Can ÇOKÇALIŞKAN, Ünal PARLAK, Beyhan SAREYYÜPOĞLU, Abdülnaci BULUT, Ayca BABAK, Fuat ÖZYÖRÜK

Şap Hastalığı Teşhisi için Enzim Immunotransfer Blot Testinin kantitatif bir yaklaşım ile iyileştirilmesi

Şap hastalığı büyük ekonomik kayıplara neden olan çok bulaşıcı bir hayvan hastalığıdır. Dünya Hayvan Sağlığı Örgütü Şap Hastalığı koduna göre canlı hayvanların ve bunların ürünlerinin ticareti, ancak, ihraç eden ülkenin hastalıktan ari olması şartıyla mümkündür. Hastalıktan ariliğin gösterilmesi için en önemli yöntemlerden biri örneklenen hedef popülasyonun Şap virüsü yapısal olmayan proteinlerine (YOP) karşı oluşan antikor seviyelerinin ölçümü temeline dayanır. Bir sürünün hastalık durumunun belirlenmesinde yaygın tarama ve Enzim bağlı Immunosorbent test (ELISA) ve Enzim bağlı Immunotransfer testi (EITB) geliştirilmiş ve WOAH (Dünya Hayvan Sağlığı Örgütü) teşhis el kitabında tanımlanmıştır. Bu çalışmada Şap virüsünün yapısal olmayan proteinleri rekombinant olarak üretilmiş, enfekte olmayan, aşılı ve doğal olarak enfekte olan hayvanlardan toplanan serumlardan oluşan panellerle, sayısal belgelemeye ve istatistik analize olanak veren, iyileştirilmiş, kantitatif bir Western blot kullanılarak test edilmiştir. YOP bant yoğunlukları, hasta ve enfekte hayvanların birbirinden ayırt edecek eşik değerlerin ve farklı YOP’lerin bu teşhiste farklı hassasiyet gösterdiğinin belirlenmesinde kullanılmıştır. Kantitatif EITB sonuçları aynı zamanda YOP-ELISA sonuçları ile korelasyon göstermiştir.

Anahtar Kelimeler:

Enzim-Bağlı İmmunoelekrotransfer Blot Testi (EITB), Şap Virüsü, Yapısal Olmayan Proteinler (YOP), YOP-ELISA, Kantitatif Western blot.

Improvement of Enzyme-Linked Immunoelectrotransfer Blot Assay by Quantitative Approach for Foot-and-Mouth Disease diagnosis

Foot-and-mouth disease (FMD) is a highly contagious animal disease that causes devastating economic losses. The trade of live animals and derived products is only possible if the exporting country is free from disease, according to the World Organization for Animal Health (WOAH) code for FMD. One of the most important ways to prove disease-free is to measure the levels of non-structural protein antibodies (NPS) of FMD virus in the target population sampled. For detection of the disease status of a herd, mass screening and assays such as Enzyme-linked immunosorbent assay (ELISA) and Enzyme-linked immunoelectrotransfer blot assay (EITB) were developed and described in the WOAH diagnostic manual. In this study, recombinant FMDV NS proteins were produced and tested with sera panels collected from uninfected and naturally infected animals using a quantitative Western blot assay as an improved EITB, which enables numerical documentation and statistical analysis. NSP band intensities were used to determine the cut-off values, differentiating infected from non-infected animals and revealing variable sensitivity among the different NSPs. The quantitative EITB results also showed a correlation with the NSP-ELISA results.

Keywords:

Enzyme-Linked Immunoelectrotransfer Blot Assay (EITB), Foot-and-Mouth Disease Virus (FMDV), Non-Structural Proteins (NSP), NSP-ELISA, Quantitative Western blot (QCWB).,

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