WO2006102099A2 - Anticorps specifiques de la proteine prp humaine et bovine - Google Patents

Anticorps specifiques de la proteine prp humaine et bovine Download PDF

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WO2006102099A2
WO2006102099A2 PCT/US2006/009752 US2006009752W WO2006102099A2 WO 2006102099 A2 WO2006102099 A2 WO 2006102099A2 US 2006009752 W US2006009752 W US 2006009752W WO 2006102099 A2 WO2006102099 A2 WO 2006102099A2
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Prior art keywords
prp
antibody
bovine
antibodies
sample
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PCT/US2006/009752
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WO2006102099A3 (fr
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Stanley B. Prusiner
Ana Veronica Serban
Jiri G. Safar
Larry Stanker
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2872—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against prion molecules, e.g. CD230
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the cell lines F20-29, F20-49, F20-80, F20-89a, F20-108a, F20-130a and F4-31 have been deposited with the American Type Culture Collection and designated Patent Deposit No. PTA-6607.
  • the deposited cell line F4-30 was assigned ATC No. PTA-6838 and the cell line F4-7 was assigned ATC No. PTA-6839.
  • This invention relates to antibodies, methods for obtaining antibodies and assays for using such antibodies, including double capture immunoassays. More specifically, the invention relates to antibodies that specifically bind to PrP from humans, bovines, and other mammals.
  • Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein.
  • PrP prion protein
  • BSE is a major socioeconomic problem.
  • Many of Canada's trading partners closed their markets to Canada's beef after a 2003 case of BSE was discovered.
  • the discovery of a BSE case in a US cow has resulted in closure of some export markets for US beef products.
  • BSE has been reported in cows from numerous countries including the United Kingdom, Japan, France, Germany, Italy, and Switzerland.
  • nvCJD Creutzfeldt- Jakob disease
  • PrP Sc is the invariant component of prion infection and is the only disease-specific diagnostic marker that can be readily detected by immunoassay in the brains of clinically ill animals and humans Meyer et al. (1986), Proc. Natl. Acad. ScL USA, 83:3693-7; Serban et al. (1990), Neurology, ⁇ 0:110-117; Taraboulos et al. (1992), Proc. Natl. Acad. ScI USA. 89:7620-7624; Grathhydrate, K. U. D., M. Horiuchi et al. (1997), Virol. Methods 54:205-216. Unfortunately, PrP Sc assays are positive only when the prion titer is high, and detection of low levels of PrP Sc has been problematic. It has also proven difficult to measure low levels of PrP Sc in the presence of high levels of PrP .
  • the antibodies are useful in numerous applications, and particularly for determining prion infection in humans and in bovines.
  • the antibodies are characterized by one or more of the following features: (1) an ability to bind to native PrP c or denatured human or bovine PrP Sc with specificity; and (2) an ability to capture various forms of PrP protein in a solids substrate format.
  • Useful capture antibodies need not have a high degree of species specificity, e.g. may cross-reactive with PrP from multiple mammalian species.
  • An important object is to provide antibodies that bind to bovine and to human PrP c and that are useful in a capture immunoassay.
  • Still another object is to provide specific methodology for high throughput screening of monoclonal antibodies for usefulness in capture assays.
  • Another object of the invention is to provide an assay for the detection of PrP Sc particularly in homogenized human or bovine brain tissue using the antibodies of the invention.
  • An advantage of the invention is that it provides a fast, cost effective assay for detecting the presence of infectious prions in the form of PrP Sc in a bovine or human sample.
  • a specific advantage is that the assay can be used as a screen for the presence of prions
  • PrP Sc PrP Sc
  • products such as pharmaceuticals (derived from natural sources) food, cosmetics, blood, or any material which might contain such prions and thereby provide further assurances as to the safety of such products.
  • Another advantage is that the antibodies can be used with a compound which denatures
  • PrP Sc to the degree needed to expose an epitope which will bind to an antibody of the invention thereby providing for a means of differentiating levels of PrP c and PrP c + PrP Sc in a sample.
  • FIG. 1 is a schematic of a double capture immunoassay (DCI).
  • Figure 2A is a graph of a DCI analysis of sera from individual mice immunized with recBoPrP 102-241.
  • Figure 2B shows a Western blot analysis of brain homogenate from PrP ablated mouse brain, PrP 0 °; mouse; Syrian hamster; Human; bovine; recombinant bovine, recBo, sheep; and mule deer. PrP was detected using serum from a mouse immunized with recBoPrP 102-241.
  • Figure 3 A is a graph depicting ELISA activity of Protein G purified F4-31 antibody.
  • Figure 4 is a graph of a DCI titration analysis of F4-31 monoclonal antibody on Human recPrP90-231 versus Bovine recPrP 102-242.
  • Figure 5 shows a Western blot analysis of brain homogenate from PrP ablated mouse brain, PrP 0 °; mouse, Mo; Syrian hamster, SHa; Human, Hu; bovine, Bo; recombinant bovine, recBo, sheep, Ov; and mule deer, MD detected with monoclonal antibody F4-31.
  • Figures 6A-6B show antibody binding to synthetic peptides.
  • Figure 7 Shows electrophoretic analysis and western blot analysis of reduced (lanes 1,
  • Figure 8 is a graph depicting results of an ELISA titration on reduced (DTT treated) and non reduced antigen.
  • the monoclonal antibodies F4-31, F20-29, F20-80 and F20-130a show loss of binding to the reduced antigen.
  • binding of the monoclonal antibodies F20-49 and F20-89 is not affected by the reduction of the antigen.
  • Figure 9 is a graph depicting the effect of GndHCl treatment of recBoPrP 102-241 or recHuPrP90-231, on subsequent antibody binding in a direct binding ELISA.
  • Figure 9A shows the results for monoclonal antibodies F4-31, F20-29, F20-49 and F20-80.
  • Figure 9B shows the results for monoclonal antibodies F20-89, F20-108a and F20-130a.
  • HuM-P is shown as a control.
  • FIG 10 panels A and B show detection B of infectious prions using a CDI incorporating antibody D-18 (a previously described antibody) versus monoclonal antibody F4-31 as the capture antibody.
  • panel A the samples analyzed were Human, CJD; Guinea Pig, F431; Mouse, RML431; and Hamster, Sc237.
  • panel B the samples were Sheep, Ov, Deer, CWD; and Bovine, BSE infectious brain homogenates.
  • Figure 11 is a graph showing the results of determining the capture affinity constant
  • Figure 12 is a graph of the results of a dissolution curve of BSE infected brain homogenate into normal cow brain homogenate.
  • an antibody includes a plurality of such antibodies and reference to “the hybridoma” includes reference to one or more hybridomas and equivalents thereof known to those skilled in the art, and so forth.
  • PrP protein protein
  • PrP protein
  • PrP protein
  • PrP Sc protein The terms "prion”, “prion protein” and “PrP Sc protein” and the like used interchangeably herein to refer to the infectious PrP Sc form of a PrP protein and is a contraction of the words “protein” and “infection” and the particles are comprised largely if not exclusively of PrP Sc molecules encoded by a PrP gene. Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and chronic wasting disease (CWD) of deer and elk.
  • BSE bovine spongiform encephalopathies
  • CWD chronic wasting disease
  • prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt- Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI).
  • CJD Creutzfeldt- Jakob Disease
  • GSS Gerstmann-Strassler-Scheinker Disease
  • FFI fatal familial insomnia
  • prion includes all forms of prions causing all or any of these diseases or others in any animals used — and in particular in humans and in domesticated farm animals.
  • PrP gene is used herein to describe genetic material that encodes PrP c proteins, including proteins having polymorphisms and mutations such as those listed herein under the subheading "Pathogenic Mutations and Polymorphisms.”
  • PrP gene refers generally to any gene of any species which encodes any form of a prion protein.
  • the PrP gene can be from any animal including the "host” and “test” animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered.
  • the protein expressed by such a gene can assume either a PrP (non-disease) or PrP Sc (disease) form.
  • standardized prion preparation which composition is obtained from brain tissue of mammals which contain substantially the same genetic material as relates to prions, e.g., brain tissue from a set of mammals which exhibit signs of prion disease which mammals (1) include a transgene as described herein; (2) have an ablated endogenous prion protein gene; (3) have a high copy number of prion protein gene from a genetically diverse species; or (4) are hybrids with an ablated endogenous prion protein gene and a prion protein gene from a genetically diverse species.
  • the mammals from which standardized prion preparations are obtained exhibit clinical signs of CNS dysfunction as a result of inoculation with prions and/or due to developing the disease due to their genetically modified make up, e.g., high copy number of prion protein genes.
  • the term "genetic material related to prions” is intended to cover any genetic material which effects the ability of an animal to become infected with prions.
  • the term encompasses any "PrP gene”, “artificial PrP gene”, “chimeric PrP gene” or “ablated PrP gene” which terms are defined herein as well as modification of such which effect the ability of an animal to become infected with prions.
  • Standardized prion preparations are produced using animals which all have substantially the same genetic material related to prions so that all of the animals will become infected with the same type of prions and will exhibit signs of infection at about the same time.
  • host animal and "host mammal” are used to describe animals which may have their genome genetically and artificially manipulated so as to include genetic material which is not naturally present within the animal.
  • host animals include mice, hamsters and rats which have their PrP gene ablated, i.e., rendered inoperative.
  • PrP PrP gene
  • the host is inoculated with prion proteins to generate antibodies.
  • Other host animals can have a natural (PrP) gene or one which is altered by the insertion of an artificial gene or by the insertion of a native PrP gene of a genetically diverse test animal.
  • test animal and "test mammal” are used to describe the animal which is genetically diverse from the host animal in terms of differences between the PrP gene of the host animal and the PrP gene of the test animal.
  • the test animal may be any animal for which one wishes to run an assay test to determine whether a given sample contains prions with the ability to infect test animal.
  • the test animal may be any bovine or mammal infected with a variant bovine prion, including human, cow, sheep, pig, horse, cat, dog or chicken, and one may wish to determine whether a particular sample includes prions which would normally infect only the test animal.
  • a mouse PrP gene is genetically diverse with respect to the PrP gene of a cow or sheep, but is not genetically diverse with respect to the PrP gene of a hamster.
  • ablated PrP protein gene means an endogenous PrP gene which has been altered (e.g., added and/or removed nucleotides) in a manner so as to render the gene inoperative.
  • non-functional PrP genes and methods of making such are disclosed in B ⁇ eler, H., et al. (1992), Nature 356, 577-582 and Weissman (WO 93/10227).
  • the methodology for ablating a gene is taught in Capecchi (1987), Cell 51:503-512, all of which are incorporated herein by reference. Preferably both alleles of the genes are disrupted.
  • hybrid animal transgenic hybrid animal
  • transgenic hybrid animal an animal obtained from the cross-breeding of a first animal having an ablated endogenous prion protein gene with a second animal which includes either (1) a chimeric gene or artificial PrP gene or (2) a PrP gene from a genetically diverse animal.
  • a hybrid mouse is obtained by cross-breeding a mouse with an ablated mouse gene with a mouse containing (1) bovine or other bovine PrP genes (which may be present in high copy numbers) or (2) chimeric mouse/bovine PrP genes.
  • hybrid includes any offspring of a hybrid including inbred offspring of two hybrids provided the resulting offspring is susceptible to infection with prions with normal infect only a genetically diverse species.
  • a hybrid animal can be inoculated with prions and serve as a source of cells for the creation of hybridomas to make monoclonal antibodies of the invention.
  • transgenic or hybrid test animal which develops a disease if inoculated with prions which would normally only infect a genetically diverse test animal.
  • the terms are used to describe a transgenic or hybrid animal such as a transgenic mouse Tg(MBo2M) which, without the chimeric PrP gene, would not become infected with a bovine prion but with the chimeric gene is susceptible to infection with bovine prions.
  • ungulate refers to any hoofed mammal. This includes, but is not limited to, cows, deer, elk, sheep and goats. For purposes of the invention a preferred ungulate is a bovine (cow).
  • antibody refers to an immunoglobulin protein that is capable of binding an antigen.
  • the archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, other avians, etc., are considered to be “antibodies.”
  • Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g. F(ab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest.
  • antibody encompasses all types of antibodies, e.g. polyclonal, monoclonal, etc., however for most purposes of the invention, reference is made to monoclonal antibodies.
  • Monoclonal antibodies are preferred for many uses because they can be reproduced by cell culture or recombinantly, and can be modified to reduce their antigenicity.
  • Particularly preferred antibodies of the invention are antibodies that have a relatively high degree of affinity for the target antigen.
  • purified antibody refers to that which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody “preferentially binds" to its cognate antigen with high specificity, and does not substantially recognize or bind to other antigenically unrelated molecules.
  • a purified antibody of the invention may be immunoreactive with, and immunospecific for, a specific species; or may be cross-reactive with PrP proteins of different mammalian species.
  • antigenic fragment of a PrP protein is meant a portion of such a protein which is capable of binding an antibody of the invention.
  • the level of affinity of antibody binding that is considered to be “specific” will be determined in part by the class of antibody, e.g. antigen specific antibodies of the IgM class may have a lower affinity than antibodies of, for example, the IgG classes.
  • the affinity will be at least about 10 '7 M, usually about 10 '8 to "9 M, and may be up to 10 "11 or higher for the epitope of interest.
  • detectably labeled antibody By “detectably labeled antibody”, “detectably labeled anti-PrP” or “detectably labeled anti-PrP fragment” is meant an antibody (or antibody fragment which retains binding specificity), having an attached detectable label.
  • the detectable label is normally attached by chemical conjugation, but where the label is a polypeptide, it could alternatively be attached by genetic engineering techniques. Methods for production of detectably labeled proteins are well known in the art.
  • Detectable labels may be selected from a variety of such labels known in the art, but normally are radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate.
  • radioisotopes e.g., fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate.
  • detectable label/substrate pairs e.g., horseradish peroxidase/diamin- Strukturzidine, avidin/streptavidin, luciferase/luciferin
  • methods for labeling antibodies, and methods for using labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)).
  • treatment means obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly humans, and bovine, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the present invention provides an antibody that specifically binds to a human or bovine
  • PrP c usually a denatured human or bovine PrP wherein all PrP has been treated in a limited manner in order to expose epitopes that bind an antibody of the invention.
  • the antibody is useful as a capture reagent, and may be cross-reactive with other mammalian PrP proteins.
  • an assay is provided for screening antibodies for use in a capture assay, depicted in Figure 1.
  • This newly developed rapid double capture immunoassay allows for early (at the first step in the screening process) identification of monoclonal antibodies that are useful as capture reagents.
  • Microtiter wells are coated with an antibody (referred to as antibody 1) that binds to the constant (Fc) region of the antibody expressed by the newly fused hybridoma cells, e.g. a goat anti-mouse IgG (Fc specific); or a goat anti-rat IgG; etc.
  • Hybridoma supernatants comprising a candidate monoclonal antibody (antibody 2) (e.g., mouse monoclonal antibodies produced by hybridomas following cell fusion) are then added to the microtiter wells and the plate incubated for a period of time sufficient to allow any antibody 2 (e.g., mouse antibodies present in the hybridoma supernatants) to specifically bind via interaction with the combining site of the immobilized antibody 1 (e.g., the goat anti-mouse antibody) .
  • antibody 2 e.g., mouse monoclonal antibodies produced by hybridomas following cell fusion
  • the plate is coated with goat-anti-mouse antibody and when hybridoma supernatants are screened to determine if they contain mouse monoclonal antibodies of interest, the hybridoma supernatant is added into the microtiter well, any mouse monoclonal antibodies present in the supernatant will specifically bind to the the anti-mouse antibody (antibody 1) by reaction of the constant region of the mouse monoclonal antibody with the combining site of the immobilized goat-anti-mouse antibody.
  • the mouse monoclonal antibodies are now immobilized on the surface of the microtiter well and are oriented so that their combining sites (in this example, that portion that binds prion protein) is available to bind antigen.
  • the supernatant is removed, the plate washed and the combining site of antibody 1 not occupied with antibodies in the hybridoma supernatants are blocked by allowing an irrelevant antibody molecule (in this example, a nonspecific mouse immunoglobulin) to bind the nonoccupied combining sites of antibody 1.
  • This second blocking step reduces background fluorescence resulting from an apparent binding of the unoccupied combining sites of antibody 1 with subsequently added reagents.
  • the plates are decanted, the cognate antigen dissolved in blocking buffer is added, and the plate incubated for a period of time sufficient for the antigen to bind to the trapped monoclonal antibodies (antibody 2). The plates are then washed, and Europium conjugated anti-prion antibody is added.
  • the methods of the invention provide for the development of antibodies that are able to recognize epitopes that are present on both PrP c and treated PrP Sc i.e. denatured prion proteins.
  • the antibody is produced by one of the hybridoma cell lines F4-31,
  • the antibodies specifically bind to a denatured human or bovine PrP Sc protein in situ with an affinity of 10 7 moles/liter or more, preferable 10 8 moles/liter or more of a single species.
  • Antibodies of the invention may have an affinity for multiple species, e.g., humans, all types of ungulates including cows, , sheep, goats, etc. or may be specific to a single species, e.g., cow or human.
  • Antibodies may be isolated, using the protocols of the present invention, with the ability to bind to all proteins coded by the different mutations and/or polymorphisms of the human and bovine PrP protein gene.
  • a battery of antibodies (2 or more different antibodies) can be provided wherein each antibody of the battery specifically binds to a protein encoded by a different mutation or polymorphism of a human or a bovine PrP gene.
  • the antibody can be bound to a support surface and used to assay a sample in vitro for the presence of a particular allele of human or of bovine PrP .
  • the antibodies of the present invention are especially useful to detect prions utilizing in vitro methods, in which the presence of PrP Sc in tissues of humans or animals (e.g, homogenated bovine brain) indicates prion infection.
  • a conformation-dependent immunoassay (CDI) such as disclosed in U.S. Patent 5,891,641 offers a rapid, specific, and highly sensitive method for the detection of bovine PrP Sc using the antibodies of the invention.
  • the assay is conformation-sensitive and can detect relatively low levels of PrP Sc in brain homogenates in which PrP c is present in a 100-fold excess relative to PrP So .
  • CDI conformation-dependent immunoassays
  • Patents 5,891,641 issued April 6, 1999, 6,617,119 and 6,620,629 are incorporated herein by reference in their entirety.
  • the selection of antibodies and resultant assays can be performed directly in samples or indirectly in the brains of animals inoculated with a sample containing prions.
  • the host mammal may be any mammal and is preferably a host mammal of the type defined herein such as a mouse, rat, rabbit, guinea pig or hamster, and is most preferably a mouse.
  • the host animal is inoculated either with PrP proteins that are endogenous to humans or to a bovine species.
  • a mouse is inoculated with a bovine PrP c peptide which may be treated to expose epitopes.
  • the antibodies will, if at all, only be generated for epitopes which differ between epitopes of the PrP protein of the host animal and epitopes of the PrP c from the genetically diverse species. This substantially limits the amount of antibodies that might be generated and decreases the ability to find an antibody that selectively binds to a human or bovine PrP c .
  • PrP protein gene i.e., a null PrP gene abbreviated as Prnp 0/0 .
  • Prnp 0/0 a null PrP gene abbreviated as Prnp 0/0 .
  • This allows antibodies to be generated against areas of bovine PrP c that are conserved between the host animal and the bovine PrP genes. Accordingly, the invention is also described in connection with the use of such "null" mammals and more specifically described in connection with "null mice.”
  • a null mouse can be created by inserting a segment of DNA into a normal mouse PrP gene and/or removing a portion of the gene to provide a disrupted PrP gene. The disrupted gene is injected into a mouse embryo and replaces the endogenous PrP gene via homologous recombination.
  • the null mouse is injected with either human or bovine PrP peptides to stimulate the formation of antibodies. Injections of adjuvants can be used in conjunction with the peptides to maximize the generation of antibodies. The mouse is then sacrificed and bone marrow and spleen cells are removed.
  • Hybridomas are generated by conventional methods. Immunization may be performed using a standard protocol, by injecting the null animal with an antigenic composition, see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. The protein, peptide, peptide-conjugate, etc. is injected into a animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the antigen may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
  • Hybridomas may be formed by isolating the stimulated immune cells, such as those from the spleen of the inoculated animal. These cells are then fused to immortalized cells, such as myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line.
  • the immortal cell line utilized is preferably selected to be deficient in enzymes necessary for the utilization of certain nutrients.
  • Many such cell lines (such as myelomas) are known to those skilled in the art, and include, for example: thymidine kinase (TK) or hypoxanthine-guanine phosphoriboxyl transferase (HGPRT) deficient lines.
  • the fusion partner is preferably altered in a manner such that they have reduced expression or no expression of the PrP protein (to which the antibody binds).
  • the immortal fusion partners utilized are derived from a line that does not secrete immunoglobulin.
  • the resulting fused cells, or hybridomas are cultured under conditions that allow for the survival of fused, but not unfused, cells and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, expanded, and grown so as to produce large quantities of antibody, see Kohler and Milstein, 1975 Nature 256:495 (the disclosures of which are hereby incorporated by reference).
  • mice Large quantities of monoclonal antibodies from the secreting hybridomas can be produced by injecting the clones into the peritoneal cavity of mice and harvesting the ascites fluid therefrom.
  • the mice preferably primed with pristane, or some other tumor-promoter, and immunosuppressed chemically or by irradiation, may be any of various suitable strains known to those in the art.
  • the ascites fluid is harvested from the mice and the monoclonal antibody purified therefrom, for example, by CM Sepharose column or other chromatographic means.
  • the hybridomas may be cultured in vitro or as suspension cultures. Batch, continuous culture, or other suitable culture processes may be utilized. Monoclonal antibodies are then recovered from the culture medium or supernatant.
  • Chimeric antibodies may be made by recombinant means by combining the murine variable light and heavy chain regions (VK and VH), obtained from a murine (or other animal- derived) hybridoma clone, with the human constant light and heavy chain regions, in order to produce an antibody with predominantly human domains.
  • VK and VH murine variable light and heavy chain regions
  • the production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659, incorporated fully herein by reference).
  • Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody.
  • immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab') 2 , or other fragments) are useful as antibody moieties in the present invention.
  • Such antibody fragments may be generated from whole immunoglobulins by f ⁇ cin, pepsin, papain, or other protease cleavage. "Fragment,” or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques.
  • Fv immunoglobulins for use in the present invention may be produced by linking a variable light chain region to a variable heavy chain region via a peptide linker (e.g., poly-glycine or another sequence which does not form an alpha helix or beta sheet motif).
  • a peptide linker e.g., poly-glycine or another sequence which does not form an alpha helix or beta sheet motif.
  • Fv fragments are heterodimers of the variable heavy chain domain (V H ) and the variable light chain domain (V L )-
  • the heterodimers of heavy and light chain domains that occur in whole IgG, for example, are connected by a disulfide bond.
  • Recombinant Fvs in which V H and VL are connected by a peptide linker are typically stable, see, for example, Huston et al., Proc. Natl.
  • derivatized immunoglobulins with added chemical linkers detectable moieties, such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties and the like, or specific binding moieties, such as streptavidin, avidin, biotin, or histidine molecules to facilitate purification, and the like may be utilized for various purposes.
  • detectable moieties such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties and the like
  • specific binding moieties such as streptavidin, avidin, biotin, or histidine molecules to facilitate purification, and the like may be utilized for various purposes.
  • the genetic material which makes up the PrP gene is known for a number of different species of animals (see Gabriel et al. (1992), Proc. Natl. Acad. Sci. USA 89:9097-9101 and U.S. Patents 5,763,740 and 5,792,901). Further, there is considerable homology between the PrP genes in different mammals. Although there is considerable genetic homology with respect to PrP genes, the differences are significant in some instances. More specifically, due to small differences in the protein encoded by the PrP gene of different mammals, a prion which will infect one mammal (e.g. a human) will not normally infect a different mammal (e.g. a mouse).
  • mice Due to this "species barrier", it is not generally possible to use normal animals, (i.e., animal which have not had their genetic material related to PrP proteins manipulated) such as mice to determine whether a particular sample contains prions which would normally infect a different species of animal such as a human.
  • the present invention provides methods for using modified, transgenic animals having bovine PrP genes or a chimeric bovine PrP gene to detect prions in samples from bovines.
  • the antibodies of the present invention provide the means by which these bovine prions can be detected in assays.
  • PrP 27-30 The major component of purified infectious prions, designated PrP 27-30, is the proteinase K resistant core of a larger native protein PrP Sc which is the disease causing form of the ubiquitous cellular protein PrP c .
  • PrP Sc is found only in scrapie infected cells, whereas PrP c is present in both infected and uninfected cells implicating PrP Sc as the major, if not the sole, component of infectious prion particles. Since both PrP c and PrP Sc are encoded by the same single copy gene, great effort has been directed toward unraveling the mechanism by which PrP Sc is derived from PrP c . Central to this goal has been the characterization of physical and chemical differences between these two molecules.
  • PrP Sc Properties distinguishing PrP Sc from PrP c include low solubility (Meyer et al.(1986), Proc. Natl. Acad. Sci. USA 83:3693-7), poor antigenicity (Kascsak et al.(1987), "Mouse Polyclonal and Monoclonal Antibody to Scrapie- Associated Fibril Proteins.” J. Virol.
  • the binding affinity of 10 7 1/mole or more may be due to (1) a single monoclonal antibody (i.e., large numbers of one kind of antibodies) (2) a plurality of different monoclonal antibodies (e.g., large numbers of each of five different monoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is also possible to use combinations of or all of (l)-(3).
  • the Conformation-Dependent Assay allows the direct measurement of the amount of PrP Sc in brain homogenates without prior digestion with proteinase K to eliminate PrP c .
  • the assay is conformation-sensitive and can detect relatively low levels of PrP Sc in brain homogenates in which PrP is present in a 100-fold excess. By selective precipitation of PrP Sc prior to differential immunoassay, PrP Sc can be measured in the presence of a 3, 000-fold excess of PrP c .
  • the assay can quantify less than 1 ng/ml of PrP Sc in brain homogenate with a dynamic range of 5 orders of magnitude (Safar, J., H. Wille et al. (1998), Nat.
  • the differential immunoassay can detect prion titers as low as 1 ID50 unit/ml.
  • the CDI allows one to distinguish multiple strains of prions by plotting the ratio of denatured/native PrP as a function of PrP Sc concentration before and after limited proteinase K digestion. In contrast, only one strain (DY) (Bessen, R. A. and R. F. Marsh (1994), J. Virol.
  • the antibodies to bovine residues 102-130 allow the CDI to detect prions in cows, deer, elk, sheep and other bovines.
  • Antibodies to human residues 90- 120 allow the CDI to detect prions in humans.
  • the high-affinity antibody reacting within epitope I of the denatured bovine PrP allow the CDI assay to detect, for example, the presence of human or bovine prions in a test sample. This epitope is critical not only for absolute, but also for conformational sensitivity of CDI. Conformational sensitivity of CDI is crucial for specificity of the assay and the ability to distinguish PrP from PrP .
  • Pathogenic mutations and polymorphisms There are a number of known pathogenic mutations in the human PrP gene. Further, there are known polymorphisms in the human, sheep and bovine PrP genes. The antibodies of the present invention may be geared to recognize specific alleles of the PrP gene. Alternatively polymorphisms or mutations known to be pathogenic in one species (e.g. human) can be added to a peptide from a bovine PrP. The following is a list of such mutations and polymorphisms:
  • the DNA sequence of the human, sheep, and cow PrP genes have been determined allowing, in each case, the prediction of the complete amino acid sequence of their respective PrP proteins.
  • the normal amino acid sequence which occurs in the vast majority of individuals is referred to as the wild-type PrP sequence.
  • This wild-type sequence is subject to certain characteristic polymorphic variations.
  • sheep PrP the gene displays two amino acid polymorphisms at residues 171 and 136, while bovine PrP has either five or six repeats of an eight amino acid motif sequence in the amino terminal region of the mature prion protein. While none of these polymorphisms are of themselves pathogenic, they appear to influence prion diseases. Distinct from these normal variations of the wild-type PrP proteins, certain mutations of the human PrP gene which alter either specific amino acid residues of PrP or the number of octarepeats have been identified which segregate with inherited human prion diseases.
  • PrP genes For example, a chicken, bovine ⁇ sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al. (1992) Proc. Natl. Acad. Sci. USA 52:9097-9101. The sequence for the Syrian hamster is published in Basler et al. (1986) Cell 46:417-428. The PrP gene of sheep is published by Goldmann et al. (1990) Proc. Natl. Acad. Sci. USA 57:2476-2480. The PrP gene sequence for bovine is published in Goldmann et al. (1991) J Gen. Virol.
  • PrP gene sequence for chicken PrP gene is published in Harris et al. (1991) Proc. Natl. Acad. Sci. USA 55:7664-7668.
  • PrP gene sequence for mink is published in Kretzschmar et al. (1992) J Gen. Virol. 73:2757-2761.
  • the human PrP gene sequence is published in Kretzschmar et al. (1986) DNA 5:315-324.
  • PrP gene sequence for mouse is published in Locht et al. (1986) Proc. Natl. Acad. Sci. USA 53:6372-6376 .
  • the PrP gene sequence for sheep is published in Westaway et al. (1994) Genes Dev. 5:959-969 .
  • Standardized prion preparations may be produced in order to test assays of the invention and thereby improve the reliability of the assay.
  • the preparation can be obtained from any animal it is preferably obtained from a host animal which has brain material containing prions of a test animal.
  • a transgenic mouse containing a bovine prion protein gene can produce bovine prions and the brain of such a mouse can be used to create a standardized bovine prion preparation.
  • the preparation is to be a "standard” it is preferably obtained from a battery (e.g., 100; 1,000, or more animals) of substantial identical animals.
  • mice all containing a very high copy number of bovine PrP genes would spontaneously develop disease and the brain tissue from each could be combined to make a useful standardized prion preparation.
  • Standardized prion preparations are described and disclosed in U.S. Patent 5,908,969 issued June 1, 1999 and U.S. Patent 6,020,537 issued February 1, 2000, both of which are incorporated herein in their entirety.
  • Standardized prion preparations can be produced using any of modified host mammals of the type described above.
  • standardized prion preparations can be produced using mice, rats, rabbits, hamsters, or guinea pigs which are genetically modified so that they are susceptible to infection with prions which prions would generally only infect genetically diverse species such as a cow, sheep, deer or horse and which modified host mammals will develop clinical signs of CNS dysfunction within a period of time of 350 days or less after inoculation with prions.
  • the most preferred host mammal is a mouse in part because they are inexpensive to use and because a greater amount of experience has been obtained with respect to production of transgenic mice than with respect to the production of other types of host animals. Details regarding making standardized prion preparation are described in U.S. Patents 6,008,435 and 6,020,537, both of which are incorporated herein by reference.
  • mice may be mice which are genetically modified by the insertion of a chimeric gene of the invention.
  • the mice might be modified by including high copy numbers of the chimeric gene and/or by the inclusion of multiple promoters in order to increase the level of expression of the chimeric gene.
  • hybrid mice of the invention could be used wherein mice which have the endogenous PrP gene ablated are crossed with mice which have a bovine PrP gene inserted into their genome. There are, of course, various subcategories of such hybrid mice.
  • the bovine PrP gene may be inserted in a high copy number an/or used with multiple promoters to enhance expression.
  • the mice could be produced by inserting multiple different PrP genes into the genome so as to create mice which are susceptible to infection with a variety of different prions, i.e., which generally infect two or more types of test animals.
  • a mouse could be created which included a chimeric gene including part of the sequence of a cow, a separate chimeric gene which included part of the sequence of a deer, and still another chimeric gene which included part of the sequence of a sheep. If all three different types of chimeric genes were inserted into the genome of the mouse the mouse would be susceptible to infection with prions which generally only infect a cow, deer and sheep.
  • the next step is to produce a large number of such mammals which are substantially identical in terms of genetic material related to prions. More specifically, each of the mice produced will include an identical chimeric gene present in the genome in substantially the same copy number.
  • the mice should be sufficiently identical genetically in terms of genetic material related to prions that 95% or more of the mice will develop clinical signs of CNS dysfunction within 350 days or less after inoculation and all of the mice will develop such CNS dysfunction at approximately the same time e.g., within ⁇ 30 days of each other.
  • mice 500 or more of such mice are produced.
  • the next step is to inoculate the mice with prions which generally only infect a genetically diverse mammal e.g., prions from an bovine such as a sheep, cow, deer or horse.
  • prions which generally only infect a genetically diverse mammal e.g., prions from an bovine such as a sheep, cow, deer or horse.
  • the amounts given to different groups of mammals could be varied.
  • the mammals are observed until the mammals exhibit symptoms of prion infection e.g., clinical signs of CNS dysfunction.
  • the brain or at least a portion of the brain tissue of each of the mammals is extracted.
  • the extracted brain tissue is homogenized which provides the standardized prion preparation.
  • mice which spontaneously develop prion related diseases can be done, for example, by including extremely high copy numbers of a cow PrP gene into a mouse genome. When the copy number is raised to, for example, 100 or more copies, the mouse will spontaneously develop clinical signs of CNS dysfunction and have, within its brain tissue, prions which are capable of infecting humans.
  • the brains of these animals or portions of the brain tissue of these animals can be extracted and homogenized to produce a standardized prion preparation.
  • the standardized prion preparations can be used directly or can be diluted and titered in a manner so as to provide for a variety of different positive controls. More specifically, various known amounts of such standardized preparation can be used to inoculate a first set of transgenic control mice. A second set of substantially identical mice are inoculated with a material to be tested i.e., a material which may contain prions. A third group of substantially identical mice are not injected with any material. The three groups are then observed. The third group, should, of course not become ill in that the mice are not injected with any material. If such mice do become ill the assay is not accurate probably due to the result of producing mice which spontaneously develop disease.
  • the assay is also inaccurate because the mice have not been correctly created so as to become ill when inoculated with prions which generally only infect a genetically diverse mammal.
  • the assay can be presumed to be accurate.
  • the second group does not become ill the test material does not contain prions and if the second group does become ill the test material does contain prions.
  • compositions containing the prions By using standardized prion preparations of the invention it is possible to create extremely dilute compositions containing the prions. For example, a composition containing one part per million or less or even one part per billion or less can be created. Such a composition can be used to test the sensitivity of the antibodies, assays and methods of the invention in detecting the presence of prions.
  • Standardized prion preparations are desirable in that they will include a constant amount of prions and are extracted from an isogeneic background. Accordingly, contaminates in the preparations will be constant and controllable. Standardized prion preparations will be useful in the carrying out of and calibration of bioassays in order to determine the presence, if any, of prions in various pharmaceuticals, products produced by using bo vines including foods, cosmetics, etc.
  • antibodies can be created which bind only to a PrP protein naturally occurring within a single bovine species or PrP from human and not bind to a PrP c protein naturally occurring within other species.
  • the antibody can be designed so as to bind only to a non-infectious form of human or a bovine prion protein ⁇ e.g., PrP c ) and not bind to an infectious form ⁇ e.g., PrP Sc ).
  • a single antibody or a battery of different antibodies can then be used to create an assay device.
  • Such an assay device can be prepared using conventional technology known to those skilled in the art.
  • the antibody can be purified and isolated using known techniques and bound to a support surface using known procedures.
  • the resulting surface having antibody bound thereon can be used to assay a sample in vitro to determine if the sample contains one or more types of antibodies.
  • An assay of the invention can use all or any of three basic types of treatment such as those discussed herein.
  • the treatments can include (1) pretreatment, (2) unfolding treatment and (3) hydrolysis treatment.
  • the conditions for pretreatment are gentle, those for unfolding treatment moderate and those for hydrolysis treatment are harsh.
  • Each type of treatment can employ the same means (e.g. proteases, time, pH, temperature, etc.) but employs each to a different degree, e.g. higher concentration, longer time, higher temperature.
  • the hydrolysis treatment must employ a compound which selectively hydrolyzes only the non- disease conformation and not the disease conformation.
  • pretreatment Before carrying out treatment or antibody testing of the sample it may be desirable to subject the sample to pretreatment.
  • the pretreatment is carried out in order to destroy or remove unrelated proteins as well as some of the non-disease form of the protein present within the sample.
  • Examples of pretreatment methodology include producing a column which includes antibodies bound to support surfaces which antibodies bind to the non-disease conformation of the protein thereby removing as much of the non-disease conformation of the proteins possible.
  • Antibodies which bind unrelated but common proteins can also be used.
  • the sample can be subjected to physical treatment such as long term hydrostatic pressure or temperature alone or in combination with chemicals such as acids or alkalines as indicated above to destroy proteins present in the sample which proteins are not related to those being assayed for or are in the non-disease conformation.
  • proteins in the non-disease and disease conformation will be destroyed.
  • a higher relative percentage of the proteins in the non-disease conformation will be destroyed because these proteins are initially in a looser conformation which is more vulnerable to destruction.
  • the pretreatment methodology results in a sample which includes a relatively lower concentration of the non-disease conformation of the protein relative to the concentration of the disease conformation of the protein. Further, the pretreated sample will have a lower concentration of unrelated proteins.
  • the unfolding treatment denatures the protein but does not hydrolyze proteins of interest and can include exposing the proteins to any physical and/or chemical means which causes the protein which is originally present in a tightened, disease related conformation (e.g., PrP.sup.Sc) to assume a more relaxed conformation which has a higher degree of binding affinity for any binding partner such as antibodies (e.g., expose an N-terminal epitope of PrP.sup.Sc).
  • the unfolding treatment involves subjecting the protein to some means which causes epitopes on the protein which were not previously exposed or partially exposed to become exposed or become more exposed so that an antibody or other binding partner can more readily bind to the newly exposed epitope.
  • Methods used for unfolding treatment may include: (1) physical, such as hydrostatic pressure or temperature, (2) chemical, such as acidic or alkaline pH, chaotropic salts, denaturing detergents, guanidine hydrochloride and proteinases such as Proteinase K and (3) combinations of above.
  • the treatment time will vary depending on the treatment used but should be carried out for sufficient time to obtain the desired effect, e.g. for unfolding treatment to expose new binding sites but not so long as to completely denature or hydrolyze the protein.
  • the temperature is raised to about 40°C. to about 80°C. for a time sufficient to obtain the desired amount of unfolding of p r p Sc Jj 16 temperature can be lower and the time shorter if the pH is raised to 12 or 13.
  • the hydrolysis treatment is a lytic treatment which is the most important treatment method used in one embodiment of the assays of the invention. After a sample has been subjected to the pretreatment treatment it is subjected to the hydrolysis treatment. This treatment will destroy or hydrolyze all or substantially all protein in the sample which is in the non-disease conformation and not hydrolyze (but unfold) the protein in the disease conformation.
  • the hydrolysis treatment is prferably via an enzyme such as a hydrolase that acts on peptide bonds, preferably a neutral protease, more preferably a metalloendopeptidase, and most preferably dispase or leucostoma peptidase A.
  • the proteases used in the method of the invention may be used alone, in combination, or in conjunction with enzymes having similar but distinct activity such as a carbohydrase, e.g. collagenase, amylase, or alkaline serine protease.
  • concentration of the treating compounds as well as the time and temperature will vary with the protein being treated and end result to be obtained.
  • PrP the treatment is carried out to hydrolyze all or substantially all non-PrP c present, but not hydrolyze (but unfold) PrP Sc present.
  • the object of this treatment is to hydrolyze as much non-disease protein as possible (preferably all) while hydrolyzing as little (preferably none) disease related protein as possible.
  • the treatment is preferably designed such that it can be quickly and completely stopped at any given time. For example, hydrolysis of PrP c with dispase or other related proteases can be stopped by adding EDTA.
  • One embodiment of the invention features assays allowing detection of PrP Sc in a human or in a bovine sample by 1) digesting the sample with an enzyme that effectively degrades PrP c and which denatures PrP Sc , or alternatively by successive treatment with an enzyme that degrades PrP c (but not PrPSc) and then an enzyme which denatures PrP Sc and 2) detecting the denatured PrP Sc using an antibody of the present invention.
  • a sample containing bovine PrP proteins i.e., PrP c and PrP Sc
  • PrP c and PrP Sc can be subjected to hydrolysis by the use of proteinase K (PK) digestion. The use of such at appropriate concentration, times and temperature will digest PrP c but not PrP Sc .
  • the sample is further digested to denature the PrP Sc , and the sample is contacted with an antibody of the present invention under suitable binding conditions.
  • the antibody is bound to a substrate and can be positioned such that the sample can be easily contacted with the substrate material having the antibody bound thereon. If material binds to the antibodies on the substrate the presence of infectious PrP ° is confirmed.
  • a sample to be tested is divided into two portions, and one is digested to denature any PrP Sc in the sample without destroying the PrP c in the sample. Both portions are contacted with an antibody of the invention, which will bind to PrP in the untreated portion and both PrP c and PrP Sc in the treated portion. Levels of PrP c or PrP c + PrP Sc are detected and the amount of PrP Sc in the sample determine from the difference in detectable signal between the two samples.
  • the antibody of the invention may be bound to a substrate support surface.
  • the denatured sample to be tested is contacted with the support surface under conditions which allow for binding. Thereafter, unreacted sites are blocked and the surface is contacted with a detection antibody.
  • the detection antibody may provide for an additional level of specificity, e.g. by specifically binding to PrP of a mammal of interest, to a specific domain of interest, or broadly cross-reactive to mammalian PrP proteins. Alternatively, the detection antibody may be non-specific.
  • the detection antibody is linked to a detectable label.
  • the detection antibody with detectable label is allowed to bind to any PrP bound to the antibodies on the support surface. If binding occurs the label can be made to become detectable such as by generating a color thereby indicating the presence of the label which indirectly indicates the presence of PrP within the sample.
  • the assay can detect denatured PrP present in an amount of 1 part per million or less, even one part per billion or less.
  • the PrP may be present in a source selected from the group consisting of (a) a pharmaceutical formulation containing a therapeutically active component extracted from an animal source, (b) food products, (c) an organ, tissue, body fluid or cells extracted from a human source, (d) an animal-based product such as injectables, orals, creams, suppositories, and intrapulmonary delivery formulations, (e) a cosmetic, and (f) a pharmaceutically active compound extracted from a mammalian cell culture.
  • a source selected from the group consisting of (a) a pharmaceutical formulation containing a therapeutically active component extracted from an animal source, (b) food products, (c) an organ, tissue, body fluid or cells extracted from a human source, (d) an animal-based product such as injectables, orals, creams, suppositories, and intrapulmonary delivery formulations, (e) a cosmetic, and (f) a pharmaceutically active compound extracted from a mammalian cell culture.
  • PrP ablated FVB female mice were immunized with either recombinant bovine PrP 102-241 protein (recBoPrP 102-241) or recombinant human PrP 90-231 protein (recHuPrP90-231) as follows. Both recBoPrP 102-241 and recHuPrP90-231 were prepared in RIBI adjuvant (Corixa, Corp.) as suggested by the manufacturer at a concentration of 1 mg/mL. Animals were immunized by subcutaneous (SC) injection of 100 ⁇ g of antigen followed by two booster immunizations at 14-day intervals.
  • SC subcutaneous
  • Hybridoma Production - Cell Fusion Chemical cell fusions were performed as previously described. Briefly, P3X63AG8U.1 myeloma cells were maintained in Dulbecco's modified Eagles Media with high glucose and L-glutamine (without sodium pyruvate) (Gibco- BRL #11965-092) supplemented with 10% fetal calf serum (Gibco-BRL #10082-147), Na pyruvate (2 niM) and penicillin/streptomycin. This media was adjusted to 15% FCS and 1 mM non-essential amino acids to maintain subsequent hybridoma cells.
  • Splenocytes were dispersed using a stainless steel mesh, the erythrocytes lysed by incubating in 0.75% NH 4 Cl for 5 min on ice. Washed myeloma cells (viability greater than 99%) were mixed with splenocytes (a ratio of 5 splenocytes to 1 myeloma cell) and the cells centrifuged at 2000 rpm for 5 min. The supernatant was removed, the pellet gently disrupted and 1 mL per 10 8 cells of a 50% polyethylene glycol solution slowly added. The cell mixture was then centrifuged again (500 x g) for 5 min, the PEG solution removed and the cell pellet slowly resuspended and incubated for 20 min at 37° C.
  • the cells were centrifuged (500 x g, 5 min), resuspended in growth medium and incubated overnight at 37°C in an 8% CO 2 atmosphere. The next day the cells were resuspended in growth medium containing hypoxanthine, aminoptrin and thymidine (HAT-medium) and dispersed over ten 96-well microculture dishes containing irradiated 3T3 fibroblast cells acting as a feeder layer. Following growth for 10-14 days, media from those wells observed to have colonies of hybridoma cells was screened for anti-prion antibodies. Monoclonal antibodies detected with this method are listed in Table 3.
  • Screening A multistep screening schedule (outlined below) was employed incorporating a direct binding ELISA, a newly developed double capture immunoassay (DCI) -and Immunoblot analyses on normal brain material from various species.
  • DCI double capture immunoassay
  • Microtiter wells of a black fluorescence assay plate are first coated by passive absorbtion with goat anti-mouse IgG Fc specific antibody (Antibody #1) (Chemicon #AP127) (50 ⁇ L/well of a 2 ⁇ g/mL solution in carbonate coating buffer, 0.1 M carbonate, pH 8.6) by incubation at 37° C for 2 h. Reactive sites on the plastic are then blocked by addition of a solution of 5% non-fat dry milk (NFDM) in Tris-Buffered Saline-tween (TBST, 0.01 M Tris- base, 0.8%NaCl, 0.05 % tween-20, pH 7.4) and the plates incubated for 1 hr at 37 °C.
  • NFDM non-fat dry milk
  • Hybridoma supernatants are then added and the plate incubated overnight at 4°C. (This step allows for attachment of any mouse monoclonal antibodies in the supernatants to the immobilized antibody #1).
  • the supernatant is removed, the plate washed and blocked a second time using 250 ⁇ L/well of a 1:1000 dilution of pure mouse IgG (Chemicon #PP54) in 5% NFDM blocking buffer. This second block is critical in order to reduce background fluorescence resulting from binding of not occupied combining sites of the goat-anti mouse antibody (Antibody #1).
  • the plates are decanted and either recBoPrP 102-241 or recHuPrP90-231 at a concentration of 2 ⁇ g/mL in blocking buffer is added, the plates sealed and then they are incubated at 37°C for 2 h.
  • the plates are then washed 3 times incorporating a 1 min soak per cycle, patted dry and 200 ⁇ L/well of Europium conjugated anti-prion antibody HuM-P(HuM-P-Eu) at a concentration of 0.25 ⁇ g/mL in assay buffer is added and the plates incubated for 2 h at 37 °C.
  • Peptide assays The ability of F4-31, F4-30, F4-7, F20-80, and F20-130a to bind the following peptides was evaluated using the direct binding ELISA where the peptides were used as antigens to coat the microtiter wells.
  • RecBoPrP 25-36 KKRPKPGGGWNT SEQ ID NO: 1 RecBoPrP 30-41 PGGGWNTGGSRY SEQIDNO:2 RecBoPrP 35-46 NTGGSRYPGQGS SEQIDNO:3 RecBoPrP 40-51 RYPGQGSPGGNR SEQIDNO:4 RecBoPrP 45-56 GSPGGNRYPPQG SEQIDNO:5 RecBoPrP 50-61 NRYPPQGGGGWG SEQIDNO:6 RecBoPrP 52-62 QGGGGWGQPHGG SEQIDNO:7 recBoPr60-71 WGQPHGGGWGQP SEQIDNO:8 RecBoPrP 65-76 GGGWGQPHGGGW SEQIDNO:9
  • the splenocytes from an animal that had received an SDS-denatured immunogen also were used in fusion experiments. Following this fusion substantially fewer growing hybridoma cells (growth in only 63 wells out of 960 wells, ten 96-well microculture plates) were observed. Supernatant from these wells was evaluated in the direct binding ELISA for the presence of anti-PrP antibodies and 7 positive wells (absorbencies greater than 0.3 OD 405 nm Units) were detected. Isotype analysis of the antibodies in each well indicated that they were of subtype IgGl with kappa light chains. These supernatants were next evaluated with the DCI and positive responses were observed from six of the supernatants.
  • Peptide Binding Binding assays to a number of short and long peptides were completed in an effort to determine the binding epitope of F4-31 F4-30, F4-7, F20-80, and F20-130a .
  • the peptides in Table 1 are short, 12 amino acid peptides with an eight amino acid overlap that span the bovine PrP molecule from residue 23 to 241.
  • Each peptide has three lysines added to the amino end to improve solubility and a single molecule of biotin was added to the terminal lysine.
  • the peptides were bound to avidin coated microassay plates for the analysis.
  • the amino acid numbering used here is based on the bovine sequence as aligned by Giles and Prusiner (2003) [Giles, K. and Prusiner, S. (2003) Prion protein and dopple (Dpi) sequences. In: Prion Biology and Diseases, (S. Prusiner, Editor) Cold Spring Harbor Press, NY, pp. 1023-1032.] Binding of F4-7, F4-30, F4-31, F20-80 ro F20-130a was not observed to any of these peptides ( Figure 6 A, results shown only for F4-31) however strong binding was observed to recBoPrP 102-241.
  • Antibody F4-7, F4-30, F4-31, F20- 80, and F20-130a binding was dramatically reduced in the DDT treated sample for each antibody tested ( Figure 7, F4-31 binding shown, compare lane 5 versus 6) demonstrating that an intact disulfide bond is necessary for binding and suggesting that the binding epitope of these antibodies has a conformational component.
  • Silver staining of these gels ( Figure 7, lanes 1-2) demonstrates that the reduced recBoPrP 102-241 migrates slower in the gel than did the non-reduced protein.
  • Antibody HuM-P is known to bind a sequential epitope distal from the disulfide bond in the PrP. Thus, when antibody HuM-P was used as the probe, as expected, it bound to both the reduced and not reduced form of the PrP protein demonstrating that the slower migrating protein observed in the reduced lane is indeed PrP 102-241 ( Figure 7, lanes 3- 4).
  • Fabs that capture with high affinity denatured BoPrP Sc but not the native conformation of the same protein in CDI-formatted ELISA.
  • the purified mAb's were used in conformation dependent immunoassay (CDI) as taught in U.S. Patent 5,891,641 to measure bovine, sheep, and deer PrP Sc and to determine the capture affinity constant.
  • CDI conformation dependent immunoassay
  • Figure 10 demonstates the increased sensitivity of ungulate prion detection obtained with a CDI assay using mAb F4-31. Capture of PrP Sc from sheep scrapie, chronic wasting disease (CWD) of deer, and BSE by monoclonal antibody F4-31 in sandwich-formated conformation-dependent immunoassay (CDI).
  • CDI sandwich-formated conformation-dependent immunoassay
  • the bars of Figure 10 represent the average signal obtained from three independent experiments using Eu-HuM-P antibody concentration 0.25 ⁇ g/ml.
  • the plates were coated with a 5 ⁇ g/ml solution of mAb F4-31 or recFab D18 in 0.2 M phosphate buffer, pH 7.4, overnight at 5 0 C. After 7 washing steps, the signal was evaluated with Discovery (Packard Inc.) time-resolved fluorescence spectroscopy.
  • Capture affinity constant (K n ) of mAb F4-31 determined against different concentrations of recombinant bovine PrP(102-241) is in the low nanomollar range.
  • Capture of recBoPrP( 102-241) by monoclonal antibody F4-31 was performed in sandwich-formated conformation-dependent immunoassay (CDI). Nintysix-well microassay plates were coated with the indicated concentration of monoclonal antibody F4-31 in 0.2 M phosphate buffer, pH 7.4, incubated overnight at 5°C, and developed with 0.25 ⁇ g/ml of EU-HuM-P. After 7 washing steps, the signal was evaluated with Discovery (Packard Inc.) time-resolved fluorescence spectroscopy. The data points shown in Figure 11 represent the average obtained from three independent experiments.

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Abstract

Cette invention concerne des anticorps qui se fixent spécifiquement avec un degré élevé d'affinité à une protéine PrPC bovine native et/ou a une protéine PrPSc bovine dénaturée, et non pas à une protéine PrPSc bovine native. Ces anticorps sont utiles comme réactifs de capture pour des immunodosages à formats de substrats solides. Ces anticorps peuvent être utilisés dans des dosages destinés à déterminer si un échantillon est infecté par des prions infectieux, c'est-à-dire la protéine PrPSc.
PCT/US2006/009752 2005-03-18 2006-03-15 Anticorps specifiques de la proteine prp humaine et bovine WO2006102099A2 (fr)

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US7863411B2 (en) 2002-12-03 2011-01-04 Pathogen Removal and Diagnostics Technologies Inc. Prion protein ligands and methods of use

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US7863411B2 (en) 2002-12-03 2011-01-04 Pathogen Removal and Diagnostics Technologies Inc. Prion protein ligands and methods of use
US8604161B2 (en) * 2002-12-03 2013-12-10 Pathogen Removal And Diagnostic Technologies Inc. Prion protein ligands and methods of use
US9678085B2 (en) 2002-12-03 2017-06-13 Pathogen Removal And Diagnostic Technologies Inc. Prion protein ligands and methods of use

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