Human prion diseases: from antibody screening to a standardized fast immunodiagnosis using automation

Abstract

Demonstration of pathological prion protein accumulation in the central nervous system is required to establish the diagnosis of transmissible subacute encephalopathies. In humans, this is frequently achieved using prion protein immunohistochemistry in paraffin-embedded tissue, a technique that requires multiple epitope retrieval and denaturing pretreatments. In addition to being time-consuming, this procedure induces tissue alterations that preclude accurate morphological examination. The aim of this study was to simplify prion protein immunohistochemistry procedure in human tissue, together with increased sensitivity and specificity. We screened a panel of 50 monoclonal antibodies produced using various immunogens (human and ovine recombinant prion protein, prion protein peptides, denatured scrapie-associated fibrils from 263K-infected Syrian hamsters) and directed against different epitopes along the human prion protein sequence. A panel of different forms of genetic, infectious and sporadic transmissible subacute encephalopathies was assessed. The monoclonal 12F10 antibody provided a high specificity and fast immunodiagnosis with very limited denaturing pretreatments. A standardized and reliable fast immunostaining procedure was established using an automated diagnostic system (Nexes, Ventana Medical Systems) and allowed prion protein detection in the central nervous system and in tonsil biopsies. It was evaluated in a series of 300 patients with a suspected diagnosis of transmissible subacute encephalopathies and showed high sensitivity and specificity.

Accuracy of diagnosis criteria in patients with suspected diagnosis of sporadic Creutzfeldt-Jakob disease and detection of 14-3-3 protein, France, 1992 to 2009

“…PRNP analysis was performed as described [ 5 , 6 ] when informed consent was obtained from either the patient or the next of kin. Western blotting and neuropathology were performed following established methods [ 8 - 10 ]: after gross examination, one hemi-brain (left or right at random) was frozen at autopsy for PrPsc displaying (Western blot), the other fixed in formalin for 1 month. Hemi-brains were kept in the brain bank.…”

“…After partial inactivation by 96% formic acid for 1 hour and paraffin embedding, 4 μm-thick sections were stained with haematein-eosin and periodic acid-Schiff techniques. Since 1998, a minimum of two sections (cerebral and cerebellar cortices) have been immunolabelled with PrP antibodies, using 12F10 monoclonal antibody (Bertin, Montigny le Bretonneux, France) [ 10 ]. Each specimen was examined by at least two neuropathologists (VS, DS, CD, J-JH), and, in most cases, neuropathologists from other centres of the French neuropathology network of Creutzfeldt-Jakob disease).…”

Prion Protein-Specific Antibodies that Detect Multiple TSE Agents with High Sensitivity

“…This is of both functional and biological significance, since data emanating from the use of antibodies with different epitopes within PrP have yielded significant advancements in prion biology relating to: diagnostic regimes for animal TSE disease (using 6H4 [22], [24], [51], [52] or human TSEs (using KG9 singly or in combination with other antibodies i.e. 3F4, ICMS35, BG4 [53]–[56]); immunological characterisation of pathology associated with established and emerging TSEs, such as immunophenotyping of atypical scrapie using an array of antibodies [2] or identification of the shared characteristic of Nor98 scrapie and human Gerstmann-Straussler-Scheinker disease (GSS) [6]; the use of sequences in the prion protein which are species specific (using 3F4 or antisera raised against synthetic peptides [14], [25], [57]); the discrimination of TSE strains in large and small animal models, for example natural scrapie, BSE and CH1641 scrapie (using P4 and 66.94ba [58], 6H4/P4 [59], P4 [60]), using triplex immunostaining with L42, 12B2 and SAF84 [61] and using multiple antibody panels [5], [41], [62]–[67]; the characterisation and classification of human prion disease [68]–[71]; the characterisation of structural elements within PrP, such as deciphering accessibility and exposure of epitopes following the conversion of PrP C to PrP Sc or those that are PrP Sc -specific i.e.…”

Region-specific protein misfolding cyclic amplification reproduces brain tropism of prion strains

“…Human tissues were selected on the basis of the availability of autopsy-obtained frozen brain material and informed consent from patients' relatives for autopsy and research use. The neuropathological examination of various brain regions was realized by standard staining methods, PrP immunohistochemistry (34), and Western blot detection of PrP Sc . Three confirmed CJD patients and three non-CJD patients were identified as providers of seed or substrate materials allowing efficient PMCA.…”