Bovine rotavirus enfeksiyonlarının çabuk teşhisinde kullanılmak üzere ELISA ve dot-ELISA sistemlerinin geliştirilmesi
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Dosyalar
Tarih
2010
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
En önemli viral gastroenterit etkenlerinden BRV, sığırlarda ekonomik kayıplara neden olan dünya üzerinde yaygın bir viral etkendir bu nedenle yenidoğan buzağı yaşamının ilk gününden itibaren hızlı test tekniklerinin uygulanması önemlidir. Bu araştırmada diyare semptomlu buzağı dışkı örneklerinde BRV antijenlerinin tespiti amacıyla monoklonal ve poliklonal antikor ile duyarlı ELISA ve Dot-ELISA geliştirilmesi hedeflenmiştir. PEG ile konsantre edilen BRV izolatı ELISA sistemlerinde pozitif kontrol antijeni olarak kullanılmıştır. Amonyum sülfat presipitasyonu ve dialize edilen tavşan antiserumu (anti-BRV poliklonal antikoru) ve ticari olarak sağlanan anti- VP6 monoklonal antikoru, ELISA sistemlerinde 100 adet dışkı örneğinin BRV antijen varlığı yönünden test edilmesi aşamasında capture antikor olarak kullanılmıştır. ELISA ve Dot-ELISA sonuçları ticari ELISA sonuçları ile sensitivite ve spesifite bakımından karşılaştırılmıştır. Elde edilen sonuçlara göre; monoklonal ve poliklonal antikor ile duyarlı ELISA ile %92 ve %96 sensitivite; monoklonal ve poliklonal antikor ile duyarlı ELISA ile %96 ve %99 spesifisite elde edilmiştir. Poliklonal antikor ile duyarlı ELISA sensitivite ve spesifisitesinin monoklonal antikor ile duyarlı ELISA sensitivite ve spesifisitesinden daha yüksek olduğu belirlenmiştir. Poliklonal antikor ile duyarlı ELISA pozitif (%96) ve negatif (%99) prediktif değerlerinin, monoklonal antikor ile duyarlı ELISA pozitif (%88) ve negatif (% 97) prediktif değerlerine göre daha yüksek olduğu belirlenmiştir. Monoklonal ve poliklonal antikor ile duyarlı Dot-ELISA ile %50 ve %46 sensitivite; monoklonal ve poliklonal antikor ile duyarlı ELISA ile %73 ve %87 spesifisite elde edilmiştir. Poliklonal antikor ile duyarlı Dot-ELISA pozitif (%52) ve negatif (%83) prediktif değerlerinin, monoklonal antikor ile elde edilen pozitif (%37) ve negatif (%82) prediktif değerlere göre daha yüksek olduğu belirlenmiştir. Dot-ELISA sensitivite, spesifisite, pozitif ve negatif prediktif değerlerinin ELISA ile elde edilen değerlere göre daha düşük olduğu belirlenmiştir. Sonuç olarak, BRV teşhisi amacıyla geliştirilen monoklonal ve poliklonal antikor ile duyarlı ELISA tekniğinin etkili, basit, ekonomik, hızlı, %95 ve %98 oranında doğruluk oranına sahip bir test tekniği olduğu belirlenmiştir. Araştırma, diyare semptomlu buzağılarda BRV enfeksiyon tespiti amacıyla ELISA ve Dot-ELISA sistemlerinin geliştirilmesine dair Türkiye'deki ilk çalışmadır.
BRV is the most important cause of viral gastroenteritis because of significant economic losses in cattle worldwide so the rapid test techniques should be delivered as early as the first few days of newborn life. The main goal of this research was to develop polyclonal and monoclonal antibody based antigen capture ELISA and Dot-ELISA for detecting BRV antigens in stool sample of diarrhoeatic calves. PEG concentrated BRV isolate was used as positive control antigen in ELISA systems. The ammonium sulphate precipatiated and dialysed rabbit antiserum (anti-BRV polyclonal antibody) and commercially available anti-VP6 monoclonal antibody was used as capture antibody in ELISA systems for detection of BRV antigen in 100 stool samples. The results were also screened by a commercial ELISA to determine the sensitivity and specificity of antigen capture ELISA and Dot-ELISA. It was evaluated that monoclonal and polyclonal antibody based antigen capture ELISA presented 92% and 96% sensitivity; monoclonal and polyclonal antibody based ELISA presented 96% and 99% specificity, respectively. The polyclonal antibody based antigen capture ELISA sensitivity and specificity were slightly superior to monoclonal antibody based antigen capture ELISA. The positive predictive (96%) and negative predictive (99%) values of the polyclonal antibody based antigen capture ELISA were slightly higher than the positive (88%) and negative predictive (97%) values of the monoclonal antibody based antigen capture ELISA. Monoclonal and polyclonal antibody based Dot-ELISA presented 50% and 46% sensitivity, respectively; monoclonal and polyclonal antibody based Dot-ELISA presented 73% and 87% specificity, respectively. The positive (52%) and negative predictive (83%) values of the polyclonal antibody based Dot-ELISA were slightly higher than the positive (37%) and negative predictive (82%) values of the monoclonal antibody based Dot-ELISA. The sensitivity, specificity, positive and negative predictive values of the Dot-ELISA was slightly lower than the antigen capture ELISA. As a result, it is concluded that an effective, simple, economic and rapid monoclonal and polyclonal antibody based ELISA technique was developed for diagnosis of BRV with a 95% and 98% accuracy rates in any research laboratory. This is the first report of developed ELISA and Dot-ELISA systems to detect of BRV infection in diarrhetic calves in Turkey.
BRV is the most important cause of viral gastroenteritis because of significant economic losses in cattle worldwide so the rapid test techniques should be delivered as early as the first few days of newborn life. The main goal of this research was to develop polyclonal and monoclonal antibody based antigen capture ELISA and Dot-ELISA for detecting BRV antigens in stool sample of diarrhoeatic calves. PEG concentrated BRV isolate was used as positive control antigen in ELISA systems. The ammonium sulphate precipatiated and dialysed rabbit antiserum (anti-BRV polyclonal antibody) and commercially available anti-VP6 monoclonal antibody was used as capture antibody in ELISA systems for detection of BRV antigen in 100 stool samples. The results were also screened by a commercial ELISA to determine the sensitivity and specificity of antigen capture ELISA and Dot-ELISA. It was evaluated that monoclonal and polyclonal antibody based antigen capture ELISA presented 92% and 96% sensitivity; monoclonal and polyclonal antibody based ELISA presented 96% and 99% specificity, respectively. The polyclonal antibody based antigen capture ELISA sensitivity and specificity were slightly superior to monoclonal antibody based antigen capture ELISA. The positive predictive (96%) and negative predictive (99%) values of the polyclonal antibody based antigen capture ELISA were slightly higher than the positive (88%) and negative predictive (97%) values of the monoclonal antibody based antigen capture ELISA. Monoclonal and polyclonal antibody based Dot-ELISA presented 50% and 46% sensitivity, respectively; monoclonal and polyclonal antibody based Dot-ELISA presented 73% and 87% specificity, respectively. The positive (52%) and negative predictive (83%) values of the polyclonal antibody based Dot-ELISA were slightly higher than the positive (37%) and negative predictive (82%) values of the monoclonal antibody based Dot-ELISA. The sensitivity, specificity, positive and negative predictive values of the Dot-ELISA was slightly lower than the antigen capture ELISA. As a result, it is concluded that an effective, simple, economic and rapid monoclonal and polyclonal antibody based ELISA technique was developed for diagnosis of BRV with a 95% and 98% accuracy rates in any research laboratory. This is the first report of developed ELISA and Dot-ELISA systems to detect of BRV infection in diarrhetic calves in Turkey.
Açıklama
Anahtar Kelimeler
Polietilen glikoller, Polyethylene glycols, Poliklonal antikor, Polyclonal antibody
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Yanbkan,S. (2010). Development of ELISA and Dot-ELISA systems for the rapid diagnosis of bovine rotavirus infections. Selçuk Üniversitesi, Yayımlanmış doktora tezi, Konya.
