Patent Application
20100196934
The invention relates to an assay method of detecting the presence of pathogenic prions in a sample. The invention also relates to kits comprising reagents used in the assay method.
Amyloid diseases are caused by the transition of soluble protein into an insoluble aggregated form. Evidence exists that this conversion is associated with a conformational change to the secondary and tertiary structure as well. There is increasing evidence that accumulation of protein deposits damage cells and tissues leading to diseases. Several diseases are associated with the deposition of specific proteins.
One group of conformational diseases is termed “prion diseases” or “transmissible spongiform encephalopathies (TSEs).” These diseases manifest in humans and animals. In humans, prion diseases include Creutzfeldt-Jakob disease (CJD), variant CJD (vCJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Fatal Familial Insomnia, and Kuru (see, e.g., Isselbacher et al., eds. (1994). Harrison's Principles of Internal Medicine. New York: McGraw-Hill, Inc.; Medori et al. (1992) N. Engl. J. Med. 326: 444-9). In animals, prion diseases include sheep scrapie, bovine spongiform encephalopathy (BSE), transmissible mink encephalopathy, and chronic wasting disease of captive mule deer and elk (Gajdusek, (1990). Subacute Spongiform Encephalopathies: Transmissible Cerebral Amyloidoses Caused by Unconventional Viruses. In: Virology, Fields, ed., New York: Raven Press, Ltd. (pp. 2289-2324)).
Recently, the rapid spread of BSE and its correlation with elevated occurrence of TSEs in humans has led to increased interest in the detection of TSEs in non-human mammals. The tragic consequences of accidental transmission of these diseases (see, e.g., Gajdusek, Infectious Amyloids, and Prusiner Prions In Fields Virology. Fields, et al., eds. Philadelphia: Lippincott-Ravin, Pub. (1996); Brown et al. Lancet, 340: 24-27 (1992)), decontamination difficulties (Asher et al. (1986) In: Laboratory Safety: Principles and Practices, Miller ed., (pp. 59-71) Am. Soc. Micro.), and concern about BSE (British Med. J. (1995) 311: 1415-1421) underlie the urgency of having both a diagnostic test that would identify humans and animals with TSEs and therapies for infected subjects.
Prions differ significantly from bacteria, viruses and viroids. The dominating hypothesis is that, unlike all other infectious pathogens, disease is caused by an abnormal conformation of the prion protein, which acts as a template and converts normal prion conformations into abnormal, aberrant conformations. The prion protein, or PrP, was first characterized in the early 1980s. (See, e.g., Bolton et al. (1982) Science 218: 1309-1311; Prusiner et al. (1982) Biochemistry 21: 6942-6950; McKinley et al. (1983) Cell 35: 57-62). Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. (See, e.g., Basler et al. (1986) Cell 46: 417-428.) The normal, cellular form of the prion protein, which is also referred to as PrPC, is a 33-35 kD protein of uncertain function and, in humans, is transcribed by a gene on the short arm of chromosome 20. The abnormally shaped prion protein is also referred to as a scrapie protein or a pathogenic prion protein (PrPSc). Deposition of PrPSc in the Central Nervous System (CNS) is associated with neuron degeneration and is always lethal. PrPSc is infectious and exposure to tissues containing infectivity could result in disease. Experimental inoculation of PrPSc into laboratory animals including primates, sheep, rodents, and transgenic mice resulted in transmission of prion disease. (See, e.g., Zhang et al. (1997) Biochem. 36(12): 3543-3553; Cohen & Prusiner (1998) Ann. Rev. Biochem. 67: 793-819; Pan et al. (1993) Proc. Natl. Acad. Sci. USA 90:10962-10966; Safar et al. (1993) J. Biol. Chem. 268: 20276-20284.)
The substantially β-sheet structure of PrPSc as compared to the predominantly α-helical folded non-diseased forms of PrPC has been revealed by optical spectroscopy and crystallography studies. (See, e.g., Wille et al. (2001) Proc. Nat'l Acad. Sci. USA 99: 3563-3568; Peretz et al. (1997) J. Mol. Biol. 273: 614-622; Cohen & Prusiner, (1999) 5: Structural Studies of Prion Proteins. In Prion Biology And Diseases, S. Prusiner, ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. (pp: 191-228.) The structural changes from PrPC to PrPSc appear to be followed by alterations in biochemical properties: PrPC is soluble in non-denaturing detergents, PrPSc is insoluble and forms oligomers that are composed of as many as 1000 molecules; PrPC is readily digested by proteases, while PrPSc is partially resistant, resulting in the formation of an amino-terminally truncated fragment known as “PrP 27-30” (27-30 kDa), “PrPres” or “PK-resistant” (proteinase K-resistant) form or core, which corresponds to the PrP fragment of amino acid residues from about 90 to about 231, numbered according to the human or Syrian Hamster PrP full-length sequence including the signal peptide. (See, e.g., Baldwin et al. (1995) J. Biol. Chem. 270:19197-19200; Prusiner et al. (1982) Biochemistry 21:6942-6950; Prusiner et al. (1983) Cell 35:349-358; Collinge, J. and Palmer, M. S. (1997) Prion Diseases, Oxford University Press, New York.) Additionally, PrPSc can convert PrPC to the pathogenic conformation. (See, e.g., Kaneko et al. (1995) Proc. Nat'l Acad. Sci. USA 92:11160-11164; Caughey (2003) Br Med. Bull. 66: 109-20; Telling et al. (1995) Cell 83:79-90; Kaneko et al. (1997) Proc. Natl. Acad. Sci. USA 94:10069-10074; DebBurman et al. (1997) Proc. Natl. Acad. Sci. USA 94: 13938-13943; Horiuchi et al. (1999) EMBO J. 18:3193-3203; Horiuchi et al. (2000) Proc. Natl. Acad. Sci. USA 97: 5836-5841; Kocisko et al. (1994) Nature 370:471-474.)
Detection of the pathogenic isoforms of conformational disease proteins in living subjects, and samples obtained from living subjects, has proven difficult. Although detection of amyloids in general can be achieved with Congo red staining, this type of staining is inaccurate and not sensitive. Specific and high affinity detection of a given protein in the presence of other proteins like in cell, tissue or homogenate is usually done with antibodies that are specific to the targeted protein. Yet, proteins that share the same sequence but differ by conformation complicate discriminatory detection by antibodies. In fact, the majority of antibodies raised against PrP bind PrPC or to both PrPC and PrPSc. (See, e.g., Matsunaga et al. (2001) Proteins: Structure, Function and Genetics 44: 110-118). In addition, the aggregation of PrPSc reduces the effective epitope concentrations for an antibody; thereby hindering efficient binding of specific antibodies to PrP. Therefore, effective immunodetection of PrPSc necessitates dissociation and denaturation of PrPSc into PrPC-like conformers while discriminating against the original PrPC molecules.
Several published immunoassay methods were based on comparing the binding level of antibodies that only bind native PrPC but not native PrPSc to the binding level of antibodies that bind both native PrPSc and PrPC in order to determine the presence of PrPSc. (See, e.g., U.S. Pat. Nos. 6,214,565 B1 and 6,406,864 B2.) Another immunoassay method was also based on comparing the antibody binding levels, but it involved a denaturing step to denature PrPSc and PrPC before binding them to the same antibody as the one used to bind to native PrPC. (See U.S. Pat. No. 5,891,641.) However, there are limitations in these assay methods because, using these methods, effective antibody detection of PrPSc can only be obtained when the amount of PrPC is very low. In an excess of PrPC, e.g., when samples are taken from subjects in the early stages of prion disease, it is difficult or even impossible to observe an increase in immunodetection due to the presence of PrPSc. For example, detection of denatured PrPSc in plasma samples with 1000 times more PrPC will not be possible because of the overwhelming signal from the detection of PrPC. Thus, simple discrimination between samples with or without PrPSc is not very feasible.
To overcome this difficulty, samples are often treated with non-specific proteases such as Proteinase K (PK) or dispase before the denaturation and detection of PrPSc. (See, e.g., U.S. Pat. No. 7,163,798 B2 and European Patent 1119 773 B1.) Because of the structure of the PrPSc isoform, PrPSc is largely resistant to protease digestion. The PrPC isoform, on the other hand, is completely degraded by treatment with such proteases. Thus, treatment of samples with a non-specific protease such as PK under some conditions (e.g., 50 μg/mL for 30 min at 37° C.) can eliminate any PrPC present and leave the protease-resistant core of PrPSc (amino acid residues from about 90 to about 231) generally intact.
One major limitation of PK treatment is the degradation of the amino-terminal, or amino-proximal residues (from about 23 to about 89) of PrPSc. This region of PrP can be useful for immunodetection since it contains a number of epitopes including epitopes in the octarepeat region. The octarepeat region contains several copies of the octarepeat sequence GQPHGG(G/S)(-/G)W (SEQ ID NO: 11). This octarepeat sequence is highly conserved in prion proteins from different species and it repeats four times between residues about 58-89, numbered according to human or Syrian hamster PrP full-length sequence, in most species except bovine PrP that has 5 repeats and monkey PrP that has 3 repeats. (See
In general, most antibodies bind an epitope that is only presented once on a protein, even though antibodies inherently contain two binding sites (i.e. two binding arms). This type of binding is termed monovalent binding. Monovalent binding is weaker than an event where both antibody-binding sites simultaneously interact with two epitopes on the same protein (i.e. multivalent binding). The overall strength of multivalent binding, called avidity, is greater than affinity, the strength of binding of a single site, since both binding sites must dissociate at the same time for the antibody to release the antigen. For example, this property is very important in the binding of antibodies to bacteria or viruses, which usually have multiple identical epitopes on their surfaces. Thus, the octarepeat region of PrP allows enhanced signal through both the uniqueness of multiple octarepeat epitopes and the multivalent binding. Since PK digests residues 23-89 of the full length mature PrPSc (amino acids 23-231), the repertoire of useful antibodies after a complete PK digestion is limited to those that bind sequences within the protease-resistant core (amino acids ˜90-231), and antibodies against the octarepeat region cannot be used. Therefore the immunoassays using PK digestion are not very sensitive.
An additional disadvantage of using non-specific proteases like PK is that PrPSc is only partially resistant; given high concentrations and enough time most or even all 90-231 residues will be digested as well. It has been shown that some conformers of PrPSc are more sensitive than others to digestion and such treatment reduces the sensitivity of detection. (See, e.g., Safar et al. (1998) Nat. Med. 4:1157-65). Consistent with this is the finding that PK reduces levels of PrPSc and prion infectivity by several logs (See McKinley et al. (1983) Cell 35:57-62).
A recently described immunoassay method addressed the limitation discussed above by controlling PK digestion conditions so that PrPC is completely digested but PrPSc is only partially digested and all or some of the octarepeat region is retained. (See U.S. Pat. No. 7,097,997 B1.) However, this solution is not ideal as the appropriate condition may vary for different samples and therefore may be laborious to achieve and hard to standardize.
Thus, there remains a need for a specific, sensitive and relatively simple or quick method and a kit using such method to detect the presence of the pathogenic prion proteins in various samples, for example in samples obtained from living subjects, in blood supplies, in farm animals and in other human and animal food supplies. This invention is directed to this, as well as other, important ends.
The present disclosure provides a new, simple, specific, and sensitive way, in the forms of assay methods and kits, to detect the presence of pathogenic prion proteins, which may be used, inter alia, in connection with methods for diagnosing a prion-related disease (e.g., in human or non-human animal subjects), for ensuring a substantially PrPSc-free blood supply, blood products supply, or food supply, for analyzing organ and tissue samples for transplantation, for monitoring the decontamination of surgical tools and equipment, as well as any other situation in which knowledge of the presence or absence of the pathogenic prion is important.
The assay methods in the present disclosure take advantage of a treatment with a site-specific protease that does not cleave in the octarepeat region of known PrP sequences or in the protease-resistant core of PrPSc after a substantially complete digestion, while cleaving away at least part of the amino acids in PrPC that correspond to the protease-resistant core in PrPSc, which makes it possible and convenient for epitopes in the octarepeat region or even those further upstream to be utilized in combination with epitopes in the protease-resistant core region for a specific and sensitive detection of PrPSc.
The present invention relates, in part, to a method for detecting the presence of PrPSc in a sample by contacting the sample with a site-specific protease under conditions in which proteolytic digestion of prion proteins is substantially complete, preventing further protease digestion of the sample, and detecting the presence of any PrPSc by binding to at least two binding partners, which include but are not limited to antibodies and aptamers, wherein one binding partner specifically binds an epitope in the amino-proximal region of PrP, preferably in the octarepeat region, and another binding partner specifically binds an epitope within the protease-resistant core region.
The invention provides a method for detecting the presence of a pathogenic form of prion protein, or PrPSc, using at least one site-specific protease, preferably trypsin or Staphylococcus aureus V8 protease (S-V8), for a substantially complete digestion of the proteins in a sample that is suspected of containing the PrPSc, wherein the sample may or may not contain a normal form of prion protein (PrPC). The PrPSc has no cleavage site for the site-specific protease within the octarepeat region or between the octarepeat region and the protease-resistant core. Therefore, a fragment of amino-proximal region including the octarepeat region remains connected to the protease-resistant core of the PrPSc, which protease-resistant core remains intact, after the substantially complete proteolytic digestion. The PrPC, if present, has at least one available cleavage site for the site-specific protease within amino acid region corresponding to the protease-resistant core of PrPSc, and the at least one available cleavage site is cleaved by the substantially complete proteolytic digestion. The site-specific proteases that are suitable for the method of the invention are further described herein.
The method further comprises a step of preventing any further proteolytic digestion following the substantially complete proteolytic digestion, which can be accomplished by adding a protease inhibitor or by removing the site-specific protease.
The method further comprises a step of denaturing the site-specific-protease-treated PrPSc after the step of preventing further proteolytic digestion, thereby providing denatured PrPSc.
The method further comprises a step of detecting the presence of PrPSc using at least two binding partners, a first binding partner and a second binding partner, which include but are not limited to antibodies and aptamers, wherein the first binding partner specifically binds a first epitope within the fragment of amino-proximal region of the PrPSc that remains connected to the protease-resistant core after the substantially complete proteolytic digestion, preferably in the octarepeat region, and the second binding partner specifically binds a second epitope within the protease-resistant core region of the PrPSc, wherein the second epitope is in a region of the PrPC that is separated from the first epitope after the substantially complete proteolytic digestion.
In certain embodiments, the PrPSc comprises a sequence selected from SEQ ID NOs. 1 to 10.
In a preferred embodiment, the first binding partner binds to an epitope that is within the octarepeat region. Exemplary first binding partners in these embodiments include monoclonal antibodies such as POM2, POM11, POM12, POM14, 3B5, 4F2, 13F10, SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, and SAF-37.
In certain other embodiments, the first binding partner binds to an epitope that is outside of the octarepeat region. Exemplary first binding partners in these embodiments include monoclonal antibodies such as BAR210, BAR231, and 14D3.
In certain embodiments, the site-specific protease used in the method is trypsin, which specifically hydrolyzes peptide bonds at the carboxyl side of Lysine (K) and Arginine (R) residues unless the amino acid on the carboxyl side of K and R is Proline (P). For these embodiments, the second binding partner can be specific to any epitope that is within the protease-resistant core of PrPSc and that has been cleaved away and thus is separated from the first epitope in PrPC by trypsin digestion. Exemplary second binding partners in these embodiments include monoclonal antibodies such as 3F4, POM1, POM4, POM5, POM6, POM7, POM8, POM9, POM10, POM13, POM15, POM16, POM17, POM19, SAF-2, SAF-4, SAF-8, SAF-9, SAF-10, SAF-12, SAF-13, SAF-14, SAF-22, SAF-24, SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-68, SAF-69, SAF-70, SAF-75, SAF-76, SAF-82, SAF-83, SAF-84, SAF-95, Pri308, Pri917, BAR215, BAR221, BAR224, BAR233, BAR234, Sha31, 11B9, 12F10, D18, 6H4, and BDI115.
In a preferred embodiment of the method in which trypsin is used, the second binding partner specifically binds to an epitope located within the globular domain of PrPC (i.e., from about amino acid 122 to about amino acid 231 of PrPC). Exemplary second binding partners in these embodiments include monoclonal antibodies such as POM1, POM4, POM5, POM6, POM7, POM8, POM9, POM10, POM13, POM15, POM16, POM17, POM19, SAF-2, SAF-4, SAF-8, SAF-9, SAF-10, SAF-12, SAF-13, SAF-14, SAF-22, SAF-24, SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-68, SAF-69, SAF-70, SAF-75, SAF-76, SAF-82, SAF-83, SAF-84, SAF-95, Pri917, BAR215, BAR221, BAR224, BAR233, BAR234, Sha31, 11B9, 12F10, D18, 6H4, and BDI115.
In certain other embodiments, the site-specific protease used in the method is S-V8 which specifically cleaves on the carboxyl side of Glutamic Acid (E) and Aspartic Acid (D) residues. For these embodiments, the second binding partner can be specific to any epitope that is within the protease-resistant core of PrPSc and that has been cleaved away in PrPC by S-V8. Exemplary second binding partners in these embodiments include monoclonal antibodies such as SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-69, SAF-70, SAF-75, SAF-76, Pri917, BAR234, Sha31, 11B9, 12F10, 6H4, and POM5.
In certain embodiments of the method, the detecting step is carried out using an ELISA, preferably a Sandwich ELISA. One such embodiment utilizes a first binding partner which binds an epitope within the amino-proximal region of PrP, particularly within the octarepeat region, as a capture binding partner and a second binding partner which binds an epitope within the protease-resistant core as a detection binding partner. In another such embodiment, the second binding partner which binds an epitope within the protease-resistant core is utilized as a capture binding partner and the first binding partner which binds an epitope within the amino-proximal region is utilized as a detection binding partner.
In certain preferred embodiments, the detection binding partners discussed above are labeled. In certain embodiments among these, the detection binding partners are labeled with an alkaline phosphatase (AP) conjugate.
In certain embodiments, the binding partners used in the method are antibodies, preferably monoclonal antibodies, specific to the first epitope and the second epitope, wherein the antibodies can be selected from, but are not limited to, the exemplary monoclonal antibodies disclosed herein.
In certain other embodiments, the binding partners used in the method are aptamers specific to the first epitope and the second epitope.
In one embodiment of the method, said site-specific protease is immobilized on a solid support.
In another embodiment, the protease inhibitor used in the method to prevent further proteolytic digestion of the sample by the site-specific protease following the substantially complete proteolytic digestion is phenylmethylsulphonyl fluoride (PMSF).
In certain embodiments, the denaturing step in the method comprises treating the digested samples with a guanidinium compound, e.g., guanidine hydrochloride (GdnHCl) or guanidine thiocyanate (GdnSCN).
In certain embodiments among the ones that used a guanidinium compound as the denaturant, a further step of diluting the samples is involved after denaturation.
In certain other embodiments, the denaturing step in the method comprises treating the digested samples with high pH or low pH and, optionally, neutralizing said high pH or said low pH after said denaturing.
In certain embodiments of the method, the samples are obtained from a living or once-living organism. Exemplary organisms in these embodiments include human, monkey, hamster, bovine, sheep, mouse, elk, and deer.
In certain embodiments, the sample used in the method is derived from the group consisting of food supply, whole blood, blood products, blood fractions, blood components, plasma, platelets, serum, cerebrospinal fluid, organs, cells, brain tissue, nervous system tissue, muscle tissue, fatty tissue, bone marrow, urine, tears, non-nervous system tissue, biopsies, necropsies, and contaminated instruments.
In certain embodiments, the sample used in the method is derived from the group consisting of whole blood, blood products, blood fractions, blood components, plasma, platelets, red blood cells, and serum.
In certain embodiments, the sample is obtained from a biopsy, autopsy or necropsy.
The present invention relates also, in part, to a kit for detecting the presence of a pathogenic form of prion protein, or PrPSc, in a sample suspected of containing the PrPSc, wherein the sample may or may not contain a normal form of prion protein (PrPC), the kit comprising reagents that are useful in the method discussed herein.
Specifically, the kit comprises at least one site-specific protease, wherein the PrPSc has no cleavage site for the site-specific protease within the octarepeat region or between the octarepeat region and the protease-resistant core, therefore a fragment of amino-proximal region of the PrPSc including the octarepeat region remains connected to the protease-resistant core after a substantially complete proteolytic digestion of PrPSc by the protease. The PrPC, if present, has at least one available cleavage site for the site-specific protease within amino acid region corresponding to the protease-resistant core of the PrPSc.
The kit further comprises at least two binding partners, a first binding partner and a second binding partner, which include but are not limited to antibodies and aptamers. The first binding partner specifically binds a first epitope which is located within the fragment of amino-proximal region that remains connected to the protease-resistant core after a substantially complete proteolytic digestion of the PrPSc by the site-specific protease in the kit. The second binding partner specifically binds a second epitope which is located within the protease-resistant core, wherein the second epitope is in a region of the PrPC that is separated from the first epitope after a substantially complete proteolytic digestion of the PrPC by the site-specific protease in the kit.
The kit further comprises an instruction for using the kit to detect the presence of any pathogenic form of prion protein.
Optionally, the kit further comprises a protease inhibitor which is capable of inhibiting the activity of the site-specific protease in the kit.
Again optionally, the kit further comprises a denaturant which is capable of denaturing the PrPSc.
In certain embodiments of the kit, the PrPSc comprises a sequence selected from SEQ ID NOs. 1 to 10.
In certain, preferred, embodiments of the kit, the first binding partners specifically binds an epitope within the octarepeat region. Exemplary first binding partners in these embodiments include monoclonal antibodies such as POM2, POM11, POM12, POM14, 3B5, 4F2, 13F10, SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, and SAF-37.
In certain other embodiments of the kit, the first binding partner specifically binds an epitope that is outside of the octarepeat region. Exemplary first binding partners in these embodiments include monoclonal antibodies such as BAR210, BAR231, and 14D3.
In certain embodiments of the kit, the site-specific protease in the kit is trypsin. For these embodiments, exemplary second binding partners in these embodiments include monoclonal antibodies such as 3F4, POM1, POM4, POM5, POM6, POM7, POM8, POM9, POM10, POM13, POM15, POM16, POM17, POM19, SAF-2, SAF-4, SAF-8, SAF-9, SAF-10, SAF-12, SAF-13, SAF-14, SAF-22, SAF-24, SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-68, SAF-69, SAF-70, SAF-75, SAF-76, SAF-82, SAF-83, SAF-84, SAF-95, Pri308, Pri917, BAR215, BAR221, BAR224, BAR233, BAR234, Sha31, 11B9, 12F10, D18, 6H4, and BDI115.
In a preferred embodiment of the kit in which trypsin is the site-specific protease, the second binding partner specifically binds to an epitope located within the globular domain of PrPC (i.e., from about amino acid 122 to about amino acid 231 of PrPC). Exemplary second binding partners in these embodiments include monoclonal antibodies such as POM1, POM4, POM5, POM6, POM7, POM8, POM9, POM10, POM13, POM15, POM16, POM17, POM19, SAF-2, SAF-4, SAF-8, SAF-9, SAF-10, SAF-12, SAF-13, SAF-14, SAF-22, SAF-24, SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-68, SAF-69, SAF-70, SAF-75, SAF-76, SAF-82, SAF-83, SAF-84, SAF-95, Pri917, BAR215, BAR221, BAR224, BAR233, BAR234, Sha31, 11B9, 12F10, D18, 6H4, and BDI115.
In certain other embodiments, the site-specific protease in the kit is S-V8. For these embodiments, exemplary second binding partners in these embodiments include monoclonal antibodies such as SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-69, SAF-70, SAF-75, SAF-76, Pri917, BAR234, Sha31, 11B9, 12F10, 6H4, and POM5.
In certain embodiments of the kit, the kit comprises an ELISA kit, preferably a Sandwich ELISA kit. Among these embodiments, certain embodiments have the first binding partner as a capture binding partner and the second binding partner as a detection binding partner. Yet certain other embodiments have the second binding partner as a capture binding partner and the first binding partner as a detection binding partner.
In certain embodiments of the kit, the detection binding partners discussed above are labeled. In certain embodiments among these, the detection binding partners are labeled with an alkaline phosphatase (AP) conjugate.
In certain embodiments, the binding partners in the kit are antibodies, preferably monoclonal antibodies, specific to the first epitope and the second epitope, wherein the antibodies can be selected from, but are not limited to, the exemplary monoclonal antibodies shown above.
In certain other embodiments, the binding partners in the kit are aptamers specific to the first epitope and the second epitope.
In one embodiment of the kit, the site-specific protease is immobilized.
In another embodiment of the kit, the optional protease inhibitor is phenylmethylsulphonyl fluoride (PMSF).
In certain embodiments of the kit, the optional denaturant is a guanidinium compound.
In certain other embodiments of the kit, the optional denaturant is a basic reagent or an acidic reagent.
The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g.; Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons); Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press); Peters and Dalrymple, Fields Virology (2d ed), Fields et al. (eds.), B. N. Raven Press, New York, N.Y.
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.
The following select terms will be discussed in the context used herein. Both the plural and singular forms of a term are included regardless of the form discussed.
“Prion,” “prion protein,” “PrP protein,” and “PrP” are used interchangeably to refer to both the pathogenic prion protein form (also referred to as scrapie protein, pathogenic protein form, pathogenic isoform, pathogenic prion and PrPSc) and the non-pathogenic prion form (also referred to as the normal form, cellular protein form, cellular isoform, nonpathogenic isoform, nonpathogenic prion protein and PrPC), as well as the denatured form and various recombinant forms of the prion protein that may not have either the pathogenic conformation or the normal cellular conformation.
Use of the terms “prion,” “prion protein,” “PrP protein,” “PrP” or “conformational disease protein” is not meant to be limited to polypeptides having the exact sequences to those described herein. It is readily apparent that the terms encompass conformational disease proteins from any of the identified or unidentified species (e.g., human, bovine) or diseases (e.g., Alzheimer's, Parkinson's, etc.). See also, co-owned U.S. Patent Publications 20050118645 and 20060035242 and PCT Publication WO 06/076687, which are incorporated herein by reference in their entireties. One of ordinary skill in the art in view of the teachings of the present disclosure and the art can determine regions corresponding to the sequences disclosed herein in any other prion proteins, using for example, sequence comparison programs (e.g., Basic Local Alignment Search Tool (BLAST)) or identification and alignment of structural features or motifs.
“Pathogenic” means that the protein actually causes the disease, or the protein is associated with the disease and, therefore, is present when the disease is present. Thus, a pathogenic protein, as used herein, is not necessarily a protein that is the specific causative agent of a disease. A “pathogenic form” of a protein means a conformation of a protein that is present when the disease is present, but it may or may not be infectious. An example of a pathogenic conformational disease protein, or a pathogenic form of a protein, is PrPSc. Accordingly, the term “non-pathogenic” or “normal form” describes a protein that does not normally cause disease or is not normally associated with causing disease. An example of a non-pathogenic or a normal form of conformational disease protein is PrPC.
“Prion-related disease” refers to a disease caused in whole or in part by a pathogenic prion protein, or a pathogenic form of prion protein (e.g., PrPSc). Examples of prion-related disease include, without limitation, scrapie, bovine spongiform encephalopathies (BSE), mad cow disease, feline spongiform encephalopathies, kuru, Creutzfeldt-Jakob Disease (CJD), new variant Creutzfeldt-Jakob Disease (nvCJD), chronic wasting disease (CWD), Gerstmann-Strassler-Scheinker Disease (GSS), and fatal familial insomnia (FFI).
The term “denature” or “denatured” has the conventional meaning as applied to protein structure and means that the protein has lost its native secondary and tertiary structure. With respect to the pathogenic prion protein, a “denatured” pathogenic prion protein no longer retains the native pathogenic conformation and thus the protein is no longer “pathogenic.” The denatured pathogenic prion protein has a conformation similar or identical to the denatured non-pathogenic prion protein.
The terms “label,” “labeled,” “detectable label,” and “detectably labeled” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, luminescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens), fluorescent nanoparticles, gold nanoparticles, and the like. Particular examples of labels that can be used include, but are not limited to fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, acridinium esters, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase and urease. The label can also be an epitope tag (e.g., a His-His tag), an antibody or an amplifiable or otherwise detectable oligonucleotide.
“Proteolytic digestion” or “protease digestion” refers to the directed digestion or degradation of a protein by a protease, through hydrolysis, or cleavage, of peptide bonds in the protein.
A “site-specific protease” refers to an enzyme that cleaves peptide bonds (a protease) at one type or a small number of different amino acid residues in a protein substrate. “Cleavage sites” for a site-specific protease refer to the specific amino acid residues in a protein substrate at which the adjacent peptide bonds are hydrolyzed, or “cleaved”, by the site-specific protease under normal proteolytic digestion conditions. The site-specific protease is distinguished from the non-specific proteases like proteinase K (which cleaves at aliphatic, aromatic and hydrophobic residues) and carboxypeptidase Y (which cleaves all residues sequentially beginning at the carboxy terminal). For example, trypsin is a site-specific protease that cleaves only at Lys and Arg residues, especially when the amino acid on the carboxyl side of Lys and Arg is not Pro. (See, e.g., Neurath and Schwert (1950) Chem. Rev. 46:70; Walsh and Neurath (1964) Proc. Natl. Acad. Sci. USA 52:884-889; Walsh (1970) Meth. Enzymol. 19:41.) Therefore, the cleavage sites for trypsin mean Lys and Arg residues when the amino acid on the carboxyl side of Lys and Arg is not Pro. The cleavage sites for trypsin are also referred to as “tryptic sites”. Another example is the protease S-V8, which specifically cleaves at Glu and Asp residues (See, e.g., Drapeau et al. (1972) J. Biol. Chem. 247:6720-6726 and Houmard and Drapeau (1972) Proc. Natl. Acad. Sci. USA 69:3506-3509). Trypsin and S-V8 are commercially available from companies such as Pierce, part of Thermo Fisher Scientific Inc., or Sigma-Aldrich, Inc.
A “substantially complete” digestion or “substantially digested” means a digestion in which a protein has been cleaved by a protease in at least 90%, preferably 99%, of all available protease cleavage sites. By “available cleavage site” is intended a site having the amino acid sequence recognized as the cleavage site by the protease and that is available for contact with the protease in the conformation of the protein. As an example, protease cleavage sites that occur within the protease-resistant core of the prion protein are generally not available to protease digestion when the prion protein is in the PrPSc conformation, and thus are not “available cleavage sites” in PrPSc.
The “octarepeat region” refers to a repeated sequence region that is found close to the N-terminal of prion proteins from all species so far identified. The octarepeat generally contains between 3 and 5, usually 4, copies of an 8 (or 9) amino acid sequence usually written as GQPHGG(G/S)(-/G)W (SEQ ID NO: 11). This sequence is highly conserved (although this sequence may vary slightly in some of the repeats) and generally occurs within about residues 58-89, numbered according to human and hamster PrP species. The octarepeat region is usually adjacent, and N-terminal proximal, to the protease-resistant region.
The “protease-resistant core” of the prion protein (sometimes called the “proteinase K resistant core”) is defined by the region of the prion protein in the PrPSc conformation that remains after exposure of the PrPSe to proteinase K under condition that are sufficient to substantially digest the prion protein in the PrPC form. In general, for most species of prion protein, the protease-resistant core region includes the regions from about amino acid 90 to about amino acid 231 numbered according to human and hamster PrP species.
The “amino acid region of PrPC corresponding to protease-resistant core” refers to the region of a PrPC that is the same as the region that forms the protease-resistant core in the pathogenic form of the prion protein with the same primary amino acid sequence of said PrPC. In general, for most species of prion protein, this region also refers to the region from about amino acid 90 to about amino acid 231 numbered according to human and hamster PrP species.
The “globular domain” of PrPC is approximately from amino acid 122 to amino acid 231 numbered according to human PrP species.
By “epitope” herein is meant the region of a target protein which is recognized by (e.g. specifically binds to) binding partners which include without limitation antibodies and aptamers. An epitope can comprise 3 or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more usually, consists of at least 8-10 such amino acids. Methods of determining spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. Furthermore, the identification of epitopes in a given protein is readily accomplished using techniques well known in the art, such as by the use of hydrophobicity studies and by site-directed serology. See, also, Geysen et al., Proc. Natl. Acad. Sci. USA (1984) 81:3998-4002 (general method of rapidly synthesizing peptides to determine the location of immunogenic epitopes in a given antigen); U.S. Pat. No. 4,708,871 (procedures for identifying and chemically synthesizing epitopes of antigens); and Geysen et al., Molecular Immunology (1986) 23:709-715 (technique for identifying peptides with high affinity for a given antibody).
“Amino-proximal region” refers to the N-terminal region of a mature PrP protein spanning from amino acid of 23 to about 89, numbered according to the human or hamster PrP sequence.
“Binding partner” refers to a molecule that can specifically recognize and bind, non-covalently, to a ligand such as an epitope. The binding partners as discussed in the current disclosure include without limitation antibodies and aptamers.
“Capture binding partner” refers to a binding partner that is coated on a solid support to specifically bind and capture a ligand, such as a PrP protein.
“Detection binding partner” refers to a binding partner that is added, in solution, to samples on a solid support to specifically detect any ligand that has been bound and captured by a capture binding partner.
In certain embodiments of the assay method, the binding partners are “aptamers.” Aptamers are nucleic acids having the molecular recognition properties of antibodies. However, aptamers are smaller and less complex than antibodies, and thus maybe easier to manufacture and modify. (See, e.g., Osborne et al. (1997) Curr. Opin. Chem. Biol. 1:5-9; Bunka et al. (2006) Nat. Rev. Microbiol. 4:588-596.) Methods of generating aptamers specific to protein epitopes are readily available (see, e.g., Tuerk and Gold (1990) Science 249:505-510; Cox et al. (1998) Biotechnol Prog. 14:845-850; Cox et al. (2002) Nucleic Acids Res. 30:e108). Aptamers can be linked to enzymes that have an easily detectable activity (see, e.g., Drolet et al. (1996) Nat. Biotechnol. 14:1021).
“Treating” or “Treatment” of a sample with a protease means that the sample and the protease are brought into contact and stay together for sufficient length of time for a reaction, particularly hydrolysis or cleavage of peptide bonds, to occur.
Prior to the present disclosure, a published patent disclosed a method using PK to digest PrPSc and retain the octarepeat region for binding with a binding partner (a ligand), which requires a controlled digestion condition for partial digestion of PrPSc but complete digestion of PrPc. (See U.S. Pat. No. 7,097,997 B1.) One problem with this previously disclosed method is that the appropriate condition may vary for different samples, and therefore, may be laborious to achieve and difficult to standardize. This previously disclosed method did not involve any use of a second binding partner to bind the protease-resistant core of PrPSc in order to differentiate it from PrPC.
To circumvent limitations associated with the use of non-specific proteases like PK, the current inventors investigated the use of other proteases, particularly site-specific proteases, that have no cleavage site within the octarepeat region of PrP. Therefore, even with a substantially complete digestion, the octarepeat region is not digested, making it consistently available for specific and strong binding by a binding partner. As compared to a partial digestion by PK, a substantially complete digestion by a site-specific protease is easier to carry out and standardize. In addition, the current disclosure includes the use of a second binding partner that binds to PrPSc but not to PrPC due to the removal of the corresponding epitope from PrPC by the protease, giving the currently disclosed method an advantage of high selectivity between the two forms of PrP.
Thus, the methods described herein relate to improvements that can increase specificity, sensitivity, ease of use and reproducibility of detection of PrPSc in a sample that may have a large amount of PrPC.
In certain embodiments of the current disclosure, the site-specific protease used is trypsin or S-V8, whose specific cleavages sites are not present in the octarepeat regions of the known PrP sequences as shown in
Prior to the present disclosure, one publication suggested using trypsin to unmask an epitope to PrPSc and eliminate availability of a corresponding epitope of PrPC. (See International Publication No. WO 99/19360 A1.) The immunoassay method suggested in WO 99/19360 A1 differs from the currently disclosed method, because the purpose or result of using trypsin in the currently disclosed method is not unmasking an epitope to PrPSc. In addition, the method suggested in WO 99/19360 A1 did not disclose binding PrPSc with an antibody specific for the octarepeat region. Therefore, the currently disclosed method offers an advantage of high sensitivity due to high avidity of binding partner's binding to the octarepeat region.
Another publication prior to the present disclosure, International Publication No. WO 03/001211 A1, disclosed a method for detecting the presence of prion proteins that involves a limited proteolysis by a protease such as PK. In one embodiment of the method described in WO 03/001211 A1, a protease such as trypsin was used briefly, prior to the steps of lysing the cells or tissues and treating the lysate with PK, and under a condition that will not disrupt the cell membrane, in order to facilitate tissue dissociation.
In addition, another publication prior to the present disclosure, International Publication No. WO 2006/088281 A1, disclosed a method for detecting a multimeric form from a monomeric form of a multimer-forming polypeptide (such as prion) that involves a treatment with trypsin. The method disclosed in WO 2006/088281 A 1 differs from the currently disclosed method. WO 2006/088281 A1 specifically promoted the using of antibodies against non-repeated sequence in prion protein over repeated sequences such as the octarepeat sequence, because antibodies against the octarepeat sequence produced higher background using the method disclosed in this publication.
In a recent publication, the protease thermolysin was used for diagnosis of prion diseases (See Owen et al., (2007) Mol. Biotechnol. 35:161-170). Thermolysin specifically cleaves at several hydrophobic residues, residues that are absent from the protease accessible amino-terminal region of PrPSc.
The present invention thus provides an assay method, comprising the steps of:
The present invention also provides a kit for detecting the presence of a pathogenic form of prion protein (PrPSc) which has a protease-resistant core and an octarepeat region in a sample suspected of containing said PrPSc, wherein said sample may or may not contain a normal form of prion protein (PrP), the kit comprising:
Site-specific proteases that are useful in the present invention are proteases that cleave peptide bonds of specific, discrete amino acid residues. Generally the site-specific proteases will cleave a protein at one type or a small number of specific amino acid residues thus allowing predictability in the cleavage of the prion protein. Examples of such site-specific proteases are: trypsin which is a site-specific protease that cleaves on the carboxyl side of Lys (K) or Arg (R) residues, when the amino acid on the carboxyl side of K and R is not Pro, and S-V8 which is a site-specific protease from S. aureus V8 strain that cleaves on the carboxyl side of Asp (D) or Glu (E) residues. For specificity of trypsin cleavage, see, e.g., Neurath et al. (1950) Chem. Rev. 46:70; Walsh and Neurath (1964) Proc. Natl. Acad. Sci. USA 52:884-889; Walsh (1970) Meth. Enzymol. 19:41. For specificity of S-V8 cleavage, see, e.g., Drapeau et al. (1972) J. Biol. Chem. 247:6720-6726, and Houmard et al. (1972) Proc. Natl. Acad. Sci. USA 69:3506-3509. Trypsin and S-V8 are commercially available from various suppliers such as Pierce, part of Thermo Fisher Scientific Inc., Rockford Ill., and Sigma-Aldrich, Inc., St. Louis, Mo.
Other such site-specific proteases can be readily selected by one of ordinary skill in the art.
In addition, to be useful in the present method, there must be a cleavage site for the site-specific protease in the prion protein in the region between the epitopes recognized by the two binding partners used in the assay method. At least one protease cleavage site will be within the protease-resistant core region (approximately amino acids 90-231 of the prion protein) and this site will be cleaved by the site-specific protease only when the prion protein is in the PrPC form and not when the prion protein is in the PrPSc form. Preferably at least one of the epitopes will be in the protease-resistant core region of the prion protein. Preferably, at least one other epitope will be located within the amino-proximal region of the prion protein, and more preferably, within the octarepeat region of the prion protein. Preferably, the site-specific protease does not cleave at a site within the octarepeat region of the prion protein. The core repeated sequence of the octarepeat region is GQPHGG(G/S)(-/G)W (SEQ ID NO: 11), which can vary slightly in prions from different species (See
As described above, the samples to be analyzed may naturally contain high levels of PrPC. PK has been used in other settings to digest the PrPC form, leaving the more resistant PrPSc form. However, PrPSc is not completely resistant to proteolysis if high concentrations of PK and/or prolonged exposure times are used as shown by the fact that PK treatment reduces infectivity of the pathogenic form. See, McKinley et al. (1983) Cell 35:57-62. Therefore, PK treatment must be carefully controlled in order to provide complete cleavage of the PrPC form but leave the resistant core of the PrPSc form intact. Too little PK digestion will leave residual PrPC form which will yield a false positive in the detection phase and too much PK digestion will cleave the PrPSc resistant core making it undetectable in the detection phase. In addition, although the particular PK digestion site(s) of PrPSc vary since the pathogenic form can adopt multiple conformations, PK digestion of PrPSc typically results in multiple protease resistant fragments around residues 90-231, which may reduce or eliminate the binding of anti-prion antibodies directed against epitopes in this region. See, Telling et al. (1996) Science 274:2079-2082.
In the current disclosure, a sample suspected of containing a pathogenic form of prion protein is treated with the selected site-specific protease under conditions in which any non-pathogenic prion protein would be digested substantially completely. One of ordinary skill in the art is competent to determine the appropriate conditions. Conditions of substantially complete digestion can readily be determined by tests using recombinant PrP. For trypsin as the site-specific protease, typically a trypsin concentration of 50 μg/ml for 1 hour at 37° C. in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20), preferably with 1% Triton X-100 and 0.2 M CaCl2, and a trypsin concentration of 10 μg/ml for 1 hour at 37° C. in TBSS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2% Sarkosyl), are both adequate.
Following substantially complete digestion of the non-pathogenic prion protein, the site-specific protease must be removed, inactivated or inhibited in order to prevent any further protease digestion, for instance, of the anti-prion antibodies that will be used for detection. The protease may be inhibited by the addition of one or more protease inhibitors. Protease inhibitors are well known in the art and include phenylmethylsulfonyl fluoride (PMSF), aprotinin, diisopropylfluorophosphate (DFP), and 1-chloro-3-tosylamido-4-phenyl-2-butanone (TLCK), among others. Alternately, some proteases are available in an immobilized form (e.g., in an agarose matrix) which can be readily removed from the reaction by conventional means (e.g., centrifugation, filtration, etc.).
Following the digestion of the sample by the site-specific protease and inhibition/removal of the protease, the digested sample which is suspected of containing PrPSc is denatured in order for a sensitive detection of epitopes.
The denaturation can be accomplished in a number of ways. In one embodiment, a chaotropic agent, preferably a guanidinium compound, e.g., guanidine thiocyanate or guanidine hydrochloride, is added to a concentration of between 3M and 6M. Addition of the chaotropic agent in these concentrations causes the pathogenic prion protein to denature. For this embodiment, the chaotropic agent must be removed or diluted before the detection is carried out because it will interfere with the binding of the binding partners to prion epitopes, e.g., anti-prion antibodies used in the ELISA.
In another embodiment, the denaturation is accomplished by either raising the pH to 12 or above (“high pH”) or lowering the pH to 2 or below (“low pH”). Details of the pH dissociation/denaturation technique are described in PCT/US2006/001437 and U.S. application Ser. No. 11/518,091, the disclosures of which are incorporated herein in their entireties. Exposure of un-denatured PrPSc to either high or low pH results in the denaturation of the pathogenic prion protein. In this embodiment, exposure of PrPSc to high pH is preferred. A pH between 12.0 and 13.0 is generally sufficient; preferably, a pH of between 12.5 and 13.0 is used; more preferably, a pH of 12.7 to 12.9; most preferably a pH of 12.9. Alternatively, exposure of PrPSc to a low pH can be used to denature the pathogenic prion protein. For this alternative, a pH of between 1.0 and 2.0 is sufficient. Exposure of the protease-digested sample to either a high pH or a low pH is carried out for only a short time e.g. 60 minutes, preferably for no more than 15 minutes, more preferably for no more than 10 minutes. Longer exposures than this can result in significant deterioration of the structure of the pathogenic prion protein such that epitopes recognized by binding partners used in the detection steps are destroyed.
After exposure for sufficient time to denature PrPSc, the pH can be readily readjusted to neutral (that is, pH of between about 7.0 and 7.5) by addition of either an acidic reagent (if high pH denaturation conditions are used) or a basic reagent (if low pH denaturation conditions are used). One of ordinary skill in the art can readily determine appropriate protocols and examples are described herein. Because the high pH or low pH used in the denaturation step can be easily readjusted to neutral by addition of small volumes of suitable acid or base, this embodiment allows the use directly in the detection step, e.g., ELISA, without any additional washes and without increasing the sample volumes significantly.
In general, to effect a high pH denaturation condition, addition of NaOH to a concentration of about 0.05 N to about 0.2 N is sufficient. Preferably, NaOH is added to a concentration of between 0.05 N to 0.15 N; more preferably, 0.1 N NaOH is used. Once the denaturation of PrPSc is accomplished, the pH can be readjusted to neutral (that is, between about 7.0 and 7.5) by addition of suitable amounts of an acidic solution, e.g., phosphoric acid, sodium phosphate monobasic.
In general, to effect a low pH denaturation condition, addition of H3PO4 to a concentration of about 0.2 M to about 0.7 M is sufficient. Preferably, H3PO4 is added to a concentration of between 0.3 M and 0.6 M; more preferably, 0.5 M H3PO4 is used. Once the denaturation of PrPSc is accomplished, the pH can be readjusted to neutral (that is, between about 7.0 and 7.5) by addition of suitable amounts of a basic solution, e.g., NaOH or KOH.
The denatured samples can be used for further detection of PrPs.
Denatured PrPSc can be detected with at least two binding partners specific for prion sequences, with one binding partner specifically binding an epitope in the amino-proximal region of denatured PrPSc, preferably in the octarepeat region, and the other binding partner specifically binding an epitope in the protease-resistant core region of denatured PrPSc which is no longer available in PrPC due to protease cleavage.
In certain embodiments of the method, the binding partners are antibodies, and a preferred embodiment among these has its detection step carried out using an ELISA.
In certain other embodiments, the binding partners used in the method are aptamers specific to the first epitope and the second epitope.
Antibodies, modified antibodies and other reagents, that bind to prions, particularly to PrPC or to the denatured PrP, have been described and some of these are available commercially (see, e.g., anti-prion antibodies described in Peretz et al. (1997) J. Mol. Biol. 273:614; Peretz et al. (2001) Nature 412:739; Williamson et al. (1998) J. Virol. 72:9413; Polymenidou et al. (2005) supra; U.S. Pat. No. 6,765,088). Some of these and others are available commercially from, inter alia, In Pro Biotechnology, South San Francisco, Calif., Cayman Chemicals, Ann Arbor Mich.; Prionics AG, Zurich; also see, WO 03/085086 for description of modified antibodies.
Preferred anti-prion antibodies will be ones that bind to a denatured form of the pathogenic prion.
Particularly preferred first antibodies will be ones for the first epitope that is located within the octarepeat region of the prion protein. Examples of such antibodies are POM2, POM11, POM12, POM14, 3B5, 4F2, 13F10, SAF-15, SAF-31, SAF-32, SAF-33, SAF-34, SAF-35, and SAF-37. (See, e.g., Polymenidou et al. (2005) Lancet Neurol. 4:805-814; Krasemann et al. (1996) Mol. Medicine. 2:725-734; Féraudet, et al. (2005) J. Biol. Chem. 280:11247-11258; U.S. Pat. No. 7,097,997 B1.)
In certain embodiments, the first antibody will bind to an epitope that is outside of the octarepeat region. Exemplary first binding partners in these embodiments include monoclonal antibodies such as BAR210, BAR231, and 14D3 (See, e.g., Krasemann et al. (1996) Mol. Medicine. 2:725-734; Féraudet, et al. (2005) J. Biol. Chem. 280:11247-11258).
Preferred second antibodies will be ones that recognize the second epitopes that are within the protease-resistant core region of PrPSc and that are no longer available in PrPC due to protease cleavage. Because the first cleavage site in the protease-resistant core region of PrPC for trypsin is different from that for S-V8 (K-106 for trypsin and D-144 for S-V8, numbered according to human PrP), there will be more useful epitopes for trypsin-digested PrPSc than for S-V8.
Exemplary second binding partners in embodiments that are digested with trypsin include monoclonal antibodies such as 3F4 (U.S. Pat. No. 4,806,627), POM1, POM4, POM5, POM6, POM7, POM8, POM9, POM10, POM13, POM15, POM16, POM17, POM19 (for POM antibodies, see, Polymenidou et al. (2005) Lancet Neurol. 4:805-814), SAF-2, SAF-4, SAF-8, SAF-9, SAF-10, SAF-12, SAF-13, SAF-14, SAF-22, SAF-24, SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-68, SAF-69, SAF-70, SAF-75, SAF-76, SAF-82, SAF-83, SAF-84, SAF-95, Pri308, Pri917, BAR215, BAR221, BAR224, BAR233, BAR234, Sha31, 11B9, 12F10 (Krasemann et al. (1996) Mol. Medicine. 2:725-734; Féraudet, et al. (2005) J. Biol. Chem. 280:11247-11258; U.S. Pat. No. 7,097,997 B1.), D18 (Peretz et al. (1997) J. Mol. Biol. 273:614), 6H4 (Liu et al. (2003) J. Histochem. Cytochem. 51:1065), and BDI115 (Biodesign International). The 3F4 antibody recognizes an epitope, MKHM, at amino acids 109-112 of human PrP. Other antibodies recognize various epitopes within the regions from about amino acid 107 to about amino acid 231 numbered according to human PrP species.
In certain embodiments of the method in which trypsin is used, the second binding partners specifically bind to epitopes located within the globular domain of PrP (i.e., from about amino acid 122 to about amino acid 231 of PrP). Exemplary second binding partners in these embodiments include monoclonal antibodies such as POM1, POM4, POM5, POM6, POM7, POM8, POM9, POM10, POM13, POM15, POM16, POM17, POM19, SAF-2, SAF-4, SAF-8, SAF-9, SAF-10, SAF-12, SAF-13, SAF-14, SAF-22, SAF-24, SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-68, SAF-69, SAF-70, SAF-75, SAF-76, SAF-82, SAF-83, SAF-84, SAF-95, Pri917, BAR215, BAR221, BAR224, BAR233, BAR234, Sha31, 11B9, 12F10, D18, 6H4, and BDI115.
Exemplary second binding partners in embodiments that are digested with S-V8 include monoclonal antibodies such as SAF-53, SAF-54, SAF-60, SAF-61, SAF-66, SAF-69, SAF-70, SAF-75, SAF-76, Pri917, BAR234, Sha31, 11B9, 12F10, 6H4, and POM5.
Other anti-prion antibodies can readily be generated by methods that are well-known in the art.
One of skill in the art will appreciate from the disclosure herein that the first and second antibodies are selected such that the first antibody specifically binds a first epitope in the amino-proximal region, preferably in the octarepeat region, and the second antibody specifically binds a second epitope within a region of the protease-resistant core of PrPSc that has been cleaved away in PrPC. In this way, following digestion with the site-specific protease, the epitopes recognized by the first and second antibodies will be present on different fragments of the PrPC (and so will not be capable of detection in the Sandwich ELISA) but these epitopes will be present on a single fragment of the PrPSc (and so will be detectable in the Sandwich ELISA).
Some anti-prion antibodies are specific for prion protein from one or a limited number of animal species, others are capable of binding prion proteins from many animal species. It will be apparent to choose suitable anti-prion antibodies based upon the samples to be analyzed and the purpose of the testing.
The protease-digested and denatured pathogenic prion proteins are preferably detected in an ELISA type assay, either as a direct ELISA or an antibody Sandwich ELISA type assay, which are described more fully below. Although the term “ELISA” is used to describe the detection with anti-prion antibodies, the assay is not limited to ones in which the antibodies are “enzyme-linked.” The detection antibodies can be labeled with any of the detectable labels described herein and well-known in the immunoassay art.
In a preferred embodiment of the method, the protease-digested and denatured pathogenic prion proteins are detected using an antibody Sandwich type ELISA. In this embodiment, the denatured prion protein is captured on a solid support comprising a capture antibody, which can be the first antibody or the second antibody in different embodiments. The solid support with the captured prion protein, is optionally washed to remove any unbound materials, and then contacted with a detection antibody, which can be the second antibody or the first antibody, depending on what the capture antibody is (the capture antibody is different from the detection antibody), under conditions that allow the detection antibody to bind to the captured prion protein. Methods for carrying out said Sandwich ELISAs are well known and are described with the Examples herein.
Suitable solid supports include any material that is an insoluble matrix and has a rigid or semi-rigid surface to which the pathogenic-prion specific reagent can be linked or attached. Exemplary solid supports include, but are not limited to, substrates such as nitrocellulose, polyvinylchloride, polypropylene, polystyrene, latex, polycarbonate, nylon, dextran, chitin, sand, silica, pumice, agarose, cellulose, glass, metal, polyacrylamide, silicon, rubber, polysaccharides, polyvinyl fluoride; diazotized paper; activated beads, magnetically responsive beads, and any materials commonly used for solid phase synthesis, affinity separations, purifications, hybridization reactions, immunoassays and other such applications. The support can be particulate or can be in the form of a continuous surface and includes membranes, mesh, plates, pellets, slides, disks, capillaries, hollow fibers, needles, pins, chips, solid fibers, gels (e.g. silica gels) and beads, (e.g., pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N-N′-bis-acryloylethylenediamine, iron oxide magnetic beads, and glass particles coated with a hydrophobic polymer. Preferred solid support for the first antibody is a microtiter plate.
Preferably, either the capture antibody or the detection antibody in the Sandwich ELISA recognizes an epitope within the octarepeat region of the prion protein. In some embodiments, the detection antibody is detectably labeled; in further embodiments, the detection antibody is enzyme labeled.
In certain other embodiments, the binding partners used in the method are aptamers. Aptamers are nucleic acids having the molecular recognition properties of antibodies. Aptamers are smaller and less complex than antibodies, and thus maybe easier to manufacture and modify. (See, e.g., Osborne et al. (1997) Curr. Opin. Chem. Biol. 1:5-9; Bunka et al. (2006) Nat. Rev. Microbiol. 4:588-596.) Methods of generating aptamers specific to protein epitopes are readily available (see, e.g., Tuerk and Gold (1990) Science 249:505-510; Cox et al. (1998) Biotechnol Prog. 14:845-850; Cox et al. (2002) Nucleic Acids Res. 30:e108). Aptamers can be linked to enzymes that have an easily detectable activity (see, e.g., Drolet et al. (1996) Nat. Biotechnol. 14:1021).
Any of the detection methods for a pathogenic prion described hereinabove can be used in a method to diagnose a prion-related disease in any sample.
For use in the methods described herein, the sample can be anything known to, or suspected of, containing a pathogenic prion protein. In some embodiments, the sample is a biological sample (that is, a sample prepared from a living or once-living organism), or a non-biological sample. In some embodiments, the sample is a biological sample. Non-limiting examples of biological samples are organs (e.g., brain, liver, and kidney), cells, whole blood, blood fractions, blood components, plasma, platelets, serum, cerebrospinal fluid (CSF), brain tissue, nervous system tissue, muscle tissue, muscle and fatty tissue (e.g., flesh), bone marrow, urine, tears, non-nervous system tissue, biopsies, necropsies, foods that are sourced from a living or once-living organism, and any other organic matter such as plant materials. In some embodiments, the biological sample comprises whole blood, blood products, blood fractions, blood components, plasma, platelets, red blood cells or serum. The biological sample can be obtained during a health related procedure such as a blood donation or screening, biopsy, autopsy, or necropsy, or during a process or procedure during food preparation such as animal selection and slaughter and quality assurance testing of finish product. In some embodiments, the sample is non-biological. Non-limiting examples of non-biological samples include pharmaceuticals, cosmetics and personal care products, contaminated instruments and foods that are not sourced from a living or once-living organism, and the like.
Suitable controls can also be used in the assays described herein. For instance, a negative control of PrPC can be used in the assays. A positive control of PrPSc (or PrPres) could also be used in the assays. Such controls can optionally be detectably labeled.
The above-described assay reagents, including the site-specific proteases, protease inhibitors (optional), denaturing agents (or denaturants, optional), anti-prion binding partners such as antibodies, etc., can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct detection assays as described above. The kit may further contain suitable positive and negative controls, as described above. The kit can also contain, depending on the particular detection assay used, suitable labels and other packaged reagents and materials (i.e., wash buffers and the like).
In order that the invention disclosed herein can be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.
The commercially available monoclonal antibody 3F4 (Covance Research Products, Inc.) was used as a capture antibody in several examples shown below. It specifically recognizes residues 109-112 (amino acids MKHM) which is located in the protease-resistant core region of human prion protein. It was diluted at 2.5 μg/mL in coating buffer (0.1 M NaHCO3, pH 8.9) and incubated at room temperature (RT) for 5 min with agitation. The diluted antibody 3F4 was added to Thermo Labsystem Micro Lite 2+ microplates at volumes of 150 μL/well, and then incubated at RT for over night in a plastic wrap sealed tray with moisture. The plates were washed 3 times with Tris-buffered Saline with Tween 20 (TBST, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) on a BioTek Elx450 washer. A blocking buffer (0.01× BlockerCasein in Tris-buffered Saline (TBS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 3% sucrose) was then dispensed to the plates at volumes of 300 μL/well of on the BioTek Elx450 washer, and incubated at RT for 1 hr. The plates were lyophilized for overnight at 16° C., and each plate was put into a pouch with 3 desiccants, double-sealed and stored at 4° C. In general, all the coated plates were blocked with 0.02% casein for 1 hr at 37° C. prior to use.
(A) Digestion of Plasma with Trypsin
To test whether trypsin degrades PrPC and renders it undetectable by ELISA, 10% normal human plasma in TBST was treated with increasing concentrations of trypsin (Table 1). The trypsin used in this and following examples was obtained from Pierce (TPCK Trypsin, Catalog No. 20233). The digestion was incubated at 37° C. for 1 hr with shaking at 200 rpm. Digestion was stopped by addition of the protease inhibitor phenylmethylsulphonyl fluoride (PMSF, final concentration 1 mM) with incubation at RT for 15 min. The trypsin-treated samples were diluted to 1% (of the normal human plasma), and 150 μL was used in each well for ELISA.
Samples were tested for the presence of PrPC by a Sandwich ELISA using 3F4 as capture antibody and detection with POM2 antibody (Polymenidou et al., supra) conjugated to alkaline phosphatase (AP) using a chemiluminescence substrate for light detection. POM2 specifically recognizes the octarepeat epitopes within residues 59-91.
Briefly, trypsin-treated plasma samples were diluted to 1% (of the original human plasma) in TBST and transferred to 3F4-coated plates at volumes of 150 μL/well. The plates were incubated at 37° C. for 1 hr with shaking at 400 rpm, and then washed 6 times with TBST. AP-conjugated POM2 detection antibody (0.01 μg/mL) was added into the plates at volumes of 150 μL/well and incubated at 37° C. for 1 hr. The plates were again washed 6 times with TBST. Finally, 150 μL/well of Lumi-Phos Plus substrate with 0.05% SDS was added to the plates and incubated at 37° C. for 30 min, and signals were read with a microplate luminometer. The results are shown in Table 1, and measurement units are defined in relative light units (RLU).
The result shows that trypsin digested PrPC in human plasma and that detection of PrPC was abolished in samples treated with higher concentration of trypsin (Table 1). We found dose response between levels of detected PrPC and trypsin concentration. At trypsin concentration of 400 μg/mL, levels of PrPC dropped 100 fold to background levels.
In this example, we tested trypsin digestion of PrPC present in normal Syrian hamster (SHa) brain homogenate (BH).
Five micro-liters of 10% SHa BH (w/v) was mixed with 5 μL of trypsin in TBST with 1% Triton X-100 and 0.2 M CaCl2, in increasing concentrations of trypsin (Table 2), for 1 hr at 37° C. with shaking at 750 rpm. Each digested sample was diluted by addition of 140 μL of TBS, and digestion was stopped by addition of 1.5 μl/well of 100 mM PMSF (for final PMSF concentration 1 mM) and incubation at RT for 10 min with shaking at 750 rpm.
The trypsin-digested SHa BH samples were tested for the presence of PrPC by a Sandwich ELISA using 3F4 as capture antibody and detection with AP-conjugated POM2 antibody as described in Example 2. The digestion solutions, 150 μL of each, were transferred into each well of the 3F4-coated microplates and incubated at 37° C. for 1 hr with shaking at 300 rpm. Plates were washed with TBST, and 150 μL/well of 0.01 μg/mL of AP-conjugated POM2 in 0.01× casein-TBST was added and incubated at 37° C. for 1 hr. Substrate was added and plates were read with a microplate luminometer as described in Example 2. The results are shown in Table 2.
Similar to the results with human plasma, trypsin digested PrPC in SHa brain homogenate and detection of PrPC was also abolished (Table 2). In contrast to plasma, efficient degradation of PrPC in brain homogenate was achieved with trypsin concentration of 25 μg/mL, much lower than the trypsin concentration needed for efficient degradation of PrPC in plasma.
In this example, we compared the effects of trypsin and PK digestion on ELISA detection of Syrian Hamster PrPSc, a pathogenic form of prion, using 4 different pairs of monoclonal antibodies and one concentration of PK and trypsin.
(A) Digestion of SHa BH with Trypsin or PK and Denaturation of Digested Samples
Two micro-liters of 5% SHa BH samples (normal or infectious) were treated with trypsin or PK at the same concentration of 25 μg/mL, each in final reaction volume of 2 μL, for 1 hr at 37° C. Digestion was stopped with PMSF (2 mM, at RT for 10 min).
Samples were denatured with guanidine hydrochloride (GdnHCl) to ensure maximum binding of antibodies to PrPSc (3 M final concentration, 1 hr at RT). Denatured samples were then diluted to 0.1 M GdnHCl and added to ELISA plates coated with various mAbs.
Microplates were coated with mAb 3F4, POM2 or POM17 at 150 μL of 2.5 μg/mL in 0.1 M NaHCO3, pH 8.9, at 4° C. overnight. The plates were blocked with 0.02% casein at 37° C. for 1 hr prior to use. POM17 recognizes an epitope within the protease resistant fragment of residues ˜90-231, in particular an epitope within the globular domain of residues ˜122-231.
The protease-digested and subsequently denatured SHa BH samples were transferred to plates coated with 3F4, POM2 or POM17 at the final volumes of 150 μL/well. The plates were incubated at 37° C. for 1 hr with shaking at 300 rpm, and then washed with TBST. AP-conjugated POM2 (POM2AP) or AP-conjugated POM17 (POM17AP), both at final concentration of 0.01 μg/mL, was added into the plates at the final volumes of 150 μL/well and incubated at 37° C. for 1 hr. The plates were again washed with TBST, Lumi-Phos Plus substrate was added and incubated at 37° C. for 30 min, and signals were read as shown in Example 2. The results of this Sandwich ELISA experiment are shown in Table 3.
We found from the results shown in Table 3 that PK reduced detection signal in all cases as compared to trypsin-digested samples. Reduction of more than 70% was observed between trypsin and PK-digested samples when we used POM2 either as detection antibody or capture antibody in the infectious brain homogenates (rows 1, 3 and 4). For example the signal obtained with 3F4/POM2AP (capture/detection) was reduced from 2370 to 527 RLU. A similar proportion in signal reduction was observed with POM2/POM17AP and POM17/POM2AP. The reduction in signal is due to the PK digestion of the octarepeat epitopes recognized by POM2 within residues 23-89.
We also noticed a drop of 20% in signal when we treated the infectious brain homogenate samples with PK and detected with 3F4/POM17AP, even though the epitopes for these antibodies are in the protease-resistant core. This drop in signal suggests that even limited PK digestion can affect the protease resistant fragment 90-231. It is worth noting that the highest readings were observed when POM2, which specifically binds to the octarepeat region of PrP, was used as the detection antibody after trypsin digestion. The next highest reading was observed when POM2 was used as the capture antibody. We conclude that, in the presence of 4 octarepeats, POM2 binds more than one epitope and therefore has stronger binding than antibodies that recognize only one epitope, such as POM17 and 3F4.
A number of anti-PrP antibodies as shown in Table 4, which includes SAF-32, a monoclonal antibody against the octarepeat region, were screened for their abilities to bind to recombinant PrP (rPrP) of different animal species and to mouse PrPSc.
aInformation from Feraudet, et al. (2005) J. Biol. Chem. 280: 11247-11258 and U.S. Pat. No. 7,097,997 B1.
(A) Coating Plates with Various PrP
Some microplates were coated with recombinant PrPs (rPrPs) of Bovine, Deer, Human, Sheep or Mouse following these steps. Each rPrP was diluted and denatured in 3M guanidine thiocyanate (GdnSCN) to 1, 0.1, 0.01, and 0.001 μg/ml, incubated at RT for 10 min, and 100 μl/well of the denatured rPrP was added in duplicate to 96-well plates. Then, to each well, 100 μl of 0.1 M NaHCO3, pH 8.8, was added, and plates were incubated at 4° C. for overnight.
Other microplates were coated with Mouse BH PrPSc following these steps. PrPSc-infected Mouse BH (4004 of 10% BH) was thawed in TBS with 1% Tween 20 and 1% Triton X-100, and precipitated by centrifugation at 20,000 rpm for 1 hr at 4° C. The precipitates were each washed with 1 mL TBS of pH 7.5, and re-precipitated by centrifugation at 20,000 rpm for 10 min at 4° C. The pellets were each re-suspended in 4 mL of 3 M GdnSCN to reach the final concentration of 1% BH, and were denatured at RT for 15 min. The denatured BH samples were diluted to 0.1%, 0.01%, and 0.001% in 3M GdnSCN, and 100 μl/well of each concentration of BH was added to the plates in duplicate. Then 100 μl/well of 0.1 M NaHCO3, pH 8.8, was added to each well of BH samples. Incubation was carried out at 4° C. for overnight.
Each plate was washed 3 times with TBST. Each well was then blocked with 200 μl, of 3% BSA in TBS and incubated at 37° C. for 1 hr. After the buffer was aspirated, 100 μL/well of primary antibody to be screened was added at 0.5 μg/ml in TBS with 1% BSA, and plates were incubated at 37° C. for 2 hr. After plates were washed 6 times with TBST, 100 μL/well of AP-conjugated secondary antibodies which bind to the screened primary antibodies, 1:5000 diluted in TBS with 1% BSA, were added. Plates were incubated at 37° C. for 1 hr, and again washed 6 times with TBST. One hundred μL/well of Lumi-Phos Plus substrate with 0.05% SDS was added and incubated at 37° C. for 30 min. Signal was read with a luminometer, and the results of two of the different coated amounts of each PrP, 0.1 ng and 1 ng per assay for each rPrP, as well as 0.1 mg and 1 mg per assay for Mouse BH PrPSc, are shown in Table 5.
The results in Table 5 show that SAF-32, which specifically recognizes the octarepeat region, bound PrP proteins of all the animal species tested—bovine, deer, human, sheep, and mouse. The strongest bindings of SAF-32 were to rPrP of bovine, deer, human and sheep. The results also showed significant binding between SAF-32 and mouse PrPSc in the brain homogenates.
In this example human normal or vCJD-infected BH samples were treated with trypsin or PK, and then were either denatured or left un-denatured before being detected with SAF-32 as the capture antibody and 3F4AP as the detection antibody.
Microplates were coated with 150 μL of 3.3 μg/mL of SAF-32 diluted in 0.1 M NaH2CO33, pH 8.9. Plates were coated for overnight at 4° C., washed with TBST, and blocked with 1% casein for 1 hour at 37° C. prior to use.
One micro-liter of 10% human normal or vCJD BH sample (w/v) was added to 1 pt of TBSTT (TBS, 1% Tween 20 and 1% Triton X-00) that has trypsin or PK for a final concentration of 25 μg/mL of trypsin or PK. Digestions by the proteases were carried out for 10 min at 37° C. and then stopped by 2 mM of PMSF. The digested samples were either left un-denatured or denatured with 4 M of GdnHCl at RT for 1 hour. The un-denatured samples were diluted with TBST to 150 μL. The denatured solutions were diluted with TBST to 150 uL for a final concentration of 0.1 M GdnHCl. Samples were added to the microplates coated with SAF-32 (Cayman Chemicals) at volumes of 150 μL/well for overnight at 4° C. Plates were washed with TBST, and 150 μL of 0.1 μg/mL of AP-conjugated 3F4 (Covance Research Products/Signet Labs) was added to each well. After incubation of 1 hr at 37° C., plates were washed, Lumi-Phos Plus substrate was added and incubated for 30 min at 37° C., and levels of signals were measured with a luminometer as shown in previous examples. Measurement units are defined in relative light units (RLU). The results are shown in Table 6 and
As shown in the results, treatment with trypsin or PK digested most or all PrPC in normal tissue and detection was only 50.4 RLU or lower after denaturation. Un-denatured vCJD tissue did not give any significant readings. Therefore, it can be concluded that efficient detection of PrPSc by SAF-32 and 3F4 antibody combination requires denaturation of PrPSc.
In addition, the results in this example showed that treatment of the vCJD BH sample with trypsin followed by denaturation resulted in a strong signal of 1653 RLU which is 32-fold higher than the signal detected in the normal BH sample. Similar treatment of the vCJD sample with PK resulted in a much lower signal of 226 RLU as compared to trypsin treatment, and the signal is 12.5-fold higher than the signal detected in the PK-treated normal sample. This example again shows that detection of PrPSc after PK digestion which cleaves within the octarepeat region in the N-terminus PrPSc is inferior to detection of PrPSc after trypsin treatment which does not affect the octarepeat region. This effect is now seen in this example and Example 4 using two different antibodies, POM2 and SAF-32, both binding the octarepeat region. This finding suggests that usage of various antibodies against the octarepeat sequence in combination with trypsin treatment will result in higher sensitivity of PrPSc detection than usage of antibodies against singular epitopes and/or PK treatment.
A recent report suggests (see U.S. Pat. No. 7,097,997 B1) that gentle treatment with PK could efficiently eliminate PrPC while preserving all or some of the octarepeat epitopes of PrPSc, providing enhanced detection through the use of antibody that binds octarepeats. The objective of the two experiments in this example is to compare the effectiveness of PK digestion in eliminating PrPC from Syrian hamster (SHa) brain homogenates (BH), while maintaining sensitivity of SHa PrPSc detection, with the method of the present invention using trypsin digestion under various digestion conditions.
Digestion of 1 μL of 10% SHa normal or scrapie-infected BH (w/v) was done with increasing amount of trypsin or PK (0 to 100 μg/mL) at 37° C. for 10 min in 50 μL of TBS with 1% Tween 20, 1% Triton X-100 and 0.02M CaCl2. Each digestion was stopped with PMSF at 2 mM, RT for 15 min, denatured with 75 μL. of 0.1 N NaOH for 10 min, and then neutralized with 304 of 0.3M NaH2PO4 for 5 min at RT.
Microplates were coated with 150 μL of 2.5 μg/mL of 3F4 diluted in 0.1 M NaH2CO3, pH 8.9, at 4° C. for overnight, washed with TBST, and blocked with 0.1% casein prior to use.
Digestion samples, 150 μL/well of each, were added to 3F4-coated plates for an ELISA assay. The plates were incubated for overnight at 4° C., washed with TBST, and 150 μL of detection antibody POM2AP (0.01 μg/mL) or POM17AP (0.1 μg/mL, in order for the assay sensitivity to be comparable to POM2) was added per well. After incubation of 1 hr at 37° C., plates were washed, substrate (Lumi-Phos Plus) was added and incubated for 30 min at 37° C., and levels of signals were measured with a luminometer as shown in previous examples. Measurement units are defined in relative light units (RLU).
The results of the experiment done with POM2AP are shown in Table 8 and
As shown in Table 8 and
As shown in Table 9 and
From the results with either POM2AP or POM17AP as summarized in Table 10, better detection of PrPSc was observed with trypsin than with PK at any given concentration of proteases, indicated by the higher S/N values of trypsin-digested samples.
When digested with PK and detected with POM2AP, detection level of PrPSc in scrapie-infected SHa BH was the lowest as indicated by the low S/N values. When the same samples were detected with POM17 the signal was about 4 times higher at optimum concentration of PK. These results are consistent with what is known and expected about the protease resistant properties of PrPSc; residues 90-231 are resistant to mild protease digestion and therefore can be detected with antibodies with epitope within this region, like POM17, while the N-terminus 23-90 is sensitive and antibodies that bind the octarepeat region, like POM2 and SAF-32, will lose their binding sites. It is also apparent that as we increase the concentration of PK the N-terminus and protease resistant core 90-231 are digested concomitantly. Thus, in order to achieve the goal as proposed in U.S. Pat. No. 7,097,997 B1, concentration of PK has to be carefully titrated. As incubation time and temperature will affect enzymatic activity, each study needs be tailored to a specific condition, making it a laborious and unstable process. Another level of complexity is the variations in enzymatic specific activities associated with different preparations, batches and sources of PK.
Treatment with trypsin does not have the same limitations as PK. Antibodies with high avidity like POM2 can be used since the octarepeat region remained intact. While PK cleaves within residues 23-89, trypsin cleaves within residues 23-48 only, leaving residues 49-89, which contains the octarepeat region, available for detection in addition to the protease-resistant core 90-231 (
In addition, PrPSc seems to be more resistant to trypsin than to PK, because at 100 μg/mL of trypsin S/N values are still over 100. This is consistent with early studies which demonstrated that PK reduced levels of PrPSc and prion infectivity by several logs while trypsin did not (McKinley et al. (1983) Cell 35:57-62). Of the samples detected with POM2AP, the S/N is 73 for samples treated with 50 μg/mL of PK, while the S/N is 448 for samples treated with the same concentration of trypsin, indicating a 6-fold improvement when using trypsin as compared to using PK. When detected with POM17AP, an improvement of more than 25 fold (415 vs. 17 RLU) was observed with 50 μg/mL of trypsin, and one of more than 2 fold (560 vs. 203 RLU) was observed with 20 μg/mL of trypsin. Thus, for both antibodies, trypsin treatment gave better detection. It should be noted that although POM2AP was used at 0.01 μg/ml, as compared to POM17AP which was used at 0.1 μg/ml, its detection was as good.
In order to confirm that trypsin digests PrPC and PrPSc differently, an immunoblot assay was carried out as described below.
A schematic of the full-length mature PrP sequence, the PK-resistant core of PrPSc, and the trypsin cleavage site map of PrP is shown in
Infectious human vCJD and sCJD BH samples were acquired from the National Institute for Biological Standards and Control (NIBSC) CJD Resource Centre in U.K. and correspond to White vCJD (MM), Red sCJD (MM), and Yellow sCJD (MV) strains, respectively. Normal brain homogenate was derived from a patient with vascular encephalopathy from the University of Zurich, corresponding to patient NRPE 327. Twenty five micrograms of normal or infectious human BH sample were digested with 50 μg/ml of trypsin or Proteinase K (PK) in 0.5×TBS with 0.5% Tween20, 0.5% TritonX-100, and 5 mM CaCl2 for 1 hr at 37° C. Samples were separated by 12% SDS-PAGE in parallel with 10 μg undigested sample for comparison, and immunoblotted with anti-PrP antibodies (3F4, POM2 or POM17). Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies were used to detect the Western blots. Chemiluminescent images were acquired via a Kodak Image Station 4000 mM. The result is shown in
In addition, normal or a vCJD human BH sample was digested with increasing concentrations of trypsin and then analyzed by immunoblotting in a similar manner as described above with POM2, 3F4 or POM1 antibody separately. POM1 recognizes an epitope within the protease-resistant core, in particular an epitope within the globular domain of residues about 122-231. The result is shown in
Immunoblot analysis of the digests indicated that trypsin-resistant PrPSc fragments (expected to be ˜49-231) were larger than PK-resistant fragments (˜90-231) with a substantial fraction of molecules preserved (
By contrast, PrPC was digested by both proteases, and was no longer detected by POM2 (residues ˜59-89) or 3F4 (residues ˜109-112) (
PK has commonly been used to characterize strain-dependent diversity in PrPSc conformations. These differences are manifested as susceptibility to protease digestion and alterations in amino-terminal cleavage sites, as observed for types 1 and 2 sCJD. Digestion with trypsin, however, has the unique advantage that cleavage sites are known, with several sites lying within well-characterized antibody epitopes (
1% BH (˜1 mg/ml total protein) diluted in TBS with 2% Sarkosyl (TBSS) was digested with 0, 1, 10, or 100 μg/ml trypsin or PK for 1 hr at 37° C. Digestions were stopped with the addition of 2 mM PMSF and a Complete Mini protease inhibitor cocktail (Roche, Indianapolis, Ind.) in four volumes of TBS. The samples were then detected by direct ELISA. Approximately 500 nl of digested 10% BH was centrifuged at 14,000 rpm for 30 minutes at 4° C. The PrPSc pellets were resuspended in 6M GdnSCN, diluted with an equal volume of 0.1M NaHCO3 pH8.9, and passively coated to ELISA plates overnight. The plates were blocked in 0.1× BlockerCasein in TBS (Pierce) and coated PrP was detected via 0.1 μg/ml 3F4, POM2 or POM17 antibodies and alkaline phosphatase (AP)-conjugated goat anti-mouse antibodies (Pierce). All samples were analyzed by ELISA in triplicate and were washed six times with TBS 0.05% Tween20 between antibody incubations. Finally, LumiphosPlus substrate (Lumigen, Southfield, Mich.) with 0.05% SDS was added to the wells and incubated for 30 minutes at 37° C. before the luminescence was measured via a Lumiskan luminometer (Thermo Electron Corporation, Waltham, Mass.). The raw data for normal and vCJD BH samples are shown in
Of note, very little PrPC-derived signal was present in the pellets of undigested samples in both normal and infectious BHs, which completely disappeared with only 1 μg/ml protease (
When examining PrPSc from a vCJD BH, we found significant differences in the exposure of epitopes to trypsin versus PK. While the 3F4 epitope was preserved at the same rate when either protease was utilized (
Interestingly, POM17 detection of the PrPSc core particle revealed that the vast majority of helix one, the region containing the epitope recognized by POM17, was resistant at every concentration of trypsin tested (
Taken together with the immunoblot analysis results shown in Example 8, these results suggest that trypsin digestion of PrPSc, especially at higher concentrations of protease, generates two populations of trypsin-resistant fragments, one population being the expected long fragments spanning residues of ˜49-231, the other population spanning residues of ˜411-231, lacking the 3F4 epitope and octarepeats. In conclusion, we find that the 3F4 epitope is equally exposed to cleavage by trypsin or PK, but that other sequences such as the octarepeat and helix one regions are uniquely preserved in trypsin digestion, making trypsin an ideal tool for analysis of PrPSc.
In this example, an experiment similar to the one described in Example 7 was carried out on several human BH samples instead of Syrian Hamster BH samples. Normal or various CJD-infected human BH samples, including vCJD (M/M), sCJD (M/M1) and sCJD (M/V2), were digested with increasing concentrations of trypsin or PK and detected by Sandwich ELISA. In order to see whether the preservation of the octarepeat region by trypsin digestion can enhance PrP detection, a detection antibody against the octarepeat region, POM2AP, was used at the same concentration as and compared to another detection antibody which is against a non-octarepeat region, POM17AP. In Example 7, POM2AP was used at one tenth of the concentration of POM17AP.
10% BH diluted 10-fold into TBS with 2% Sarkosyl (TBSS) was digested with 0, 1, 10, or 100 μg/ml trypsin or PK for 1 hr at 37° C. Digestions were stopped by adding 2 mM PMSF and Complete Mini protease inhibitor cocktail (Roche, Indianapolis, Ind.) in four volumes of TBS. The samples were then detected by Sandwich ELISA. Two hundred and fifty nl of digested 10% BH was denatured with 0.1M NaOH for 10 minutes at 25° C., neutralized with NaH2PO4 pH 4.3, and PrP was captured with 3F4-coated plates (375 ng/well) and detected with 0.02 μg/ml AP-conjugated POM2 or POM17. All samples were analyzed by ELISA in triplicate and were washed six times with TBS 0.05% Tween20 between antibody incubations. Finally, LumiphosPlus substrate (Lumigen, Southfield, Mich.) with 0.05% SDS was added to the wells and incubated for 30 minutes at 37° C. before the luminescence was measured via a Lumiskan luminometer (Thermo Electron Corporation, Waltham, Mass.). The raw data for normal and various CJD BH samples are shown in
As shown in
As those skilled in the art will appreciate, numerous changes and modifications can be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention. It is also intended that each of the patents, applications, and printed publications, including books, mentioned in this patent document be hereby incorporated by reference in its entirety.
This application claims the benefit of U.S. Ser. Nos. 60/921,951, filed Apr. 4, 2007 and 61/066,704, filed Feb. 21, 2008, the disclosures of which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2008/004454 | 4/3/2008 | WO | 00 | 4/9/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60921951 | Apr 2007 | US | |
| 61066704 | Feb 2008 | US |
