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

Yıl 2017, , 7 - 10, 03.04.2017

Uğur Değirmenci , Haşmet Çağırgan

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.

Anahtar Kelimeler

ELISA, biotin-avitin, alabalık, VHSV

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

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.

Anahtar Kelimeler

ELISA, biotin-avidin, VHSV, Oncorhynchus mykiss

Kaynakça

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