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. 2000 Sep;38(9):3254-9.
doi: 10.1128/JCM.38.9.3254-3259.2000.

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue

K I O'Rourke  1 , T V Baszler, T E Besser, J M Miller, R C Cutlip, G A Wells, S J Ryder, S M Parish, A N Hamir, N E Cockett, A Jenny, D P Knowles
Affiliations

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue

K I O'Rourke et al. J Clin Microbiol. 2000 Sep.

Abstract

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97. 6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.

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Figures

FIG. 1
FIG. 1
Immunohistochemical detection of PrP-Sc in third eyelid lymphoid tissue with a cocktail of MAbs F89/160.1.5 and F99/97.6.1. (a) Third eyelid biopsy specimen from a sheep with preclinical scrapie. Multiple lymphoid follicles (arrowheads) have punctate PrP-Sc immunoreactivity (red). Bar, 250 μm. (b) Third eyelid of a sheep with preclinical scrapie. The lymphoid follicle (F) within the germinal center (red) has multifocal, coarse, granular PrP-Sc immunoreactivity. The top is the conjunctival surface. Bar, 30 μm. (c) Third eyelid of an uninfected sheep. No PrP-Sc immunoreactivity was observed in the lymphoid follicle (F). The top is the conjunctival surface. Bar, 30 μm.
FIG. 1
FIG. 1
Immunohistochemical detection of PrP-Sc in third eyelid lymphoid tissue with a cocktail of MAbs F89/160.1.5 and F99/97.6.1. (a) Third eyelid biopsy specimen from a sheep with preclinical scrapie. Multiple lymphoid follicles (arrowheads) have punctate PrP-Sc immunoreactivity (red). Bar, 250 μm. (b) Third eyelid of a sheep with preclinical scrapie. The lymphoid follicle (F) within the germinal center (red) has multifocal, coarse, granular PrP-Sc immunoreactivity. The top is the conjunctival surface. Bar, 30 μm. (c) Third eyelid of an uninfected sheep. No PrP-Sc immunoreactivity was observed in the lymphoid follicle (F). The top is the conjunctival surface. Bar, 30 μm.
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