PrP及其缺失突变体与GFAP体外相互作用的初步研究

目的研究PrP蛋白与GFAP蛋白是否发生分子间相互作用以及PrP蛋白多肽链中与GFAP蛋白相互作用的区域。方法制备仓鼠脑组织匀浆上清,通过原核表达系统以及体外翻译系统表达了全长的小鼠GFAP蛋白、人PrP蛋白以及各种缺失突变体,利用Pull-down及免疫共沉淀实验检测PrP与GFAP的分子间相互作用。结果不仅重组的GFAP与PrP发生分子间的相互作用,而且脑组织中的GFAP与PrPC及PrPSc也发生相互作用。PrP与GFAP蛋白相互作用的区域位于PrP的C端第91至231位氨基酸。结论PrP及其缺失突变体与GFAP在体外能够发生分子间的相互作用,提示GFAP可能参与朊蛋白的正常生理功能或者朊病毒病的病理过程。     

Increased expression of the normal cellular isoform of prion protein in inclusion-body myositis, inflammatory myopathies and denervation atrophy

The cellular isoform of the prion protein (PrPc) is a glycosylphosphatidylinositol-anchored glycoprotein, normally expressed in neural and non-neural tissues, including skeletal muscle. In transmissible spongiform encephalopathies, or prion diseases, PrPc, which is soluble in nondenaturing detergent and sensitive to proteinase K (PK)-treatment, represents the molecular substrate for the production of a detergent-insoluble and PK-resistant isoform, termed PrP(Sc). In human prion diseases, PrP(Sc) accumulation occurs only in brain tissues, with the exception of new variant Creutzfeldt-Jakob disease, where PrP(Sc) is also detected in lymphoid tissues. Increased amounts of prion protein expression and deposition have been described in pathological muscle fibers of two human muscle disorders, called sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy, but it is unknown whether accumulated prion protein reflects normal PrPc or PrP(Sc). We investigated the biochemical characteristics of prion protein in normal human muscle, s-IBM, other inflammatory myopathies and denervation atrophy. We report that 1) both the glycoform profile and size of the normal muscle PrPc are different from those of human brain PrPc; 2) in addition to s-IBM, increased PrPc expression is seen in polymyositis, dermatomyositis and neurogenic muscle atrophy, but PrPc glycoforms are unchanged; 3) only the normal PrPc isoform, and not PrP(Sc), is detected in s-IBM. The present results exclude that s-IBM is a prion disease.     

Detection of ultra-low levels of pathologic prion protein in scrapie infected hamster brain homogenates using real-time immuno-PCR

Pathologic prion protein (PrP(Sc)), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrP(Sc). The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrP(C) was consistently detected at 1 fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10(-8) (approximately 10-100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1 million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrP(Sc) in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrP(C) in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive.     

Effect of the E200K mutation on prion protein metabolism. Comparative study of a cell model and human brain

The hallmark of prion diseases is the cerebral accumulation of a conformationally altered isoform (PrP(Sc)) of a normal cellular protein, the prion protein (PrP(C)). In the inherited form, mutations in the prion protein gene are thought to cause the disease by altering the metabolism of the mutant PrP (PrP(M)) engendering its conversion into PrP(Sc). We used a cell model to study biosynthesis and processing of PrP(M) carrying the glutamic acid to lysine substitution at residue 200 (E200K), which is linked to the most common inherited human prion disease. PrP(M) contained an aberrant glycan at residue 197 and generated an increased quantity of truncated fragments. In addition, PrP(M) showed impaired transport of the unglycosylated isoform to the cell surface. Similar changes were found in the PrP isolated from brains of patients affected by the E200K variant of Creutzfeldt-Jakob disease. Although the cellular PrP(M) displayed some characteristics of PrP(Sc), the PrP(Sc) found in the E200K brains was quantitatively and qualitatively different. We propose that the E200K mutation cause the same metabolic changes of PrP(M) in the cell model and in the brain. However, in the brain, PrP(M) undergoes additional modifications, by an age-dependent mechanism that leads to the formation of PrP(Sc) and the development of the disease.     

Influence of guanidine on proteinase K resistance in vitro and infectivity of scrapie prion protein PrP(Sc)

As the scrapie prion protein PrP(Sc) is rich in beta-sheets it aggregates into prion rods, which show infectivity and proteinase K (PK) resistance. Consequently, dissociation of prion rods and breakdown of beta-sheets in PrP(Sc) by denaturation results in loss of both infectivity and PK-sensitivity. In this study, the effects of guanidine (Gdn), which solubilizes and denatures proteins by breaking down their higher structure, on the solubility, the PK-resistance in vitro and the infectivity of PrP(Sc) of scrapie strain 263K was examined. The infectivity was assayed by intracerebral inoculation into hamsters. Brain tissues of scrapie-infected hamsters were used for preparation of homogenates and crude extracts of PrP(Sc). A treatment of PrP(Sc) with Gdn enhanced its PK-sensitivity in a dose-dependent manner. The PK-resistance in vitro of PrP(Sc) denatured with lower concentrations of Gdn (<2.5 mol/l) could partially resume by renaturation. Gdn markedly reduced or, at higher concentrations, even destroyed the infectivity of PrP(Sc). On the other hand, the infectivity of PrP(Sc) inactivated by denaturation could be partially restored by renaturation. These results confirmed our assumption that all the alternations in the PK-resistance and the infectivity of PrP(Sc) caused by Gdn resulted from changes in its higher structure. However, it should be emphasized that a complete loss of PK-resistance of PrP(Sc) may not necessarily mean its full non-infectivity.     

The possible role of protein X, a putative auxiliary factor in pathological prion replication, in regulating a physiological endoproteolytic cleavage of cellular prion protein

The posttranslational conformational conversion of the cellular isoform of prion protein PrP(C) into its scrapie isoform PrP(Sc) is the fundamental process underlying the pathogenesis of prion disease. Based on several transgenic data, it has been postulated that a putative auxiliary factor denoted protein X functions as a molecular chaperone through its unfolding activity of PrP(C) during the formation of PrP(Sc). However, the assumption that protein X therefore exists exclusively in prion diseases appears improbable and thus, it should have some simultaneous physiological role. We, hereby, propose a novel concept - a characteristic role of protein X in supporting a physiological endoproteolytic cleavage of PrP(C). The events corresponding to the formation of the physiologically metabolized PrP(C) or the pathologically transformed PrP(Sc) are mutually exclusive. Amino acid residues that are critical in terms of the target site of protein X for the pathological alteration into PrP(Sc) overlap at the cleavage site. These amino acid residues tend to have a hydrophobic property and are most probably found buried inside the native protein structure. Therefore, a putative molecular chaperone identical to protein X may target the same hydrophobic residues in PrP(C) and work in conjunction with either PrP(Sc) in prion disease or PrP proteases during the physiological state. This postulation may help explain in a relatively simple manner these two mutually exclusive phenomena, viz. the physiological endoproteolytic cleavage of PrP(C) and its pathological conversion into PrP(Sc).     

一种朊蛋白错误折叠循环扩增方法的建立

目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术(PMCA),用于朊病毒病脑组织中PrPSc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合,经反复孵育/超声,共10~15个循环。WesternBlot检测扩增产物中蛋白酶K抗性PrPSc信号。结果在本研究试验体系下,263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrPSc-PMCA技术可检测到10-5稀释的毒株原液中的PrPSc。与常规的脑组织免疫印记方法相比,敏感度提高了105~106倍。研究还显示PrPSc还可利用小脑和脑干为基质进行体外扩增复制。结论成功建立了PrPSc-PMCA技术,为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。     

Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins

Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrP(C), and the pathological isoform, PrP(Sc). In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were 'orphan' clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (K(d)) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrP(C). Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrP(Sc) following guanidinium denaturation.     

Generation of antibodies against bovine recombinant prion protein in various strains of mice

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, belong to a group of neurodegenerative disorders affecting humans and animals. To date, definite diagnosis of prion disease can only be made by analysis of tissue samples for the presence of protease-resistant misfolded prion protein (PrP(Sc)). Monoclonal antibodies (MAbs) to the prion protein provide valuable tools for TSE diagnosis, as well as for basic research on these diseases. In this communication, the development of antibodies against recombinant bovine prion protein (brecPrP) in four strains of mice (BALB/c, ND4, SJL, and NZB/NZW F(1)) is described. Immunization of autoimmunity-prone NZB/NZW F(1) and SJL mice with brecPrP was applied to overcome self-tolerance against the prion protein. ND4 and SJL mice did not develop an immune response to brecPrP. BALB/c mice produced antibody titers of 1:1,000 to 1:1,500 in an enzyme-linked immunosorbent assay (ELISA), while NZB/NZW F(1) mice responded with titers of 1:7,000 to 1:11,000. A panel of 71 anti-brecPrP MAbs recognizing continuous and discontinuous epitopes was established from BALB/c and NZB/NZW F(1) mice. Seven anti-brecPrP MAbs reacted with both the cellular form of PrP and protease K-resistant PrP(Sc) from sheep brain in Western blot assays. The epitope specificity of these MAbs was determined, and applicability to immunohistochemical detection of prions was studied. The MAbs generated will be useful tools in the development of TSE immunochemical diagnosis and for research. This is the first report of the development of anti-PrP MAbs by use of autoimmune NZB/NZW F(1) mice as an alternative approach for the generation of PrP-specific MAbs.     

CpG oligodeoxynucleotide-enhanced humoral immune response and production of antibodies to prion protein PrPSc in mice immunized with 139A scrapie-associated fibrils

Prion diseases are characterized by conversion of the cellular prion protein (PrP(C)) to a protease-resistant conformer, the srapie form of PrP (PrP(Sc)). Humoral immune responses to nondenatured forms of PrP(Sc) have never been fully characterized. We investigated whether production of antibodies to PrP(Sc) could occur in PrP null (Prnp(-/-)) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrP(Sc) antibody levels in wild-type (Prnp(+/+)) mice was also investigated. Prnp(-/-) and Prnp(+/+) mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp(-/-) mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies to an N-terminal epitope, which were only present in the immune repertoire of mice receiving ODN 1826. mAb 6D11, derived from such a mouse, reacts with the N-terminal epitope QWNK in native and denatured forms of PrP(Sc) and recombinant PrP and exhibits a K(d) in the 10(-)(11) M range. In Prnp(+/+) mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrP(Sc) in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrP(Sc) and suggest methods for optimizing the generation of mAbs to PrP(Sc), many of which could be used for diagnosis and treatment of prion diseases.     

Retinal cell types are differentially affected in sheep with scrapie

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases characterized microscopically by spongiform lesions (vacuolation) in the neuropil, neuronal loss, and gliosis. Accumulation of the abnormal form of the prion protein (PrP(Sc)) has been demonstrated in the retina of natural and non-natural TSE-affected hosts, with or without evidence of microscopically detectable retinal pathology. This study was conducted to investigate the effect of PrP(Sc) accumulation on retinal neurons in a natural host lacking overt microscopical evidence of retinal degeneration by comparing the distribution of retinal cell type-specific markers in control and scrapie-affected sheep. In retinas with PrP(Sc)-immunoreactivity, there was disruption of the normal immunoreactivity patterns of the alpha isoform of protein kinase C (PKCalpha) and vesicular glutamate transporter 1 (VGLUT1), markers of retinal bipolar cells. Altered immunoreactivity was also observed for microtubule-associated protein 2 (MAP2), a marker of a subset of retinal ganglion cells, and glutamine synthetase (GS), a marker of Müller glia. These results demonstrate alterations of immunoreactivity patterns for proteins associated with specific cell types in retinas with PrP(Sc) accumulation, despite an absence of microscopical evidence of retinal degeneration.     

In vitro amplification and detection of variant Creutzfeldt-Jakob disease PrPSc

Variant Creutzfeldt-Jakob disease (vCJD) poses a serious risk of secondary transmission and the need to detect infectivity in asymptomatic individuals is therefore of major importance. Following infection, it is assumed that minute amounts of disease-associated prion protein (PrP(Sc)) replicate by conversion of the host cellular prion protein (PrP(C)). Therefore, methods of rapidly reproducing this conversion process in vitro would be valuable tools in the development of such tests. We show that one such technique, protein misfolding cyclic amplification (PMCA), can amplify vCJD PrP(Sc) from human brain tissue, and that the degree of amplification is dependent upon the substrate PRNP codon 129 polymorphism. Both human platelets and transgenic mouse brain are shown to be suitable alternative substrate sources, and amplified PrP(Sc) can be detected using a conformation-dependent immunoassay (CDI), allowing the detection of putative proteinase K sensitive forms of PrP(Sc).     

Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity

The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrP(Sc), associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrP(Sc) that recognizes both PrP(C) and PrP(Sc). This scFv fragment had a dissociation constant (K(D)) with recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrP(Sc) but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrP(Sc) and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs.     

Correlation of polydispersed prion protein and characteristic pathology in the thalamus in variant Creutzfeldt-Jakob disease: implication of small oligomeric species

The vacuolation, neuronal loss and gliosis that characterize human prion disease pathology are accompanied by the accumulation of an aggregated, insoluble and protease-resistant form (termed PrP(Sc)) of the host-encoded normal cellular prion protein (PrP(C)). In variant Creutzfeldt-Jakob disease the frontal cortex and cerebellum exhibit intense vacuolation and the accumulation of PrP(Sc) in the form of amyloid plaques and plaque-like structures. In contrast the posterior thalamus is characterized by intense gliosis and neuronal loss, but PrP(Sc) plaques are rare and vacuolation is patchy. We have used sucrose density gradient centrifugation coupled with conformation dependent immunoassay to examine the biochemical properties of the PrP(Sc) that accumulates in these different brain regions. The results show a greater degree of PrP(Sc) polydisperal in thalamus compared with frontal cortex or cerebellum, including a subpopulation PrP(Sc) molecules in the thalamus that have sedimentation properties resembling those of PrP(C). Much effort has focused on identifying aspects of PrP(Sc) biochemistry that distinguish between different forms of human prion disease and contribute to differential diagnosis. Here we show that PrP(Sc) sedimentation properties, which can depend on aggregation state, correlate with, and may underlie the distinct neurodegenerative processes occurring in different regions of the variant Creutzfeldt-Jakob disease brain.     

Detection of type 1 prion protein in variant Creutzfeldt-Jakob disease

Molecular typing of the abnormal form of the prion protein (PrP(Sc)) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrP(Sc) molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resulting from infection with a single strain of the agent (bovine spongiform encephalopathy). Here we show for the first time that the PrP(Sc) that accumulates in the brain in variant Creutzfeldt-Jakob disease also contains a minority type 1 component. This minority type 1 PrP(Sc) was found in all 21 cases of variant Creutzfeldt-Jakob disease tested, irrespective of brain region examined, and was also present in the variant Creutzfeldt-Jakob disease tonsil. The quantitative balance between PrP(Sc) types was maintained when variant Creutzfeldt-Jakob disease was transmitted to wild-type mice and was also found in bovine spongiform encephalopathy cattle brain, indicating that the agent rather than the host specifies their relative representation. These results indicate that PrP(Sc) molecular typing is based on quantitative rather than qualitative phenomena and point to a complex relationship between prion protein biochemistry, disease phenotype and agent strain.     

Use of a new immunoassay to measure PrP Sc levels in scrapie-infected sheep brains reveals PrP genotype-specific differences

The diagnosis of prion diseases, such as scrapie and BSE, has traditionally relied upon the identification of the disease-associated form of the prion protein, PrP(Sc), based on its resistance to digestion by proteinase K (PK). A more recent development is the conformation-dependent immunoassay (CDI), which distinguishes between PrP Sc and normal PrP (PrP C) based on their differing solubility in guanidine hydrochloride rather than resistance or sensitivity to PK. We have developed a CDI-formatted sandwich immunoassay for the measurement of PrP Sc in sheep brain, which discriminates between clinically affected scrapie cases (natural or experimental) and uninfected controls of the same PrP genotype. Using this method, we have shown for the first time that, in sheep, the PrP genotype has a significant influence on the amount of PrP Sc deposited in the brains of animals experimentally infected with scrapie.     

Expression of PrP(C) as HIS-fusion form in a baculovirus system and conversion of expressed PrP-sen to PrP-res in a cell-free system

Conversion of the PrP cellular form (PrP(C)) to the pathogenic form (PrP(Sc)) is the key step in the pathogenesis of transmissible spongiform encephalophathies. Although the mechanism of conformational conversion of PrP proteins remains uncertain, the cell-free conversion reaction and other in vitro PrP amplification tests allow it to be studied under the much quicker and simpler conditions than those of transmission bioassay in vivo. Using baculovirus expression system, wild-type hamster (HaPrP) and human PrP (HuPrP), as well as D178N mutated human PrP (HuPrPm178) were expressed in HIS-fusion form. After 35S-methionine labeling and purification with Ni-NTA agarose affinity chromatography, individual expressed PrP proteins were mixed with PrP(Sc) isolated from hamster brain tissue infected with scrapie 263K. Protease-resistant isoform was detected in the homologous HaPrP reaction, but not in the two heterologous HuPrP preparations, implying a species-specific molecular recognition between PrP(C) and PrP(Sc). HIS-tag in HIS-HaPrP seems to have little effect on the formation of protease-resistant protein in this preparation. This system proposes a simple and protein productive-enriched way for cell-free conversion of prion proteins, as the replacement of native or genetic engineering expressed sole PrP(C) from mammalian or non-mammalian sources.     

Efficient in vitro amplification of a mouse-adapted scrapie prion protein

Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie prion protein (PrP(Sc)), a major protein component of the infectious agents associated with prion diseases. Although exponential in vitro amplification of hamster scrapie PrP(Sc) has been established, the PMCA used was unsuccessful in achieving good amplification of PrP(Sc) from other animals. Here, we have investigated the cause of the insufficient PrP(Sc) amplification in mice and have developed an improved method suitable for amplification of the PrP(Sc) of the mouse-adapted scrapie prion strain Chandler. Mouse PrP(C), the cellular form of the prion protein, tends to become resistant to proteases during incubation independent of sonication. By adding digitonin to the reaction buffer as a lipid detergent, accumulation of the protease-resistant PrP(C) was inhibited; hence, mouse PrP(Sc) could be amplified to infinite levels. The present study is the first report describing effective amplification of PrP(Sc) of the mouse-adapted scrapie prion and this improved PMCA technique will contribute to prion research that uses mice as experimental animals.     

Follicular dendritic cell dedifferentiation reduces scrapie susceptibility following inoculation via the skin

Transmissible spongiform encephalopathies (TSEs) are a group of subacute infectious neurodegenerative diseases that are characterized by the accumulation in affected tissues of PrP(Sc), an abnormal isoform of the host prion protein (PrPc). Following peripheral exposure, TSE infectivity and PrP(Sc) usually accumulate in lymphoid tissues prior to neuroinvasion. Studies in mice have shown that exposure through scarified skin is an effective means of TSE transmission. Following inoculation via the skin, a functional immune system is critical for the transmission of TSEs to the brain, but until now, it has not been known which components of the immune system are required for efficient neuroinvasion. Temporary dedifferentiation of follicular dendritic cells (FDCs) by treatment with an inhibitor of the lymphotoxin-beta receptor signalling pathway (LTbetaR-Ig) 3 days before or 14 days after inoculation via the skin, blocked the early accumulation of PrP(Sc) and TSE infectivity within the draining lymph node. Furthermore, in the temporary absence of FDCs before inoculation, disease susceptibility was reduced and survival time significantly extended. Treatment with LTbetaR-Ig 14 days after TSE inoculation also significantly extended the disease incubation period. However, treatment 42 days after inoculation did not affect disease susceptibility or survival time, suggesting that the infection may have already have spread to the nervous system. Together these data show that FDCs are essential for the accumulation of PrP(Sc) and infectivity within lymphoid tissues and subsequent neuroinvasion following TSE exposure via the skin.     

Single chain Fv antibodies directed against the 37 kDa/67 kDa laminin receptor as therapeutic tools in prion diseases

Transmissible spongiform encephalopathies are a group of neurological disorders associated with the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(c). The 37 kDa/67 kDa laminin receptor (LRP/LR) has been identified as a prion receptor and several lines of evidence strongly suggest that this protein plays a role during prion pathogenesis. Here we report the selection of recombinant single chain antibodies (scFvs) directed against LRP from na?ve and synthetic phage scFv libraries for therapeutic application. Western blotting and FACS analysis confirmed a specific LRP/LR recognition pattern of the two selected scFvs S18 and N3. Both scFvs specifically interfered with the PrP/LRP interaction in vitro. High yield production of the scFvs of approx. 1mg/l of culture medium was achieved in E. coli. Passive immunotransfer of the scFv S18 antibody reduced PrP(Sc) levels by approx. 40% in the spleen of scrapie infected C57BL/6 mice 90 days post scFv injection, suggesting that scFv S18 interferes with peripheral PrP(Sc) propagation, without a significant prolongation of incubation and survival times.