosorbent assay (ELISA). J. Appl. Ichthyol. 4:182- 189pp.
  • Wolf K., (1988). Viral Hemorrhagic Septicemia, Fish Viruses and Fish Viral Diseases. Cornell University Pres/Ithaca and Landon 217-249 pp.

Ayrıntılar

Konular Fen
Bölüm Makaleler
Yazarlar

Uğur DEĞİRMENCİ>
EGE UNIV
Türkiye


Haşmet ÇAĞIRGAN Bu kişi benim
EGE UNIV
Türkiye

Yayımlanma Tarihi 3 Nisan 2017
Gönderilme Tarihi 13 Ocak 2017
Kabul Tarihi 13 Şubat 2017
Yayınlandığı Sayı Yıl 2017, Cilt 2, Sayı 1

Kaynak Göster

APA Değirmenci, U. & Çağırgan, H. (2017). The Development of Enzyme-Linked Immunosorbent Assay (ELISA) Method for Diagnosis of Viral Hemorrhagic Septicemia (VHS) Disease in Rainbow Trout (Oncorhynchus mykiss, Walbaum) . Journal of Anatolian Environmental and Animal Sciences , 2 (1) , 7-10 . DOI: 10.35229/jaes.288116


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