Clostridium perfringens Epsilon Toksoid İçeren Aşılarda MDCK Hücre Hatlarında Toksisite Analizi ile Potensin Belirlenmesi

Epsilon toksoid içeren klostridium aşılarının potens kontrolü Toksin Nötralizasyon Testi (TNT) ile yapılmaktadır. TNT standart bir metot olmakla birlikte hayvan refahı ve metot doğrulama çalışmaları açısından dezavantajları olan bir testtir. Bu nedenle alternatif metotların geliştirilmesi ve uygulanması teşvik edilmektedir. Ancak serolojik tabanlı in vitro metotlarda antijen, antikor ve referans standartların saflaştırılması ve standardizasyonu halen önemli bir sorun olarak durmaktadır. Bu bakımdan benzer reaktifler ile gerçekleştirilen hücre kültürü analizleri klostridial aşıların potens testlerinde olası alternatifler içinde görülmektedir. Bu yaklaşımla aşılanan tavşan serumlarında epsilon antitoksin düzeyi fare TNT ve Madin-Darby Canine Kidney (MDCK) hücre kültürü sitotoksisite analizi ile belirlenmiştir. Hücre kültüründeki toksisite yanıtı farelerdeki yanıta benzerdir. İki metodun sonuçları arasında yüksek bir korelasyon ve iyi bir lineer ilişki gözlenmiştir. Non-spesifik reaksiyon olmamıştır. Bu sonuçlar Clostridium perfringens epsilon toksoid içeren aşıların potens testlerinde, in vivo fare TNT yerine, in vitro MDCK hücre hattı seronötralizasyon analizinin başarıyla kullanılabileceğini göstermektedir.

Anahtar Kelimeler:

Klostrial aşı, Epsilon toksin, MDCK, Potens, TNT

Determining the Potency of Vaccines Containing Clostridium perfringens Epsilon Toxoid via Toxicity Analysis in MDCK Cell Lines

The potency control of the clostridium vaccines containing epsilon toxoid is performed with the Toxin Neutralization Test (TNT). Although TNT is a standard method, it has some disadvantages in terms of animal welfare and method validation studies. For this reason, the development and application of alternative methods are encouraged. However, purification and standardization of antigen, antibody and reference standards in serological-based in vitro methods still remain a significant problem. In this respect, cell culture analyses performed using similar reagents are considered possible alternatives in potency tests of the clostridial vaccines. Based on this approach, epsilon antitoxin levels in the sera of vaccinated rabbits were determined by cytotoxicity assay in mouse TNT and Madin-Darby Canine Kidney (MDCK) cell cultures. The toxicity response in cell culture is similar to the response in mice. A high correlation and a good linear relationship were observed in the results of the two methods. There were no non-specific reactions. These results show that, in potency tests of vaccines containing Clostridium perfringens epsilon toxoid, in vitro MDCK cell line seroneutralization assay can be successfully used instead of in vivo mouse TNT.

Keywords:

Clostridial vaccine, Epsilon toxin, MDCK, Potency, TNT,

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