Anti-prion Antibodies Research Articles

Distribution of the misfolded isoform of the prion protein in peripheral tissues and spinal cord of Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring chronic wasting disease.

Chronic wasting disease (CWD) is an infectious transmissible spongiform encephalopathy of cervids associated with the presence of a misfolded prion protein (PrPCWD). Progression of PrPCWD distribution has been described using immunohistochemistry and histologic changes in a single section of brain stem at the level of the obex resulting in scores from 0 (early) to 10 (terminal) in elk with naturally occurring CWD. Here we describe the spread and distribution of PrPCWD in peripheral tissues and spinal cord in 16 wild and 17 farmed Rocky Mountain elk (Cervus elaphus nelsoni) with naturally occurring CWD and correlate these findings with obex scores. Spinal cord and approximately 110 peripheral tissues were collected, processed, stained with hematoxylin and eosin, and immunolabeled with the anti-prion protein monoclonal antibody F99/97.6.1. The medial retropharyngeal and tracheobronchial lymph nodes were the first tissues to accumulate PrPCWD, followed by other lymphoid tissues, myenteric plexus, spinal cord, and finally tissues outside of the lymphatic and neural systems. However, the only significant histological lesion observed was mild spongiform encephalopathy in the dorsal column of the lower spinal cord in elk with an obex score of ≥9. Initial exposure to CWD prions may be through the respiratory system and spread appears to occur primarily via the autonomic nervous system. Therefore, we suggest using obex scores as a proxy for stage of disease progression and verifying with key peripheral tissues.

Therapeutic anti-amyloid β antibodies cause neuronal disturbances.

Recent published clinical trial safety data showed that 41% of Alzheimer patients experienced amyloid-related imaging abnormalities (ARIA), marks of microhemorrhages and edema in the brain, following administration of Biogen's Aduhelm/aducanumab (amino acids 3-7 of the Aβ peptide). Similarly, Janssen/Pfizer's Bapineuzumab (amino acids 1-5 of the Aβ peptide) and Roche's Gantenerumab (amino acids 2-11/18-27 of the Aβ peptide) also displayed ARIA in clinical trials, including microhemorrhage and focal areas of inflammation or vasogenic edema, respectively. The molecular mechanisms underlying ARIA caused by therapeutic anti-Aβ antibodies remain largely unknown, however, recent reports demonstrated that therapeutic anti-prion antibodies activate neuronal allergenic proteomes following cross-linking cellular prion protein. Here, we report that treatment of human induced pluripotent stem cells- derived neurons (HSCN) from a non-demented donor, co-cultured with human primary microglia with anti-Aβ1-6, or anti-Aβ17-23 antibodies activate a significant number of allergenic-related proteins as assessed by mass spectrometry. Interestingly, a large proportion of the identified proteins included cytokines such as interleukin (IL)-4, IL-12, and IL-13 suggesting a type-1 hypersensitivity response. Following flow cytometry analysis, several proinflammatory cytokines were significantly elevated following anti-Aβ1-6, or anti-Aβ17-23 antibody treatment. These results justify further and more robust investigation of the molecular mechanisms of ARIA during immunotherapy study trials of AD. Allergenic-related proteins are often linked with Alzheimer's disease (AD). We investigated the effects of amyloid beta (Aβ) immunotherapy on stem cell derived neurons and primary neuronal cells co-cultured with microglia. Anti-Aβ antibody treatment of neurons or neurons co-cultured with microglia led to activation of a substantial number of allergenic-related genes. These allergenic-related genes are associated with endothelial dysfunction possibly responsible for ARIA.

A high-content neuron imaging assay demonstrates inhibition of prion disease-associated neurotoxicity by an anti-prion protein antibody

There is an urgent need to develop disease-modifying therapies to treat neurodegenerative diseases which pose increasing challenges to global healthcare systems. Prion diseases, although rare, provide a paradigm to study neurodegenerative dementias as similar disease mechanisms involving propagation and spread of multichain assemblies of misfolded protein (“prion-like” mechanisms) are increasingly recognised in the commoner conditions such as Alzheimer’s disease. However, studies of prion disease pathogenesis in mouse models showed that prion propagation and neurotoxicity can be mechanistically uncoupled and in vitro assays confirmed that highly purified prions are indeed not directly neurotoxic. To aid development of prion disease therapeutics we have therefore developed a cell-based assay for the specific neurotoxicity seen in prion diseases rather than to simply assess inhibition of prion propagation. We applied this assay to examine an anti-prion protein mouse monoclonal antibody (ICSM18) known to potently cure prion-infected cells and to delay onset of prion disease in prion-infected mice. We demonstrate that whilst ICSM18 itself lacks inherent neurotoxicity in this assay, it potently blocks prion disease-associated neurotoxicity.

The Cellular Prion Protein: A Promising Therapeutic Target for Cancer.

Studies on the cellular prion protein (PrPC) have been actively conducted because misfolded PrPC is known to cause transmissible spongiform encephalopathies or prion disease. PrPC is a glycophosphatidylinositol-anchored cell surface glycoprotein that has been reported to affect several cellular functions such as stress protection, cellular differentiation, mitochondrial homeostasis, circadian rhythm, myelin homeostasis, and immune modulation. Recently, it has also been reported that PrPC mediates tumor progression by enhancing the proliferation, metastasis, and drug resistance of cancer cells. In addition, PrPC regulates cancer stem cell properties by interacting with cancer stem cell marker proteins. In this review, we summarize how PrPC promotes tumor progression in terms of proliferation, metastasis, drug resistance, and cancer stem cell properties. In addition, we discuss strategies to treat tumors by modulating the function and expression of PrPC via the regulation of HSPA1L/HIF-1α expression and using an anti-prion antibody.

Recent developments in antibody therapeutics against prion disease.

Preclinical evidence indicates that prion diseases can respond favorably to passive immunotherapy. However, certain antibodies to the cellular prion protein PrPC can be toxic. Comprehensive studies of structure-function relationships have revealed that the flexible amino-terminal tail of PrPC is instrumental for mediating prion toxicity. In a first-in-human study, an anti-prion antibody has been recently administered to patients diagnosed with sporadic Creutzfeldt-Jakob's disease, the most prevalent human prion disease. Moreover, large-scale serosurveys have mapped the prevalence of naturally occurring human anti-prion autoantibodies in health and disease. Here, we provide a perspective on the limitations and opportunities of therapeutic anti-prion antibodies.

Intermolecular crosslinking of abnormal prion protein is efficiently induced by a primuline-sensitized photoreaction

In prion diseases, infectious pathogenic particles that are composed of abnormal prion proteins (PrPSc) accumulate in the brain. PrPSc is biochemically characterized by its protease-resistance core (PrPres), but its structural features have not been fully elucidated. Here, we report that primuline, a fluorescent dye with photosensitization activity, dramatically enhances UV-irradiation-induced SDS-resistant PrPSc/res oligomer formation that can be detected by immunoblot analysis of prion-infected materials. This oligomer formation occurs specifically with PrPSc/res but not with normal prion protein, and it was demonstrated using purified PrPSc/res as well as unpurified materials. The oligomer formation proceeded in both primuline-dose- and UV irradiation time-dependent manners. Treatment with urea or formic acid did not break oligomers into monomers. Neither did the presence of aromatic amino acids modify oligomer formation. Analysis with a panel of anti-prion protein antibodies showed that the antibodies against the N-terminal region of PrPres were less reactive in the dimer than the monomer. These findings suggest that the primuline-sensitized photoreaction enhances intermolecular crosslinking of PrPSc/res molecules at a hydrophobic area of the N-terminal region of PrPres. In the screening of other compounds, photoreactive compounds such as luciferin exhibited a similar but lower activity with respect to oligomer formation than primuline. The enhanced photoreaction with these compounds will be useful for evaluating the structural features of PrPSc/res, especially the interactions between PrPSc/res molecules.

Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

Enhanced detection of infectious prions by direct ELISA from the brains of asymptomatic animals using DRM2-118 monoclonal antibody and Gdn-HCl

In this report we describe the use of a novel anti-prion monoclonal antibody (DRM2-118) for the direct detection of infectious prions by ELISA. Epitope mapping using overlapping hamster (SHa) prion peptides indicates DRM2-118 binding occurs between residues 93-100 and at the 310-helix (residues 163-170) between alpha helix-A and -B. This antibody shows broad species binding to endogenous prions from brain homogenates and corresponding recombinant prion proteins. To evaluate the performance of this MAb for the detection of prion proteins we performed an animal time course and evaluated prion detection from both crude brain homogenates and lipid raft fractions (DRM) by direct ELISA. Prion detection was significantly enhanced by the addition of the chaotropic guanidine-HCl (Gdn-HCl) during protein immobilization with detection of PK-resistant prion from asymptomatic animal brains at (45-DPI) and from lipid rafts at (24-DPI). Our data demonstrates enhanced prion detection from brain lipid rafts of asymptomatic animals by a simple direct ELISA using the DRM2-118 MAb combined with Gdn-HCl.

Coexistence of two forms of disease-associated prion protein in extracerebral tissues of cattle infected with H-type bovine spongiform encephalopathy.

H-type bovine spongiform encephalopathy (H-BSE) is an atypical form of BSE in aged cattle. H-BSE is characterized by the presence of two proteinase K-resistant forms of disease-associated prion protein (PrPSc), identified as PrPSc #1 and PrPSc #2, in the brain. To investigate the coexistence of different PrPSc forms in the extracerebral tissues of cattle experimentally infected with H-BSE, immunohistochemical and molecular analyses were performed by using N-terminal-, core-region- and C-terminal-specific anti-prion protein antibodies. Our results demonstrated that two distinct forms of PrPSc coexisted in the various extracerebral tissues.

Exploring the cause of initially reactive bovine brains on rapid tests for BSE

ABSTRACTBovine spongiform encephalopathy (BSE) is an invariably fatal prion disease of cattle. The identification of the zoonotic potential of BSE prompted safety officials to initiate surveillance testing for this disease. In Canada, BSE surveillance is primarily focused on high risk cattle including animals which are dead, down and unable to rise, diseased or distressed. This targeted surveillance results in the submission of brain samples with a wide range of tissue autolysis and associated contaminants. These contaminants have the potential to interfere with important steps of surveillance tests resulting in initially positive test results requiring additional testing to confirm the disease status of the animal.The current tests used for BSE screening in Canada utilize the relative protease resistance of the prion protein gained when it misfolds from PrPC to PrPSc as part of the disease process. Proteinase K completely digests PrPC in normal brains, but leaves most of the PrPSc in BSE positive brains intact which is detected using anti-prion antibodies. These tests are highly reliable but occasionally give rise to initially reactive/false positive results. Test results for these reactive samples were close to the positive/negative cut-off on a sub set of test platforms. This is in contrast to all of the previous Canadian positive samples whose numeric values on these same test platforms were 10 to 100 fold greater than the test positive/negative cut-off. Here we explore the potential reason why a sample is repeatedly positive on a sub-set of rapid surveillance tests, but negative on other test platforms.In order to better understand and identify what might cause these initial reactions, we have conducted a variety of rapid and confirmatory assays as well as bacterial isolation and identification on BSE positive, negative and initially reactive samples. We observed high levels of viable bacterial contamination in initially reactive samples suggesting that the reactivity may be related to bacterial factors. Several bacteria isolated from the initially reactive samples have characteristics of biofilm forming bacteria and this extracellular matrix might play a role in preventing complete digestion of PrPC in these samples.

Tumor necrosis factor-α reduces beta-amyloid accumulation primarily by lowering cellular prion protein levels in a brain endothelial cell line

Disruption of beta-amyloid (Aβ) transport across the blood–brain barrier is thought to cause Aβ accumulation in the brain, thus leading to the development of Alzheimer’s disease (AD). As AD patients show increased serum tumor necrosis factor-α (TNFα) levels, we examined the effect of TNFα on the function and expression of Aβ transport-related proteins including cellular prion protein (PrPC) in the mouse brain microvascular endothelial cell line MBEC4. TNFα decreased PrPC levels and intracellular radiolabeled Aβ. Similarly, anti-prion protein antibody also decreased radiolabeled Aβ. These results suggest that TNFα lowers PrPC levels, which in turn, reduces Aβ in the brain endothelium.

Potential candidate camelid antibodies for the treatment of protein-misfolding diseases

Protein-misfolding diseases (PMDs), including Alzheimer's disease would potentially reach epidemic proportion if effective ways to diagnose and treat them were not developed. The quest for effective therapy for PMDs has been ongoing for decades and some of the technologies developed so far show great promise. We report here the development of antibodies by immunization of camelids with prion (PrioV3) and Alzheimer's (PrioAD12, 13 & 120) disease-derived brain material. We show that anti-PrP antibody transmigration across the blood–brain barrier (BBB) was inhibited with phosphatidylinositol-specific phospholipase C (PIPLC). Our camelid anti-prion antibody was also shown to permanently abrogate prion replication in a prion-permissive cell line after crossing the artificial BBB. Furthermore, anti-Aβ/tau antibodies were able to bind their specific immunogens with ELISA and immunohistochemistry. Finally, both PrioV3 and PrioAD12 were shown to co-localize with Lamp-1, a marker of late endosomal/lysosomal compartments.These antibodies could prove to be a valuable tool for the neutralization/clearance of PrPSc, Aβ and tau proteins in cellular compartments of affected neurons and could potentially have wider applicability for the treatment of PMDs.

Prion protein-specific antibodies that detect multiple TSE agents with high sensitivity.

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94–233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays.

Protein misfolding and neurodegenerative diseases.

Protein misfolding and neurodegenerative diseases.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

Immunotherapeutic Approaches in Prion Disease: progress, Challenges and Potential Directions

Therapeutic trials utilizing animal models of prion disease have explored a variety of compounds and a number of approaches with varying success, including several immunotherapeutic strategies, such as passive immunization through the delivery of viruses carrying nucleic acid inserts encoding prion protein-specific immunoglobulin. Targeted, organ-specific cellular production of therapeutic proteins is a relatively unexplored approach in the treatment of neurodegeneration despite many successful experimental outcomes in animal models and human trials of other diseases of the CNS. Emphasizing studies utilizing mouse models of disease, this review outlines developments and limitations of immunological approaches to the treatment of prion diseases. In addition, the authors discuss the potential of an experimental therapeutic strategy, utilizing hybridoma cells injected directly into the CNS to establish long-term production of anti-prion antibodies in vivo within the organ associated with the greatest pathogenic change in prion disease, the brain.

Abstract 5207: Targeting prion protein as a potential oncogene in pancreatic cancer.

Abstract Background: Human prion protein (PrP) has been found to be up-regulated in gastric, breast, pancreatic cancers and its expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Current literature suggests that overexpression of PrP induces inherent resistance to cancer therapeutics, while repression of PrP induces sensitization to cancer therapeutics. Our previous studies using expression microarray have revealed that PrP is not detected in normal human pancreatic ductal cells but is expressed in human pancreatic ductal adenocarcinoma (PDAC) cell lines and 40% of pancreatic cancer specimens. Further, expression of PrP confers a worse prognosis in PDAC patients. The poor prognosis of pancreatic cancer demonstrates needs for better target and drug development. Our recent studies also found that PrP is universally expressed in fetal pancreatic precursor ductal cells, and co-expresses with pancreatic stem cell markers, such as Pdx1 and Notch1, suggesting that PrP is a marker of pancreatic stem cells and may cross-talk with Notch1 during development and pancreatic tumorigenesis. However, the role for PrP in the establishment or progression of cancer is unknown, and whether PrP cross-talks with oncogenes such as Notch in these processes need to be elucidated. Methods: We applied immunoprecipitation (Abcam, Anti-Prion protein antibody 8H4) and western blotting to investigate the binding partners for PrP. Knockdown and over-expression of PRNP gene were carried out on various pancreatic cell lines, followed by in vitro proliferation assay and apoptotic activity analysis of these cells. Results: Pancreatic cancer cell lines express different levels of PrP, e.g. BxPC3 and Pan02.03 have higher PrP expression when compared to Capan-1. Western blot analysis revealed that PrP can bind with Notch1 protein in both BxPC-3 and CAPAN-1 cell lines. Moreover, down-regulation of PrP led to decreased cell proliferation and accelerated apoptosis, accompanied by down-regulation of cleaved/activated Notch1 (ICN1) and Notch2 (ICN2) expression in BxPC3 cells, but had lesser effect on CAPAN-1 cells. In comparison, over-expression of PrP significantly increased the proliferation rate of CAPAN-1 cells and dramatically upregulated the expression of ICN1. In conjunction with our previous findings that PrP exists as a pro-prion that binds filamin A in pancreatic cancer cells, these findings suggest that PrP may mediate pancreatic cancer cell progression through regulation of Notch activity and other cell mechanic and signaling integrators. This hypothesis is being further tested by xenograft analysis. Elucidating the mechanisms for PrP mediated Notch activation in PDAC will further our long-term goal of understanding the role of oncogenic PrP in conferring pancreatic cancer aggressiveness, and potentially will lead to novel therapeutic options for treatment of pancreatic cancer. Citation Format: Yiwei Wang, Lan Zhou, Wei Xin. Targeting prion protein as a potential oncogene in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2013-5207

Therapeutic effect of peripheral administration of an anti-prion protein antibody on mice infected with prions

Therapeutic effect of peripheral administration of an anti-prion protein antibody on mice infected with prions

Structural studies on the folded domain of the human prion protein bound to the Fab fragment of the antibody POM1

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrP(c) into a pathogenic isoform PrP(sc). Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrP(c)) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrP(c). POM1 Fab forms a 1:1 complex with huPrP(c) and the measured K(d) of 4.5 × 10(-7) M reveals moderately strong binding between them. Structural comparisons have been made among three prion-antibody complexes: POM1 Fab-huPrP(c), ICSM18 Fab-huPrP(c) and VRQ14 Fab-ovPrP(c). The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion protein in vivo. However, both of the glycosylation sites on huPrP(c) are positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion protein in vivo.

Utilização de biópsias da terceira pálpebra e mucosa retal em ovinos para diagnóstico de scrapie em uma propriedade da região sul do Brasil

Scrapie é uma encefalopatia espongiforme transmissível (EET) que causa lesões cerebrais degenerativas em ovinos e caprinos. Caracteriza-se pelo acúmulo, no tecido encefálico e linforreticular, da forma anormal da proteína priônica (PrP Sc) que provoca a morte maciça de neurônios e células gliais, além de vacuolização intensa no tecido afetado. Esse trabalho descreve a utilização da técnica de imuno-histoquímica (IHQ) para proteína priônica em tecido linforreticular de biópsias de terceira pálpebra e mucosa retal, como método diagnóstico de scrapie em ovinos. Realizaram-se exames de IHQ para scrapie em amostras de uma propriedade de origem de um ovino com diagnóstico dessa enfermidade. Utilizaram-se anticorpos monoclonais antipríon para diagnóstico ante mortem pela técnica de IHQ. Nas 318 amostras de biópsias analisadas, encontrou-se 19 resultados positivos para PrP Sc nos folículos de terceira pálpebra e não foi obtida marcação no tecido linfático de mucosa retal em nenhuma das amostras coletadas. Realizaram-se 18 necropsias dos animais positivos anteriormente por biópsia e 21 necropsias de ovinos parentes dos positivos de scrapie. Confirmou-se o resultado de scrapie pela IHQ após a necropsia dos animais positivos nas biópsias de terceira pálpebra. Nesses animais, os órgãos com maior número de cortes positivos foram a terceira pálpebra (18/18) e a tonsila (8/18). Nos ovinos com parentesco com os positivos, nenhum resultado de scrapie ocorreu. A utilização de tecidos linfoides no diagnóstico de scrapie por IHQ através de biópsias mostrou-se um método viável e eficaz para o diagnóstico pré-clínico.