Ahmet Kürşat AZKUR, Emel AKSOY, Murat YILDIRIM, Kader YILDIZ

Optimisation of Indirect ELISA by Comparison of Different Antigen Preparations for Detection of Antibodies Against Schmallenberg Virus

Schmallenberg virus (SBV) infection, discovered in 2011, was reported in Europe including Turkey, Africa and recently in some Asian countries. Commercial enzyme-linked immunosorbent assay (ELISA) kits were widely used by researchers in many epidemiological studies and SBV diagnosis. The aim of this study was to optimise indirect in-house ELISA that is based on different antigen preparations of cell-culture derived whole SBV particle. Antigen preparations were maintained with various methods: PEG precipitation, ultracentrifugation, dialysis, and antigen inactivation. Following antigen optimisation, steps of antigen coating, blocking, conjugate and stop solution were optimised and in-house ELISA was compared to commercial indirect SBV ELISA kit. The best result in ELISA antigen preparation for SBV was gained by 30% PEG purification method followed by formaldehyde inactivation. Although results of this study demonstrated that in-house ELISA for detection of SBV specific antibodies was equally sensitive and specific as commercial kit, purified SBV antigen based in-house ELISA development could increase S/P ratios.

Schmallenberg Virüs Antikorlarının Belirlenmesi İçin İndirekt ELISA’nın Farklı Antijenler Karşılaştırılarak Optimizasyonu

Schmallenberg virüs (SBV) enfeksiyonu 2011 yılında keşfedilmiş ve enfeksiyon Türkiye dahil Avrupa kıtasında, Afrika’da ve bazı Asya ülkelerinde bildirilmiştir. Ticari ELISA kitleri çok sayıda epidemiyolojik çalışmada ve SBV tanısında sıklıkla kullanılmaktadır. Bu çalışmanın amacı, hücre kültüründen elde edilen tam SBV partikülünün farklı antijen hazırlama yöntemleri kullanılarak indirekt in-house ELISA optimizasyonudur. Antijen hazırlamak için; PEG presipitasyon, ultrasantrifüjleme, diyaliz ve antijen inaktivasyonu gibi farklı yöntemler kullanıldı. Antijen optimizasyonu sonrasında, antijen kaplama, bloklama, konjugat ve durdurma solüsyonları optimize edildi ve geliştirilen in-house ELISA ticari ELISA kiti ile kıyaslandı. ELISA antijen hazırlanmasında en iyi sonuçlar %30 PEG presipitasyon sonrasında formaldehit ile inaktivasyon sonucunda elde edildi. SBV özgül antikorların belirlenmesinde in-house ELISA ticari kit kadar duyarlı ve özgül olsa da saf SBV antijeni temelli in-house ELISA geliştirilmesinin S/P oranını yükseltebileceği düşünüldü.

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