WO2012099884A1 - Methods for amplification and detection of prions - Google Patents
Methods for amplification and detection of prions Download PDFInfo
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- WO2012099884A1 WO2012099884A1 PCT/US2012/021561 US2012021561W WO2012099884A1 WO 2012099884 A1 WO2012099884 A1 WO 2012099884A1 US 2012021561 W US2012021561 W US 2012021561W WO 2012099884 A1 WO2012099884 A1 WO 2012099884A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2828—Prion diseases
Definitions
- the present disclosure relates to methods and compositions for the detection of infectious proteins or prions in samples, including the diagnosis of prion related diseases.
- TSEs The transmissible spongiform encephalopathies
- CJD human Creutzfeldt-Jakob disease
- BSE bovine spongiform encephalopathy
- CWD cervid chronic wasting disease
- TSE transmissible mink encephalopathy
- the infectious agent, or prion, of the TSEs appears to be composed primarily of an abnormal, misfolded, oligomeric, and usually partially protease-resistant form of prion protein (e.g., PrP-res, PrP ⁇ 0 , PrP Sc ).
- PrP-res is formed post-translationally from the normal cellular prion protein (PrP ) (Borchelt et al, J Cell Biol, 110, 743- 752, 1990;Caughey and Raymond, J Biol Chem, 266, 18217-18223, 1991). PrP-res, which in purified form can resemble amyloid fibrils, induces the polymerization and conformational conversion of PrP c to infectious PrP-res/PrP Sc (Castilla et al.
- PrP normal cellular prion protein
- PrP-res can self-propagate, and although the mechanism is not fully understood, it appears to be a seeded or templated polymerization (Gadjusek, Infectious amyloids: Subacute Spongiform Encephalopathies as Transmissible Cerebral Amyloidoses. In Fields,B.N.,
- Prions can be found in a wide variety of tissues and accessible bodily fluids from infected mammalian hosts, including blood (Brown et al., Transfusion, 38, 810-816, 1998; Manuelidis et al., Science, 200, 1069-1071, 1978; Mathiason et al., Science, 314, 133-136, 2006; Saa et al., 2006a; Terry et al., J Virol, 83, 12552-12558, 2009; Thorne and Terry, / Gen Virol, 89, 3177-3184, 2008), breast milk (Konold et al, BMC Vet Res, 4, 14, 2008; Lacroux et al, PLoS Pathog, 4, el000238, 2008), saliva (Mathiason et al., Science, 314, 133
- Methods are disclosed for detecting prion proteins. These methods provide sensitive and specific identification of prions in both biological and environmental samples. These methods include the use of both immunopreciptiation and an amplification assay that uses shaking in the absence of sonication, such as QuIC or RT-QuIC.
- methods for detecting prion protein that include contacting a sample with an effective amount of an antibody that specifically binds a PrP-res for sufficient time to form an immune complex, and mixing the immune complex with purified recombinant prion protein (rPrP ) to make a reaction mixture.
- the immune complex can be separated from the sample.
- An amplification reaction is performed, that includes incubating the reaction mixture to permit coaggregation of the PrP-res with the rPrP in the reaction mixture and maintaining incubation conditions that promote coaggregation of the rPrP with the PrP-res to result in a conversion of the rPrP c to rPrP-res (Sc) while inhibiting (e.g., preventing) development of spontaneously formed rPrP-res (spon) .
- the reaction mixture is agitated, wherein agitating comprises shaking the reaction mixture without sonication.
- rPrP-res (Sc) is detected in the reaction mixture, wherein detection of rPrP-res (Sc) in the reaction mixture indicates that PrP-res is present in the sample.
- amounts of rPrP-res (Sc) in the reaction mixture can be quantitated.
- detecting rPrP-res (Sc) in the reaction mixture includes the use of Thioflavin T (ThT).
- the rPrP can be replenished by adding additional rPrP substrate prior to detecting in the reaction mixture.
- the immune complex can be pre-incubated, such as with a buffer comprising a detergent, such as sodium dodecyl sulfate, prior to performing the amplification reaction.
- the antibody that specifically binds PrP-res can be bound to a solid substrate, including but not limited to, magnetic beads.
- the antibody is 15B3.
- the rPrP c can be a chimeric rPrP c , such as a chimeric hamster-sheep rPrP .
- the assay can detect vCJD and other forms of STEs.
- methods for detecting prion protein in a biological sample include contacting the biological sample, such as plasma, blood, serum, cerebral spinal fluid or a tissue sample with an effective amount of antibody 15B3 coupled to a solid substrate for sufficient time to form an immune complex on the solid substrate.
- the immune complex on the substrate is separated from the other components of the biological sample.
- the immune complex on the solid substrate is incubated with a buffer comprising about 0.01% to about 0.05 % sodium dodecyl sulfate.
- the immune complex on the solid substrate is mixed with purified recombinant prion protein (rPrP c ), such as hamster sheep chimeric recombinant prion protein (rPrP c ), and
- the amplification reaction includes: (a) incubating the reaction mixture to permit coaggregation of the PrP-res with the rPrP that are present in the reaction mixture; (b) maintaining incubation conditions that promote coaggregation of the rPrP with the PrP-res to result in a conversion of the rPrP c to rPrP-res (Sc) while inhibiting development of rPrP-res (spon) ; (c) agitating aggregates formed during step (i), wherein the reaction mixture is shaken and then not shaken for a substantially equal period of time, such as shaken for about 60 seconds and then not shaken for about 60 seconds, or shaken for about 30 seconds and then not shaken for about 30 seconds; (d) adding additional recombinant prion protein (rPrP ), such as hamster sheep chimeric prion protein, to the reaction mixture prior to the formation of
- steps (c) and (d) can optionally be repeated.
- he rPrP-res (Sc) in the reaction mixture is detected using ThT fluorescence, wherein fluorescence of the reaction mixture indicates that PrP-res was present in the sample.
- the rPrP c can be replenished by adding additional rPrP c substrate prior to detecting rPrP-res (Sc) in the reaction mixture.
- Dilutions of human non-prion (tumor, T) control or vCJD brain homogenates were spiked into 500 ⁇ of human plasma to give final dilutions of 4 x 10 "7 (T); and 4 x 10 "7 , 4 x 10 "9 , and 4 x 10 "10 (vCJD; containing ⁇ 10 pg , 100 fg and 10 fg PrP-res, respectively).
- PrP vCJD was immunoprecipitated and subjected to S-QuIC as described in Materials and Methods.
- the first S-QuIC round was at 50 ° C for 8 hour (h) (Fig. la) and 1/10 of the first-round reaction volume was used to seed the 2 nd round (45°C for 10 h) (Fig. lb).
- Plasma-free positive and negative control reactions were seeded directly with 2 ⁇ of 5 x 10 " dilutions of hamster uninfected (N) or scrapie (Sc) brain, the latter containing -100 fg PrP res seed.
- Hamster rPrP 23-231 was used as a substrate in all reactions and comigrated with the 25 kDa marker. PK-digested products were analyzed by immunoblot using the polyclonal R20 antibody as previously reported (24). Open circles mark 17-kDa fragments and brackets indicate the lower molecular weight bands (10-13 kDa).
- Figs 2a-2b IP-S-QuIC detection of endogenous PrP Sc in plasma of scrapie-infected hamsters by IP-S-QuIC.
- Fig. 2a Plasma samples from scrapie 263K and uninfected (N) hamsters (500 ⁇ ) were subjected to IP-S-QuIC as described in the Examples Section with the 1 st round S-QuIC at 50°C for 10 hours (h) and the 2 nd round (Fig. 2b) at 50°C for 8 h, except for lanes marked with asterisks which show the 1 st round products seeded with sample #6 for comparison. Plasma- free positive and negative control reactions, rPrP 23-231 substrate and analysis of PK-digested products were as described for Figure 1. Open circles mark 17-kDa fragments and brackets indicate the lower molecular weight bands (10-13 kDa).
- Figs. 3a-3b IP-RT-QuIC detection of endogenous PrP Sc in plasma and serum of scrapie-infected hamsters.
- FIG. 3a IP-RT-QuIC analyses of plasma samples from a scrapie 263K and a normal hamster, and a serum sample from a scrapie 263K hamster.
- FIG. 3b Analyses of plasma samples from nine scrapie 263K and one uninfected hamster. In all cases, 500 ⁇ samples were immunoprecipitated using 15B3-coated beads for -20 h at 37°C. One fifth of the beads was pre- incubated with 0.05% SDS in PBS at room temperature for -20 minutes and used to seed RT-QuIC containing 300 mM NaCl. RT-QuIC reactions were incubated at
- Figs. 4a-4b eQuIC detection of human PrP vCJD spiked into human plasma. Dilutions of human non-prion (tumor and Alzheimer's disease) control or vCJD brain tissues were spiked into 500 ⁇ of human plasma to give final dilutions of 4 x 10 "7 (tumor and Alzheimer's disease); and 4 x 10 "12 , 4 x 10 "13 and 4 x 10 "14 (vCJD; containing ⁇ 100 ag, 10 ag and 1 ag PrP-res, respectively). PrP ⁇ 0 was immunoprecipitated using 15B3-coated beads (Fig. 4a) or mock anti-IgM-coated beads (Fig.
- Figs. 5a-5b eQuIC detection of endogenous PrP Sc in plasma of scrapie- infected hamsters.
- Fig. 5a eQuIC analysis of plasma samples (without preclearing) from 8 uninfected hamsters and 6 scrapie-infected hamsters, with one collected at 30 dpi (preclinical) and 5 at 80 dpi (near- terminal).
- the vertical axis indicates the average fluorescence from 4 replicate wells and the fractions on the right indicate the positive/total replicate reactions associated with the adjacent traces. Although all replicate reactions seeded with the scrapie samples were positive, submaximal average fluorescence observed for 3 of the samples at 60 h.
- Fig. 6 Schematic diagram of potential mechanisms of substrate replacement effect.
- Figs. 7a-7b Better IP-S-QuIC sensitivity and consistency of PrP Sc detection in spiked human plasma using 15B3 vs. mock beads. (Fig. 7a)
- Beads were resuspended in 10 ⁇ of PBS. One fifth of the beads was used to seed a 1 st round S- QuIC at 50°C for 10 h and 1/10 of the 1 st round reaction volume was used to seed the 2 nd round (50°C for 10 h).
- Fig. 7b 15B3 beads with 20-h IP from 500 ⁇ plasma and single-round S-QuIC. Dilutions of N or Sc brain homogenates were spiked into
- Figs. 8a-8b SDS pre-treatment of 15B3-bound PrP Sc accelerates RT- QuIC detection. Dilutions of hamster N or Sc 263K brain homogenates were spiked into 500 ⁇ of human plasma to give final brain dilutions of 2 x 10 "
- Figs 9a-9c Improved RT-QuIC detection of 15B3-bound human PrP vCJD with hamster-sheep chimeric rPrP c (Ha-S rPrP c ) vs. human rPrP c 23- 231 with NaCl variation.
- Dilutions of human non-prion (tumor) control or vCJD brain tissues were spiked into 500 ⁇ of human plasma to give final dilutions of 4 x
- Figs. 10a- 10b Comparison of 15B3 beads to Magnabeads in eQuIC. Dilutions of human non-TSE Alzheimer's disease (AD) control or vCJD brain tissues were spiked into 0.5 ml of human plasma to give final dilutions of 4 x 10 " (AD); and 4 x 10 ⁇ 7 , 4 x 10 ⁇ 10 , 4 x 10 "13 and 4 x 10 "14 (vCJD; containing ⁇ 10 pg, 10 fg, 10 ag and 1 ag PrP-res, respectively). PrP ⁇ 0 was immunoprecipitated using 1.6 x 10 total 15B3-coated beads (Fig. 10a) or an equivalent number of
- MAGNABINDTM beads (Fig. 10b) for -20 h at 37°C using immunoprecipitation buffer (Prionics). Beads were washed twice with wash buffer (Prionics) and resuspended in 10 ⁇ of PBS. The remainder of the protocol was as described in Material and Methods, starting with the preincubation in 0.05% SDS in PBS. The vertical axis indicates the average fluorescence from four replicate wells and the fractions on the right indicate the positive/total replicate reactions associated with the adjacent traces. Similar results were obtained using the MAGNABINDTM PrP Sc capture conditions (Miller and Supattapone, 2011, J.Virol. 85:2813-2817).
- Fig. 11 Improved speed & sensitivity of IP-RTQ using higher 15B3 content on beads.
- Dilutions of hamster normal (NBH) or scrapie 263K brain homogenates were spiked into 500 ⁇ of human plasma to give final brain tissue dilutions of 2 x 10 "9 (NBH); and 2 x 10 "9 and 2 x 10 "10 (263K; containing -100 or 10 fg PrP-res, respectively).
- PrP-res was immunoprecipitated using 40 ⁇ of 15B3 beads for -20 hours at 37°C. Beads were washed twice with 0.2% Sarkosyl/TBS and resuspended in 10 ⁇ of PBS. Following a 0.05% SDS pre-treatment, one fifth of the beads was used to seed RTQ reactions.
- Hamster rPrP 90-231 was used as a substrate in all reactions. The vertical axis indicates the average fluorescence from 2 replicate wells.
- Fig. 13 eQuIC detection of ARQ sheep brain homogenate in spiked sheep plasma.
- the assay provided detection of > ⁇ 100ag PrP-res (5 x 10 - " 13 dilution of brain tissue) in 500ul of sheep plasma.
- Fig. 14 eQuIC detection of endogenous PrP-res in plasma of scrapie positive sheep.
- the assay detected endogenous PrP-res in plasma from three slinically affected scrapie-infected sheep. No prions were detected in plasma samples from four non-infected sheep.
- Fig. 15 Sensitivity of detection of sCJD brain homogenate spiked into plasma by e-QuIC.
- the assay detected sCJD brain homogenate spikes containing as little as ⁇ 10ag of PrP res in 0.5mL of human plasma.
- Fig. 16 Sensitivity of detection of sCJD brain homogenate spiked into cerebrospinal fluid (CSF) by e-QuIC.
- the assay detected sCJD brain tissue dilutions containing as little as ⁇ lOag of PrPres in 0.5 ml of human cerebrospinal fluid.
- Fig. 17 Sensitivity of detection of mouse-adapted RML scrapie brain homogenate spiked into plasma by e-QuIC.
- the assay detected down to 10 "13 RML scrapie brain tissue dilutions (containing ⁇ 100ag_ of PrP-res) in 0.5 ml of plasma.
- Fig. 18 eQuIC detection of endogenous PrPres in plasma of scrapie positive wild type (WT) & GPI- mice.
- the assay detected endogenous PrP-res in plasma from a wild-type mouse and a transgenic mouse expressing only PrP-sen that lacks the glycophosphatidylinositol anchor (GPI ). No prions were detected in a plasma sample from a non-infected wild type normal mouse.
- GPI- mice glycophosphatidylinositol anchor
- nucleic and amino acid sequences listed are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822.
- nucleic acid sequences only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
- SEQ ID NO: 1 is an amino acid sequence of a recombinant Syrian golden hamster proteinase K-sensitive prion protein.
- SEQ ID NO: 2 is an amino acid sequence of a recombinant mouse (Prnp-a) proteinase K-sensitive prion protein.
- SEQ ID NO: 3 is an amino acid sequence of a recombinant human (129M) proteinase K-sensitive prion protein.
- SEQ ID NO: 4 is an amino acid sequence of a recombinant human (129V) proteinase K-sensitive prion protein.
- SEQ ID NO: 5 is an amino acid sequence of a recombinant bovine (6- octarepeat) proteinase K-sensitive prion protein.
- SEQ ID NO: 6 is an amino acid sequence of a recombinant ovine (136 A).
- SEQ ID NO: 7 is an amino acid sequence of a recombinant Deer (96G 132M 138S) proteinase K-sensitive prion protein.
- SEQ ID NO: 8 is an amino acid sequence of a full-length Syrian golden hamster proteinase K-sensitive prion protein.
- SEQ ID NO: 9 is an amino acid sequence of a full-length mouse (Prnp-a) proteinase K-sensitive prion protein.
- SEQ ID NO: 10 is an amino acid sequence of a full-length human (129M) proteinase K-sensitive prion protein.
- SEQ ID NO: 11 is an amino acid sequence of a full-length human (129V) proteinase K-sensitive prion protein.
- SEQ ID NO: 12 is an amino acid sequence of a full-length chimeric Hamster-Sheep (H-S) proteinase K-sensitive prion protein wherein residues 23-137 are of the Syrian hamster sequence and the remaining residues 138-231 were homologous to sheep residues 141-234 (R154,Q171 polymorph).
- H-S Hamster-Sheep
- the methods disclosed herein allow testing for prion contamination, diagnostics and/or surveillance in a number of biological samples, including blood, blood fractions, blood products, urine, nasal fluids, saliva, cerebral spinal fluid, feces, muscle biopsies, lymphoid tissues, skin samples, samples of tissues for transplantation, amongst others. These methods have medical and veterinary applications, and also can be used to test biotechnology products and environmental samples (such as water, soils, plants, landfills, sewage) and agriculture samples
- the presently disclosed methods can be used for prion-free herd/flock certification, such as in cattle, sheep, and cervids.
- the methods disclosed herein can also be used to detect spontaneous Creutzfeldt-Jacob disease.
- the cell culture based standard scrapie cell assay can also be used to measure infectivity levels by end-point and limiting dilution methods (Klohn et al., 2003).
- the SSCA offers several advantages over animal bioassays, but it still requires weeks to perform and has been limited to a few mouse-adapted scrapie strains.
- An analogous cell-based assay for cervid prions (designated CPCA) has also been reported (Bian et al., J Virol, 84, 8322-8326, 2010).
- the limitations of the animal bioassay, SSCA and CPCA mean that more practical assays for prion quantitation are needed.
- Fluorescence correlation spectroscopy can be used to detect femtomolar concentrations of PrP-res aggregates in cerebral spinal fluid (CSF) samples treated with fluorescently tagged antibodies (Bieschke et al., supra, 2000).
- CSF cerebral spinal fluid
- rPrP c fluorescently labeled recombinant PrP c
- synthetic prion protein aggregates allowed their ultra-sensitive detection by FACS analyses and a similar approach allowed the discrimination of sera of several BSE-infected and non-infected cattle (Trieschmann et al., 2005).
- the speed and practicality of PMC A assays has also been improved by the use of rPrP c (Atarashi et al., supra, 2007) and by substituting shaking for the sonication step as described for the quaking-induced conversion (QuIC) reactions (Atarashi et al., supra, 2008; Orru et al., supra, 2009).
- the standard QuIC (also called "SQ") assay can detect sub-femtogram amounts of PrP-res (less than one lethal intracerebral dose) in hamster brain homogenates (BH) within a single day.
- the effectiveness of the SQ for prion detection was demonstrated by its ability to discriminate normal from prion-infected hamsters using 2- ⁇ 1 samples of CSF
- the readout for SQ and PMCA assays is the detection of specific protease- resistant prion-seeded rPrP products by immunoblotting, which is difficult to adapt to automated high-throughput formats.
- An alternative, and potentially higher- throughput approach was used for the amyloid seeding assay (ASA) in which the fluorescent dye thioflavin T (ThT) was used to detect prion seeding of rPrP polymerization (Colby et al., Proc Natl Acad Sci U S A, 104, 20914-20919, 2007, incorporated herein by reference).
- the ASA can also detect protease sensitive disease-causing prions and has a 98% correlation with neuropathological signs of prion disease (Colby et al., PLoS Pathog, 6, el000736, 2010).
- a potentially confounding aspect of ASA is the frequent spontaneous formation of rPrP fibrils (without seeding by prions) within about twice the lag phase of prion- seeded reactions (Colby et al., Proc Natl Acad Sci U SA, 104, 20914-20919, 2007).
- RT-QuIC real-time-QuIC
- prion seeding activity was measured in the nasal fluids and CSF of prion- infected hamsters.
- the RTQ can rapidly determine relative prion concentrations with a sensitivity that rivals that of animal bioassays, but with greatly reduced time and cost.
- the presently described methods substantially improve the sensitivity and applicability of prion seeding/amplification assays such as the SQ and RTQ, in part by integrating them with novel prion/PrP-res/PrP Sc immunoprecipitation and treatment protocols.
- the methods enable the capture and detection of extremely low levels of prions in various fluids or tissue extracts, including complex biological specimens such as blood plasma, which can contain strong inhibitors of prions.
- Aggregate More than one molecule in association, such as dimers, multimers, and polymers of prion proteins, for instance aggregates, dimers, multimers, and polymers of PrP-res or rPrP-res (Sc) .
- Agitation Introducing any type of turbulence or motion into a mixture or reaction mix, for examples by sonication, stirring, or shaking.
- agitation includes the use of force sufficient to fragment rPrP-res (Sc) aggregates, which disperses rPrP-res (Sc) aggregates and/or polymers to facilitate further amplification.
- fragmentation includes complete fragmentation, whereas in other examples, fragmentation is only partial, for instance, a population of aggregates can be about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% fragmented by agitation.
- Exemplary agitation methods are described in the Examples section below.
- Antibody A polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen or a fragment thereof.
- An antibody can specifically bind PrP- res/PrP Sc .
- Antibodies can be composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V H ) region and the variable light (V L ) region. Together, the V R region and the V L region are responsible for binding the antigen recognized by the antibody.
- antibody includes intact immunoglobulins and the variants and portions of them well known in the art, such as Fab' fragments, F(ab)' 2 fragments, single chain Fv proteins ("scFv”), and disulfide stabilized Fv proteins ("dsFv").
- scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
- the term also includes genetically engineered forms such as chimeric antibodies (for example, humanized antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J.,
- a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
- H heavy chain
- L light chain
- ⁇ lambda
- k kappa
- IgM immunoglobulin heavy chain classes
- Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”).
- the heavy and the light chain variable regions specifically bind the antigen.
- Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called “complementarity-determining regions” or "CDRs".
- CDRs complementarity-determining regions
- the extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference).
- the Kabat database is now maintained online.
- the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
- the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
- the CDRs are primarily responsible for binding to an epitope of an antigen.
- the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
- a V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
- a V L CDRl is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
- An antibody that binds an antigen of interest has a specific V H region and the V L region sequence, and thus specific CDR sequences.
- Antibodies with different specificities due to different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).
- immunoglobulin heavy chain including that of an Fv, scFv, dsFv or Fab.
- V L refers to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
- a "monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected, or a progeny thereof. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
- Antibody binding affinity Affinity of an antibody for an antigen, such as PrP-res. In one embodiment, affinity is calculated by a modification of the
- binding affinity is measured by an antigen/antibody
- a high binding affinity is measured by a competition radioimmunoassay.
- a high binding affinity is at least about 1 x 10 - " 8 M.
- a high binding affinity is at least about 1.5 x 10 ⁇ 8 M, at least about 2.0 x 10 ⁇ 8 M, at least about 2.5 x 10 "8 M, at least about 3.0 x 10 "8 M, at least about 3.5 x 10 "8 M, at least about 4.0 x 10 "8 M, at least about 4.5 x 10 "8 M, or at least about 5.0 x 10 "8 M.
- Antigen A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal.
- An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
- the term "antigen” includes all related antigenic epitopes.
- Epitopes or “antigenic determinant” refers to a site on an antigen to which B and/or T-cells respond.
- T-cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule.
- Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.
- Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
- An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.
- An antigen can be a tissue- specific antigen, or a disease- specific antigen, such as PrP-res. These terms are not exclusive, as a tissue- specific antigen can also be a disease specific antigen.
- Conservative variant In the context of a prion protein, refers to a peptide or amino acid sequence that deviates from another amino acid sequence only in the substitution of one or several amino acids for amino acids having similar
- prion protein variants can have no more than 1, 2, 3, 4, 5, 10, 15, 30, 45, or more conservative amino acid changes.
- a conservative variant prion protein is one that functionally performs substantially like a similar base component, for instance, a prion protein having variations in the sequence as compared to a reference prion protein.
- a prion protein or a conservative variant of that prion protein will aggregate with PrP-res (or PrP Sc ), for instance, and will convert rPrP c to rPrP-res (Sc) (or will be converted to rPrP-res (Sc) ).
- the prion protein and the conservative variant prion protein do not have the same amino acid sequences.
- the conservative variant can have, for instance, one variation, two variations, three variations, four variations, or five or more variations in sequence, as long as the conservative variant is still complementary to the corresponding prion protein.
- a conservative variant prion protein includes one or more conservative amino acid substitutions compared to the prion protein from which it was derived, and yet retains prion protein biological activity.
- a conservative variant prion protein can retain at least 10% of the biological activity of the parent prion protein molecule from which it was derived, or alternatively, at least 20%, at least 30%, or at least 40%.
- a conservative variant prion protein retains at least 50% of the biological activity of the parent prion protein molecule from which it was derived.
- the conservative amino acid substitutions of a conservative variant prion protein can occur in any domain of the prion protein.
- Contacting includes in solution and solid phase, for example contacting a sample with a specific binding agent, such as an antibody that specifically binds PrP-res.
- Conditions sufficient to detect Any environment that permits the desired activity, for example, that permits an antibody to bind an antigen, such as PrP-res, and the interaction to be detected.
- such conditions include appropriate temperatures, buffer solutions, and detection means such as and digital imaging equipment.
- Detect To determine if an agent (such as a signal or protein, for example PrP-res) is present or absent. In some examples, this can further include
- quantification for example the quantification of the amount of PrP-res in a sample, such as a serum sample, or a fraction of a sample.
- Identifying the presence or nature of a pathologic condition such as, but not limited to, identifying the presence of PrP-res, such as in
- Creutzfeldt-Jacob disease Diagnostic methods differ in their sensitivity and specificity.
- the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives).
- the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
- Prognostic is the probability of development (for example severity) of a pathologic condition.
- Disaggregate To partially or complete disrupt an aggregate, such as an aggregate of PrP-res or rPrP-res (Sc) .
- Encode Any process whereby the information in a polymeric
- macromolecule or sequence is used to direct the production of a second molecule or sequence that is different from the first molecule or sequence.
- the term is construed broadly, and can have a variety of applications.
- the term "encode” describes the process of semi-conservative DNA replication, wherein one strand of a double- stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase.
- a DNA molecule can encode an RNA molecule (for instance, by the process of transcription incorporating a DNA-dependent RNA polymerase enzyme).
- an RNA molecule can encode a peptide, as in the process of translation.
- the term “encode” also extends to the triplet codon that encodes an amino acid.
- an RNA molecule can encode a DNA molecule, for instance, by the process of reverse transcription incorporating an RNA-dependent DNA polymerase.
- a DNA molecule can encode a peptide, where it is understood that "encode” as used in that case incorporates both the processes of transcription and translation.
- Fluorophore A chemical compound, which when excited by exposure to a particular stimulus, such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light).
- Fluorophores are part of the larger class of luminescent compounds.
- Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminesce, but rather use a chemical source of energy.
- chemiluminescent molecules such as aequorin
- an external source of electromagnetic radiation such as a laser
- fluorophores that can attached to antibodies that specifically binds PrP Sc are provided in U.S. Patent No. 5,866,366 to Nazarenko et ah, such as 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4- anilino-l-naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7- amino-4-trifluoromethylcouluar
- diethylenetriamine pentaacetate 4,4'-diisothiocyanatodihydro-stilbene-2,2'- disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5- carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-d
- Malachite Green isothiocyanate 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (CibacronTM Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of s
- TAMRA N,N,N',N'-tetramethyl-6-carboxyrhodamine
- HEX -6- carboxy-fluorescein
- TET Tetramethyl fluorescein
- ROX 6-carboxy-X-rhodamine
- Texas Red 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein
- JE Cy3, Cy5, VIC® (Applied Biosystems)
- LC Red 640 LC Red 705, Yakima yellow amongst others.
- fluorophores include those known to those skilled in the art, for example those available from Molecular Probes (Eugene, OR).
- a fluorophore is used as a donor fluorophore or as an acceptor
- a fluorophore is detectable label, such as a detectable label attached to an antibody.
- Immunoassay A biochemical test that measures the presence or
- a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from a subject
- concentration of a substance in a sample such as a biological sample, for example a serum sample obtained from
- IP Immunoprecipitation
- Isolated An "isolated" biological component, such as a peptide or assembly of polypeptides (for example PrP Sc ), cell, nucleic acid, or serum samples has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extrachromosomal DNA and RNA, and proteins.
- Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a cell as well as chemically synthesized peptide and nucleic acids.
- an isolated peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
- a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or even at least 99% of the peptide or protein concentration.
- Nucleic acid molecule A polymeric form of nucleotides, which can include both sense and anti sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
- a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide.
- a "nucleic acid molecule” as used herein is synonymous with "nucleic acid” and “polynucleotide.”
- a nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single and double stranded forms of DNA.
- a nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non naturally occurring nucleotide linkages.
- Prions A type of infectious agent composed mainly of protein. Prions cause a number of diseases in a variety of animals, including bovine spongiform encephalopathy (BSE, also known as mad cow disease) in cattle and Creutzfeldt- Jakob disease in humans. All known prion diseases affect the structure of the brain or other neural tissue, and all are untreatable and fatal.
- BSEs bovine spongiform encephalopathy
- TSEs transmissible spongiform encephalopathies
- TSEs transmissible spongiform encephalopathies
- TSEs human Creutzfeldt- Jakob disease
- BSE bovine spongiform encephalopathy
- sheep scrapie sheep scrapie
- TAE transmissible mink encephalopathy
- Prions are believed to infect and propagate by refolding abnormally into a structure that is able to convert normal molecules of the protein into the abnormally structured forms (for instance, PrP Sc in scrapie or PrP vCJD in variant CJD), which are usually partially resistant to proteinase K digestion, and hence will be designated generically herein as PrP-res for PrP-resistant.
- PrP Sc in scrapie or PrP vCJD in variant CJD
- PrP-res for PrP-resistant.
- Most, if not all, known prions can polymerize into amyloid fibrils rich in tightly packed beta sheets. This altered structure renders them unusually resistant to denaturation by chemical and physical agents, making disposal and containment of these particles difficult.
- PrP-res the pathological, typically protease-resistant form of prion protein, PrP-res, appears to propagate itself in infected hosts by inducing the conversion of its normal host-encoded protease-sensitive precursor, PrP-sen or PrP , which is sensitive to proteinase K digestion, into PrP-res.
- PrP-sen PrP
- PrP-sen is a monomelic glycophosphatidylinositol-linked glycoprotein that is low in ⁇ -sheet content, and highly protease-sensitive.
- PrP-res e.g. PrP Sc
- aggregates are high in ⁇ -sheet content and partially protease-resistant.
- the recombination protein, rPrP-res (Sc) is a generic term for the prion-induced rPrP conversion product, regardless of the species and strain of origin of the prions.
- the recombinant protein, rPrP-res (Sc) is not infectious.
- PMCA or Protein Misfolding Cyclic Amplification A method for amplifying PrP-res in a sample by mixing PrP with the sample, incubating the reaction mix to permit PrP-res to initiate the conversion of PrP to aggregates of
- PrP-res fragmenting any aggregates formed during the incubation step (typically by sonication), and repeating one or more cycles of the incubation and fragmentation steps.
- Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred.
- polypeptide or protein as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
- polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
- polypeptide fragment refers to a portion of a polypeptide which exhibits at least one useful epitope.
- polypeptide refers to all fragments of a polypeptide that retain an activity of the polypeptide.
- Biologically functional fragments for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
- QuIC or Quaking Induced Conversion A particular type of PrP amplification assay, in which shaking of the reaction vessels is performed instead of sonication to disrupt aggregated rPrP c and rPrP-res (Sc) .
- Real Time (RT)-QuIC An assay that includes intermittent shaking to disrupt aggregated PrP and PrP-res and includes the use of a fluorescent readout, such as the fluorescent dye thioflavin T (ThT).
- a fluorescent readout such as the fluorescent dye thioflavin T (ThT).
- Exemplary protocols are disclosed, for example, in Wilham et al., PLOS Pathog. 6(12): el001217, pages 1-15.
- this assay uses PrP as a substrate, intermittently shaken reactions, predominantly detergent-free (such as ⁇ 0.002% of SDS) or detergent-free, and chaotrope-free reactions conditions, and ThT-based fluorescent detections of prion seeded rPrP amyloid fibrils.
- Sample A biological sample obtained from a subject, such as a human or veterinary subject, which contains for example nucleic acids and/or proteins.
- biological samples include all clinical samples useful for detection of PrP-res/prions in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; extracted galls; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrow aspirates.
- the biological sample is obtained from a subject, such as in the form of a blood sample, such as serum sample.
- Samples also include environmental samples, such as soil or water samples.
- Sequence identity The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences. Methods for aligning sequences for comparison are described in detail below, in section IV E of the Detailed Description.
- rPrP-res can be amplified in a sample, by mixing the sample with purified rPrP to make a reaction mix; performing an amplification reaction that includes (i) incubating the reaction mix to permit coaggregation of the rPrP with the PrP-res that may be present in the reaction mix, and maintaining incubation conditions that promote coaggregation of the rPrP with the PrP-res and results in a conversion of the rPrP c to rPrP-res (Sc) while inhibiting development of rPrP-res (spon) (protease-resistant rPrP products that are generated spontaneously in the absence of prions or PrP-res) (ii) agitating aggregates formed during step (i); (iii) optionally repeating steps (i) and (ii) one or more times.
- rPrP-res is detected in the reaction mix, wherein detection of rPrP-res (Sc) in the reaction mix indicates that PrP-res was present in the sample.
- Additional substrate rPrP
- rPrP can be added during the reaction, such as during the lag phase (between the addition of the sample and the formation of detectable of rPrP-res (Sc) ).
- a portion of the reaction mix is not removed and incubated with additional rPrP in a separate reaction mixture.
- Sonication The process of disrupting or dispersing biological materials using sound wave energy.
- Specific binding agent An agent that binds substantially only to a defined target.
- a specific binding agent is an antibody that specifically binds PrP-res but not PrP .
- the term "specifically binds” refers to the preferential association of an antibody or other ligand, in whole or part, with an antigen. Specific binding may be distinguished as mediated through specific recognition of the antigen. Although selectively reactive antibodies bind antigen, they may do so with low affinity. On the other hand, specific binding results in a much stronger association between the antibody (or other ligand) and antigen (or cells bearing the antigen) than between the bound antibody (or other ligand) and another protein (or cells lacking the antigen).
- Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody or other ligand (per unit time) to a cell or tissue expressing the target epitope as compared to a cell or tissue lacking this epitope.
- a variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein.
- solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
- affinity purification such as
- a quaking induced conversion assay (QuIC) or a Real-Time quaking induced conversion assay (RT-QuIC) to detect prions in a sample, such as a biological sample.
- QIC quaking induced conversion assay
- RT-QuIC Real-Time quaking induced conversion assay
- biotechnology products and environmental samples such as water, soils, plants, landfills, sewage
- agriculture samples such as animal-based foods, animal-based feeds & nutritional supplements, animal waste products, byproducts, carcasses, slaughterhouse wastes, specified risk materials
- the presently disclosed methods also can be used for prion-free herd/flock certification, such as in cattle, sheep, and cervids.
- affinity purification such as immunopreciptiation
- QuIC or RT-QuIC provide an unexpectedly superior sensitivity and specificity for the detection of PrP-res.
- TSEs The transmissible spongiform encephalopathies
- BSE bovine spongiform encephalopathy
- TAE transmissible mink encephalopathy
- CWD chronic wasting disease
- EUE exotic ungulate encephalopathy
- CJD Creutzfeldt-Jakob disease
- iCJD iatrogenic Creutzfeldt-Jakob disease
- vCJD variant Creutzfeldt-Jakob disease
- fCJD familial Creutzfeldt- Jakob disease
- sCJD sporadic Creutzfeldt-Jakob disease
- TSEs have incubation periods of months to years, but after the appearance of clinical signs often are rapidly progressive, untreatable, and invariably fatal. Attempts at TSE risk reduction have led to profound changes in the production and trade of agricultural goods, medicines, cosmetics, and biotechnology products.
- PrP Sc or PrP-res appears to propagate itself in infected hosts by inducing the conversion of its normal host-encoded precursor, PrP-sen, also known as PrP , into PrP-res.
- PrP is a monomeric glycophosphatidylinositol-linked glycoprotein that is low in ⁇ -sheet content, and highly protease-sensitive. Conversely, PrP-res aggregates are high in ⁇ - sheet content and partially protease-resistant. Mechanistic details of the conversion are not well understood, but involve direct interaction between PrP-res and PrP c , resulting in conformational changes in PrP c as the latter is recruited into the growing PrP-res multimer (reviewed in Caughey & Baron
- PrP-res and prion infectivity also could be amplified using PrP purified from brain tissue as long as polyanions such as RNA were added (Deleault et al., (2007) Proc Natl Acad Sci C/SA.104(23):9741-6).
- PrP c Recombinant PrP c (rPRP c , also called rPrP-sen) from E. coli lacks glycosylation and the GPI anchor can be induced to polymerize into amyloid fibrils spontaneously or when seeded by preformed rPrP fibrils.
- TSE infectivity (prions) by rapid methods.
- the most commonly used marker for TSE infections is PrP-res, and the PMCA reaction allows extremely sensitive detection of PrP-res at levels below single infectious units in infected tissue.
- vCJD affects young patients with an average age of 27 years, and causes a relatively long illness (14 months compared with 4.5 months for sCJD). Because of insufficient information available about the incubation time and the levels of exposure to contaminated cattle food products, it is difficult to predict the future incidence of vCJD. In animals, there is little evidence for inherited forms of the disease, and most cases appear to be acquired by horizontal or vertical transmission.
- the clinical diagnosis of sCJD is based on a combination of rapidly progressive multifocal dementia with pyramidal and extrapyramidal signs, myoclonus, and visual or cerebellar signs, associated with a characteristic periodic electroencephalogram (EEG).
- EEG periodic electroencephalogram
- a key diagnostic feature of sCJD that distinguishes it from Alzheimer's disease and other dementias is the rapid progression of clinical symptoms and the short duration of the disease, which is often less than 2 years.
- the clinical manifestation of fCJD is very similar, except that the disease onset is slightly earlier than in sCJD.
- Family history of inherited CJD or genetic screening for mutations in the prion protein gene are used to establish fCJD diagnosis, although lack of family history does not exclude an inherited origin.
- Variant CJD appears initially as a progressive neuropsychiatric disorder characterized by symptoms of anxiety, depression, apathy, withdrawal and delusions, combined with persistent painful sensory symptoms and followed by ataxia, myoclonus, and dementia. Variant CJD is differentiated from sCJD by the duration of illness (usually longer than 6 months) and EEG analysis (vCJD does not show the atypical pattern observed in sCJD). A high bilateral pulvinar signal noted during
- MRI is often used to help diagnose vCJD.
- a tonsil biopsy can be used to help diagnose vCJD, based on a number of cases of vCJD have been shown to test positive for PrP vCJD staining in lymphoid tissue (such as tonsil and appendix).
- GSS is a dominantly inherited illness that is characterized by dementia, Parkinsonian symptoms, and a relatively long duration (typically, 5-8 years).
- GSS is similar to Alzheimer's disease, except that is often accompanied by ataxia and seizures. Diagnosis is established by clinical examination and genetic screening for prion protein mutations. FFI is also dominantly inherited and associated with prion protein mutations. However, the major clinical finding associated with FFI is insomnia, followed at late stages by myoclonus,
- the present disclosure provides an assay system that uses immunopreciptiation, followed by a second detection method, such as QuIC and RT-QuIC, to provide a sensitive and specific method for detecting prions.
- a second detection method such as QuIC and RT-QuIC
- pre-emptive replenishment of rPrPC substrate in the QuIC or RT-QuIC assay is utilized.
- this combined assay provides unanticipated increases in sensitivity, and surprising reductions in assay time.
- the methods disclosed herein include contacting a sample, such as a biological sample, with an antibody that specifically binds only the disease related conformation of a prion protein (e.g. PrP Sc , PrP ⁇ 0 or PrP-res).
- a sample such as a biological sample
- the sample is contacted with a capture-monoclonal antibody (or epitope-binding fragment thereof), which can be immobilized on a solid substrate.
- Monoclonal antibodies can be selected that specifically bind an epitope that is expressed on PrP-res, but not on PrP .
- the monoclonal antibodies that specifically bind PrP-res or PrP Sc can be from any species, such as murine antibodies.
- the monoclonal antibodies can be produced by known monoclonal antibody production techniques. Typically, monoclonal antibodies are prepared by recovering spleen cells from immunized animals with the protein of interest and immortalizing the cells in conventional fashion, for example, by fusion with myeloma cells or by Epstein-Barr virus transformation, and screening for clones expressing the desired antibody. See, for example, Kohler and Milstein Eur. J. Immunol. 6:511 (1976). Monoclonal antibodies, or the epitope-binding region of a monoclonal antibody, may
- chimeric or humanized forms of a monoclonal antibody are utilized, wherein the antibody of use includes the complementarity determining regions (CDRs) of an antibody that specifically binds PrP-res or PrP Sc .
- CDRs complementarity determining regions
- the monoclonal antibody can be a murine monoclonal antibody that is generated by immunizing "knock out” mice with recombinant normal mouse cellular protein (PrP ). Spleen cells (antibody producing lymphocytes of limited life span) from the immunized mice can then be fused with non-producing myeloma cells (tumor lymphocytes that are "immortal") to create hybridomas. The hybridomas can then be screened for the production of antibody specific to PrP-res or PrP Sc and the ability to be propagated in tissue culture.
- PrP normal mouse cellular protein
- hybridomas can then be cultured to provide a permanent and stable source for the specific monoclonal antibodies.
- monoclonal antibodies produced by this method are disclosed in U.S. Pat. No. 6,528,269.
- These monoclonal antibodies include 2F8, 5B2, 6H3, 8C6, 8H4 and 9H7 produced by cell lines PrP2F8, PrP5B2, PrP6H3, PrP8C6, PrP8H4 and PrP9H7, that can specifically bind human PrP-res, and also bind PrP-res from mouse, cow, sheep and other species, see also U.S. Published Patent Application No. 2005/0118720, which is incorporated herein by reference.
- the methods disclosed herein can also utilize monoclonal antibody 15B3, which is described in U.S. Published Patent Application No. 2008/0220447, published September 1, 2008, which is incorporated herein by reference.
- the antibody 15B3 is available from Prionics AG, Zurich, Switzerland and methods to generate this antibody are disclosed in PCT Publication No. WO 98/37210, which is incorporated herein by reference.
- This PCT Publication also describes antibodies that bind PrP-res but not PrP c .
- PCT Publication No. WO 98/37210 discloses that hybridomas that produce antibody 15B3 were deposited in accordance with the Budapest treaty at DSMZ— Deutsche Sammlung von Mikroorganismen und
- the IgM monoclonal antibody 15B3 specifically recognizes the disease- associated form of the prion protein (i.e., PrP-res or PrP Sc ) and is capable of detecting abnormal PrP in brain homogenates without the need of PK digestion (Korth et al., Nature 1997; 390:74-77, 1997, herein incorporated by reference).
- the antibody 15B3 was shown to bind to protease- sensitive forms of PrP Sc in a transgenic mouse model of Gerstmann Straussler Scheinker syndrome which is of considerable importance as it was shown that infectivity in blood is sensitive to protease digestion (Nazor et al., EMBO J. 24(13):2472-80, 2005; Yakovleva et al., Transfusion 44: 1700-5, 2004).
- the capture-monoclonal antibody (such as 15B3, Ig 261, Ig W226 or 262) can be immobilized on a solid phase by insolubilizing the capture-monoclonal antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (U.S. Pat. No. 3,720,760, herein incorporated by reference in its entirety) or non-covalent or covalent coupling, for example, using glutaraldehyde or
- the solid phase used for immobilization may be any inert support or carrier that is essentially water insoluble and useful in immunometric assays, including supports in the form of, for example, surfaces, particles, porous matrices, sepharose, etc.
- supports in the form of, for example, surfaces, particles, porous matrices, sepharose, etc.
- commonly used supports include small sheets, Sephadex, polyvinyl chloride, plastic beads, magnetic beads, and assay plates or test tubes manufactured from polyethylene, polypropylene, polystyrene, and the like including 96-well microtiter plates and 384-well microtiter well pates, as well as particulate materials, such as filter paper, agarose, cross-linked dextran, and other
- the immobilized capture-monoclonal antibodies are coated on a microtiter plate, and in particular the solid phase can be a multi-well microtiter plate.
- the multi-well microtiter plate can be a microtest 96-well ELISA plate.
- the solid phase can be a magnetic bead, such as DYNABEADS® (Invitrogen) or other magnetic beads, such as those available from NEW ENGLAND BIOLABS® or DYNAL® magnetic beads.
- the capture-monoclonal antibody (such as, but not limited to,
- the solid substrate can also have an antibody, such as a rabbit anti-mouse antibody or a rabbit anti-human antibody covalently linked to the solid substrate.
- the antibody attached to the solid substrate can then be incubated with a second antibody of interest (such as a mouse or human antibody) to achieve attachment of the second antibody to the solid substrate.
- a rabbit anti-mouse antibody is coupled to the solid substrate, which is then incubated with a second antibody that specifically binds a prion protein, such as, but not limited to, 15B3, IgG W226 or IgG 261.
- cross-linking agents for attaching a capture-monoclonal antibody to the solid phase substrate include, for example l,l-bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, esters with 4- azido salicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-l,8-octane.
- Derivatizing agents, such as methyl-3-[(p- azidophenyl)dithio]propioimidate yield photoactivatable intermediates capable of forming cross-links in the presence of light.
- micro-titer well plates e.g., 96-well plates or 384- well plates
- they can be coated with affinity purified capture monoclonal antibodies (typically diluted in a buffer) at, for example, room temperature and for about 2 to about 3 hours.
- the plates can also be coated with the antibody that specifically binds PrP- res or PrP Sc directly.
- the plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.
- the beads can be coated with the antibody using any procedures known in the art.
- the DYNABEADS® are suspended in a vial using vortexing, and then an appropriate amount of the
- DYNABEADS® is moved to a polypropylene or polystyrene tube.
- the tube is placed on a magnet for a short period of time, and then removed from the magnet.
- a coating buffer is added, and the beads are mixed, such as by using a vortex.
- a coating buffer comprising about 0.01 to 1%, such as about 0.1% bovine serum albumin in phosphate buffered saline is utilized.
- additional blocking agents for the coating buffer might include, but are not limited to egg albumin, casein, and non-fat milk.
- the antibody of interest is added (such as, but not limited to, 15B3, IgG W226 or IgG 261), and the DYNABEADS® are incubated with the antibody of interest with gentle mixing for a sufficient time for the antibody to adhere to the beads.
- a magnet can then be used to separate the coated beads from the supernatant, and a coating buffer can be added.
- the antibody can be coupled to the substrate as about 5 ⁇ g antibody per 100 ⁇ DYNABEADS®.
- the antibody (such as, but not limited to, 15B3) can be coupled to the substrate as per lxlO "6 DYNABEADS® per ⁇ g of 15B3 antibody.
- 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or 30-fold more antibody can be utilized, such as 30-50 ⁇ g, such as 36 ⁇ g of antibody, for example 15B3 per 100 ⁇ DYNABEADS® (for example, 4 x 10 8 beads/ml).
- lng to 10 ⁇ g of antibody can be used for 1 x 10 beads.
- 100-300 ⁇ g of antibody for example 15B3 per 1 x 10 ⁇ 8 DYNABEADS® (for example, 4 xlO 8 beads/ml) can be utilized.
- the concentration of the antibody on the magnetic beads is about 10-500 ⁇ g of 15B3 per 1 x 10 beads.
- Coated plates or beads optionally can be treated with a blocking agent that binds non-specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
- a blocking agent that binds non-specifically to and saturates the binding sites to prevent unwanted binding of the free ligand to the excess sites on the wells of the plate.
- appropriate blocking agents for this purpose include gelatin, bovine serum albumin, egg albumin, casein, and non-fat milk.
- a sample to be analyzed is added to the immobilized antibody.
- the sample can be a biological sample or an environmental sample.
- the sample can be homogenized (such as for a tissue sample, such as a brain sample), and appropriately diluted with, for example, a lysis buffer (e.g., phosphate-buffered saline (PBS) with 1% Nonidet P-40, 0.5% sodium deoxycholate, 5 mM EDTA, and pH 8.0).
- PBS phosphate-buffered saline
- Other detergents can be used, such as anioinic, cationic or non-ionic detergents, including but not limited to sodium dodecyl sulfated (SDS) to homogenize a sample.
- SDS sodium dodecyl sulfated
- the biological sample can be a blood, serum, plasma, or a sample of another biological fluid, such as, but not limited to cerebral spinal fluid or nasal fluid.
- the sample can be a tissue sample, such as a brain sample or a lymphoid tissue sample (such as tonsils).
- the sample can be diluted, such as in buffer, for example a buffer including bovine serum albumin.
- the sample is diluted in a buffer, such as tris buffered saline (TBS) or phosphate buffered saline (PBS), optionally including a detergent.
- TBS tris buffered saline
- PBS phosphate buffered saline
- the detergent can be a cationic, anionic or non-ionic detergent.
- the detergent is Sarkosyl.
- the beads can be contacted with the sample in the presence of about 0.1% to about 1%, such as about 0.4 % Sarkosyl in TBS.
- the beads can be contacted with the sample in the presence of about 0.1% to about 1% Sarkosyl in TBS, such as 0.4% to about 1% Sarkosyl in a buffer, such as TBS or PBS.
- about 0.1%, about 0.4%, about 1%, about 2%, about 3% or about 4% Sarkosyl in a buffer, such as TBS or PBS is utilized.
- the amount of sample added to the immobilized capture monoclonal antibody can be such that the immobilized capture monoclonal antibodies are in molar excess of the maximum molar concentration of the conformational altered protein anticipated in the biological sample after appropriate dilution of the sample.
- the conditions for incubation of the biological sample and immobilized monoclonal antibody are selected to maximize sensitivity of the assay and to minimize dissociation.
- the incubation is accomplished at fairly constant temperatures, ranging from about 0° C to about 40° C, such as at about 4 °C, room temperature (e.g., about 25° C), about 35 °C to about 39 °C, or at about 37 °C, or about 35 °C to 40 °C. In some embodiments he temperature is about 19 to about 40 °C, such as at room temperature.
- the time for incubation can be for example, 2 hours to 12 hours, such as overnight.
- the incubation period is 2, 4, 6, 8, 10, 12, 20 or 24 hours, for example overnight at about 0° C to about 40° C, such as at about 4 °C, room temperature (e.g., about 25° C), or 37 °C.
- the incubation is about 2 hours at room temperature or overnight at 4 °C, such as about 12 hours at 4 °C or for about 10 to 20 hours at room temperature, such as 20 hours at room temperature or 37 °C.
- the washing medium is generally a buffer (“washing buffer”) with a pH determined using the considerations and buffers typically used for the incubation step.
- the washing may be done, for example, one, two, three or more times.
- the washing can be performed at any temperature, such as from about 0°C to about 40° C, such as at room temperature (e.g., 25 °C) or at 37 °C.
- the method comprises using SDS in a buffer, such as 0.01% to 0.1% SDS, such as about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06% or 0.07% SDS, for example 0.04% to 0.06% SDS, such as about 0.05% SDS.
- washing buffers include, but are not limited to, phosphate buffered saline (PBS) and Tris buffered saline (TBS), optionally including Sarkosyl, such as about 0.05-0.5% Sarkosyl, such as 0.1%, 0.2%, 0.3% OR 0.4% Sarkosyl.
- PBS phosphate buffered saline
- TBS Tris buffered saline
- Sarkosyl such as about 0.05-0.5% Sarkosyl, such as 0.1%, 0.2%, 0.3% OR 0.4% Sarkosyl.
- One exemplary washing buffer is 0.2% Sarkosyl in TBS.
- the solid substrate such as magnetic beads that have been contacted with the sample, can then be processed to detect bound prion protein, such as using a Standard QuIC (SQ) reaction or a real-time QuIC (RTQ) reaction, as discussed below.
- prion proteins e.g. PrP Sc
- the reaction mix including both the solid substrate comprising the antibody and the prion proteins are directly used in an assay to detect PrP-res or PrP Sc , such as, but not limited to, SQ or RTQ.
- the immune complexes comprising the antibody that specifically binds PrP-res are not separated from the reaction mixture, but used directly in a SQ or RTQ assay.
- PMCA protein misfolding cyclic amplification
- incubation and sonication are alternated over a period of approximately three weeks, and intermittently a portion of the reaction mix is removed and incubated with additional PrP in order to serially amplify the PrP-res in the sample. Following the repeated incubation/sonication/dilution steps, the resulting PrP-res is detected in the reaction mix.
- brain extract-based PMCA is a very sensitive assay for detecting PrP-res, it has a number of limitations, notably the time required to achieve optimal sensitivity (2-3 weeks) and the use of brain-derived PrP as the amplification substrate. This method also uses sonication.
- PrP-res amplification methods that utilize rPrP , one that uses sonication (rPrP-PMCA) (Atarashi et al., (2007) Nat Methods, 4, 645-650) and one that utilizes shaking (QuIC) (Atarashi et al., (2008) Nat Methods, 5, 211- 212). These methods facilitate fundamental studies of the structure and conversion mechanism of PrP-res. Site-directed mutations can allow precise labeling of rPrP with a variety of probes that can report on conformational changes, and both inter- molecular and intra-molecular distances within rPrP-res (Sc) aggregates.
- RTQ allows detection of the amplification product using thioflavin T (ThT).
- ThT thioflavin T
- rPrP is preemptively replenished before much detectable (ThT- positive) polymerization has occurred (such as before 24 hours of incubation), while retaining the existing rPrP-res (Sc) .
- the QuIC and RT-QuIC methods generally involve mixing a sample (for example a tissue sample, CSF sample, or plasma sample that is suspected of containing prions or PrP-res) with purified rPrP to make a reaction mix, and performing a primary reaction to form and amplify specific forms of rPrP-res (Sc) in the mixture.
- a sample for example a tissue sample, CSF sample, or plasma sample that is suspected of containing prions or PrP-res
- Sc rPrP-res
- This primary reaction includes incubating the reaction mix to permit the PrP-res to initiate the conversion of rPrP to specific aggregates or polymers of rPrP-res (Sc) ; fragmenting any aggregates or polymers formed during the incubation step; and repeating the incubation and fragmentation steps one or more times, for instance from about 1 to 2 times, 1 to 4 times, 1 to 10 times, or 10 to about 50 times.
- serial amplification is carried out by removing a portion of the reaction mix and incubating it with additional rPrP .
- additional rPrP is added to the reaction, such as during the lag phase (prior to the formation of detectable rPrP-res (Sc) , such as prior to 24 hours of the reaction), and the incubation and fragmentation steps are repeated.
- the method is performed without serial
- PrP-res can be amplified in a sample, by mixing the sample with purified rPrP to make a reaction mix; performing an amplification reaction that includes (i) incubating the reaction mix to permit coaggregation of the rPrP with the PrP- res/PrP Sc that may be present in the reaction mix, and maintaining incubation conditions that promote coaggregation of the rPrP with the PrP-res and results in a conversion of the rPrP c to rPrP-res (Sc) while inhibiting development of rPrP-res (spon) ; (ii) agitating aggregates formed during step (i); (iii) optionally repeating steps (i) and (ii) one or more times.
- rPrP-res is detected in the reaction mix, wherein detection of rPrP-res (Sc) in the reaction mix indicates that PrP-res was present in the sample.
- Additional substrate rPrP
- rPrP can be added during the reaction, such as during the lag phase between the addition of the sample and the detection of rPrP-res (Sc) formation.
- the rPrP c can be replenished by adding additional rPrP c substrate to the reaction mix.
- the reaction includes the use of shaking in the absence of sonication (the QuIC reaction), and the use of cycles of shaking/rest that are about 1: 1 in duration.
- the reaction alternates 60 seconds of shaking and 60 seconds of no shaking (rest).
- the reaction alternates 30 seconds of shaking and 30 seconds of no shaking (rest).
- the times can be varied, such as 45 seconds of shaking and 45 seconds of no shaking or 70 seconds of shaking and 70 seconds of no shaking.
- the period of rest and the period of shaking are equal.
- the period of rest and the period of shaking are about 120 seconds in length for the total cycle.
- the reaction includes or 90 seconds of shaking and 30 seconds of no shaking, or 100 seconds of shaking and 20 seconds of no shaking, or 80 seconds of shaking and 40 seconds of rest.
- the total cycle time is about 60, 70, 80, 90, 100, 110 or 120 seconds in length and includes at least 30 seconds, at least 40, or at least 50 seconds of shaking.
- Reactions have also been found to be particularly efficient at 37-60° C, for example 45-55° C, such as about 50° C, or at about 42°C to 46°C. These conditions are particularly effective at promoting the formation of rPrP-res (Sc) (notably the 17 kDa PK-resistant species), while reducing rPrP-res (spon) formation within the first 24 hours of unseeded reactions.
- the reaction can be performed for 3 to 12 hours, such as 6 to 12 hours, such as 8 to 10 hours.
- longer amplification reactions of 14 hours, 16 hours, 20 hours, 24 hours, such as at least 45 hours, 48 hours or even 65 or 96 hours can also provide excellent results, depending on the reaction temperature.
- the reaction is performed for 3 to 96 hours.
- the reaction can be performed for no more than 12 hours, no more than 24 hours, no more than 36 hours, no more than 48 hours, no more than 72 hours, no more than 96 hours or no more than 120 hours. In some examples the reaction is performed from about 5 hours to about 120 hours.
- the reaction is performed using sodium chloride (NaCl) at a concentration of 100 mM to 500mM, such as about 100 mM, 200 mM, 300 mM, 400 mM NaCl. In other embodiments, the reaction is performed using 200 to 400 mM NaCl.
- NaCl sodium chloride
- ThT is used to detect rPrP-res (Sc) .
- the solid substrate is a bead, such as magnetic beads, the beads and any associated prions or prion- induced RTQ conversion products tend to cling to the bottom of reaction vessel, such as a well.
- the reaction fluid can easily be changed, and the substrate replenished in its pre-RTQ state, without removing many beads or bound reaction products from the well.
- the rPrP substrate can be replenished preemptively during the lag phase, such as before ThT positivity indicated much consumption by conversion to prion- seeded amyloid product.
- IP-RTQ is highly sensitive, such that the overall sensitivity of the RTQ was increased by at least 1000- fold and overall reaction time is greatly reduced.
- the concentration of substrate is generally 0.1 mg/ml.
- QuIC reaction can be an RT-QuIC reaction, and thus can include thioflavin T (ThT) which allows detection of the rPrP-res (Sc) .
- the RT-QuIC assay incorporates rPrP c as a substrate, intermittent shaking of the reactions such as in 96- well plates, detergent- and chao trope-free reaction conditions and ThT-based fluorescence detection of prion-seeded rPrP c amyloid fibrils.
- ThT thioflavin T
- Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils (see Khurana et al., J. Structural Biol. 151: 229-238, 2005), and is commonly used to detect amyloid fibrils.
- the prion-initiated rPrP-res ( c) in the reaction mix is detected.
- ThT is included in the reaction (RT-QuIC)
- rPrP-res (Sc) can be detected using fluorescence at 450 +/- 10 nM excitation and 480 +/- 10 nm emission (see for example, Wilham et al., PLOS Pathogens 6(12): 1-15, 2010, incorporated herein by reference.)
- ThT can be included directly in the amplification mixture.
- the reaction mix does not include chaotropes or detergents.
- the reaction mix does not include chaotropic agents or detergents that can alter the rPrP-res (Sc) -sensitivity of ThT.
- the final concentration of ThT in each reaction is 1 mM.
- ThT is used at a final concentration of about 0.1 to 1 mM in the reaction.
- the sodium chloride (NaCl) concentration can be varied in the reaction.
- a concentration of about 200-400 mM NaCl allows sensitive detection of hamster, sheep, deer and human PrP-res while reducing the incidence of spontaneous conversion of the substrate.
- detergent at a concentration of greater than 0.002% is not included in an RTQ reaction.
- PrP-res can be detected by means other than ThT fluorescence, for example, using an antibody (see below).
- the reaction mix is digested with proteinase K (which digests the remaining rPrP in the reaction mix) prior to detection of the rPrP-res (Sc) .
- proteinase K which digests the remaining rPrP in the reaction mix
- Sc rPrP-res
- Two types of mis-folded prion protein can be generated in QuIC reactions, one occurring spontaneously (rPrP- res (spon) ) and the other initiated by the presence of prions (rPrP-res (Sc) ) in the test sample.
- RT-QuIC which includes thioflavin T
- optimal conditions that support specific prion/PrP-res-seeded SQ include the use of a detergent, such as an ionic and/or a non-ioinic detergent.
- a detergent such as an ionic and/or a non-ioinic detergent.
- the conditions can include the use of about 0.05- 0.1% of an ionic detergent, such as SDS.
- the conditions also can include the use of about 0.05-0.1% of a nonionic detergent such as TX-100 in the reaction mixture;
- PrP substrate it has also been found that the SQ and RTQ assays can perform cross-species amplification of target PrP-res.
- rHaPrP and chimeric rPrP can be used in SQ and RTQ reactions for amplification of human prions.
- rHaPrP appears to have a structure that promotes the formation of these aggregates with minimal formation of rPrP-res (spon) byproduct.
- rHaPrP and chimeras including HaPrP components, can be used to amplify target PrP-res in a sample taken from a species other than a hamster, such as a sample taken from a human, sheep, cow or cervid.
- the PrP in used in the reaction can be recombinant prion protein, for example prion protein from cells engineered to over express the protein. Any prion protein sequence can be used to generate the rPrP , for instance: Xenopus laevis (GENBANK® Accession No: NP001082180), Bos Taurus (GENBANK®
- rPrP c only a partial prion protein sequence is expressed as rPrP c .
- rPrP c includes amino acids 23-231 (SEQ ID NOS: 1, 2) of the hamster (SEQ ID NO: 8) or mouse (SEQ ID NO: 9) prion protein sequences, or the corresponding amino acids of other prion protein sequences, for instance amino acids 23-231 (SEQ ID NO: 3)of human (129M) prion protein (SEQ ID NO: 10), amino acids 23-231 (SEQ ID NO: 4) of human (129V) prion protein (SEQ ID NO: 11), amino acids 25-241 (SEQ ID NO: 5) of bovine (6-octarepeat) prion protein, amino acids 25-233 (SEQ ID NO: 6) of ovine (136A 154R 171Q) prion protein, or amino acids 25-234 (SEQ ID NO: 7) of deer (96G 132M 138S) prion
- the partial prion protein sequence expressed as rPrP can correspond to the polypeptide sequences of the natural mature full-length PrP molecule, meaning that the rPrP polypeptide lacks both the amino-terminal signal sequence and carboxy-terminal glycophosphatidylinositol-anchor attachment sequence.
- amino acids 30-231 , 40-231 , 50-231 , 60-231 , 70-231 , 80-231 , or 90-231 of any one of human, human 129V, bovine, ovine, or deer are utilized in the assays described herein.
- One of skill in the art can readily produce these
- polypeptides using the sequence information provided in SEQ ID NOs: 1-11, or using information available in GENBANK® (as available on July 20, 2007).
- the rPrP c can be a chimeric rPrP c , wherein a portion of the protein is from one species, and a portion of the protein is from from another species, can also be utilized.
- about 10 to about 90%, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% about 80% or about 90% of the rPrP is from one species, and, correspondingly, about 90%, about 80%, about70%, about 60%, about 50%, about 40%, about 30%, about 20% or about
- Chimeric proteins can include, for example, hamster rPrP c and rPrP c from another species, such as human PrP c .
- the chimeric protein includes hamster PrP c and sheep PrP c .
- a chimeric hamster- sheep rPrP construct can be used, such as, but not limited to, for the detection of human vCJD prions.
- a chimeric rPrP c (designated Ha-S PrP c ) is used, wherein the chimeric molecule includes residues 23-137 were of the Syrian hamster sequence and the remaining residues 138-231 were homologous to sheep residues 141-234 (R154.Q171 polymorph).
- host cells are transformed with a nucleic acid vector that expresses the rPrP , for example human, cow, sheep or hamster rPrP , or a chimeric form thereof.
- a nucleic acid vector that expresses the rPrP , for example human, cow, sheep or hamster rPrP , or a chimeric form thereof.
- These cells can be mammalian cells, bacterial cells, yeast cells, insect cells, or whole organisms, such as transgenic mice.
- Other cells also can serve as sources of the PrP .
- the cell is a bacterial cell, such as an E. coli cell.
- Purified rPrP c from rPrP c expressing cells or, in some cases, raw cell lysates, can be used as the source of the non-pathogenic protein.
- the recombinant protein is fused with an additional amino acid sequence. For example, over expressed protein can be tagged for purification or to facilitate detection of the protein in a sample.
- fusion proteins include histidine tags, Glutathione S- transferase (GST), Maltose binding protein (MBP), green fluorescent protein (GFP), and Flag and myc-tagged rPrP
- GST Glutathione S- transferase
- MBP Maltose binding protein
- GFP green fluorescent protein
- Flag and myc-tagged rPrP additional sequences can be used to aid in purification and/or detection of the recombinant protein, and in some cases are subsequently removed by protease cleavage.
- coding sequence for a specific protease cleavage site can be inserted between the PrP coding sequence and the purification tag coding sequence.
- One example for such a sequence is the cleavage site for thrombin.
- fusion proteins can be cleaved with the protease to free the PrP from the purification tag.
- plasmids or viral vectors can be used. These vectors can be introduced into cells by a variety of methods including, but not limited to, transfection (for instance, by liposome, calcium phosphate,
- Recombinant PrP also can include proteins that have amino sequences containing substitutions, insertions, deletions, and stop codons as compared to wild type sequences.
- a protease cleavage sequence is added to allow inactivation of protein after it is converted into prion form. For example, cleavage sequences recognized by Thrombin, Tobacco Etch Virus (Life).
- Changes also can be made in the pPrP protein coding sequence, for example in the coding sequence for mouse, human, bovine, sheep, goat, deer and/or elk prion protein (GENBANK® accession numbers NM_011170, NM_183079, AY335912, AY723289, AY723292, AF156185 and AY748455, respectively, all of which are incorporated herein by reference, July 20, 2007).
- mutations can be made to match a variety of mutations and polymorphisms known for various mammalian prion protein genes (see, for instance, Table 1).
- chimeric PrP molecules comprising sequences from two or more different natural PrP sequences (for instance from different host species or strains) can be expressed from vectors with recombinant PrP gene sequences, and such chimeras can be used for RT-QuIC and QuIC detection of prion from various species.
- Cells expressing these altered prion protein genes can be used as a source of the rPrP , and these cells can include cells that endogenously express the mutant rPrP gene, or cells that have been made to express a mutant rPrP protein by the introduction of an expression vector.
- mutated rPrP can be advantageous, because some of these proteins are more easily or specifically converted to protease-resistant forms, or are less prone to spontaneous (prion-independent) conversion, and thus can further enhance the sensitivity of the method.
- cysteine residues are placed at positions 94 and 95 of the hamster prion protein sequence in order to be able to selectively label the rPrP at those sites using sulfhydryl-reactive labels, such as pyrene and fluorescein linked to maleimide-based functional groups.
- these tags do not interfere with conversion but allow much more rapid, fluorescence-based detection of the reaction product.
- pyrenes in adjacent molecules of rPrP-res (Sc) are held in close enough proximity to allow eximer formation, which shifts the fluorescence emission spectrum in a distinct and detectable manner.
- Recombinant prion proteins can be produced by any methods known to those of skill in the art.
- in vitro nucleic acid amplification such as polymerase chain reaction (PCR)
- PCR polymerase chain reaction
- PCR is a standard technique that is described, for instance, in PCR Protocols: A Guide to Methods and Applications (Innis et al., San Diego, CA:Academic Press, 1990), or PCR Protocols, Second
- a representative technique for producing a nucleic acid sequence encoding a recombinant prion protein by PCR involves preparing a sample containing a target nucleic acid molecule that includes the prion protein-encoding sequence.
- a target nucleic acid molecule that includes the prion protein-encoding sequence.
- DNA or RNA can serve as a suitable target nucleic acid molecule for PCR reactions.
- RNA is the initial target
- the RNA is reverse transcribed (using one of a myriad of reverse transcriptases commonly known in the art) to produce a double- stranded template molecule for subsequent amplification. This particular method is known as reverse transcriptase (RT)-PCR.
- RT reverse transcriptase
- RT-PCR RT-PCR Protocols, A Guide to Methods and Applications, Innis et al. (eds.), 21-27, Academic Press, Inc., San Diego, California, 1990).
- primers typically, at least 10 consecutive nucleotides of prion- encoding nucleic acid sequence
- amplification conditions may be required to accommodate primers and amplicons of differing lengths and composition; such considerations are well known in the art and are discussed for instance in Innis et al. (PCR Protocols, A Guide to Methods and Applications, San Diego, CA: Academic Press, 1990). From a provided prion protein-encoding nucleic acid sequence, one skilled in the art can easily design many different primers that can successfully amplify all or part of a prion protein-encoding sequence.
- nucleic acid sequences As described herein, a number of prion protein-encoding nucleic acid sequences are known. Though particular nucleic acid sequences are disclosed, one of skill in the art will appreciate that also provided are many related sequences with the functions described herein, for instance, nucleic acid molecules encoding conservative variants of a prion protein.
- sequence identity a measure of similarity between two nucleic acid sequences or between two amino acid sequences expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.
- NCBI National Center for Biotechnology Information
- BLASTTM Basic Local Alignment Search Tool
- Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx.
- sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx A description of how to determine sequence identity using this program is available on the internet under the help section for BLASTTM.
- Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, r at least 98%, or at least 99% sequence identity to the prion sequence of interest.
- prion protein-encoding nucleic acid variants hybridize to a disclosed (or otherwise known) prion protein-encoding nucleic acid sequence, for example, under low stringency, high stringency, or very high stringency conditions. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na +
- Hybridization 5x SSC at 65°C for 16 hours
- Prion protein variants that include the substitution of one or several amino acids for amino acids having similar biochemical properties (so-called conservative substitutions), can also be used in the presently described methods.
- Conservative amino acid substitutions are likely to have minimal impact on the activity of the resultant protein, such as its ability to convert to PrP-res. Further information about conservative substitutions can be found, for instance, in Ben Bassat et al. (J.
- prion protein variants can have no more than 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservative amino acid changes.
- the following table shows exemplary conservative amino acid substitutions that can be made to a prion protein, for instance the recombinant prion proteins shown in SEQ ID NOs: 1-7, such that thy can still be used in the presently claimed assays.
- the composition is subjected to fractionation to remove various other components from the composition.
- Various techniques suitable for use in protein purification are well known. These include, for example, precipitation with ammonium sulfate, PTA, PEG, antibodies and the like, or by heat denaturation followed by centrifugation; chromatography steps such as metal chelate, ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity, and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.
- the samples analyzed using the methods described herein can include any composition capable of being contaminated with a prion.
- Such compositions can include tissue samples, biopsy samples, or bodily fluids including, but not limited to, plasma, blood, lymph nodes, brain, spinal cord, tonsils, spleen, skin, muscles, appendix, olfactory epithelium, cerebral spinal fluid, urine, feces, milk, intestines, tears and/or saliva.
- the sample can be a human sample or a veterinary sample, such as, but not limited to a sample from a cow, sheep or deer.
- the presently disclosed methods also can be used for prion-free herd/flock certification, such as in cattle, sheep, and cervids.
- compositions from which samples can be taken for analysis include food stuffs, pharmaceutical agents (such as animal-derived biological agents), drinking water, forensic evidence, surgical implements, and/or mechanical devices.
- samples that can be tested include biotechnology products and environmental samples (such as water, soils, plants, landfills, sewage) and agriculture samples (such as animal-based foods, animal-based feeds and nutritional supplements, animal waste products, byproducts, carcasses,
- rPrP-res (Sc) can be detected in the reaction mixture.
- Direct and indirect methods can be used for detection of rPrP-res (Sc) in a reaction mixture. Detection using ThT is described above.
- separation of newly-formed rPrP-res (Sc) from remaining rPrP usually is required. This typically is accomplished based on the different natures of rPrP-res (Sc) versus rPrP c .
- rPrP-res typically is highly insoluble and resistant to protease treatment. Therefore, in the case of rPrP- res (Sc) and rPrP c , separation can be by, for instance, protease treatment. Lateral flow assays or SOPHIA can also be used.
- reaction mixtures are incubated with, for example, Proteinase K (PK).
- PK Proteinase K
- An exemplary protease treatment includes digestion of the protein, for instance, rPrP , in the reaction mixture with 1-20 ⁇ g/ml of PK for about 1 hour at 37° C. Reactions with PK can be stopped prior to assessment of prion levels by addition of PMSF or electrophoresis sample buffer. Depending on the nature of the sample, incubation at 37° C with 1-50 ⁇ g/ml of PK generally is sufficient to remove rPrP c .
- rPrP-res also can be separated from the rPrP c by the use of ligands that specifically bind and precipitate the misfolded form of the protein, including conformational antibodies, certain nucleic acids, plasminogen, PTA and/or various peptide fragments.
- reaction mixtures fractioned or treated with protease to remove rPrP c are then subjected to Western blot for detection of rPrP-res (Sc) and the discrimination of rPrP-res (Sc) from rPrP-res (spon) .
- Typical Western blot procedures begin with fractionating proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The proteins are then electroblotted onto a membrane, such as nitrocellulose or PVDF and probed, under conditions effective to allow immune complex (antigen/antibody) formation, with an anti-prion protein antibody.
- Exemplary antibodies for detection of prion protein include the 3F4 monoclonal antibody, monoclonal antibody D13 (directed against residues 96-106 (Peretz et al. (2001) Nature 412, 739-743)), polyclonal antibodies R18 (directed against residues 142-154 ), and R20 (directed against C-terminal residues 218-232) (Caughey et al. (1991) J. Virol. 65, 6597-6603).
- the membrane is washed to remove non- complexed material.
- An exemplary washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
- the immunoreactive bands are visualized by a variety of assays known to those in the art.
- the enhanced chemoluminesence assay (Amersham, Piscataway, N.J.) can be used.
- prion protein concentration can be estimated by Western blot followed by densitometric analysis, and comparison to Western blots of samples for which the concentration of prion protein is known. For example, this can be accomplished by scanning data into a computer followed by analysis with quantitation software. To obtain a reliable and robust quantification, several different dilutions of the sample generally are analyzed in the same gel.
- immunoassays in their most simple and direct sense are binding assays.
- Specific non-limiting immunoassays of use include various types of enzyme linked immunosorbent assays (ELISAs), immunochromatographic strip assays, radioimmunoassays (RIA), and specifically conformation-dependent immunoassays.
- anti-PrP antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a reaction mixture suspected of containing prion protein antigen is added to the wells. After binding and washing to remove non- specifically bound immune complexes, the bound prion protein can be detected. Detection generally is achieved by the addition of another anti-PrP antibody that is linked to a detectable label.
- ELISA is a simple "sandwich ELISA.” Detection also can be achieved by the addition of a second anti-PrP antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
- the reaction mixture suspected of containing the prion protein antigen is immobilized onto the well surface and then contacted with the anti-PrP antibodies. After binding and washing to remove non- specifically bound immune complexes, the bound anti-prion antibodies are detected. Where the initial anti-PrP antibodies are linked to a detectable label, the immune complexes can be detected directly. Again, the immune complexes can be detected using a second antibody that has binding affinity for the first anti-PrP antibody, with the second antibody being linked to a detectable label.
- Another ELISA in which protein of the reaction mixture is immobilized involves the use of antibody competition in the detection.
- labeled antibodies against prion protein are added to the wells, allowed to bind, and detected by means of their label.
- the amount of prion protein antigen in a given reaction mixture is then determined by mixing it with the labeled antibodies against prion before or during incubation with coated wells.
- the presence of prion protein in the sample acts to reduce the amount of antibody against prion available for binding to the well and thus reduces the ultimate signal.
- the amount of prion in the sample can be quantified.
- ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non- specifically bound species, and detecting the bound immune complexes. These are described below.
- a plate with either antigen or antibody In coating a plate with either antigen or antibody, one generally incubates the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antibodies. These include bovine serum albumin, casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, and thus reduces the background caused by nonspecific binding of antibodies onto the surface.
- a secondary or tertiary detection means rather than a direct procedure with ELISAs, though this is not always the case.
- the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
- Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin, milk proteins, and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
- Suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° C to 27° C, or can be overnight at about 4° C or so.
- An exemplary washing procedure includes washing with a solution such as PBS/Tween or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes can be determined.
- the second or third antibody generally will have an associated label to allow detection.
- this is an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
- the first or second immune complex is contacted and incubated with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (for instance, incubation for two hours at room temperature in a PBS-containing solution such as PBS-Tween).
- the amount of label is quantified, for instance, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid) and H 2 0 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, for instance, using a visible spectra spectrophotometer. D. rPrP Labeling
- the recombinant rPrP substrate protein can be labeled to enable high sensitivity of detection of protein that is converted into rPrP- res (Sc) .
- rPrP c can be radioactively labeled, epitope tagged, or fluorescently labeled. The label can be detected directly or indirectly. Radioactive labels include, but are not limited to 125 1, 32 P, 33 P, and 35 S.
- the mixture containing the labeled protein is subjected to an amplification assay, such as QuIC, and the product detected with high sensitivity by following conversion of the labeled protein after removal of the unconverted protein for example by proteolysis.
- the protein can be labeled in such a way that a signal can be detected upon the conformational changes induced during conversion.
- FRET technology in which the protein is labeled by two appropriate fluorophores, which upon refolding become close enough to exchange fluorescence energy (see for example U.S. Pat. No. 6,855,503).
- cysteine residues are placed at positions 94 and 95 of the hamster prion protein sequence in order to be able to selectively label the rPrP at those sites using sulfhydryl-reactive labels, such as pyrene and fluorescein linked to maleimide-based functional groups.
- these tags do not interfere with conversion but allow much more rapid, fluorescence-based detection of the reaction product.
- pyrenes in adjacent molecules of rPrP-res (Sc) are held in close enough proximity to allow eximer formation, which shifts the fluorescence emission spectrum in a distinct and detectable manner.
- rPrP-res (Sc) amplification reaction can be run in a multiwell plate, digested with proteinase K, and then eximer fluorescence can be measured to rapidly test for the presence of rPrP-res (Sc) .
- Sites 94 and 95 are chosen for the labels because the PK-resistance in this region of constituent PrP molecules distinguishes rPrP-res (Sc) from rPrP-res (spon) , giving rise to the 17 kDa rPrP-res (Sc) band.
- rPrP-res (Sc) fragment 17-kDa rPrP-res (Sc) fragment from all rPrP-res (spon) fragments
- Sc 17-kDa rPrP-res
- Spon rPrP-res
- the use of a fluorescently-tagged rPrP substrate for the reaction is combined with the use an immunochromatographic strip test with an immobilized rPrP-res (Sc) specific antibody (for example, from Prionics AG, Schlieren-Zurich, Switzerland). Binding of the rPrP-res (Sc) to the antibody is then detected with a fluorescence detector.
- TSEs are largely unbeatable and difficult to diagnose definitively prior to irreversible clinical decline or death.
- the transmissibility of TSEs within and between species highlights the need for practical tests for even the smallest amounts of infectivity.
- most in vitro methods have major limitations that would preclude their use in routine diagnostic or screening applications.
- TSEs or prion diseases in medicine, agriculture, and wildlife biology is the development of practical tests for prions that are at, or below, infectious levels.
- tests capable of detecting prions in blood components such as plasma but blood typically has extremely low prion concentrations and contains inhibitors of the most sensitive prion tests.
- coupling of immunoprecipitation and an improved real time QuIC reaction dramatically enhanced detection of variant Creutzfeldt- Jakob disease (vCJD) brain tissue diluted into human plasma. Dilutions of 10 14 -fold, containing ⁇ 2 ag/ml of proteinase K- resistant prion protein, were readily detected, indicating ⁇ 10,000-fold greater sensitivity for vCJD brain than has previously been reported.
- eQuIC enhanced QuIC
- Vortex Rat anti-Mouse IgM DYNABEADS® (Invitrogen, cat. No. 110.39D) for 30 seconds.
- reaction tubes 0.5 ml conical microcentrifuge tubes with screw caps (Fisher 02-681-334)
- X and Y are variables: X is the concentration of the rPrP which can vary depending on the specific preparation, and Y is the volume of protein added to the reaction to have a final concentration of 0.1 mg/ml
- the final reaction volume is 100 ⁇ , so once the volume of rPrPC is established that needs to be added along with all the other components (e.g. NaCl, PBS), the remaining volume to get to lOOul is water.
- Vortex first three components for 5 s prior to adding the rPrP c . Add the rPrP c gently, to avoid creation of bubbles. Cap reaction tubes, but do not vortex.
- reaction mixture in fresh reaction tubes similar to 1 st round described above. Note: gently vortex sample tubes to evenly suspend any pellet just prior to transferring volume to seed the 2 nd round reaction tube.
- reaction plates 96 well Optical Bottom Plate
- RQB Real-Time QuIC Buffer
- Thioflavin T Thioflavin T
- EDTA ethylenediaminetetraacetic acid tetrasodium salt
- Each reaction in each well has a final volume of 100 ⁇ .
- "Y” is the volume of rPrPC that is added to each reaction to have a final concentration of 0.1 mg/ml. This volume varies depending on the concentration of the protein added. Thus the volume of MilliQ water added per reaction (“X”) -varies depending on the volume of rPrP added to that specific reaction.
- Shaking program 1 minute shaking at 700 rpm Double Orbital, then 1
- Substrate replenishment step :
- Plasma sample collection and tissue homogenate preparation were inoculated intracerebrally with 50 ⁇ 1% brain homogenate (BH)
- Plasma samples were centrifuged at 3000 rpm in a Beckman J6-HC centrifuge for 15 minutes (min). Plasma was transferred to a new tube and stored at -80°C. Hamster serum samples were collected in a similar fashion but with no sodium citrate. When designated (i.e., Figure 2B), plasma samples were centrifuged at 16k relative centrifugal force (rcf) for 30 seconds (s) after thawing and immediately prior to the immunoprecipitation step (using the plasma supernatant). Pooled human plasma (Innovative Research) was stored at -20°C.
- rcf 16k relative centrifugal force
- BH dilution spiking experiments human plasma aliquots were thawed over night at 4°C and subjected to a 10 min 2000 x g spin to eliminate precipitated fraction.
- Hamster and human 10% (w/v) BH were made as previously reported (Saa et al, J.Biol.Chem. 281:35245-35252, 2006), aliquoted and stored at -80°C.
- BH was serially diluted in either 1% or 0.1% SDS in phosphate buffered saline (PBS) with 130 mM NaCl and N2 media supplement (Gibco) (20,21,24), for S-QuIC or RT-QuIC assays, respectively.
- PBS phosphate buffered saline
- Nibco phosphate buffered saline
- Two microlilters of the designated BH dilutions were used to spike 0.5 ml of human plasma.
- MA GNABiNDTM beads MAGNABINDTM beads (Pierce,
- Immunoprecipitation Buffer (Prionics) was added. Next, 500 ⁇ of BH-spiked human plasma or 500 ⁇ of hamster plasma from uninfected or scrapie-positive animals was added to the beads. Samples were incubated with "end-over-end” rotation at room temperature or 37°C over night (ON). Subsequently, samples were incubated on the magnet for 2 min, plasma-buffer mix was discarded, and beads were washed twice with 500 ⁇ of Wash Buffer (Prionics). All beads were resuspended into 10 ⁇ of PBS and used fresh.
- S-QuIC and RT-QuIC The S-QuIC assay was performed as previously described (Atarashi et al., Nat.Methods 5:211-212, 2008; Orru et al., Protein Eng Des Sel 22:515-521, 2009). 15B3 coated- or mock bead S-QuIC reactions were each seeded with 2 ⁇ of beads in phosphate buffered saline (PBS). The RT-QuIC was performed as previously described (Wilham et al., PLoS.Pathog. 6:el001217, 2010) except for a few modifications.
- the 15B3 IP was adapted to detection by RT-QuIC (designated IP-RT- QuIC).
- the RT-QuIC assay uses intermittent shaking of reactions in 96-well plates, rPrP c as the substrate, virtually detergent- ( ⁇ 0.002% SDS) and chaotrope-free conditions, and ThT-based detection of prion-seeded amyloid fibrils (Wilham et al., supra, 2010; Atarashi et al, Nat.Med. 17: 175-178, 2011). Positive reactions are indicated by an enhancement of ThT fluorescence in the presence of rPrP amyloid fibrils, which can be plotted as the average fluorescence from replicate wells.
- the IP-RT-QuIC protocol detected -10 "10 dilutions of scrapie brain in human plasma, but was less sensitive for vCJD brain (Fig. 9C).
- hamster rPrP 90-231 was used as a substrate.
- vCJD it was found that a chimeric rPrP molecule, comprised of Syrian hamster residues 23-137 followed by sheep residues 141-234 (R 154 ,Q 1 7 1 polymorph), provided for greater sensitivity and less spontaneous (prion-independent) conversion to ThT-positive products than was observed with the homologous human PrP 23-231 construct (Fig. 9).
- the 15B3-coupled beads provided for >10 6 -fold more sensitive eQuIC detection than superparamagnetic nanoparticles that were reported recently to have prion-binding capacity (Miller et al., J.Virol. 85:2813-2817, 2011) (Fig. 10). These results showed the ability of the 15B3-based eQuIC to detect extremely low concentrations of prions spiked into human plasma.
- the results showed that prions can be captured from a complex inhibitor-laden biological fluid in a manner that is compatible with ultrasensitive detection by in vitro prion amplification assays.
- the eQuIC assay in particular provided a practical, high-throughput and rapid means of testing for amounts of PrP- res (1 ag) that was several orders of magnitude below those typically required to cause prion disease by intracerebral inoculation into animals.
- the ability of eQuIC to detect prions in plasma samples raises indicates that this assay can be used to improve prion disease diagnosis in humans and animals, and to screen the blood supply for prion contamination.
- the two-stage substrate addition disclosed herein for the eQuIC differs from serial (multiple-round) amplification steps that were used in protein misfolding cyclic amplification (PMCA) (Saa et al., Science 313:92-94, 2006; Saa et al., J.Biol.Chem.
- PMCA protein misfolding cyclic amplification
- the results suggest that at least two processes are occurring during the initial lag phase of the eQuIC reaction, specifically between the addition of seed and substrate replacement (Fig. 6).
- the rPrP can be moving into a pool that is less rapidly accessible to prion-seeded fibril assembly, such as an off-pathway oligomer (00); otherwise, the addition of fresh rPrP after 20 h, but before the initial substrate is converted to detectable ThT-positive fibrillar products, would not accelerate the reaction.
- fibril assembly might be hastened by the pre-alignment or scaffolding of rPrP substrate or amyloidogenic intermediate (AI) along the sides of an existing fibril, either with or without the need for a similarly aligned seed.
- AI amyloidogenic intermediate
- the more effective rPrP substrate for the RT-QuIC is not always one that is most homologous with the type of prion/PrP-res being assayed.
- the substrate that worked best for the detection of human vCJD was the chimeric hamster-sheep construct (Ha-S rPrP c ), rather than a human rPrP c molecule.
- the IP-RT-QuIC assay offers considerable advantages when compared to other ultrasensitive prion/PrP Sc assays.
- the eQuIC not only allows for prion detection in inhibitor-laden samples such as plasma, but also enhances the sensitivity for vCJD brain homogenate dilutions into human plasma by at least 10,000-fold.
- the IP-RT-QuIC is more rapid for a given sensitivity level, more practical by using bacteria rather than brain as the source of PrP substrate, more easily replicated by using shaking rather than sonication, and more amenable to high-throughput analyses due to multiwell plate-based reactions and fluorescence detection.
- vCJD PrP-res detection assay which includes prion capture on stainless steel beads and an ELISA detection method.
- this capture-ELISA assay detected 10 10 -fold dilutions of vCJD brain homogenate in whole blood
- the eQuIC assay disclosed herein detected 10 14 -fold dilutions in plasma.
- the Edgeworth assay detected PrP vCJD in blood from 15 symptomatic patients with a -70 % sensitivity and 100 % specificity, which is nearly as effective as the RT-QuIC in diagnosing sporadic CJD using CSF samples (Atarashi et al., Nat.Med.
- the -10,000-fold greater sensitivity of the eQuIC assays that are disclosed herein in detecting brain-derived vCJD seeding activity provides improved sensitivity of vCJD and sCJD diagnosis using blood, plasma, CSF or other samples.
- the assays disclosed herein also have use in a wide variety of materials such as foods, feeds, transplanted tissues, medical devices, agricultural wastes and byproducts, soils, water sources, and other environmental samples.
- prion seeding activity could be eluted from the beads with 1 M NaCl prior to SDS treatment and addition to RTQ reactions.
- the eluted material required a pre-incubation with 0.05% SDS for 15- 20 min, at room temperature, to get enhanced prion specific seeding activity in the RTQ.
- the amount of 15B3 antibody loaded onto the beads was doubled ("20X") to determine if increased concentration of the antibody on the beads would increase sensitivity of the assay for sheep ARQ brain homogenate (containing 100 fg or 10 pg PrP-res) spiked into 0.5 ml sheep plasma.
- 20X indicates 200 ⁇ g 15B3 incubated with 1 x 10 8 total beads and "10X” indicates 100 ⁇ g 15B3 incubated with 1 x 10 8 total beads).
- Ha-S rPrP C was used as the substrate. Improved sensitivity (see the dilution containing 100 fg PrP-res) was achieved with higher loading of 15B3.
- the "20X" eQuIC conditions were used to detect sheep ARQ scrapie brain homogenates (containing down to 100 ag PrP-res) spiked into 0.5 ml plasma (see Fig. 13, four replicate reactions).
- the same eQuIC condition were used to test whether the assay can detect prion seeding activity endogenous to 0.5 ml plasma samples from scrapie infected sheep (ARQIVRQ, VRQWRQ), as opposed to brain homogenate spiked in plasma.
- ARQIVRQ, VRQWRQ scrapie infected sheep
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RU2013111962A (en) * | 2013-03-18 | 2014-09-27 | Олег Ильич Эпштейн | METHOD FOR DETERMINING THE EXPRESSION OF MODIFICATION ACTIVITY ASSOCIATED WITH A CARRIER |
EP3563158A1 (en) | 2016-12-30 | 2019-11-06 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Methods for the detection of tau protein aggregates |
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EP3694531A4 (en) * | 2017-10-13 | 2021-07-21 | Amira Medical Technologies Inc. | Products and methods associated with multiple sclerosis as a transmissible protein misfolding disorder |
KR102037059B1 (en) * | 2018-01-22 | 2019-10-30 | 대한민국 | Detecting method of abnormal prion from soil |
JP7173532B2 (en) * | 2018-08-28 | 2022-11-16 | 学校法人順天堂 | Diagnosis of α-Synucleinopathy |
US20240183865A1 (en) * | 2021-03-31 | 2024-06-06 | Kaneka Corporation | Method for measuring aggregating proteins, method for visualizing aggregation, and kit used therefor |
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