BG64291B1 - Method and kit for extracting prion protein - Google Patents

Abstract

A method for extracting prion protein from a biological material, e.g., an animal tissue or product. In a specific example, abnormal prion protein is extracted from homogenized sheep brain with hexafluoro-2-propanol. The hexafluoro-2-propanol is separated from the aqueous brain preparation by increasing the ionic strength of the aqueous solution. Prion protein in the organic extract can be further purified, or the extract can be tested, e.g., by immunoassay, for the presence of prion protein, and more particularly abnormal prion protein. The extraction process permits testing for the presence of abnormal prion protein, e.g., for diagnoses or transmissible spongioform encephalopathies (TSE).

Description

Technical field

The invention relates to a method for extracting a prion protein from a biological material, such as animal tissue or biological fluid. The extraction process allows testing for abnormal prion protein, for example for the diagnosis of transmissible spongiform encephalopathies.

BACKGROUND OF THE INVENTION

Prion diseases or transmissible spongiform encephalopathies (TSEs) cause progressive central nervous system degenerative disorders leading to death (Prusiner, Med. Res. Rev. 16: 487, 1996; Weissman, FEBS Letters 289: 3, 1996). Scrapie, TSE in sheep, was first described 200 years ago (Pattisson, Vet. Rec. 123: 661, 1988) and is a prototype of these diseases. There are no known treatments for these diseases and no known antemortem (post-mortem) tests for the presence of the disease in an animal. Prion diseases are caused by a conformational change in the normal host prion protein into an abnormal structure that forms aggregates. Due to the recent outbreak of bovine spongiform encephalopathy in the UK and the relationship between this TSE and the new variant, Creutzfeld-Jakob (Bruce et al., Nature 389: 498, 1997), human TSE, there is a need for novel methods that are both sensitive and accurate for diagnosing TSEs. Ideally, this diagnosis can be used to test animals before they show clinical symptoms and before they enter the human food chain or pharmaceutical preparations for human use.

Most of the methods used to prepare and purify the causative agents of TSEs include a complex sequence of enzyme and surfactant treatment and centrifugation (Bolton et al., J. Virol. 53: 596, 1985). Anor small prion protein is completely soluble in conventional biological buffers. One method of producing purified abnormal prion protein is hydrophilic interaction chromatography (HILIC) (Alpert, J., Chromatogr. 499: 177, 1990), which is the inversion of reverse phase chromatography. It is usually started with 70-85% organic solvent and a descending organic gradient is released. The elution is in the direction from the smallest to the highest polarity. The most mobile organic phases of HILIC are competitive with proteins that are not normally found free in aqueous solution, such as membrane proteins (Jeno et al., Anal. Biochem. 215: 292, 1993), β-amyloid peptide (1-43) ( Alpert et al., Eighth Symposium of the Protein Society, July 1994, San Diego, CA) and histones (Lindner et al., J. Chromatogr. A. 782: 55, 1997). Surfactants and other denaturants elute in or near empty volume, while proteins and peptides are generally well stored.

After HILIC purification, the prion protein can be detected by capillary electrophoretic immunological assay (Schmerr and Jenny, Electrophoresis 19: 409, 1998) or by capillary isoelectric focusing (Schmerr et al., J. Chromatogr. A. 802: 135, 1998).

As noted above, current analytical methods for the detection of abnormal prion protein are generally used after post mortem, so a pre-mortem test for abnormal prion protein is required. In addition, a method for isolating an abnormal prion protein without ultracentrifugation steps is required, requiring equipment that is not always available for veterinary diagnostic laboratories. The presence of abnormal prion protein as aggregates, the large size of which facilitates the formation of sediment in centrifuge tubes, requires centrifugation. Such aggregates are difficult to dissolve and detect in subsequent steps. The use of centrifugation also jeopardizes the possibility of detecting a monomeric abnormal prion protein, potentially reducing the sensitivity of any sample. There is even greater need for a rapid test bridge, such as a qualitative immunological test, to test the herd for TSE infection. Thus, in the art, there is a need for an effective, simple method of extracting abnormal prion protein.

In addition, the antibodies that are produced detect the abnormal prion protein in their monomeric form, except for the antibodies produced against the native abnormal prion protein (Korth et al., Nature 390: 74, 1997). As a result, the abnormal prion protein should be deaggregated with strong surfactants or denaturing agents; the latter should then be removed before most immunological studies are performed. Thus, there is a need in the art for a fast, simple method of extracting prion protein free of detergents or denaturing agents for immunoassays.

The present invention provides a new method for extracting all sizes of the abnormal prion protein, whether in aggregate or monomeric form. The invention makes it possible to test for abnormal prion protein in live animal samples, for example using immunological studies. For example, the diagnosis may be based on blood samples that will allow the testing of live animals and will facilitate the removal of infected animals from flocks or herds and prevent possible contamination of the products for consumption.

SUMMARY OF THE INVENTION

The invention provides a method for extracting an abnormal prion protein from a biological material that is believed to contain an abnormal prion protein. The method involves incubating a mixture of the extraction solvent and an isotonic or hypotonic aqueous preparation of the biological material under conditions effective for extracting an abnormal prion protein from the biological material into the extraction solvent. The extraction solvent is a polar organic solvent in which the abnormal prion protein can be dissolved and can be mixed with a hypotonic or isotonic aqueous solution but cannot be mixed with a lyotropic aqueous solution. The lyotropic activity of the mixture is increased so that said extraction solvent is separated as a distinct phase from the aqueous preparation of the biological material to obtain an extraction solvent containing any abnormal prion protein from the biological material.

The invention further provides a method for detecting the presence of an abnormal prion protein in an animal, comprising testing an individual extraction solvent prepared as described above for an abnormal prion protein.

A kit for isolating an abnormal prion protein from a biological sample is also proposed. The kit comprises an extraction solvent having the characteristics set forth above and a lyotropic salt or aqueous solution of lyotropic salt to be added to an aqueous preparation of a biological sample so that the organic solvent becomes immiscible with the aqueous preparation. In another embodiment of the invention, the kit comprises a prion protein test sample, preferably an abnormal prion protein assay.

Thus, it is an object of the invention to provide a rapid method of isolating an abnormal prion protein from a biological sample.

It is another object of the invention to provide a method for the early detection of organisms infected with an abnormal prion protein.

It is another object of the invention to provide an extraction technique for the isolation of an abnormal prion protein for a biological sample.

It is another object of the invention to simplify the analytical testing of an animal or human biological material for the presence of an abnormal prion protein.

It is another object of the invention to provide an extract containing an abnormal prion protein for further testing or purification.

These or other objects of the invention are set out in more detail in the accompanying figures and the detailed description of the invention.

Description of the attached figures

Figure 1 is a chromatogram of ¹²⁵ 1-prion protein HILIC as determined by radioactivity (open circles) and by absorption at 280 nm (solid line);

Figure 2 - antibody binding of HILIC fractions to ¹²⁵ 1-prion protein.

Description of the invention

The invention provides a method for extracting an abnormal prion protein from a biological material. The extracted product can be tested by immunoassay for abnormal prion protein. This extraction of abnormal prion protein, in combination with an immunological assay, can be used to diagnose living organisms for infection with TSE, and will be widely used to test TSE infected animals and humans. Thus, the present invention advantageously permits testing for prion protein in clinical and veterinary laboratories lacking expensive centrifuges and allows field testing.

An aqueous preparation of the biological material is combined with an extraction solvent to form a mixture. The extraction solvent is a polar organic solvent in which the abnormal prion protein is soluble and can be mixed with a non-lyotropic isotonic aqueous solution but is immiscible with a lyotropic aqueous solution. In a particularly preferred embodiment, the extraction solvent is hexafluoro-2-propanol (also called hexafluoroisopropanol or HFIP). Although the volumes of the extraction buffer and aqueous preparation of biological material are almost identical in the examples below, any ratio can be used to give two clearly distinct phases as described above.

In a preferred embodiment, the mixture is incubated at a temperature in the range of about 20 ° C to about 100 ° C. However, any temperature at which both the extraction solvent and the aqueous preparation of the biological material are in the liquid phase, i.e., between the freezing point and the boiling point, may be used.

The extraction solvent-biological material mixture is incubated under conditions effective for extracting an abnormal prion protein from the biological material into the extraction solvent. After incubation, the lyophilic activity of the mixture was increased so that the extraction solvent was separated from the aqueous preparation. The extraction solution containing prion protein is removed from the aqueous preparation of the biological material.

According to the invention, the lyotropic activity of the solvent-aqueous extraction mixture can be increased by the addition of a lyotropic salt. The salt may be added in solid form directly to the mixture or as a concentrated aqueous solution. Preferred examples of lyotropic salts include sodium and ammonium sulfate. In the specific embodiment given below, the ionic strength is increased by adding about 0.5: 1 M sodium sulfate to the mixture at a ratio of about 1: 1 (v / v).

The invention has the extreme advantage of not requiring the preparation of necropsy or necropsy material. According to the invention, the biological material may be a sample of a living animal. The method of the invention offers the extraction of an abnormal prion protein from a biological fluid or an organic biopsy for analytical testing. Conversely, biological material may be obtained from necropsy or necropsy, for example of animal products for use in food, pharmaceuticals, cosmetics or other products for human use or with other animals. In another embodiment, the method of the invention can be used to remove prion protein from such materials to ensure that infectious prions are not transmitted.

The extraction method of the invention can be used to extract both normal prion protein and abnormal prion protein. By pretreatment of a biological sample with proteinase-K, normal prion protein can be degraded. Thus, where only abnormal prion protein is desired, the aqueous preparation of the biological material may be pretreated with proteinase-K before being mixed with the extraction solvent.

An extraction solution containing any prion protein may be dried to obtain an extraction precipitate. The prion protein in solution or extraction residue may be further purified, for example by hydrophilic interaction chromatography.

The abnormal prion protein in an animal can be detected by testing the material in the separated extraction precipitate for its presence. Preferably, the test method is an immunological test for an abnormal prion protein. The sample can be treated with proteinase-K before isolation of the prion protein so that no normal prion protein is isolated.

In another aspect, the invention provides kits for isolating an abnormal prion protein from a biological sample. In one embodiment, the kit comprises an extraction solvent, for example hexafluoro-2-propanol. The kit further includes a lyotropic salt or an aqueous solution of the lyotropic salt for adding to the aqueous preparation of a biological sample so that the extraction solvent becomes miscible with the aqueous preparation.

The invention further provides a kit for detecting the presence of an abnormal prion protein from a biological sample. In addition to the components of the kit described above, this kit includes a sample to detect abnormal prion protein. The preferred sample for detecting the presence of an abnormal prion protein is an immunoassay.

Although this extraction method and the whale were first developed for the diagnosis of scrapie (TSE in sheep), they are applicable to the diagnosis of other TSEs in other vertebrates, in particular in mammals and birds such as humans, cattle, pigs , moose, roe deer, poultry and rodents. Abnormal prion protein can be detected in animal and human tissues up to two weeks after infection. An important application of the method of this invention is the detection of abnormal prion protein in human blood. This technique can also be used to extract an abnormal prion protein from a processed material used to produce pharmaceuticals for humans or other products intended for human use, including food substitutes.

As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given degree or interval.

Organic materials

The present invention allows the extraction of prion protein from a biological material. Generally, prion proteins are found in vertebrates as discussed above. Therefore, in most circumstances, the biological material will be animal or human. Prion protein can also be produced during eukaryotic fermentation processes. It can be expressed as a recombinant prion protein. Of greater interest is the possibility of incidental expression of an endogenous prion protein by cells that have been recombinantly modified to express another protein. This possibility is more likely if the cells are of neural origin, such as PC 12 cells. In this case, the biological material may be a fermentation product, for example recombinant protein.

Examples of biological materials from animals include, but are not limited to, tissues such as the brain, muscles (including heart), liver, appendix, pancreas, gastrointestinal organs, skin, and lymphoid tissues such as thymus, spleen, tonsils, lymph nodes and more. Conversely, the biological material may be a biological fluid. The term biological fluid refers to cerebrospinal fluid, blood, serum, plasma, milk, urine, saliva, tears, mucous secretions, sweat, semen, and body fluids containing these components. It may also refer to a culture fluid (or culture medium) used to produce recombinant proteins or containing cells in suspension prior to transplantation. The term "biological material" covers products made from animal organs or tissues, including serum proteins (such as albumin and immunoglobulin), hormones, food and processed foods, food substitutes, bone meat, animal feed, extracellular matrix proteins , gelatin and other animal by-products used for production or end products.

When the biological material is woven or solid, it must first be dissolved or suspended in an aqueous solution so that it is suitable for the extraction process. For example, brain tissue may be suspended in a sugar solution (e.g. 0.32 M sucrose) at 10% by weight. by volume. Other hypotonic or isotonic solutions include 5% dextrose, phosphate buffer solution, tri-buffer solution, HEPES buffer solution or any of the above buffers. The biological material may be homogenized, primed or otherwise destroyed to maximize contact between the extraction solvent and the biological material. However, if the biological fluid is the biological material, the addition of a liquid is probably not necessary except to dilute the ionic strength of the biological fluid to allow the extraction solvent to be miscible.

Prion protein

The term "prion protein" as used herein refers to native protein expressed in neutral tissue, in particular the brain, and at lower levels in lymphoid tissues and all other tissues. In some circumstances, the prion protein adopts a pathogenic conformation, which is here called an abnormal prion protein. Certain prion gene mutations in some individuals seem to predispose the prion protein to adopt the pathogenic conformation. Exposure of an organism to a transmissible infectious agent, prion, can also induce a conformational change leading to pathology.

Abnormal prion protein is less susceptible to proteolysis than normal prion protein. Treatment of biological material with proteinase, in particular proteinase-K, degrades normal protein but not abnormal prion protein.

In specific examples below, sheep abnormal prion protein (PrP ^sc ) is extracted by the method of the invention. However, other prion proteins of other species, in particular those noted above, can also be extracted using the method of the invention.

The category of abnormal prion protein also includes human prion proteins found in neurodegenerative diseases Kuru, Creutzfeld-Jakob Disease (CJD), Gerstmann-Straussler Syndrom (GSS), and fatal familial insomnia. Some cases of CJD and GSS are associated with known prion gene mutations. CJD is also associated with exposure to TSEs. For example, as noted above, CJD has been associated with bovine spongiform encephalopathy. The present invention allows, for the first time, the extraction of an abnormal prion protein from patients prior to necropsy. Establishing an extracted abnormal prion protein can be used to diagnose any of these diseases.

Scrapie (sheep, goats) and bovine spongiform encephalopathy (cows) are animal diseases of abnormal prion protein. Prion proteins are also isolated in chickens, minks, pigs, mice, hamsters and guinea pigs. Moreover, mouse, hamster and guinea pig can develop spongiform encephalopathy by exposure to human prions or other animal sources. The prion protein from any of these sources can be detected or extracted by the method of the invention.

Extraction solvent and conditions The extraction solvent for use in the present invention should be capable of separation as a distinct phase from water or aqueous solution under lyotropic conditions. At the same time, the prion protein should be soluble in the extraction solvent. Some polar organic solvents meet these criteria. The preferred polar organic solvent is hexafluoro-2-propanol. Other solvents that may be used include isopropanol; 1,1,1 Trifluoro-2-propanol (TFIP); 2,2,3,3-tet refluoro-1-propanol (tetrH); perfluoro-butyl alcohol (PFtBA); 1,1,1,3,3,3-hexafluoroacetone (HFA); trifluoroacetic acid (TFA); 2,2,2-Trifluoro-1-ethanol (TFE); 2,2,3,3,4,4,4-heptafluoro-2-propanol (HFB); 1,1,1,3,3,4,4,4-octafluoro-2-butanol (OFIB); 1-methyl-2-pyrrolidinone (NMR); see Wille et al., J. Mol. Biol., 259: 608, 1996. Other possible solvents that can be evaluated include DMSO; tetrahydrofuran; and similar. Conversely, a solvent that is immiscible with water but in which the prion protein is soluble may be used.

In a specific embodiment, the solvent is mixed with water, for example at physiological ionic strength (isotonic aqueous solution) or lower than physiological ionic strength (hypotonic aqueous solution). However, when the buffer contains lyotropic salts in concentrations (or ionic strength) above the threshold value, the extraction solvent is insoluble in aqueous solution: the two phases are separated into a layer of extraction solvent and a layer of aqueous solution. They are referred to here as "lyotropic states". The ionic strength of a lyotropic salt of an aqueous solution that reaches lyotropic states may vary depending on the extraction solvent selected and the salt (s) used. It can be readily determined by titration or other systematic variation of the concentration of the lyotropic salt, by testing the compatibility or immiscibility of the extraction solvent with the aqueous solution. As used herein, an aqueous solution having an ionic strength of a lyotropic salt in which the extraction solvent is separated from water is referred to as a lyotropic aqueous solution. Aqueous solutions containing lower concentrations of lyotropic or physiological salts, such as isotonic buffers, are considered non-lyotropic solutions.

In general, almost equal volumes of the extraction solvent and the aqueous preparation of the biological material are used in the soluble extraction process. However, the ratio of the extraction solvent to the aqueous preparation may range from about 5: 1 to about 1: 5, preferably from about 3: 1 to about 1: 3.

After mixing the extraction solvent with the aqueous preparation, the mixture can be incubated for the same period of time and at a specified temperature to increase the extraction of the prion protein in the extraction buffer. The incubation time can range from 1 min to 1 h, and can be determined by analyzing the extracted material for the presence of prion protein. Once the amount of prion protein in the extraction material reaches the plateau over time, which can be tested by chromatographic or immunological techniques, or both, as described in the examples, further incubation will have no effect on the yield of prion protein. In a specific embodiment, the incubation time is 5 min.

In addition, the incubation temperature can be increased to increase the extraction efficiency, provided that the extraction solvent and the aqueous solution are both liquids at the selected temperature. Higher temperatures, i.e., above room temperature, are preferred insofar as they increase the solubility of the prion protein in the extraction solvent. Particularly applicable are temperatures within the range of about 50 C60 C. As for other variations, such as the ionic strength of the aqueous preparation and the incubation time, the optimum temperature can be determined by routine experimentation and testing.

Phase separation

Different lyotropic salts can be used to increase the ionic strength of the aqueous solution, thereby inducing phase separation. Preferred salts are sodium sulfate and ammonium sulfate, which are lyotropic. Both are used at a concentration below which proteins precipitate. For example, in a specific embodiment, to an equal volume of the extraction solvent / aqueous mixture, a 0.5 M sodium sulfate solution is added, resulting in a final concentration of 0.25 M sodium sulfate. This concentration is sufficient to induce phase separation of HFIP and water. Other salts may also be used provided that they reach the required lyotropic activity at a concentration at which they are soluble. The term "lyotropic activity" is used herein to refer to the structure-forming properties of a solution of a lyotropic salt. Lyotropic activity is achieved by reaching a sufficient concentration of the lyotropic salt to induce phase separation of the organic and aqueous phases. Protein precipitating concentrations of the lyotropic salt are avoided.

"Liotropic" or "structure-forming" salts, also known as cosmotropes, promote the order of the aqueous solution, thus excluding the dissolved organic substances. If the organic solute is the extraction solution, phase separation occurs. If it is a protein, it is bound to the salt outside the solution. Good structure-forming salts include sodium or ammonium sulfate, phosphates, citrates and the like (Washabaugh and Collins, J. Biol. Chem., 261: 12477, 1986). (Structure-destroying salts, or chaotropes, include guanidine hydrochloride, sodium perchlorate, sodium bromide, etc. They have the opposite effect, directing organic solutes into aqueous solution). The ionic strength of the aqueous solution is a function of the total number of ions in the solution as to whether they are structure-forming or structure-destroying ions. The term "ionic strength" refers to the concentration of one salt.

As discussed above, the lyotropic salt may be added as a solid or a concentrated liquid, provided that the concentration of the final lyotropic salt is effective in inducing phase separation. Preferably, the final ratio of the extraction solvent to the aqueous phase (which includes the aqueous preparation of biological material and solution of any salt) after increasing the lyotropic activity of the mixture is about 1:10 to about 10: 1, preferably (and as described below in the Examples) about 1: 3 to about 3: 1 provided that at a lower ratio of the extraction solvent to the aqueous phase, the final salt concentration is still high enough to induce phase separation .

After increasing lyotropic activity, the extraction solvent, which now contains any prion protein contained in the biological material, is separated from the aqueous preparation. The separation process takes several minutes and ends when the two phases are clear and a discrete separation between them is observed. After the two phases have been completely separated, the extraction solvent, now containing any prion protein, can be removed or withdrawn, for example by drawing with a pipette or funnel, or with a separation flask.

An extraction solvent containing any prion protein (hereinafter called "extract") may be dried, for example, by evaporation, lyophilization or vacuum centrifugation to obtain a highly concentrated or dry extract. The extract may contain other components, including cellular lipids, lipid membrane-binding proteins and other more hydrophobic cellular components. Optionally, the prion protein can be isolated or purified from these components, for example by hydrophilic interaction chromatography as described in the examples below, or other chromatographic techniques (cation exchange chromatography, gel permeation chromatography, reverse phase chromatography and affinity chromatography, for example on an antibody column).

Conversely, the concentrated or dried extract material can be analyzed directly as described below to detect abnormal prion protein.

Prion protein detector; immunoassays

Various prion protein assay methods are known in the art, including testing for selective detection of abnormal prion protein. Capillary gel electrophoresis has been shown to be an effective analytical tool for abnormal prion protein (Schmerr and Jenny, Electrophoresis, 19: 409, 1998). The preferred method is immunological, for example as described by Schmerr and Jenny, above. The antiserum described in this source is specific for the abnormal prion protein, as it has been found to respond in Western staining with scrapie-infected but not normal brain. Another prion protein reactive antiserum is well known in the art. A preferred immunoassay is an ELISA (e.g., Grathwohl et al., J. Virol. Methods, 64: 205, 1997).

Thus, in some cases, the detection of the presence of a prion protein, and in particular an abnormal prion protein, is based on the biophysical and chemical characteristics of the prion protein. These include resistance to proteinase (in particular proteinase K) and profile degradation (whether with proteolytic enzymes, glycolytic enzymes, chemicals, heating, denaturants, etc.). The effects of such treatments on the actual molecular weight and isoelectric point and different binding samples can be evaluated. Proteinase resistance and degradation profile can be established by chromatography, gel electrophoresis and other molecular weight sensitive techniques. The isoelectric point can be measured using capillary isoelectric focusing (IEF) or gel isoelectric focusing, although capillary IEF is able to measure prion pl of 3 more effectively than most gels. Moreover, the qualitative determination of total charge (acidity or basicity) can be determined by ion exchange chromatography. Other biophysical techniques known in the art can also be used to identify the prion protein.

Examples of prion protein detection studies include the actual molecular weight and isoelectric point of the protein, including after heating, cyanogen bromide cleavage, neuraminidase treatment, and the like. (Bolton et al., J. Virol. 53: 596, 1985); glycosidase treatment and lectin binding (Sommerville and Ritchie, J. Gen. Virol., 71: 883, 1990); resistance to proteinase-K (Race et al., Am. J. Vet. Res., 53: 883, 1992); and immunological assay (Farquhar et al., J. Virol. Methods, 24: 215,1989).

Alternatively, sequencing or micro-sequencing of the extracted, and preferably purified, prion protein allows unambiguous confirmation of its identity.

Prion protein immunological assays can be performed by techniques known in the art, for example radioimmunological, ELISA, sandwich immunoassays, immunoradiometric samples, gel diffusion precipitation, immunodiffusion and immunodiffusion tests using colloidal gold, enzymatic or radioisotope markers, for example), Western blots, precipitation reactions, agglutination reactions (eg gel agglutination tests), hemagglutination tests, tests s The complement fixation, immunofluorescence samples, the protein A and protein G, imunoelektroforetichni tests, measuring levels thereof in appropriate physiological samples, etc.. In one embodiment, antibody binding is established by detecting a primary antibody marker. In another embodiment, the primary antibody is detected by detecting the binding of a secondary antibody or reagent to the primary antibody.

The extraction method of the invention provides an inexpensive source of prion protein that can be used to generate additional antibodies. Moreover, since the extraction conditions of the invention are very different from the usual extraction conditions, the prion protein extracted in accordance with the invention may have a different conformation and exhibit a different population of antibodies if used for immunization.

This method shortens the extraction time to 1 to 2 hours. Moreover, because of its simplicity, it can be automated. The method extracts prion protein with different molecular weights so that it is not limited. It also dissolves the abnormal prion protein so that most immunoassays can be used to detect it. Moreover, and not insignificantly, it reduces the inefficiency of the abnormal prion protein, making the process safer.

Whales

The components for practicing the present invention may be provided in a convenient kit form. In its simplest embodiment, the kit of the invention provides an extraction solvent, preferably HFIP, and a lyotropic salt (or concentrated lyotropic salt solution) to enhance the lyotropic activity of the extraction solvent-aqueous mixture. The quantity of each component can be pre-measured to provide a specific number of tests. In another embodiment, the kit will include a sample container, preferably of plastic or material, treated to avoid non-specific binding of the prion protein.

As used herein, the term container has a broader meaning, i.e., any receiver for placing material or reagent. It may be factory-made from glass, plastic, ceramics, metal or any other material commonly used to contain reagents. However, acceptable material should not be reactive with the content it is supposed to have.

The kit may also include proteinase-K for the degradation of normal prion protein in a biological sample.

In another embodiment, the kit comprises a sample container with a volume indicator for the aqueous sample of the biological sample. In this embodiment, the polar organic solvent and the lyophilic salt are optimally offered in pre-measured units connected to the sample container. The biological sample preparation may be placed in the sample container. The pre-measured unit of extraction solvent may be added, followed by mixing. Then, the pre-measured unit of the lyotropic salt can be added to induce the separation of the phases from the extraction solution and water. In yet another embodiment, Proteinase-K is provided in the kit for treating the aqueous preparation of the biological sample, preferably in a pre-measured unit.

A tissue prion protein extraction kit may include a dilution buffer, such as 0.32 M sugar solution or phosphate buffer solution, to homogenize the tissue for the aqueous preparation of the biological material.

In another embodiment, in which the kit is a kit for detecting the presence of an abnormal prion protein in a biological material or sample, the kit provides a detector for an abnormal prion protein or assay as described above. Immunological assays, as described above, are preferred to detect abnormal prion protein extracted in accordance with the invention.

In yet another embodiment, the kit comprises an immunochromatographic membrane or surface. An extraction solvent containing any prion protein may be applied to the surface directly, or a dried extract may be administered, for example, upon re-dissolution. Under suitable conditions, the prion protein can penetrate the surface. It can be captured, for example, by an immobilized anti-prion antibody and the immobilized prion protein is detected. A variety of methods and equipment known in the art for immunochromatographic testing can be used in the invention. Immunochromatographic tests are especially applicable in field conditions where laboratory equipment is not available. Examples of such tests are provided in US patents 5,248,619, 5,451,504, 5,500,375, 5,624,809 and 5,658,801.

The kit of the invention preferably includes packaging and instructions for its use, for example on the package or in the package insert.

Examples of carrying out the invention

The present invention can be better understood by reference to the following non-limiting examples which are proposed in the invention.

Example 1. Analysis of abnormal prion protein extracted from infected sheep brain and lymph nodes

Brain tissue or lymph nodes from every second scrapie infected sheep were homogenized in 10% sarcosyl and treated with proteinase K to break down the normal host prion protein but not the altered abnormal prion protein form. Equal volumes (0.5 mM) of homogenate and HFIP were mixed and incubated for 5 min at 56 ° C. To this mixture was added 0.5 mM 0.5 M Na ₂ SO ₄ and the mixture was incubated for an additional 5 min. Under these conditions, the HFIP layer is separated from the aqueous layer. The HFIP layer was separated and dried on a centrifuge. The dried samples were resuspended and mixed in 25 μΐ of distilled water. 10 μΐ of the sample was mixed with 5μ1 of 20% SDS buffer and boiled for 5 min at 100 ° C.

Western blot analysis was performed on a 10% to 15% gradient polyacrylamide gel. The protein was transferred from the polyacrylamide gel to nitrocellulose under standard conditions. Nitrocellulose was blocked by incubation with 5% fish gelatin solution and washed with Tris-Tween buffer. Nitrocellulose was incubated overnight with rabbit antiprion protein (an antibody created against full prion protein diluted 1 to 2500; (Kascak et al., Immunol. Invest., 26: 259, 1997; see also Miller et al., J. Vet. Diagn. Invest 5: 309; Kascak et al., J. Virol., 59: 676, 1986) After incubation with an antiprion antibody, nitrocellulose is washed with Tris-Tween and then reacted with anti-rabbit IgG-HRP ( husk peroxidase conjugate and incubate for 1 h. The nitrocellulose is then washed extensively and developed with a chemiluminescent reagent (Pierce UltraSuperSignal ^R ). Peroxidase activity is detected by x emiluminescent transducer (Chemi-Imager-4000; Alpha, Innotech).

Extracts from both scrapie-infected sheep, containing 2.75 μΐ, produce material indicative of abnormal prion protein for both brain and lymph node tissues. Brain tissue extract containing 1.5 μΐ material from one of the scrapie infected sheep produces a band indicative of abnormal prion protein. Western staining of similar extracts from normal (uninfected) sheep did not produce any streaks indicative of abnormal prion protein.

Example 2. Analysis of abnormal prion protein extracted and purified from infected sheep brain and lymph nodes

This example shows further purification and analysis of abnormal (scrapie) prion protein (PrPSC) using hydrophilic interaction chromatography (HILIC). Tissue samples including sheep brain and lymph nodes were obtained with surfactant and proteinase K as described previously. The resulting extracts were applied to a HILIC column and eluted with a descending gradient of acetonitrile in 0.1% trifluoroacetic acid and 50 mM hexafluoro-2-propanol. The recovery from the column is approximately 75% as determined by the radioactive iodine prion protein. After drying, the collected peaks were resuspended in water and tested with antibodies specific for prion protein. The method allows for the effective purification of the prion protein as well as immunoassay testing as the interfering surfactants are removed.

Example 3. Analysis of abnormal prion protein extracted from infected sheep brain

Obtaining material from sheep brain

Scrapie-infected sheep brains are derived from field cases that are positive for the abnormal prion protein by Western blot (Race et al., Am. J. Vet. Res. 53: 883, 1992). The sample was drawn from 3 positive brains. The same sample was used for all the experiments presented here. Normal brains come from sheep from scrapless flocks and are negative for abnormal Western blot prion protein. Brain chromatography material was obtained by modifying the method of Bolton et al. (J. Virol. 53: 596, 1985). Briefly, brainstems were dissected, weighed and placed in 0.32 M sucrose (10% w / v). The material was then homogenized for 60 s with Brinkman Polytron (Kinematica AG, Lucerne Switzerland) using a 0.7 cm high speed steel generator. The homogenate was centrifuged at 10,000 g for 20 min to remove particles and the resulting supernatant was centrifuged at 230,000 g for 1 h. This precipitate is then subjected to a series of washes and ultracentrifugations as above. The sample was treated with 10 mM Tris pH 7.4 containing 10% sodium lauryl sulfate and proteinase-K (50 pg / ml). After final centrifugation, the sample was resuspended in 10 mM Tris pH 7.4 (200 μΐ / g from the original brain sample).

Hydrophilic Interaction Chromatography (HILIC)

The sample was dissolved in 0.01 M Tris HCl, pH 8.00, containing 2 mM EDTA, 5% SDS and 10% hexafluoro-2-propanol at 100 ° C for 10 min. After treatment with SDS, the sample was placed in a solvent consisting of 100% acetonitrile containing 0.1% TFA acid and 50 mM hexafluoro-2-propanol (buffer A) and applied to a hydrophilic interaction column. All columns used are from PolyLC, Inc. (Columbia, MD, USA) with dimensions 200 x 4.6-mm; 5 pm; 300-A. Three loads were investigated, PolyWAX LP ™ (ion exchange material), PolyHYDROXYETHYLA ™ (neutral material), and PolySULFOETHYL A ™ (strong cation exchange material) (all three trademarks are owned by PolyLC, Inc.). The flow rate was 0.5 ml / min. The elution conditions of Prp ^sc are: 100% A for 8 min and then a linear gradient to 100% water containing 0.1% trifluoroacetic acid and 50 µM hexafluoro2-propanol (buffer B) in 15 min, then 100% B for 10 min . Peak fractions were collected and dried in a vacuum centrifuge (Savant Instruments, Inc., Farmingdale, NY, USA). The fractions were resuspended in 10 μΐ deionized H ₂ O and the fraction tested positive by immuno-staining for PrP ^c was used in a capillary electrophoresis sample.

Marking a prion protein with ^{| 25} 1.

PrP ^sc was labeled with ^12ί Ι using IODOGEN ™ (Pierce, Rockford, IL, USA). The labeled protein was separated from the free ¹²⁵ l by passing it through a solid phase extraction cassette containing PolyWAX LP ™ (PolyLC, Inc.) equilibrated with buffer A. The labeled PrP ^sc was eluted from the cartridge using buffer B. Unbound ¹²⁵ 1 is obtained on the cartridge and could be disposed of as solid radioactive waste. The fractions containing the labeled PrP ^sc were dried in a vacuum centrifuge, dissolved in water, diluted 1/10 in buffer A and loaded onto the HPLC column.

Drip Colors

Equal portions of one Ιμΐ of the peak fractions of HILIC chromatography were applied to nitrocellulose paper, dried and then incubated in 20 mM Tris, pH 7.5 containing 500 mM NaCl, 0.05% Tween 20 (TTBS) and 5% fish gelatin in 1 h. The staining was washed 2x with TTBS and then incubated with 1/500 dilution antibodies created against prion protein peptides for 3 h at 25 ° C (rabbit antibodies against peptide 142-154). After incubation, the staining was washed 2x with TTBS and then incubated with biotinylated protein G (Bio-Rad Laboratories, Hercules, CA, USA) for 1 h. Again the staining was washed as above. Horseradish peroxidase bound to NeutrAvidin ™ (Pierce, Rockford, IL, USA) was added dropwise and incubated for 1 h at 25 ° C. After incubation, the staining was washed 6x with TTBS. After washing, the incubation was incubated in a SupersignaF Substrate system (Pierce) for 10 min and then exposed to a Kodak XOMAT AR (Eastman Kodak Company, Rochester, NY, USA) UV film for 15 s.

Connection sample for ¹²⁵ IprP ^sC

Fractions containing ^{| 25} l of the PolyWAX LP column were tested for binding activity to an antibody that was produced against the peptide corresponding to residues 142-154 of the prion protein. The radioactivity-containing tubes were dried in a Savant vacuum centrifuge at 42 ° C and resuspended in 10 μΐ H ₂ O and then diluted with buffer-containing salts in 0.1% BSA. The PVC dish was coated with the antibody in 0.1 M Na ₂ CO ₃ , pH 9.0. After washing with the above buffer, 100 μΐ of ¹²⁵ 1PrP ^sc were incubated on plates at 37 ° C for 2 h and then overnight at 4 ° C. The dish is washed and cut into individual wells and counted. Subtract background from sub in wells.

Capillary electrophoresis conditions

Capillary Electrophoresis Free Zone (Schmerr and Jenny, Electrophoresis 19: 409, 1998) was submitted to Beckman P / ACE

5500 (Beckman Instruments, Fullerton, CA, USA). Laser induced fluorescence (LIF) detection using an air-cooled argon laser (Beckman Instruments) with excitation at 488 nm and emission at 520 nm. Unmodified capillaries were obtained from Beckman Instruments. A 20 cm capillary (length to detector) x 201 pm ID with 200 mM Trisin, pH 8.0 was used. This buffer contains 0.1% N-octylglucoside (Boehringer Mannheim GmbH, Indianopolis, IN, USA) and 0.1% BSA (Sygma Chemical Co., St. Louis, MO, USA). In the separation preparation, the capillary was washed for 1 min with 0.25 M NaOH, washed for 2 min with H ₂ O, and then washed with buffer for 2 min. The separation conditions were 30 KV for 3 min at 20 ° C. The current is about 20 μΑ. The sample was injected for 15 s followed by 5 s injection of the leaked buffer. The sample volume is about 0.95 nl. The washes are carried out at high pressure and the injection of the sample is carried out at low pressure.

Immunocomplex and prion binding assays μΐ fluorescein-labeled peptide containing about 2 pmoles of fluorescently labeled peptide were mixed with affinity-purified rabbit IgG to demonstrate antibody binding to fluorescein-labeled peptide. Fractions of one μΐ of HILIC chromatography were added to the sample. After mixing the components, the samples were incubated at 25 ° C for 10 min.

Results

In FIG. 1 shows the chromatogram of PrP ^sc after purification and iodination. The radioactivity (cpm) of the ^{| 25} l prion-labeled protein coincides with absorption at 280 nm except for the last peak detected by absorption. The yield of an abnormal protein based on the recovery of ¹ L cpm loaded on the column is about 76%. The cpm and A280 absorption peaks coincide with the peaks showing antibody activity in the binding assay (Fig. 2). The peak in this test at about 25 min coincides with the peaks for ^1M 1-PrP and A280 absorption at 25 min in FIG. 1. Similar results were obtained for the chromatogram (not shown) of extraction (unpurified) of scrapie infected sheep brains.

A wide range of pl values have been reported in the literature (Schmerr and Jenny, supra; Safar et al., Proc. Natl. Acad. Sci. USA 87: 6373, 1990; Somerville et al., J. Gen. Virol. 70: 25 , 1989) for an abnormal prion protein, pl of this protein would affect the binding of this protein to the column fillers. There is no big difference between the retention time on a positively loaded PolyWAX LP column and the neutral PloyHYDROXYETHYL A column. This suggests that the protein may be acidic. Samples of abnormal prion protein containing SDS were eluted from a negatively charged PolySULFOETHYL A column over a wide range. Accordingly, the abnormal prion protein purified on a PolyWAX LP column is again skipped on PolySULFOETHYL A. Column Eluting it in or near the empty volume indicates that it is indeed acidic. This has been confirmed by both isoelectric focusing and capillary electric focusing (Schmerr et al., Chromatogr. A. 802: 135, 1998). pl values range from 3 to 6, in most species at 3.00. These results suggest that neutral or anion-exchange material is required for hydrophilic interaction chromatography.

In capillary immunoelectrophoresis using HILIC-purified samples, the resultant electrophoregrams (not shown) mean that samples from infected sheep respond, whereas samples from normal (uninfected) sheep do not respond. Because SDS inhibits typical immunoassays, including capillary electrophoretic samples, the removal of SDS is required in order to conduct such tests. A competitive sample using capillary electrophoresis could be performed after HILIC chromatography, as SDS eluted at or near the empty volume (Jeno et al., Anal. Biochem., 215: 292,1993).

Example 4. Analysis of abnormal prion protein extracted from infected sheep blood

Centrifuge fractions of TSE infected sheep blood samples were diluted with Tris buffered saline (10% tissue; 90% buffer). Samples were then treated with proteinase K to degrade not the normal host prion protein but not the altered abnormal prion protein form. After digestion, the treated sample was mixed with an equal volume of hexafluoro-2-propanol (HFIP) and incubated at 56 C for 5 min. An equal volume of 0.5 M sodium sulfate was added and the phases were allowed to separate. The HFIP containing layer was removed and the sample was dried in a vacuum centrifuge.

The precipitate was resuspended in water and the suspension was placed in an organic chromatographic mobile phase containing 95% acetonitrile, 5% water, 0.1% trifluoroacetic acid and 50 mM HFIP. The mobile phase was applied to a solid phase extraction cassette of PolyHYDROXYETHYL Aspartamide ™ (PolyLC, Inc.). Abnormal prion protein was eluted from this surface with 100% water, 0.1% trifluoroacetic acid and 50 mM HFIP, and then dried and resuspended in water.

The presence of abnormal prion protein is detected by capillary immunoelectrophoresis. Abnormal prion protein is not detected in TFE-free electrophoregrams for control of blood samples.

Example 5. Analysis of abnormal prion protein extracted from infected black-tailed deer blood

The procedure of Example 4 was repeated with blood samples from TSE infected black-tailed deer. The presence of abnormal prion protein was detected by capillary immunoelectrophoresis. Abnormal prion protein has not been detected in electrophoregrams for blood samples not infected with TSE.

Example 6 Analysis of Abnormal Prion Protein Extracted from Infected Moose Blood

The procedure of Example 4 was repeated with blood samples from a TSE infected moose. The presence of abnormal prion protein has been established by capillary immunoelectrophoresis. Abnormal prion protein has not been detected in electroferograms for TSE non-infected blood samples.

All patents, patent descriptions, test protocols and publications cited herein are incorporated by reference in their entirety.

The present invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein become apparent to one skilled in the art from the foregoing description and the accompanying figures. Such modifications are contemplated to be within the scope of the appended claims.

Claims (5)

1. A method for extracting an abnormal prion protein from a biological material that is presumed to contain an abnormal prion protein, characterized in that it includes: a) incubating a mixture of extraction solvent and an isotonic or hypotonic preparation of biological material under conditions effective to extract an abnormal prion protein from said biological material into said extraction solvent, wherein said extraction solvent is: i) a polar organic solvent in which said abnormal prion protein is soluble, and j) miscible with non-lyotropic aqueous solution but not miscible with lyotropic aqueous solution; and b) increasing the lyotropic activity of the mixture for separating said extraction solvent from said aqueous preparation of said biological material to produce an extraction solvent containing any abnormal prion protein from said biological material. 2. The process of claim 1, wherein said extraction solvent is hexafluoro-2-propanol. 3. The method of claim 1, wherein said biological material is a vertebral tissue or biological fluid. The method of claim 3, wherein said tissue is brain tissue. 5. The method of claim 3, wherein said bio-