Meryem IŞIK, Zülal BİLİCİ, Naci ÇİNE, Selma ÖZTÜRK

Antikor Bazlı BoNT Tespit Sistemi için Peptit Antijenlerin Kullanımı

Amaç: Bu çalışmada, doğal yapılı BoNT A toksininin kendisi yerine biyoinformatik araçlar kullanılarak tasarlanan peptitleri kullanarak BoNT'ye özgü bir bağışıklık tepkisi ortaya çıkarmayı ve bu anti-peptit antikorlarının bir toksin tespit sisteminde kullanımını göstermeyi amaçladık. Yöntem: BoNT A'nın LC, HN ve HC bölgelerine özgü sentetik epitopik bölgeler, IEDB'den B hücresi "Epitop Tahmin Araçları" kullanılarak antijen olarak seçildi ve bir yazılımla (Discovery Studio 4.0) BoNT A yüzeyinde olduğu gösterildi. Seçilen peptidler, fare bağışıklamalarında kullanıldı ve geliştirilen anti-peptid antikorları ile doğal yapılı BoNT A arasındaki ilişki incelendi. Bulgular: Farelerde üç farklı peptide (P1, P2 ve P3) karşı geliştirilen anti-peptid antikorları ile mililitrede pikogram seviyelerinde doğal yapılı BoNT A'nın saptanması gerçekleştirildi. Sonuç: Bu çalışma, sentetik peptitlerin, toksinlere karşı yüksek afiniteli antikorları geliştirmek için en az doğal toksin veya toksoidin kendisi kadar etkili olduğunu göstermektedir. Ayrıca botulizmin hızlı teşhisine duyulan ihtiyaç ve halihazırda kullanılan test sistemlerinde çok sayıda deney hayvanının kurban edildiği göz önüne alındığında, bu sonuçlar hem hayvan sayısını hem de toksin kullanım miktarını azaltmak için sentetik peptit immünojenlerinin kullanılmasının gerekliliğini ortaya koymaktadır.

Anahtar Kelimeler:

Botulinum Nörotoksin A, Toksin Tespiti, Sentetik Peptitler, Botulizm, antikor üretimi

Usage of Peptide Antigens for Antibody-Based BoNT Detection System

Objective: In the current study, we aimed to elicit a BoNT-specific immune response via using peptides designed by using bioinformatics tools instead of using intact native BoNT A toxin itself and to demonstrate the usage of these anti-peptide antibodies in a toxin detection system. Methods: Synthetic epitopic regions specific to LC, HN and HC regions of BoNT A were selected as antigen using B cell “Epitope Prediction Tools” form IEDB and shown to be on the surface of BoNT A with a software (Discovery Studio 4.0). Selected peptides were used in mice immunizations and the interaction between developed anti-peptide antibodies and the native intact BoNT A was examined. Results: The detection of native intact BoNT A at picogram levels per milliliter was performed with anti-peptide antibodies developed against three different peptides (P1, P2, and P3) in mice. Conclusion: The current study shows that synthetic peptides are at least as effective as the native toxin or the toxoid itself for raising high-affinity antibodies against toxins. In addition, considering the need for a quick diagnosis of botulism and, already used test systems in which many experimental animals are sacrificed, these results demonstrate the necessity of synthetic peptide immunogens usage to reduce both the number of animals and the amount of toxin usage.

Keywords:

Botulinum Neurotoxin A, Toxin Detection, Synthetic Peptides, Antibody Generation, Botulism,

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