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. 2011 Jan;75(1):69-72.

Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method

Hongsheng Huang  1 , Andrei Soutyrine, Jasmine Rendulich, Katherine O'Rourke, Aru Balachandran
Affiliations

Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method

Hongsheng Huang et al. Can J Vet Res. 2011 Jan.

Abstract
in English, French

Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

Des tissus non-convenables sont souvent soumis pour être éprouvés par examens immuno-histochimique et histopathologique pour le diagnostic de la maladie débilitante chronique chez les cervidés (CWD) et la tremblante chez les moutons. Dans la présente étude nous avons examiné les effets d’une autolyse expérimentale sur la détection de protéine prion anormale (PrPsc) dans les tissus des systèmes lymphoïde et nerveux (CNS) de wapitis et de moutons. La PrPsc a été détectée par une épreuve d’immunobuvardage (WB) suite à un enrichissement de la PrPsc par précipitation à l’acide phosphotungstique sodique (PTA-WB). Une épreuve immuno-enzymatique commerciale (ELISA) a été utilisée comme épreuve de référence pour la mesure quantitative. L’étude a démontré que la quantité de PrPsc dans les tissus lymphoïde et du CNS des wapitis et des moutons a diminué graduellement à cause de l’autolyse, mais la PrPsc était toujours détectable par PTA-WB après 5 et 15 j d’incubation à 37 °C à partir de tous les échantillons de tissus lymphoïde et de CNS. Les résultats de l’ELISA confirment ceux du PTA-WB, particulièrement pour les échantillons du CNS. En conclusion, l’autolyse à 37 °C pendant 15 j n’affecterait pas de manière significative la détection par WB et ELISA de PrPsc dans les tissus lymphoïde et du CNS, et l’épreuve PTA-WB est une épreuve de confirmation utile et alternative pour détecter PrPsc dans des échantillons de tissus autolysés provenant du système lymphoïde et du CNS.

(Traduit par Docteur Serge Messier)

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Figures

Figure 1
Figure 1
Effects of experimental autolysis (using scrapie positive specimens as examples) on abnormal prion protein (PrPsc) of central nervous system (CNS) tissues. The results showed in this image were obtained using the same sample from the sheep indicated as sheep 1b in Table I. Lanes 1 to 3, day 0 (PTA enriched, crude extract, and PK-, respectively); lanes 4 to 6, day 5 (PTA enriched, crude extract, and PK-, respectively), and lanes 7 to 9, day 15 (PTA enriched, crude extract, and PK-, respectively), showing slight decreasing of PrPsc band intensity in crude extract (lanes 2, 5, and 8).
Figure 2
Figure 2
Effects of experimental autolysis (using scrapie positive specimens as examples) on abnormal prion protein (PrPsc) of lymphoid tissues. The result shown in this image was obtained using the same sample from the sheep indicated as sheep 1 in Table I: Lanes 1 to 3, day 0 (PTA enriched, crude extract, and PK-, respectively); lanes 4 to 6, day 5 (PTA enriched, crude extract, and PK-, respectively), and lanes 7 to 9, day 15 (PTA enriched, crude extract, and PK-, respectively), showing significant decreasing of PrPsc band intensity in PTA enriched (lanes 1, 4, and 7).
See this image and copyright information in PMC

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