Monoclonal Antibody against a Peptide of Human Prion Protein Discriminates between Creutzfeldt-Jacob's Disease-affected and Normal Brain Tissue

Abstract

Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrPC) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrPSc by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrPSc. In cryostat sections from Creutzfeldt-Jacob's disease (CJD) patients' brains, V5B2 selectively labels various deposits of PrPSc without any pretreatment for removal of PrPC. V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrPSc-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrPSc aggregates forms a structural epitope whose conformation is distinct from that of PrPC. Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrPC) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrPSc by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrPSc. In cryostat sections from Creutzfeldt-Jacob's disease (CJD) patients' brains, V5B2 selectively labels various deposits of PrPSc without any pretreatment for removal of PrPC. V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrPSc-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrPSc aggregates forms a structural epitope whose conformation is distinct from that of PrPC. A conformational transition of the normal cellular prion protein (PrPC) 1The abbreviations used are: PrPprion proteinPrPCcellular PrPPrPScpathological PrP isoformrecPrPrecombinant PrPBSEbovine spongiform encephalopathyTSEtransmissible spongiform encephalopathyBSAbovine serum albuminCJDCreutzfeldt-Jacob's diseasesCJDsporadic CJDvCJDvariant of CJDELISAenzyme-linked immunosorbent assayGdnHClguanidine HClHRPhorseradish peroxidaseIHCimmunohistochemistryKLHkeyhole limpet hemocyaninmAbmonoclonal antibodyPBSphosphate-buffered saline.1The abbreviations used are: PrPprion proteinPrPCcellular PrPPrPScpathological PrP isoformrecPrPrecombinant PrPBSEbovine spongiform encephalopathyTSEtransmissible spongiform encephalopathyBSAbovine serum albuminCJDCreutzfeldt-Jacob's diseasesCJDsporadic CJDvCJDvariant of CJDELISAenzyme-linked immunosorbent assayGdnHClguanidine HClHRPhorseradish peroxidaseIHCimmunohistochemistryKLHkeyhole limpet hemocyaninmAbmonoclonal antibodyPBSphosphate-buffered saline. into a pathological prion isoform (PrPSc) is believed to be responsible for the development of transmissible spongiform encephalopathies (TSE) (1Griffith J.S. Nature. 1967; 215: 1043-1044Google Scholar, 2Prusiner S.B. Science. 1982; 216: 136-144Google Scholar). PrPSc forms characteristic insoluble aggregates, is partially resistant to protease digestion, and differs in secondary structure from PrPC (1Griffith J.S. Nature. 1967; 215: 1043-1044Google Scholar, 2Prusiner S.B. Science. 1982; 216: 136-144Google Scholar, 3Prusiner S.B. Scott M.R. Annu. Rev. Genet. 1997; 31: 139-175Google Scholar). The importance of TSE arose in the last decade with the epidemic of bovine spongiform encephalopathy (BSE) and the appearance of a new variant of Creutzfeldt-Jacob's disease (vCJD), predominantly but not exclusively in the United Kingdom. A specific, rapid, and sensitive test for detecting PrPSc is required to minimize the possible risk of prion contamination. Currently available methods for immunological diagnosis of TSE rely on the use of antibodies that react with both forms of PrP (4Will R.G. Ironside J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4738-4739Google Scholar) and, thus, depend on prior elimination of PrPC by proteinase K digestion for positive identification of the remaining PrPSc (5Giaccone G. Canciani B. Puoti G. Rossi G. Goffredo D. Iussich S. Fociani P. Tagliavini F. Bugiani O. Brain Pathol. 2000; 10: 31-37Google Scholar) as its partially digested product, PrPres. Because it has been established that the conformation of PrPSc differs from that of PrPC, the aim of our work was to identify the epitope specific for PrPSc that would enable better insight into the differences between the two isoforms and provide a tool for analyzing biological samples. An earlier study reported production of a monoclonal antibody (mAb) specific for the disease-associated form of PrP (6Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. Schulz-Schaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Google Scholar). It was later stated by the same authors that the availability of PrP knock-out mice and recombinant PrP (recPrP) were some of the prerequisites for successful immunization and generation of the specific antibodies (7Korth C. Streit P. Oesch B. Methods Enzymol. 1999; 309: 106-122Google Scholar). Our approach for generating PrPSc-selective mAbs was to immunize BALB/c mice with carefully chosen peptides based on previous investigations on the structure and mechanism of PrPSc formation. We have prepared a mAb that reacts with an epitope specific for the native PrPSc and show that this epitope could not be generated in vitro from recombinant PrP. We suggest that the C-terminal part of PrPSc folds into a distinct conformation, which may be stabilized by interaction with other molecules under disease conditions. prion protein cellular PrP pathological PrP isoform recombinant PrP bovine spongiform encephalopathy transmissible spongiform encephalopathy bovine serum albumin Creutzfeldt-Jacob's disease sporadic CJD variant of CJD enzyme-linked immunosorbent assay guanidine HCl horseradish peroxidase immunohistochemistry keyhole limpet hemocyanin monoclonal antibody phosphate-buffered saline. prion protein cellular PrP pathological PrP isoform recombinant PrP bovine spongiform encephalopathy transmissible spongiform encephalopathy bovine serum albumin Creutzfeldt-Jacob's disease sporadic CJD variant of CJD enzyme-linked immunosorbent assay guanidine HCl horseradish peroxidase immunohistochemistry keyhole limpet hemocyanin monoclonal antibody phosphate-buffered saline. Peptides—Peptides based on the human PrP sequence P1 (214–226; CITQYERESQAYY), P2 (167–179; DEYSNQNNFVHDC) and P3 (139–150; CIHFGSDYEDRYY Cys was added for conjugation to KLH) were made by solid state synthesis, conjugated to keyhole limpet hemocyanin (KLH) (Bachem, Bubendorf, Switzerland), and used for immunization. Preparation of Monoclonal Antibodies—Hybridomas were prepared in BALB/c mice according to the procedures described in Ref. 16Price K.M. Ritter M.A. Ladyman H.M. Monoclonal Antibodies: Production, Engineering, and Clinical Applications. Cambridge University Press, Cambridge, UK1995: 60-82Google Scholar, and mAbs were selected with indirect ELISA against peptide, peptide-KLH, and KLH. Brain Tissue Sample Preparation—Human brain tissue samples (nine sCJD positive and two non-CJD) were homogenized (HT1000 Potter homogenizer) in ice-cold PBS containing 0.5% Nonidet P-40 and 0.5% deoxycholate to give a 10% (w/v) final suspension. Homogenates were centrifuged for 10 min at 5000 × g at 4 °C. Supernatants of brain homogenates were aliquoted and stored at -80 °C. Dot Blot Analysis—50 μlof10×,20×,or40× diluted supernatants of brain homogenates were loaded on 0.2-μm nitrocellulose membranes using the dot blot (Bio-Rad). The membranes were blocked with 5% (w/v) nonfat dry milk (Amersham Biosciences) in Tris-buffered saline/Tween 20 (TBS-T) at 4 °C overnight and then incubated with primary mAbs (5 μg/ml V5B2 or 0.2 μg/ml 3F4 in 1% milk/TBS-T) for 1 h with shaking at room temperature. Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)-labeled anti-mouse secondary antibody (Amersham Biosciences; 1:1500 in 1% milk/TBS-T) at room temperature for 1 h. Reaction was detected using chemiluminescence detection reagents (ECL, Amersham Biosciences). Western Blot Analysis—Native or proteinase K-digested (final proteinase K concentration 5 μg/ml for 30 min at 37 °C, heat inactivated) supernatants of brain homogenates, denatured with SDS-PAGE sample buffer (Novex), were loaded on SDS-Tris-glycine polyacrylamide gels (7.5% gel) and run for 60 min at 120 V with 25 mm Tris, 0.32 m glycine, and 0.16% SDS (w/v), pH 8.3. The samples were blotted on 0.2 μm nitrocellulose membranes (Bio-Rad) for 60 min at 200 mA using 25 mm Tris, 192 mm glycine, and 20% methanol buffer. The membranes were blocked, and proteins were detected as described above. Sandwich ELISA with Brain Tissue Homogenates—Microtiter plates (Nunc, Roskilde, Denmark) were coated with 5 μg/ml mAb 6H4 (Prionics, Zürich, Switzerland) in 50 mm carbonate/bicarbonate buffer, pH 9.6. The plates were blocked with 1% BSA and 50 μl of sCJD or non-CJD brain homogenates in 1% BSA/PBS/Tween20 were applied to the wells. After 90 min of incubation at 37 °C, V5B2, conjugated with HRP and diluted 1:1000 in 1% BSA/PBS/Tween 20, was added to the wells. After 90 min incubation at 37 °C reaction was detected as an indirect ELISA. Immunoprecipitation—mAb V5B2 was covalently coupled to carboxyl-terminated magnetic beads (Sigma) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a bifunctional coupling reagent as suggested by the manufacturer. Beads were preincubated in 1% BSA/PBS at room temperature for 1 h and washed in binding buffer (3% Nonidet P-40, 3% Tween, and 250 mm NaCl in 20 mm potassium phosphate buffer, pH 7.4). 30 μl of brain homogenate containing ∼270 μg of protein was added to a suspension containing 50 μl of magnetic beads and 150 μl of binding buffer. Suspension was shaken for 1 h at room temperature and washed three times with washing buffer (2% Nonidet P-40, 2% Tween 20, and 350 mm NaCl in 20 mm potassium phosphate buffer, pH 7.4). Bound proteins were eluted with SDS loading buffer and heated at 95 °C before application to gel electrophoresis and detection by either the 3F4 or the 6H4 antibody conjugated to horseradish peroxidase. Cryosections of Fresh Frozen Tissue Samples—Brains of clinically suspected CJD cases were collected at autopsy and, prior to whole brain formalin fixation, pieces of cerebral and cerebellar tissue were frozen rapidly in liquid nitrogen and stored at -80 °C until final pathological diagnosis of CJD. Brain cryosections of two sCJD cases and one non-CJD patient (cause of death was myocardial infarction) were used in this study. Sections were cut in the cryostat at 10 μm, thaw-mounted to SuperFrost Plus slides (Menzel Gläser, Germany), and labeled without prior fixation. Paraformaldehyde-fixed, Paraffin-embedded Tissue Samples—Brain tissues from 16 cases of sporadic CJD were used. Following 14 days of buffered 4% paraformaldehyde fixation, tissue blocks were immersed in 96% formic acid for 1 h and embedded in paraffin. 4-μm-thick sections were either microwaved (96 °C) in EDTA buffer (pH 8) for 10 min (“mild” pretreatment) or autoclaved for 30 min (121 °C), cooled, immersed for 5 min in 96% formic acid, and washed prior to immunohistochemistry (IHC). A sample of hematoxylin-eosin stained section of a vCJD case brain was destained and labeled immunohistochemically without any pretreatment. Indirect IHC—V5B2 was tested in manual IHC on CJD and non-CJD cryostat brain sections at concentrations ranging from 0.3 to 30 μg/ml. Commercially available anti-PrP antibody 3F4 (Dako, Glostrup, Denmark) was used for comparison. Manual IHC was performed on slide-mounted sections using the avidin-biotin method with a peroxidase substrate as described (8Bresjanac M. Antauer G. Exp. Neurol. 2000; 164: 53-59Google Scholar). IHC on paraffin-embedded sections was performed on an automated machine (Ventana Medical Systems, Tucson, AZ) using Ventana kits and prescribed protocols at every step of the procedure. In addition to non-CJD brain tissue that served as a control for V5B2 selectivity for PrPSc, controls of IHC labeling were performed by omitting primary antibody from the IHC protocol carried out on adjacent sections as described above. Hematoxylin (Dako) was used to lightly counterstain the cell nuclei on all IHC-labeled slides. The slides were analyzed under a microscope (Eclipse E600, Nikon). Images were acquired with an attached digital camera (DXM 1200 digital camera, Nikon) linked to a PC. V5B2 conjugated with HRP (V5B2-HRP) was used following the same antigen retrieval procedure as described above for direct IHC. The concentration of V5B2-HRP was 5 μg/ml. Preparation of peptide with a was into Ref. A. Scholar) to the protein was in in the form of were in m pH against and the was used in human PrP and forms were in in form of and R. C. Wuthrich K. 1997; Scholar). Preparation of of structural forms of human were to test the of V5B2 was by overnight incubation at room temperature in m mm and 16 mm pH was to 50 mm pH to the in a was performed by the protein for min at 95 °C. of PrP with a of structure were by the following reaction 1 or m m 50 mm pH 4 or M. 1 m 50 mm pH 4 W. M. P. 2000; or in 4 m pH 4 A. H. Science. 1999; and PrP in or acid, pH Scholar). Monoclonal V5B2 against a in BALB/c the three peptides based on the sequence of human PrP used for immunization the were with a peptide from the C-terminal part of human PrP We have selected this of PrP it is part of the protease-resistant structure as by with mAb raised against the peptide M. B. 1999; Scholar). part of the has been to be for its and (6Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. Schulz-Schaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Google Scholar, C. Streit P. Oesch B. Methods Enzymol. 1999; 309: 106-122Google Scholar, H. H. M. M. K. M. K. 1999; Scholar). that the sequence of our peptide is to prion and the the human at of antibodies against a peptide from this of the bovine with PrPC but not with PrPSc H. H. M. M. K. M. K. 1999; Scholar). described as specific for bovine PrPSc, have been one of which is in the same of the protein (6Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. Schulz-Schaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Google Scholar, C. Streit P. Oesch B. Methods Enzymol. 1999; 309: 106-122Google Scholar). mice and the recombinant bovine prion protein were a for an mAb with this the of and and three of in our has been reported to be in production of specific antibodies K.M. Ritter M.A. Ladyman H.M. Monoclonal Antibodies: Production, Engineering, and Clinical Applications. Cambridge University Press, Cambridge, UK1995: 60-82Google Scholar). with this peptide in a in all the BALB/c were prepared from three and, of the that antibodies against three were to between PrPSc and PrPC. dot Western and IHC were used to mAb V5B2 from a of mAbs of for PrPC or PrPSc. PrPSc of was to between normal and CJD brain homogenates in dot blot is in to mAb 3F4 and the antibody for prion detection R. M. R. H.M. H. which with non-CJD as as pathological brain homogenate Sandwich available mAb 6H4 (6Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. Schulz-Schaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Google Scholar) as the antibody and V5B2 as the detection antibody, a with the brain homogenate from that from non-CJD brain without proteinase K digestion that the of V5B2 in tissue is PrPSc characteristic for the prion disease and is not in normal V5B2 reaction on Western blot with from CJD and non-CJD brain to SDS-PAGE as for a conformational mAb 3F4 was used as a positive V5B2 conjugated to magnetic beads PrP from but not normal brain homogenate the of ELISA discriminates between and and human brain homogenates tested with sandwich ELISA of 6H4 as the and the V5B2-HRP as the antibody, the specific of V5B2 with PrPSc in samples without with proteinase blot of and human brain homogenates under conditions. were or with proteinase K. was performed with antibodies 3F4 and selectively and homogenates from and loaded to SDS-PAGE and from and brain homogenates by V5B2 to magnetic beads Homogenates of protein in controls and as as in homogenates used in V5B2 magnetic beads were a control without brain homogenate. PrP was detected by direct with of the selectivity of V5B2 for PrPSc and a of its from its and labeling of PrPSc aggregates in thaw-mounted cryosections of human brain tissue from CJD cases labeling with normal human brain tissue or with brain tissue from other as disease and disease In labeling of tissue in tissue cryosections of both CJD and non-CJD brain was with mAb The conformational epitope was by V5B2 paraformaldehyde and could be detected in sections from paraffin-embedded a direct method of was used in which V5B2 was conjugated with HRP without of the the secondary antibody and the avidin-biotin of a formic acid incubation step in the antigen retrieval protocol V5B2 with the CJD brain detection of prion deposits V5B2 was tested on paraffin-embedded tissue sections from it to prion aggregates We have that mAb V5B2 be used to and in the brain tissue of and not mAb V5B2 with of not bind to human or bovine in either dot blot or ELISA the of the peptide sequence at the it to the peptide and its to KLH. test the antibody would the prepared human in the form of insoluble but was reaction with V5B2 as a was reaction with 3F4 have the peptide to the of to test the against PrP be to its in the form. protein in with that some of the of the peptide that react with V5B2 in the native conformation of the and PrPC as as in denatured PrP. that the conformation of the peptide in the form that reacts with V5B2 differs from that in and PrPC as as in denatured PrP. and and the of the of the peptide by Scholar) that the peptide does not an structure but is in it is part of the protein it is stabilized in a conformation by has been to a of that were to a transition in vitro to a conformation with a of with subsequent M. Scholar, A. H. Science. 1999; Scholar, Scholar). or concentrations of and and the of or of in any with V5B2 for the and is an for that antibodies that react with the PrPSc without any pretreatment. Sandwich as described in the and which the PrPSc-specific antibody V5B2, between samples from normal and and is a tool for In to other as the assay H. D. H. M. S.B. the of PrPSc is from the in between the native and denatured sample or by degradation of PrPC by proteinase our assay the of PrPSc in the of PrPC. The sequence of the peptide partially with the conformational epitope of the reported PrPSc-specific (6Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. Schulz-Schaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Google Scholar, C. Streit P. Oesch B. Methods Enzymol. 1999; 309: 106-122Google Scholar). the mAb has been available for either or and has not been to work in detection of PrPSc. our was under PrPSc-specific antibodies M. S. M. A. M. R. Ironside K. A. Scholar). report that antibodies against the for the conformation of PrP. A sequence is at the of our peptide, that both antibodies may the same of the the conformational transition in PrP is believed to in the part of the the of PrP from which the peptide of this study has The differences in the conformation of the of PrPC from at the of the C-terminal which is in in human and bovine PrP R. A. Riek R. C. Billeter M. G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; Scholar, M. Riek R. G. Hornemann S. Glockshuber R. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). investigations of the PrP have that the with the at the of the R. A. Riek R. C. Billeter M. G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; Scholar). Our provide that the conformation at the of PrPSc differs from the conformation of PrPC. of in PrPC that any structural of which is believed to be would the its D. Scholar). have that PrP an of the of which part of the peptide of this study A. 1999; Scholar). It is that this the on removal of which the of the epitope for The C-terminal to our peptide was to be in both PrPSc and PrPC D. H. C. R. S.B. 1997; which that part of the is in conformation that an of were mAbs have been that against both PrPC and denatured PrPSc, that the specific conformation of PrPSc and that on removal of to a conformation D. H. C. R. S.B. 1997; Scholar). V5B2 in this to PrPSc but not PrPC or denatured PrPSc. The conformation of PrPSc be disrupted with that its M. M.A. S.B. Proc. Natl. Acad. Sci. U. S. A. Scholar). We have that the specific conformation of PrPSc was disrupted and the with V5B2 was by with SDS and but not by not which have been to or the of PrPSc S.B. Science. 1982; 216: 136-144Google Scholar). It has been suggested that the of the and the to not for the of the of in as a may be for the C-terminal part of PrP in the conformation by of this in the and interaction with a has been suggested Scott M. R. M. S.B. Scholar, K. Scott M. S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar). for of to the at the of P1 M. S. B. K. C. R. Scholar). the in and in which in the peptide, against of the disease K. P. S.B. Proc. Natl. Acad. Sci. U. S. A. Scholar, S. Neurol. Scholar). from the and antibodies against this have been to of PrP aggregates in M. B. and, in of the of the peptide reported in this it as a We show that mAb V5B2 recognizes the conformation of the C-terminal of the PrPSc which differs from the PrPC. V5B2 does not react with recombinant PrP, of The PrPSc-specific mAb V5B2 detection of PrPSc by procedures on tissue from CJD patients' in dot blot or ELISA. production does not the use of knock-out mice (6Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. Schulz-Schaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Google Scholar, C. Streit P. Oesch B. Methods Enzymol. 1999; 309: 106-122Google and, it is for procedures and for TSE in as as in the show that V5B2 is a tool for TSE and may to identify and the of PrP, which a in conformation the transition to the pathological form. We Zürich, Switzerland) for the of for of human PrP, Ironside from the CJD for sections of vCJD brain and for and for of the and We for and for