Patent Grant
9441050
The present invention relates to the enrichment and purification of prion proteins and the generation of antibodies to infectious prion proteins.
Prion diseases are a family of progressive, fatal neurodegenerative disorders caused by the accumulation of the alternatively folded prion protein PrPSc. In the CNS, prions produce neuronal cell death, spongiform vacuolation and gliosis (1). The PrPSc protein is extractable from diseased tissue and biochemically distinguished from endogenous PrPC by partial protease resistance and detergent insolubility (2). Both PrPC and PrPSc share the same amino acid sequence, but PrPSc adopts an abnormal conformation that is transmissible and serves as a template for the conversion of host PrPC into the pathogenic prion isoform (3;4). The mechanism responsible for the transmission, conformational conversion of PrPC to PrPSc, and subsequent disease progression remains enigmatic.
Detection of infectious prions relies on combined use of immunoassay and histopathological assessment of brain tissue from infected animals (5). Current immunoassays are dependant on antibodies that recognize both the normal and abnormal isoforms of PrP. To distinguish abnormal PrPSc from normal PrPC requires limited digestion with proteinase-K (PK) to hydrolyze PK-sensitive PrPC while retaining the PK-resistant PrPSc (PrP 27-30). The PrP 27-30 protein is smaller than PrPC and intact PrPSc and thus can be recognized by a mobility shift following SDS-PAGE and Western blot detection with anti-PrP antibodies (6;7). Yet prion accumulation in the brain is progressive and infected, asymptomatic animals pose significant sampling challenges as minimal accumulation of PrPSc is localized to other more accessible tissue or fluid compartments (8;9). Moreover, variability in the efficacy of prion proteolysis of samples confounds detection of low-level PrPSc (10).
There remains an acute need for a sensitive and selective prion immunodiagnostic assay capable of pre-clinical assessment of infected animals from accessible tissues or fluids (11). Most immunoassay detection limits are insufficient to detect low-level prion contamination that can transmit disease by bioassay. Current assays are confounded by reliance on removal of PK-sensitive PrPC as no antibody has emerged that can selectively distinguish infectious PrPSc from PrPC (12). The need to remove PrPC protein from samples often diminishes immunoassay sensitivity by reducing the amount of PrPSc and increasing assay background. Moreover, the occurrence of PK-sensitive PrPSc isoforms poses additional concerns for many immunodiagnostic assays (13).
The difficulty of prion antibody generation is underscored by the identical primary structure of normal and abnormal PrP protein isoforms and isolation of purified infectious prion. The use of synthetic PrP peptides or recombinant PrPC has been successful in generating anti-PrP antibodies for detection of both PrPC and PrPSc proteins, but use of a PrPC derivative cannot yield an antibody that selectively bind the structurally distinct PrPSc (14;15). Since the primary structure of PrPSc is identical to PrP, a recombinant PrPSc protein cannot be generated. Moreover, the PrPC antigen has proven to be a poor immunogen as endogenous PrPC protein negates a robust immune response (16;17). The immunogenicity of PrPC antigen has been improved by using Prnp-null mice (Prnp0/0) with resulting production of high-affinity anti-PrP antibodies (14). However, the use of a PrPC antigen invariably leads to production of antibodies that recognize PrPC with a low probability of generating a PrPSc selective antibody capable of directly discriminating between normal PrPC and infectious PrPSc.
The most common methods for the diagnostic confirmation of prion disease involve clinical assessment, followed by post-mortem histopathological evaluation of brain tissue along with biochemical detection of PrP 27-30 (21;22). Several problems have confounded the pre-clinical diagnostic detection of prion. First, accumulation of PrPSc increases progressively over time; second, most PrPSc resides in the brain which imposes biopsy challenges. Third, prion concentrations below current immunoassay detection limits can transmit disease in animal bioassay (23;24). Fourth, no direct detection method has been developed that can distinguish PrPSc from PrPC without enzymatic or chemical manipulation to render endogenous PrPC undetectable while retaining PrPSc activity. Indeed, no antibody has emerged that can selectively bind PrPSc but not PrP, moreover, no surrogate analyte has been identified that can identify prions in preclinical animals (22;25). Finally, species and prion strain variability presents additional detection challenges as a result of distinct tissue distribution and availability (26;27).
Useful biochemical methods have emerged for the enrichment of PrPSc from brain homogenates that take advantage of differences in sedimentation and solubility (28;29). Yet, these preparative methods have proven insufficient to yield PrPSc enriched fractions suitable for crystal formation or as immunogen for the generation of PrPSc selective antibodies. Several factors likely contribute to the inability to generate a PrPSc selective antibody. First, the choice and preparation of inoculum have favored the generation of PrPC antibodies. The use of recombinant PrPC invariably yields antibodies that recognize PrP. Moreover, preparation of a native PrPSc is often confounded by contaminating proteins including PrP. Second, wt animals expressing endogenous PrPC may provide a less robust system for the generation of PrPSc antibodies (30). Third, the method used for screening antibodies requires the selective discrimination of those that bind PrPC from those that bind PrPSc. A method that yields that yields abundant PrPSc from diseased tissue and demonstrates a progressive increase in specific infectivity of prions and generation of high-titer antisera with selective activity to PrP is therefore desired.
Method for prion enrichment in biological tissue or fluids wherein the prion enriched samples serves as antigen for detection of prion proteins.
Method for purifying infectious prion protein from biological tissue and fluids wherein the purified prion serves as inoculum for antibody generation.
Method for generation and use of Prnp0/0 Balbc/J and Balb/c Bailey mice.
Method of identifying hybridoma cells producing prion specific monoclonal antibodies.
Method of generating prion specific antisera and monoclonal antibodies against prion protein.
Mouse hybridoma cells and resultant high-affinity monoclonal anti-prion antibodies.
The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated, the numerical properties set forth in the following specification and claims are approximations that may vary depending on the desired properties sought to be obtained in embodiments of the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from error found in their respective measurement.
Herein is described a novel method to purify infectious prion from biological tissue and fluids wherein the purified prion serves as inoculums for antibody generation or target of detection. The purification and enrichment of prion proceeds through (a) Prion isolation with detergent resistant membranes (DRM), (b) Proteinase-K (PK) enzymatic treatment and phosphotungstic acid (PTA) protein precipitation and (c) Size exclusion chromatography and PTA concentration. Alternatively, the purification and enrichment of prion proceeds through (a) Prion isolation with detergent resistant membranes (DRM), (b) dialysis with >300 kDa molecular weight cut-off (MWCO), (c) protein concentration. Post-translational processing of PrPC includes the addition of a GPI-anchor that targets the protein to lipid rafts of the plasma membrane (31;32). This localization is important for the conformational conversion of PrPC into infectious prion (33-36). Biochemically these lipid-rich membrane domains can be isolated by sucrose density centrifugation as buoyant detergent resistant membranes (DRMs) containing both PrPC and PrPSc proteins (10;31). Exploitation of this methodology allows for a significant enrichment and purification, effectively separating >99% of brain protein from prion in a single step. The resulting DRM fraction is enriched in PrPSc (>40-fold) relative to crude brain homogenate and represents <1% total brain proteins. Prions in the DRM fraction remain soluble and are highly infectious in bioassay. Treatment of DRM fractions with PK effectively removes PrPC and results in PrPSc truncation to form PrP 27-30. Abnormal infectious prions (PrPSc) form large molecular weight aggregates that differ from normal PrPC and dialysis with high molecular weight cut-off can be used to separate aggregate PrPSc from PrPC.
Phosphotungstic acid (PTA) has been used effectively to precipitate prion protein (37;38). The application of PTA to precipitate PrPSc after PK-digestion from DRMs provides an effective method to both concentrate and separate prion from lipids in the enriched DRM preparation. In addition, we found PrPSc in brain DRM fractions is highly aggregated with a molecular mass of >400 kDa. We exploited this property by using size exclusion chromatography to further separate PrPSc from residual PK (<30 kDa) and other protein fragments. Chromatography over Sephadex G100 allowed us to rapidly collect the majority of PrPSc in the column void fraction while residual proteins <100 kDa were retarded by the gel. The resulting PrPSc fraction (PrPSc DRM-PK-PTA-G100) retained infectivity as determined in bioassay. The PrPSc fractions from multiple column separations were pooled and concentrated by a second PTA precipitation to yield a highly purified PrPSc pellet (PrPSc DRM-PK-PTA-G100-PTA). This method (outlined in
An additional embodiment of the aforementioned method is non PK based using dialysis with a high molecular weight cut off after prion isolation with DRM. Dialysis with high molecular weight cutoff (HMWCO), greater than 300 kDa, of biological samples can be used in the diagnostic detection of diseases associated with protein aggregation, such as transmissible spongiform encephalopathies, Alzheimer's and Parkinson's. Dialysis with HMWCO can be used with protein concentration methods Dialysis with HMWCO can be used with protein concentration methods, such as PTA and centrifuge with concentration by small MWCO membranes, to enhance detection of abnormal aggregate proteins such as prions, Tau, β-amyloid, Synuclein and other aggregate proteins associated with disease as part of diagnostic detection assays. Dialysis with HMWCO can be used to concentrate and remove infectious prions or other aggregate proteins from environmental and biological samples and can be used to concentrate and separate full-length infectious (PrPSc) from normal native (PrPC) prion from biological samples blood, urine, and tissue extracts for enhanced detection and disease diagnosis. Dialysis with HMWCO can be used to concentrate and separate aggregate forms of disease associated proteins from non-aggregate normal counterparts for enhanced detection and disease diagnosis. HMWCO combined with prion enrichment methods can be used to isolate full-length infectious prion (PrPSc) aggregates for biochemical analysis such as immunogen preparation, structural analysis, the identification of prion-binding compounds, the identification of amino acid modifications, and disease infectivity).
An embodiment of the invention is the generation of Prnp-null Balb/c JAX and Balb/c Bailey mice wherein the Balb/c Bailey strain is useful for generation of a prion ablated myeloma cell line that can be used as a hybridoma fusion partner resulting in novel hybridomas producing monoclonal anti-prion antibodies. The mice would provide a molecular genetic tool for selective prion immuno-response. Additionally, wild-type mice from different genetic backgrounds could provide a suitable host for immunization with the described purified prion immunogen and hybridoma selection screen for the generation of anti-prion antibodies.
The development of a Prnp0/0/Balbc/J mouse provides a useful genetic background to promote a robust immune response to prions and production of monoclonal antibodies. These mice offer a syngenic background for myeloma-spleenocyte fusion and subsequent hybridoma generation. Indeed, our data shows that despite the purity of our prion inoculum it serves as a poor immunogen in wt Balb/cJ mice. However, the lack of detection of any other brain DRM proteins using the antisera from immunized wt Balbc/J mice supports the purity of our prion inoculum. In sharp contrast, immunization of Prnp0/0/Balb/cJ mice elicits a robust immune response that resulted in production of high-titer anti-prion specific antiserum. The response appears comparable by Western blot to that observed with established monoclonal anti-PrP antibodies. Additionally, no antibodies against other brain DRM proteins were detected in the antisera from these mice again suggesting a highly purified inoculum.
Antisera from the Prnp0/0/Balb/cJ mice recognized both PK-sensitive and -resistant PrP proteins. However, the model outlined here is well suited for the generation of hybridomas and isolation of monoclonal antibodies. Herein is described a method for the enrichment of infectious prions from lipid rafts and their subsequent purification in sufficient quantity to elicit a robust and selective immune response in Prnp0/0/Balb/cJ mice.
The robust immune response of Prnp0/0/Balb/cJ mice to purified prion immunogen resulted in selective high-titer anti-prion antisera. Hybridoma technology combined with comparative screening was effective in isolating cells that produce only anti-prion monoclonal antibodies. These DRM antibodies show high-affinity and selectivity for prion protein and are useful for immunoassay detection of infectious prions by ELISA and Western blot. Moreover, combined with prion enrichment in detergent resistant membranes we could detect prions in asymptomatic animals as early as 34 days post infection by Western blot.
Another embodiment of the invention is a method of identifying hybridoma cells producing prion specific antibodies using differential comparative direct binding ELISA for antibody screening and selection. The hybridoma selection and use of detergent resistant membrane binding assay proceeds through (a) a primary screen of hybridoma conditioned media on prion containing PK-treated brain derived DRMs (PrPSc DRM+PK). Positive binders are indicated by greater than 3-fold above background selected and (b) a secondary screen of select hybridomas by comparative binding against infectious and normal (PrP) as well as PrPC and PrPSc plus and minus PK treatment and recombinant derived PrP peptide. The binding phenotype of putative anti-PrPSc monoclonal antibodies are as follows: (+)PrPSc DRM, (+) PrPScDRM+PK, (−) PrPC DRM, (−) PrPC DRM+PK, (−) recombinant PrP protein, (−) proteinase-K. Combinations of the antigens listed above can be used during different phases of the screening strategy for selection of hybridomas producing anti-prion monoclonal antibodies.
A further embodiment of the invention is the generation of prion specific antisera, hybridomas and monoclonal antibodies via immunization of animals with purified prion derived from DRM fractions as well as the generation of a hybridoma cell lines producing anti-prion monoclonal antibodies. Three hybridoma cell lines have been cloned, monoclonal anti-prion antibodies isolated, and binding epitopes to prion proteins defined. Hybridoma cell lines making anti-prion monoclonal antibodies include: 1) DRM1-31 which binds a discontinuous epitope corresponding to amino acids 159-170 (NQVYYRPVDQYN), SEQ ID NO:1 and 215-226 (CTTQYQKESQAY), SEQ ID NO:2 of the Ha PrP protein sequence; 2) DRM1-60 which binds to amino acids 159-170 (NQVYYRPNDQYN), SEQ ID NO:3 corresponding to Ha PrP protein sequence; and 3) DRM2-118 which binds to amino acids 88-92 (GWGQGG), SEQ ID NO:4 corresponding to Ha PrP protein sequence.
Methods for diagnostic detection of prion diseases in environmental or biological samples using DRM prion enrichment and/or generated anti-prion antibodies. A significant enrichment of prion concentration in biological or environmental samples would aid in detection of infectious prions by immunoassay or other diagnostic approaches. Prions localized in DRM fractions provide novel source of infectious prions as target for detection, antibody screening, source of inoculum for antibody generation.
Methods for therapeutic intervention in prion diseases using identified monoclonal anti-prion antibodies. Humanized monoclonal anti-prion antibodies can be infused in patients with prion diseases as a therapeutic treatment.
Animals
All animals were housed in pairs on a 12 h light-dark cycle and provided continual access to food and water. All protocols were approved by the USDA animal care and use committee and experimental procedures conducted in certified BL2 laboratory. Hamster-passaged Sc237 scrapie prions were propagated in female Syrian Golden hamsters (LVG; Charles Rivers Laboratory, MA) beginning at 4 weeks of age. Prion-infected hamsters were sacrificed when clinical symptoms included; increased startle response, ataxia, and >5s righting reflex. Prnp0/0/Balb/cJ mice were generated at the University of California, San Francisco under approved animal protocols by speed congenic backcrossing 129/SvJ/C57-BL6 Prnp0/0 to inbred Balb/cJ mice and homozygosity verified by PCR as previously described (14;18). Antisera was obtained from anesthetized mice following transcardiac puncture with a 20-gauge needle attached to a 3 mL syringe, transferred to a BD Vacutainer SST tube (BD Biosciences), allowed to clot and sera collected after centrifugation.
Inoculation
Infectious PrPSc was propagated by serial passage in hamster brain following 40 μL intracerebral inoculation of a 1% brain homogenate in 320 mM sucrose using a 27-gauge needle inserted into the right parietal lobe. Detergent resistant membrane (DRM) fractions were diluted in sucrose to a final concentration of 320 mM and inoculated as described. Phosphotungstic acid (PTA) precipitated protein pellets were solubalized in n-octyl-glucoside to final concentration of 60 mM, diluted in sucrose and inoculated as described. Incubation time assay was used to calculate ID50 using the equation Log T=17+[Log D]−(0.138*Y) and used for calculation of specific infectivity (ID50/mg inoculum). Onset of clinical scrapie was determined by occurrence of two symptoms in days post-inoculation as defined above. Prnp0/0/Balb/cJ mice starting at 25d were inoculated (i.p.) with 100 μL antigen using the following regime: two inoculations containing purified PrPSc in RIBI adjuvant (Sigma-Aldrich, MO; Sigma Adjuvant System) separated by 10 days. Sera was collected 3 days after the final inoculation and evaluated for anti-PrP immunoreactivity.
Reagents
All reagents were of the highest grades commercially available. All antibodies were diluted in 10 mM Tris Buffered Saline with 1% Tween-20 (TBST) containing 1% IgG-free BSA (Jackson Immuno Chemical, PA). Primary antibodies used include: Caveolin-1 rabbit polyclonal diluted 1:1K (Santa Cruz, Calif.; N20), Flotillin-1 rabbit polyclonal diluted 1:1K (Santa Cruz; H-104), IPC1 anti-prion monoclonal diluted 1:10K (Sigma). Secondary antibodies include: goat-anti-mouse-HRP and goat-anti-rabbit-HRP diluted 1:10K (Pierce, Ill.). Recombinant Syrian hamster (recSHa) PrP(90-231) was generated at UCSF as previously described (19).
Isolation of Detergent Resistant Membranes
Hamster brains were homogenized (10% w/v) on ice in 25 mM MES (pH 6.5) with 150 mM NaCL, 1% Triton X-100, 60 mM n-octyl-glucoside, 10 mM PMSF, and protease inhibitors (Complete mini; Roche, C H). The homogenate was pre-cleared by centrifugation (1000×g) at 4° C. and supernatant mixed with equal volume of 80% sucrose in 25 mM MES (pH 6.5) with 150 mM NaCL. A 12 mL discontinuous sucrose gradient was formed by applying 4 mL of the 40% brain-sucrose in the bottom of a clear ultra-centrifuge tube (14×89 mm; Beckman, Calif.) followed by a 4 mL layer of 30% MES-Sucrose then 4 mL 5% MES-Sucrose. Tubes were placed in a SW-40T rotor and centrifuged at 39,000 RPM at 4° C. for 18 h in a L8-70M class H ultra-centrifuge (Beckman). A visible lipid-rich band corresponding to the detergent resistant membrane (DRM) fraction was observed within the 30-5% sucrose zone and collected (˜1 mL/gradient).
Linear Sucrose Sedimentation Gradient
DRM fractions obtained from hamster brain homogenates were mixed with n-octyl-glucoside to a final concentration of 60 mM and incubated at 4° C. for 15 min with rotation. A cushion of 250 μL of 50% sucrose in 25 mM MES (pH 6.5) with 150 mM NaCL and 60 mM n-octyl-glucoside was place in the bottom a clear ultra-centrifuge tubes (11×60 mm; Beckman) and a 50-5% linear sucrose gradient (4 mL) formed using a mixing gradient maker. The DRM fraction was loaded (300 μL) to the top of the gradient and tubes centrifuged in a SW60 rotor at 50,000 RPM for 10 h at 4° C. in a L8-70M class H ultra-centrifuge. 12×0.35 mL fractions were collected and analyzed. The gradient was calibrated with known molecular standards as previously described (20).
Western Blotting
Protein concentration was quantified using a micro-BCA assay (Pierce). Proteinase-K (PK; Roche) treatment was used at a final concentration of 25 μg/mL for brain homogenates and 150 μg/mL for DRM fractions for 1 h at 60° C. and inactivation of PK was by denaturation in LDS sample buffer or by addition of PMSF to 10 mM. Electrophoresis was performed on heat denatured samples in LDS buffer normalized by BCA and loaded on 4-12% Bis-Tris gels electrophoresed with MOPS running buffer (Novex; Invitrogen). Gels were transferred to nitrocellulose (Bio-Rad), washed in TBST, blocked with 10% non-fat dry milk, probed with antibodies, protein bands resolved by ECL (Supersignal; Pierce) and imaged on a Flurochem HD documentation system (Alpha Innotech, Calif.). Gel staining was performed with Coomassie blue (R250; Sigma) or Silver (ProteoSilver Plus; Sigma) and imaged on a light box.
Direct ELISA
Samples with equivalent protein concentration were diluted in 0.1 M sodium bicarbonate buffer (pH 9.4) and 100 μL absorbed to 96-well maxisorb plates (NUNC, NY) overnight at 4° C. Plates were washed in TBST, blocked in 10% non-fat milk for 1 h at 37° C., incubated 1 h with primary antibody, washed, incubated 1 h with HRP-conjugated secondary antibody, washed, resolved by chemiluminescence (Supersignal; Pierce) detection using Victor2 plate reader (PerkinElmer, MA) and expressed as relative light units (RLU).
Phosphotungstic Acid Protein Precipitation (PTA)
A stock of sodium phosphotungstate hydrate (Aldrich, Wis.) was dissolved at 4% in PBS (pH 7.4) with 170 mM MgCl2. PTA was added to samples to a final concentration of 0.3% with 13 mM MgCL2 and incubated at room temperature for 10 min. Precipitated protein was centrifuged at 10,000×g for 20 min at 4° C., pellets washed repeatedly with 200 mM EDTA in PBS followed by centrifugation with a final wash in ddH2O with remaining water aspirated after centrifugation. PTA pellets were solubalized with n-octyl-glucoside to a final concentration of 60 mM in buffer.
Size Exclusion Chromatography
A 15 mL gel bed of Sephadex G100 (Superfine grade, Sigma) was poured in a glass column and equilibrated in 25 mM Tris-HCL (pH 7.4). Column calibration was performed with gel filtration standards (Bio-Rad, CA; #151-1901) and samples loaded at 250 μL in 25 mM Tris-HCL (pH 7.4) with 60 mM n-octyl-glucoside. Proteins were fractionated with 25 mM Tris-HCL (pH 7.4) at a flow rate of 100 μL/min in 1 mL fractions. Column void was defined at 5 mL with detectable high molecular weight standards (>100 kDa) eluted.
Differential Hybridoma Screen
Following immunization of animals with a purified prion brain DRM derived prion preparation and spleenocyte-myeloma fusion resulting hybridomas are sequentially screen by comparison of supernatant binding to normal PrPC and PrPSc proteins. To identify hybridoma cells producing monoclonal antibodies that selectively bind to the infectious prion isoform an initial screen of hybridoma supernatant binding to proteinase-K treated prion infected brain DRM fractions on a microtiter plate is evaluated. Hybridomas that bind to PK-resistant prion in brain DRM fractions are expanded then supernatant is evaluated in a secondary screen for binding activity to PrPC, PrPSc+PK and recombinant PrP. Those that bind all three antigens are producing anti-prion monoclonal antibody that is not selective but recognizes both the PrPC and PrPSc isoforms. Those that bind on the PrPSc+PK antigen are producing prion selective monoclonal antibody that recognizes only the infectious PK-resistant PrPSc isoform.
Hybridoma Cell Cloning and Antibody Purification
Hybridoma cells were isolated by limiting dilution and clones expanded for monoclonal antibody production. Cloned hybridoma cells are inoculated into mice and ascites obtained and purified by protein-G affinity chromatography. Five anti-prion monoclonal antibodies were identified and assigned the designation DRM. All antibodies identified specifically recognized prion proteins, three of these antibodies; DRM1-31 (Ha PrP amino acids 159-170 (NQVYYRPVDQYN), SEQ ID NO:1 and 215-226 (CTTQYQKESQAY), SEQ ID NO:2, DRM1-60 (Ha PrP amino acids 159-170 (NQVYYRPNDQYN), SEQ ID NO:3, DRM2-118 (Ha PrP amino acids 88-92 (GWGQGG), SEQ ID NO:4 were shown to have distinct genetic sequences in their IgG domain variable region.
Results
Prion Isolation with Detergent Resistant Membranes
A sucrose density gradient effectively separates soluble proteins from those that are detergent resistant at 4° C. by exploiting differences in buoyancy properties due to lipid-association of some proteins. A measure of total protein following fractionation of crude brain homogenate (12×1 mL) showed that the majority of proteins (>99%) were retained at the bottom of the sucrose gradient between fractions #8-12 (40% sucrose), whereas a small protein peak (<1%) migrated to the zone of the 5/30% sucrose in fractions #4-5 corresponding to detergent resistant membrane (DRM) derived from lipid rafts (
Prion Enrichment
Prion enrichment was evaluated by comparing levels of PK-resistant PrPSc in crude brain homogenate to DRM gradient fractions. At equal concentration of total protein the DRM fraction contained ˜50-fold more detectable prion relative to crude brain homogenate by direct ELISA (
Prion in DRM is High Molecular Weight
Isolation of Prion Protein
Limited PK-digestion of PrPSc positive DRM fractions resulted in the proteolytic degradation of PrPC and the N-terminal truncation of PrPSc to form PrP 27-30. Importantly, the PK-digested samples also included peptide fragments, PK, and lipids. To isolate PrPSc from these contaminants, we exploited preferential binding and precipitation of PrPSc by PTA. Following PK-digestion of PrPSc DRM fraction, proteins were PTA precipitated then solubilized and fractionated over Sephadex G100. A major protein peak emerged at the column void fraction #5 (void >100 kDa;
Purified Prion Retains Infectivity
Using the hamster bioassay we showed that the DRM fraction (PrPSc DRM) isolated from prion-infected brain homogenate remained infectious (
Dialysis with High Molecular Weight Cut Off—Detection of Abnormal (PrPSc)
Normal and prion infected hamster brains were homogenized, detergent resistant membranes (DRM) isolated, dialyzed against water using a 300 kDa molecular weight cut off (>300K MWCO), and dilute fraction (−) or phosphotungstic acid (PTA) concentrate (+) were evaluated by SDS-PAGE Western blot using anti-prion monoclonal antibody DRM2-118 (1 μg/mL) with chemiluminescent detection. At equivalent protein concentration only prion infected hamster brain had detectable prion protein. Native non-aggregate PrPC protein was effectively dialyzed out of the DRM fraction, whereas the high molecular weight aggregate PrPSc (see
Dialysis with high molecular weight cutoff (>300K MWCO) of brain DRM fraction followed by sample concentration by centrifugation through 5 kDa MWCO filter effectively retains infectious PrPSc protein. 5K MWCO concentration results in a supernatant and protein precipitate. PrPSc protein was detectable in both the supernatant and precipitate fraction by Western blot using DRM1-31-HRP (1 μg/mL) at 5, 10, 20 μg/well () by chemiluminescence. Silver stained protein bands were observed in the gel following SDS-PAGE corresponding to PrPSc detected by Western blot. See
Purified PrPSc is Immunogenic in Prnp0/0/Balb/cJ Mice
Prnp0/0/Balb/cJ mice were immunized with a PrPSc fraction enriched for prion infectivity according to the purification scheme outlined in
Further characterization of antisera binding to protein in crude brain homogenate and brain-derived DRM fractions from normal (PrPC) and disease (PrPSc) hamster brain with (+) and without (−) PK-digestion is shown from a representative immunized Prnp0/0/Balb/cJ mouse by Western blot (
Spleenocytes from prion immunized Prnp0/0/Balb/cJ mice were fused with myeloma cells and hybridomas screened for anti-prion monoclonal antibody production. Hybridoma cells making anti-prion monoclonal antibodies were identified and cells cloned by limiting dilution. Five hybridoma clones were selected for ascites production in mice and resulting DRM antibodies purified by protein-G affinity chromatography. Anti-prion monoclonal antibodies were characterized by ELISA and Western blot and shown to bind with high affinity to proteinase-K resistant prion protein in hamster brain DRM (
This application is a divisional of application Ser. No. 13/157,216, filed Jun. 6, 2011, which claims priority to U.S. Provisional Patent Application Ser. No. 61/353,480 filed Jun. 10, 2010 herein incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 7344842 | Garssen et al. | Mar 2008 | B1 |
| Entry |
|---|
| MABN780, Anti-PrP, clone DRM1-31 Antibody product sheet, EMD Millipore Corp. Retrieved from internet May 21, 2016. |
| MABN768, Anti-PrP, clone DRM2-118 Antibody product sheet, EMD Millipore Corp. Retrieved from internet May 27, 2016. |
| MABN772, Anti-PrP, clone DRM1-60 Antibody product sheet, EMD Millipore Corp. Retrieved from internet May 27, 2016. |
| Stanker LH et al. Novel epitopes identified by anti-PrP monoclonal antibodies produced following immunization of Prnp o/o Balb/cJ mice with purified scrapie prions. Hybridoma, 2012, 31(5):314-324. |
| Number | Date | Country | |
|---|---|---|---|
| 20140065723 A1 | Mar 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61353480 | Jun 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13157216 | Jun 2011 | US |
| Child | 13949905 | US |
