B. GÜVEN, S. ÖZSAR, E. SABAN, S. ÖZDEMİR

Progesteron için enzimimmunoassay (EIA) tekniğinin geliştirilmesi

Bu çalışmada total sütte progesteron tayini için çift antikorlu mikrotitrasyon plak EIA yöntemi geliştirildi. 6 $\beta$ -OH-progesteron-hemisuccinate, HRP enzimi ile işaretlendi ve sephadex G-25 kolon kromatografisinden geçirilerek saflaştırıldı. Tavşanlarda progesteron-7 $\infty$ -CTE-BTG'ye karşı üretilmiş olan antikor testte kullanıldı. Keçilerde tavşan IgG'sine karşı ikinci antikor üretildi ve antiserumdan IgG fraksiyonu affinite kromotografisinden geçirilerek saflaştırıldı. Testin hassasiyetinin 2 pg/kuyu, deney içi ve deneyler arası varyasyon katsayılarının %14'den düşük olduğu bulundu. Östrus siklusundaki bir inekten toplanan süt örnekleri RIA ve EIA ile analiz edildiğinde iki teknik arasındaki korelasyon %96 olarak tesbit edildi. Spektrofotometrik olarak ölçülebilen sonuçlar göz ile de değerlendirilebilmekte olup 80 örneğin analizi 4 saate tamamlanabilmektedir.

Anahtar Kelimeler:

immunoenzim teknikleri, sığır, süt, progesteron

Development of enzymeimmunoassay (EIA) for progesterone

In this study a double antibody microtitration plate EIA was developed for the determination of progesterone in whole milk. 6 $\beta$ -OH-Progesterone-hemisuccinate was labelled with HRP and purified through the column chrom- atography (sephadex G-25). Antiserum produced against progesterone-7 $\infty$ -CTE-BTG in rabbits was used. A sec¬ond antibody against rabbit IgG was produced in goats and IgG fraction of antiserum was isolated through affinity chromatography. The sensitivity of the assay was 2 pg/well. The intra and inter assay coefficients of variation were <14%. Milk samples collected from a cow in estrus cycle were analysed using both EIA and RIA, the correlation between two techniques was obtained as 96%. The resulting color is measured by using spectrofotometer as well as by naked eye and it is possible to analyse 80 samples within 4 hours.

Keywords:

immunoenzyme techniques, cattle, milk, progesterone,

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