JP4049135B2 - Method for detecting pathogenic prion protein, method for concentrating the same, and kit for concentrating or detecting the same - Google Patents

Description

  The present invention provides a method for detecting pathogenic prion protein from animal tissue-derived substances, and further concentrates the pathogenic prion protein-derived protein to be detected in carrying out this detection method. The present invention relates to a method, and its concentration or detection reagent kit.

  Scrapie, a prion disease, has been known as a serious disease in western Europe for more than 200 years in sheep. In recent years, scrapie-infected sheep were administered as an unheated cattle feed in the UK, resulting in the outbreak of mad cow disease (Bovine Spongiform Encephalopathy).

  It has also been pointed out that there is a causal relationship between eating mad cow beef curd meat and a new type of Creutfelt Jakob Disease (CJD), one of human prion diseases. In other words, it is no exaggeration to say that critical contamination is progressing with respect to mutton, its milk, beef and milk, which are important animal protein resources for humankind.

  However, unlike infectious bacteria and viral diseases that have been reported so far, prion disease is a protein that is originally present in the living body, has a new transmission function, and is relatively Development of effective diagnostic methods and preventive methods has been delayed because of having a long time and difficult pathogenic inactivation.

  As the most sensitive pathogenic prion protein (abnormal prion protein) detection method currently used, Western blotting (WB method; Western method) for detecting pathogenic prion protein at a low concentration before disease on sheep scrapie Blotting) has been developed.

  However, this method is difficult to apply to cattle due to differences in pathogen accumulation sites, the time taken to implement this method, and the number of animals treated. That is, rapid processing is difficult.

  In addition to the methods described above, the present situation is that detection methods for infected cattle by immunohistochemical staining using pathogenic prion protein antibodies and pathological findings are widely implemented.

  However, these methods are effective for livestock that have developed significant neurological symptoms or have died after the onset of disease, and are considered to be the safety of livestock in the incubation period, in other words, to apparently normal cattle, even to slaughterhouses. Safety could not be secured.

  In recent years, there have been reports on enzyme-linked immnosorbent assay (ELISA) and diagnostic methods using urine and blood from overseas, but their sensitivity and specificity have been questioned.

  That is, the detection sensitivity can be improved if the pathogenic prion protein contained in the target pathogen is concentrated in the measurement sample preparation stage and efficiently adsorbed to the microtiter plate for the ELISA method. The conventional methods have limitations on detection sensitivity.

  Bovine spongiform encephalopathy (BSE: Wales et al., 1987) is caused by meat and bone marrow fed as a diet and scrapie-contaminated sheep scrape contained in cattle feed. It was clear that newly infected cattle material was recycled (Virus Miss et al., 1993).

  Since then, in the UK, several captive ungulates (Wyatt et al., 1991) have developed spongiform encephalopathy as well as felines. In all these cases, BSE is thought to have originated through contaminated feed

  A report by Will et al. (1996) on a special case of Creutfeldt-Jakob disease (CIJ) in the UK reported one of the human variants in this group of infectious spongiform encephalopathy (TSE) or prion disease. However, it suggests that BSE may be transmitted to humans (Wil et al., 1996). For this reason, all TSEs are generally considered to occur across species (Dillinger, 1995).

  Current research priorities are to detect animals and materials infected with scrapie and BSE, and develop methods to prevent the spread of infection and entry into the food chain system.

  Unfortunately, to date, these prevention methods and management measures, as well as scrapie and BSE eradication programs, have not progressed well due to diagnostic difficulties.

The most common diagnostic methods currently in use are histopathological methods (Fraser, 1976) for diagnosing infection when a representative cancellous change in the central nervous system is noticeable, and proteinase K It is partially resistant to the treatment method (Bolton et al., 1982; Dillinger et al., 1983) and cannot be extracted with a neutral surfactant (Meyer et al., 1986; Bolton et al., 1987). These are two methods for detecting the scrapie special isoform (PrP ^sc ) of prion protein, which can be distinguished from normal prion protein (PrP ^c ) due to its characteristics.

Recently, a method for detecting PrP ^sc in reticulolymphoid organs by immunohistochemical assay using biopsy tonsil tissue of sheep has been reported (Schruder et al., 1996). However, this method is not suitable for cattle because abnormal prion accumulation is not significant in reticulolymphoid organs.

On the other hand, since histopathology occurs later in the incubation period, the pathological changes of the central nervous system occur later. Therefore, when compared with the PrP ^sc detection method, both experimental scrapie and BSE (Bolton et al. 1991; Gendroska et al., 1991), the effectiveness of its use is reduced.

  Recently, the importance of prion diseases has increased, and there is a need for a more sensitive diagnostic method for screening sheep and cattle at the time of slaughter.

The ELISA method can be said to be a suitable screening method, but at present, this method is only used in the basic research of TSE (Kasukuzak et al., 1987, Safar et al., 1990; Serban et al., 1990). In these studies, only high-purity PrP ^sc is adsorbed on the microtiter plate, but the use of raw tissue extract is also necessary for diagnosis.

  As described above, bovine spongiform encephalopathy (BSE) is one of the most serious diseases among humans among prion diseases.

  This disease is triggered by an exogenous pathogenic prion protein, accumulates the pathogenic prion protein in the central nervous system of livestock, etc., and causes neurological symptoms resulting in death. Pathogenic prion protein as a pathogenic substance Accumulated concentration and disease progression are significantly proportional.

  Therefore, since the accumulated concentration of the pathogenic substance is low during the incubation period after morbidity, a specific and highly sensitive detection method is required. In addition, considering the number of cattle processed worldwide, the measurement sample preparation method (ie, concentration method) and detection method must be simple, accurate, rapid, economical, etc. Needless to say.

  On the other hand, this disease is strongly suggested to be transmitted to humans by eating central nervous system organs of affected cows. In order to solve these problems, it is effective to conduct quarantine surveys, find sheep and cattle affected by this disease, and eliminate them at the early stage of the food chain.

  The present invention has been made in view of the above-described conventional situation, and its object is to quickly and easily at a relatively low concentration from a substance derived from an animal tissue, and highly sensitive and tissue-specific pathogenic prion protein. And a method for concentrating the pathogenic prion protein-derived protein to be detected when the detection method is carried out.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor uses a method for detecting pathogenic prion protein from animal tissue-derived substances according to the type of animal tissue-derived substance to be detected. It has been found that the pathogenic prion protein can be detected quickly, simply and with high sensitivity even at a relatively low concentration by appropriately selecting a preparation agent (particularly a surfactant), a preparation method, and a detection method.

That is, the present invention provides a method for detecting a pathogenic prion protein from an animal tissue-derived substance,
A first step of leveling the animal tissue-derived substance using a surfactant according to the type of the animal tissue-derived substance;
A second step of decomposing the homogenized product obtained in the first step using a decomposing enzyme containing a microbial protease;
A third step of obtaining a concentrate containing a pathogenic prion protein-derived protein from the homogenized product decomposed in the second step, and a concentration step of the pathogenic prion protein-derived protein,
A fourth step of dissolving a concentrate containing the pathogenic prion protein-derived protein in a solvent to obtain a lysate of the concentrate;
A fifth step of binding the pathogenic prion protein-derived protein in the lysate to the adsorption surface;
A pathogenic prion protein detection method (hereinafter referred to as the detection method of the present invention), which has a sixth step of coloring the pathogenic prion protein-derived protein bound in the fifth step. It is.

  According to the detection method of the present invention, first, in the step of concentrating the pathogenic prion protein-derived protein, the first to third steps described above are included, and in particular, depending on the type of animal tissue-derived substance. Since this is homogenized using a surfactant, the pathogenic prion protein-derived protein can be sufficiently concentrated even if the accumulated concentration of the pathogenic prion protein accumulated in the animal tissue-derived substance is relatively small. it can.

  Further, since this is detected based on an immunoassay having the fourth to sixth steps, for example, an enzyme immunosorbent assay (ELISA method; hereinafter the same), the pathogenic prion protein-derived protein is specifically identified. And firmly (fixed), and this can be detected quickly, easily and with high sensitivity.

  That is, according to the detection method of the present invention, for example, cattle and sheep can be diagnosed and selected at the early stage of infection with prion disease (scrapie or BSE), and this can be performed in large quantities and quickly. . In particular, a biopsy using a lymph node is possible in sheep, but according to the present invention, it is considered that a biopsy using a lymph node can also be performed, for example, in cattle.

Further, the present invention provides a method for concentrating a pathogenic prion protein-derived protein to be detected in carrying out a method for detecting a pathogenic prion protein from an animal tissue-derived substance.
A first step of leveling the animal tissue-derived substance using a surfactant according to the type of the animal tissue-derived substance;
A second step of decomposing the homogenized product obtained in the first step using a decomposing enzyme containing a microbial protease;
And a third step of obtaining a concentrate containing the pathogenic prion protein-derived protein from the homogenized product decomposed in the second step (hereinafter referred to as the first method of the present invention). (Referred to as a concentration method).

Furthermore, the present invention provides a method for concentrating a pathogenic prion protein-derived protein to be detected in carrying out a method for detecting a pathogenic prion protein from an animal tissue-derived substance.
A first step of leveling the animal tissue-derived substance using a surfactant according to the type of the animal tissue-derived substance;
A second step of decomposing the homogenized product obtained in the first step using a decomposing enzyme containing a microbial protease;
A third step of obtaining a concentrate containing the pathogenic prion protein-derived protein from the homogenized product decomposed in the second step;
Washing of the concentrate containing the pathogenic prion protein-derived protein obtained in the third step with a surfactant composed of N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate A concentration method comprising the steps (hereinafter referred to as the second concentration method of the present invention).

  According to the first concentration method and the second concentration method of the present invention, the pathogenic prion protein-derived protein is sufficiently obtained even if the accumulated concentration of the pathogenic prion protein accumulated in the animal tissue-derived substance is relatively small. It can be concentrated.

  Here, the animal tissue-derived substance is an animal's central nervous system tissue, reticulolymphoid tissue or bone, and further a substance derived from these tissues (for example, food, dura mater for transplantation, medical collagen) Etc. (hereinafter the same). The pathogenic prion protein means an abnormal prion protein that is considered to be the cause of prion disease. Examples of the prion disease include CJD, scrapie, and BSE described above. The detection method of the present invention, the first concentration method of the present invention, and the second concentration method of the present invention are not limited to sheep scrapie and BSE, and can cope with various prion diseases.

According to the detection method of the present invention, in the method for detecting pathogenic prion protein from animal tissue-derived substances,
A first step of leveling the animal tissue-derived substance using a surfactant according to the type of the animal tissue-derived substance;
A second step of decomposing the homogenized product obtained in the first step using a degrading enzyme containing a microbial protease;
A third step of obtaining a concentrate containing a pathogenic prion protein-derived protein from the homogenized product decomposed in the second step, and a concentration step of the pathogenic prion protein-derived protein,
A fourth step of dissolving a concentrate containing the pathogenic prion protein-derived protein in a solvent to obtain a lysate of the concentrate;
A fifth step of binding the pathogenic prion protein-derived protein in the lysate to the adsorption surface;
And detecting the pathogenic prion protein derived from the pathogenic prion protein by a method comprising: detecting the pathogenic prion protein-derived protein bound in the fifth step; In the concentration step, the above-mentioned first to third steps are included, and in particular, this is homogenized using a surfactant according to the type of animal tissue-derived substance. Even if the accumulation concentration of the pathogenic prion protein accumulated in the derived substance is relatively small, the pathogenic prion protein-derived protein can be sufficiently concentrated.

  Furthermore, since this is detected based on the enzyme immunosorbent assay having the fourth to sixth steps, the pathogenic prion protein-derived protein is specifically and firmly bound (immobilized). It can be detected quickly, conveniently and with high sensitivity.

  That is, according to the detection method of the present invention, for example, cattle and sheep can be diagnosed and selected at the early stage of infection with prion disease (scrapie or BSE), and this can be performed in large quantities and quickly. .

  In addition, according to the first concentration method and the second concentration method of the present invention, the pathogenicity to be detected when the pathogenic prion protein detection method for detecting the pathogenic prion protein from the animal tissue-derived substance is performed. In the method for concentrating a prion protein-derived protein, an effective concentration method capable of sufficiently concentrating even if the accumulation concentration is relatively small can be provided.

  First, the detection method of the present invention will be described.

  As the first step in the detection method of the present invention, since there is a homogenization step of homogenizing the animal tissue-derived substance using a surfactant according to the type of the animal tissue-derived substance, the animal Thoroughly dissolves tissue-derived substances, solubilizes non-specific substances in the presence of the surfactant according to the type, and sufficiently homogenizes the animal tissue-derived substances containing pathogenic prion protein can do.

  Therefore, even if the proportion of the pathogenic prion protein in the animal tissue-derived substance is relatively low, it can be uniformly homogenized, and thus a good pathogenic prion protein-derived protein concentrate can be obtained. Can do. That is, a homogenate effective for separating and extracting a pathogenic prion protein-derived protein can be obtained.

  In the first step, when the animal tissue-derived substance is a central nervous system tissue (for example, brain tissue or spinal cord tissue), the surfactant is Zwittergent 3-12 [trade name: Calvi. Manufactured by O Chemical Co .: N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate: molecular weight 336.6] or triton It is desirable to use a surfactant made of (Triton) X-100 [trade name: manufactured by Sigma: t-octylphenoxypolyethoxyethanol]. In addition to the surfactant, for example, Zudentent 3-0, 3-10, 3-14, 3-16, Nonidet P-40 (octylphenoxypolyethoxyethanol) manufactured by Calbio Chemical Co., Ltd., etc. may be used. .

  By using each of the above surfactants, non-specific substances (normal prion protein and other proteins: the same applies hereinafter) in the brain tissue can be sufficiently solubilized. In particular, it is more desirable to use brain tissue as the central nervous system tissue.

As a mechanism for improving the tissue specificity of the detection sensitivity by selecting a surfactant, for example, in brain tissue, Cercosyl having a concentration of about 0.5% and a pH of about 7.5 (trade name: manufactured by Sigma (molecular formula C ₁₅ H ₂₅ NO ₃ Na)], the loss of PrP ^sc is small, and protein can be extracted non-specifically. On the other hand, removal of non-specific contaminating proteins may be insufficient in lymph and spleen tissues. This is considered to be because it is desirable to selectively extract PrP ^sc again with the high concentration of the cercosyl.

  When the animal tissue-derived substance is reticulolymphoid tissue (for example, spleen, lymph node, bone marrow, etc.), the surfactant is a nonionic surfactant made of t-octylphenoxypolyethoxyethanol. It is desirable.

  By using the surfactant, the nonspecific substance in the spleen tissue can be sufficiently solubilized. In particular, it is more desirable to use spleen tissue as the reticulolymphoid tissue.

  Next, as the second step, there is a decomposition treatment step of decomposing the homogenized product obtained in the first step using a decomposing enzyme containing a microbial protease. It is possible to sufficiently extract the target pathogenic prion protein by sufficiently decomposing and digesting a substance (especially chromosome and DNA) containing the sex prion protein.

  In general, pathogenic prion protein is considered to be on a gene in a chromosome. Therefore, in order to specifically extract this protein, it is required to decompose the protein containing the protein. This 2nd process is operation which decomposes | disassembles the protein containing pathogenic prion protein while decomposing | disassembling a nonspecific substance.

  Here, the homogenate is decomposed using collagenase (collagenase) and DNA-degrading enzyme (DNase) as the degrading enzyme, and further using a proteolytic enzyme (proteinase or protease). It is desirable to decompose.

  Next, the third step includes a separation step of obtaining a concentrate containing the pathogenic prion protein-derived protein from the homogenized product decomposed in the second step. The substance containing the pathogenic prion protein-derived protein sufficiently homogenized and decomposed in the steps 2 and 2 can be efficiently separated.

  In this separation step, for example, separation and concentration can be performed using means such as centrifugation (ultracentrifugation).

  The above is the basic configuration of the pathogenic prion protein-derived protein concentration step. In the concentration step in the detection method of the present invention, in addition to the first to third steps described above, for example, It is desirable to add a process such as

  For example, during the concentration step, the concentrate containing the pathogenic prion protein-derived protein obtained in the third step is decomposed using a degrading enzyme containing microbial protease, and then subjected to a salting-out treatment after separation. It is desirable to further include a step.

That is, the in order to improve the solubility of the pathogenic prion protein derived from the protein further, for example, Sakoshiru (Sarkosyl, trade name: _{Sigma: C 15 H 25 NO 3 Na} ) dissolving treatment using such separation process And the separated extract obtained is salted out using NaCl, for example, and then subjected to a separation treatment, whereby a concentrate containing a more concentrated pathogenic prion protein-derived protein can be obtained.

  Moreover, it is desirable that the concentration step further includes a washing step of washing the concentrate containing the pathogenic prion protein-derived protein obtained in the third step with a surfactant.

  That is, as an additional washing step of the concentrate (for example, pellets), the concentrate is washed with a surfactant to further remove unwanted substances (non-specific substances) in the concentrate. Many can be removed.

  Here, as a surfactant to be used, an interface comprising N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate (eg, Twittergent 3-12, 3-08, 3-10, etc.). An activator is desirable.

  Next, a measurement method (from the fourth step to the sixth step) based on the detection method of the present invention will be described.

  Here, as a measurement method performed in the fourth to sixth steps, an immunoassay method, for example, enzyme-linked imsorbent assay (ELISA) is also called an enzyme antibody method, and an antibody is arranged on a specific adsorption surface. In this method, an antigen and an antibody are bound to form a complex, and this is detected.

  First, as the fourth step, there is a step (dissolution step) in which a concentrate containing the pathogenic prion protein-derived protein is dissolved in a solvent to obtain a lysate of the concentrate. A concentrate of protein derived from pathogenic prion protein that can be easily adsorbed on the adsorption surface in the adsorption step can be produced.

  Here, it is desirable to use guanidine thiocyanate (GdnSCN) as the solvent.

  Guanidine thiocyanate is considered to have an effect of facilitating adsorption of the concentrate in the adsorption step in the next stage. This is thought to be due to the expression of an antigenic site that increases the immunoreactivity of the anti-prion protein antibody by guanidine thiocyanate. By dissolving the guanidine thiocyanate, the antigen-antibody complex is strongly and specific. Reaction can be detected.

  The guanidine thiocyanate is preferably 1 to 5 molar (M). This concentration is more preferably 3 to 4 molar. Moreover, it is desirable to dissolve the concentrate in the above-described guanidine thiocyanate using a buffer such as phosphate buffered saline (PBS; pH ≦ 5).

  Next, as the fifth step, there is a step (binding step) of binding the pathogenic prion protein in the lysate to the adsorption surface, and the pathogenic prion in the lysate dissolved in the previous stage A protein-derived protein-derived protein can be adsorbed on, for example, a microtiter plate. Therefore, strong and specific reactions for the formation of antigen-antibody complexes can be detected by this method.

  In addition, the adsorption surface can be formed with a primary antibody, and an antigen-antibody complex of the primary antibody and the pathogenic prion protein-derived protein can be bound to the adsorption surface. In general, the ELISA method generates an antigen-antibody complex between an antigen to be measured and an antibody for immobilization thereof, and this is detected by, for example, a color development method using an enzyme-labeled antibody and a substrate. .

  Next, the sixth step includes a step of coloring the pathogenic prion protein-derived protein bound in the fifth step by reacting an enzyme-labeled antibody with a chromogenic substrate (coloring step). Therefore, this can be easily detected by a colorimeter or a spectrophotometer. That is, by detecting the degree of color development, the presence or absence of pathogenic prion protein and the accumulated concentration thereof can be examined.

  In addition, 2,2-azizo-bis (3-ethyl-benzthiazoline-6-sulfonate) or the like can be used as a coloring reagent (chromogenic substrate). As is well known, fluorescence measurement can be performed using an enzyme-labeled antibody and a fluorescent substrate. Luminescence can be measured using an enzyme-labeled antibody and a luminescent substrate.

  As this color development method, an avidin-biotin complex that observes color development based on the formation of a complex of avidin (avidin) that binds to the pathogenic prion protein-derived protein and biotin (biotin) that binds to the chemiluminescent substance The method (ABC method) and the indirect method (HRP method) using horseradish peroxidase-conjugated donky anti-rabbit IgG can be used.

An outline of this detection method is, for example, that PrP ^sc bound to a well specifically binds to polyclonal antibody B103 and binds with a secondary antibody (anti-rabbit IgG) -biotin complex that specifically recognizes the antibody, This is a method in which adivine is bound thereto, followed by binding of horseradish peroxidase (HRP) -biotin, and a substrate of HRP is reacted to cause color development.

  In general, the results obtained by the avidin-biotin complex method are more reproducible than the indirect method, but in particular, when detecting pathogenic prion protein in spleen tissue as the animal tissue-derived substance, the avidin-biotin complex It is desirable to use the body method.

  As described above, according to the enzyme immunosorbent method (ELISA method) of the present invention, even when the pathogenic prion protein to be measured is at a relatively low concentration, the pathogenic prion protein-derived protein is strongly and specifically The pathogenic prion protein can be detected quickly and easily.

  In addition, the detection by ELISA method shows the sensitivity at least equivalent compared with the western blotting method (WB method) mentioned above, and also the measurement is practical and quick. In addition, the advantage of detection by ELISA is that many samples can be analyzed at one time, potentially infected animals can be diagnosed and selected in large quantities, and prion disease caused by infection (especially BSE). It can be used for a wide range of applications such as controlling

  Next, the above-described first concentration method of the present invention will be described.

  According to the first concentration method of the present invention, the method for concentrating the pathogenic prion protein-derived protein to be detected in carrying out the method for detecting the pathogenic prion protein from the animal tissue-derived substance, As a step, the animal tissue is obtained using a surfactant such as N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate, t-octylphenoxypolyethoxyethanol according to the type of the animal tissue-derived substance. Since there is a homogenizing step for homogenizing the derived material, the animal tissue-derived material is sufficiently dissolved, and N-dodecyl-N, N-dimethyl-3 is used as a surfactant according to the type. -Solubilizing non-specific substances in the presence of surfactants consisting of amino-1-propanesulfonate or t-octylphenoxypolyethoxyethanol It can be sufficiently equalized the animal tissue derived materials containing pathogenic prion protein.

  Therefore, even if the proportion of the pathogenic prion protein in the animal tissue-derived substance is relatively low, it can be uniformly homogenized, and thus a good pathogenic prion protein-derived protein concentrate can be obtained. Can do. That is, a homogenate effective for separating and extracting a pathogenic prion protein-derived protein can be obtained.

  Here, it is desirable that the animal tissue-derived substance is a central nervous system tissue. More preferably, the central nervous system tissue is a brain tissue.

  In addition, as the second step, there is a decomposition treatment step of decomposing the homogenized product obtained in the first step using a decomposing enzyme containing a microbial protease. A substance containing a protein (particularly, a chromosome) can be sufficiently decomposed and digested to sufficiently extract the target pathogenic prion protein-derived protein.

  As the degrading enzyme, it is desirable to decompose the homogenized product using a collagen degrading enzyme and a DNA degrading enzyme, and further using a proteolytic enzyme containing a microbial protease.

  Further, the homogenized product can be decomposed using the above-mentioned microbial protease as the decomposing enzyme.

  Next, as the third step, the method includes a separation step of obtaining a concentrate containing the pathogenic prion protein-derived protein from the homogenized product decomposed in the second step. The substance containing the pathogenic prion protein-derived protein sufficiently homogenized and decomposed in the step and the second step can be efficiently separated.

  In this separation step, for example, separation and concentration can be performed using means such as centrifugation (ultracentrifugation).

  The above is the basic configuration of the first concentration method of the present invention. In the first concentration method of the present invention, as in the detection method of the present invention described above, the first to third steps are performed. In addition to the steps, for example, it is desirable to add the following steps.

  For example, during the concentration step, the concentrate containing the pathogenic prion protein-derived protein obtained in the third step is decomposed using a degrading enzyme containing microbial protease, and then subjected to a salting-out treatment after separation. It is desirable to further include a step.

  That is, in order to further improve the solubility of the pathogenic prion protein-derived protein, for example, dissolution treatment and separation treatment are performed using sarkosyl or the like, and the obtained separated extract is salted using, for example, NaCl or the like. After the analysis, a concentrate (for example, a pellet) containing the pathogenic prion protein-derived protein further concentrated can be obtained by performing a separation treatment.

  Moreover, it is desirable that the concentration step further includes a washing step of washing the concentrate containing the pathogenic prion protein-derived protein obtained in the third step with a surfactant.

  That is, as an additional washing step of the concentrate (for example, in the form of pellets), by washing the concentrate using the surfactant, unwanted substances (non-specific substances) in the concentrate are removed. More can be removed. As the surfactant, a surfactant comprising N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate (for example, the above-mentioned Twittergent 3-12, 3-08, 3-10, etc.). It is desirable to use

  Next, the second concentration method of the present invention described above will be described.

  According to the second concentration method of the present invention, the first step in the method for concentrating the pathogenic prion protein-derived protein to be detected in carrying out the method for detecting the pathogenic prion protein from the animal tissue-derived substance is performed. As having a homogenization step of homogenizing the animal tissue-derived substance using a surfactant according to the type of the animal tissue-derived substance, sufficiently dissolve the animal tissue-derived substance, The non-specific substance can be solubilized in the presence of the surfactant corresponding to the type, and the animal tissue-derived substance containing the pathogenic prion protein can be sufficiently homogenized.

  Therefore, even if the proportion of the pathogenic prion protein in the animal tissue-derived substance is relatively low, it can be uniformly homogenized, and thus a good pathogenic prion protein-derived protein concentrate can be obtained. Can do. That is, a homogenized product effective for separating and extracting the pathogenic prion protein-derived protein can be obtained.

  In the first step, it is desirable that the animal tissue-derived substance is a reticulolymphoid tissue. In particular, when the reticulolymphoid tissue is spleen tissue, as described above, the non-specific substance in the spleen tissue can be sufficiently solubilized by using the surfactant.

  Next, as a second step, since there is a decomposition treatment step of decomposing the homogenized product obtained in the first step using a degrading enzyme, the pathogenic prion protein in the homogenized product is included. Substances (particularly chromosomes) can be sufficiently decomposed and digested to sufficiently extract the target pathogenic prion protein-derived protein.

  Here, it is desirable that the homogenized product is degraded using a collagen degrading enzyme and a DNA degrading enzyme as the degrading enzyme, and then further degrading using a proteolytic enzyme containing a microbial protease.

  Next, as the third step, the method includes a separation step of obtaining a concentrate containing the pathogenic prion protein-derived protein from the homogenized product decomposed in the second step. The substance containing the pathogenic prion protein-derived protein sufficiently homogenized and decomposed in the step and the second step can be efficiently separated.

  In this separation step, for example, separation and concentration can be performed using means such as centrifugation (ultracentrifugation).

  Next, as the washing step, the concentrate containing the pathogenic prion protein-derived protein obtained in the third step is converted to an interface comprising N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate. Since it further has a washing step of washing with an active agent, it is possible to remove more unwanted substances (non-specific substances; such as normal prion protein and other proteins) in the concentrate. .

  According to the first concentration method and the second concentration method of the present invention described above, the pathogenic prion protein-derived protein can be obtained even if the accumulated concentration of the pathogenic prion protein accumulated in the animal tissue-derived substance is relatively small. Can be sufficiently concentrated. In addition, the concentrate containing the pathogenic prion protein-derived protein obtained by each of the above-described concentration methods may be used in an ELISA method at a later stage, or may be used in a WB method, an electrophoresis method, or the like.

  Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited to the following examples.

This embodiment, PrP ^sc crude tissue extracts from scrapie infected mice (pathogenic prion protein, hereinafter the same) is intended for concentrating the detection, and PrP ^sc from protein to be detected in.

1. In this example, SIc / ICR mice were used. This mouse was brain-infected with Obihiro species scrapie (Shinagawa et al., 1985), and brain tissue and spleen tissue were taken out from the mouse that clearly developed scrapie.

2. Concentration of PrP ^sc from brain tissue and spleen tissue Samples were prepared by eight different enrichment methods shown in FIGS. All eight methods were: digesting a tissue sample minced with scissors with a degrading enzyme (second step) and extracting soluble non-specific material in the presence of a surfactant; Common in performing homogenization (first step) for the purpose of centrifugation (third step).

Hereinafter, with respect to the method for concentrating PrP ^{sc -derived} protein, Method 1 to Method 8 will be briefly described with reference to FIGS.

Method 1
First, 8% Twittergent 3-12 (surfactant), Cercosyl, 100 mM sodium chloride (NaCl), 5 mM magnesium chloride (MgCl ₂ ), 50 mM, Tris-HCl buffer (pH = 7.5) Tris-HCl) was added to homogenize the brain tissue to prepare a homogenate (homogenate) (first step).

  The homogenized product is then used with 0.5 mg / 100 mg collagenase [3,4,24,3] (Collagenase) and 40 μg / 100 mg DNase [3,1,21,1] (DNase) at a temperature of 37. Decomposition (digestion) treatment at 4 ° C. for 4-12 hours, and further with 50 μg / 100 mg proteinase [3,4,21,14] (Proteinase K) at 37 ° C. for 0.5-2 hours (Digestion) treatment was performed (second step). This decomposition (digestion) treatment is performed for enzymatic decomposition of a measurement contaminant other than the pathogenic prion protein in the tissue.

Then, after stopping the reaction, the mixture was centrifuged at 15,000 rpm and room temperature for 20 minutes. This was heated and dissolved in 5% SDS (sodium dodecyl sulfate) for 10 minutes (third step) to obtain a PrP ^{sc -derived} protein-containing concentrate (FIG. 1).

Method 2
First, as in Method 1, the brain tissue was homogenized to produce a homogenized product (first step). Next, the homogenized product was decomposed (digested) using bromelin [3,4,22,23] (Bromelain) at a temperature of 45 ° C. for 0.5 to 2 hours (second step).

Then, after stopping the reaction, the mixture was centrifuged at 15,000 rpm and room temperature for 20 minutes. This was heated and dissolved in 5% SDS (sodium dodecyl sulfate) for 10 minutes (third step) to obtain a PrP ^{sc -derived} protein-containing concentrate (FIG. 1).

  When bromelin is used, it is desirable that the working temperature is 45 to 80 ° C. and the working time is about 1 to 10 hours. As a result of using bromelin, only one type of enzyme is used, and the operation is simplified (reduction of operation time and cost reduction). The measurement results were comparable (similar) to the sensitivity when using three types of enzymes (collagenase, DNanase, and proteinase).

Method 3
5-8 times the tissue weight of 4% Triton X-100 (nonionic surfactant), 0.5% Sarcosyl, 100 mM sodium chloride (NaCl), 5 mM magnesium chloride (MgCl ₂ ), 50 mM , PH = 7.5 and Tris-HCl buffer were added to homogenize the spleen tissue and brain tissue (first step).

  Subsequently, the decomposition | disassembly (digestion) process similar to the method 1 was performed (2nd process). Further, the same separation treatment as in method 1 was performed (third step).

  Next, the precipitate obtained in the separation process (third step) was suspended and decomposed using 6.25% Sarkosyl and 10 mM, Tris-HCl buffer solution of pH = 9.2 (decomposition step). .

Next, this was ultrasonically crushed and then centrifuged at 15,000 rpm, and then NaCl was added to the supernatant of the solution obtained by centrifugation at a final concentration of 12% and stirred (salting out step). Thereafter, it ultracentrifuged at a rotational speed 55,000 rpm, dissolved by heating with 5% SDS, to obtain a concentrate of PrP ^sc from protein-containing (Fig. 2).

Method 4
Similarly to the method 3 and the separation treatment (third step), after separating the spleen tissue and brain tissue, the obtained precipitate (pellet shape) is heated and dissolved using 5% SDS. Thus, a PrP ^sc derived protein-containing concentrate was obtained (FIG. 2).

Method 5
In the same manner up to the method 3 and the separation treatment (third step), the spleen tissue was separated, and the resulting precipitate (pellet shape) was converted into 8% Twittergent 3-12 (surfactant). ) And 10 mM, pH = 7.5 Tris-hydrochloric acid buffer solution, and homogenized to prepare a homogenized product (washing step).

Next, the mixture was centrifuged at 15,000 rpm, and the resulting precipitate (pellet) was heated and dissolved using 5% SDS to obtain a PrP ^sc- derived protein-containing concentrate (FIG. 2).

Method 6
In Method 3, instead of performing degradation (digestion) treatment using collagenase and DNase and further proteinase in the degradation treatment step (second step), bromelain similar to Method 2 is used. A PrP ^{sc -derived} protein-containing concentrate was obtained in the same manner as in Method 3 except that the measurement contaminants other than the pathogenic prion protein in the tissue were enzymatically degraded (FIG. 3).

Method 7
In Method 4, instead of performing degradation (digestion) treatment using collagenase and DNase, and also proteinase in the degradation treatment step (second step), bromelin similar to that in Method 2 is used in the tissue. A PrP ^sc- containing protein concentrate was obtained in the same manner as in Method 4 except that the measurement contaminants other than the pathogenic prion protein were enzymatically degraded (FIG. 3).

Method 8
In Method 5, instead of performing degradation (digestion) treatment using collagenase and DNase, and also proteinase in the degradation treatment step (second step), bromelin similar to that in Method 2 is used in the tissue. A PrP ^sc- containing protein concentrate was obtained in the same manner as in Method 5 except that the measurement contaminants other than the pathogenic prion protein were enzymatically degraded (FIG. 3).

  That is, in Method 3, Method 4 and Method 5, 5-8 times the volume of 4% (wt / vol) Triton X-100 is added to the homogenization buffer, and in Method 1, 8% (wt / vol) Twitter. Gent 3-12 was added instead of Triton X-100.

  The Twittergent 3-12 used in Method 5 was centrifuged (TLA 100.3 rotator) in the third step (rotation speed 15,000 rpm) in order to remove more unwanted materials (non-specific substances). This was used as an additional washing step for pellets obtained with an Optima TLX desktop ultracentrifuge, manufactured by Beckman.

In Method 3, to increase the solubility of PrP ^sc from protein, pH = 9.2 by the PrP ^sc protein derived from 6.25% (wt / vol) Sakoshiru (which is a value higher than the pH using conventional) Extracted with. Sakoshiru after extraction, to reduce the solubility of PrP ^sc proteins derived again, 10% (vol / vol) returned to neutral pH with HCl, followed by 12% (wt / vol) salting out of the PrP ^sc proteins derived by NaCl If, it performs a final centrifugation at 55,000 rpm, to obtain pellets containing the more concentrated PrP ^sc proteins derived. This Sarcosyl extraction and salting out of the PrP ^sc derived protein with NaCl were omitted in Method 4, Method 5 and Method 1 to simplify the sample preparation process.

3. Western blot analysis Brain and spleen of mice infected with scrapie were used as samples. The brain was prepared by Method 1 and the spleen was prepared by Method 3. Samples were diluted 2 ¹¹ times 2 ³ times. Prior to the WB method, each diluted sample was subjected to SDS-PAGE (electrophoresis) and transferred to a PVDF membrane. 5% skim milk was used for blocking, and B103 polyclonal antibody was used for detection. Detection was performed by ELC-Western blot detection system (Amersham).

4). ELISA Method As shown in FIG. 4, in order to examine the appropriate adsorption conditions to the microtiter plate, first, brain tissue and spleen tissue extracts were heated and boiled in 5% SDS: sodium dodecyl sulfate for 10 minutes (1 This was precipitated in ice-cold methanol over 10 volumes (2). This tissue extract usually contained 10-40 mg of raw tissue. However, the (numerical value) corresponds to the (numerical value) in FIG. 4 (the same applies hereinafter).

  Subsequently, after the centrifugation (3), the pellet-like precipitate obtained was separated into 100 μl of guanidine thiocyanate (guanidine thiocyanate: GdnSCN) at different concentrations (1 to 5 M), PBS (phosphate buffered saline: final pH). ≦ 5) was ultrasonically dissolved (4). Steps (1) to (4) up to this point correspond to the fourth step described above.

  Moreover, as shown in FIG. 5, in order to perform the measurement when SDS is used as a solvent, the pellets are changed to the use of GdnSCN, and the pellets have different concentrations of 0.1 to 4% (vol / vol). Dissolved in SDS, PBS (final pH ≦ 5).

  Each solution was then distributed on a 96-well round-bottom maxisorp immunoplate (trade name: Maxisorp immunoplate: Nunc), and cultured overnight at room temperature under shaking (5) (for example, Corning) An ELISA plate high coupling type 430452 may be used).

  The plate was then washed 3 times with PBS (6) and blocked (closed) at 37 ° C. for 1 hour in PBS-5% skim milk (7).

  The plate was then washed 3 times with PBS containing 0.05% Tween 20 (hereinafter referred to as PBST) (8).

  Subsequently, using an initial volume of PBS as a primary antibody of sputum antiserum B-103 (Horiuchi et al., 1995), 2,000-fold in PBST-0.5% skim milk after precipitation with 33% ammonium sulfate After dilution, 100 μl was distributed in the well (the above-mentioned hole: the same applies hereinafter). The plate was cultured under shaking for 1 hour at room temperature to form an antigen-antibody reaction complex (9). Here, the steps (5) to (9) correspond to a fifth step.

  The plate was washed 3 times with PBST, and then revealed by the Avidin-Biotin complex (ABC) method as described below. As a result, the antigen-antibody complex became visible. It was. However, Vector Stain Elite ABC kit, manufactured by Vector Laboratories (Vectastain Elite ABC kit, Vector Laboratories) was used as a kit for the ABC method.

  Biotin (vitamin H) -modified anti-rabbit IgG was diluted 1,500 times in PBST-0.5% skim milk, and 100 μl of the well was incubated at room temperature for 1 hour under shaking ( 10).

  Next, after washing 4 times with PBST and 1 time with PBS (11), each of component A (avidin) and component B (biotin) of the kit was added to PBS at a dilution ratio of 200 times in the wells. (12). Here, the kit is a set of tools and preparations for carrying out the ABC method.

  Cultivation and washing were then performed on the biotinylated antibody (ie conjugated antibody) (13).

  The color was developed by incubation with 100 μl of a substrate solution [100 μl / ml 2,2′-azizo-bis (3-ethyl-benzthiazoline-6-sulfonate): ABTS] and then a buffer composed of 0.05 M citrate-phosphate. Performed in the dark with 0.04% hydrogen peroxide at room temperature for 1 hour in the liquid (14).

  Next, color development at a wavelength of 405 nm was confirmed with a microplate reader [Model 2550, Bio-Rad Laboratories] (15).

  Instead of this color development method based on the ABC method, horseradish peroxidase-conjugated donky anti-rabbit IgG (Amersham) 100 μl in PBST-0.5% skim milk 800 times Wells were cultured at a dilution of (16). Thereafter, the plate was washed 4 times with PBST and once with PBS (17). Hereinafter, this method is referred to as an indirect method.

  In addition, culture and washing were performed on the biotinylated antibody. The cut-off line was determined by the sum of the average optical density (OD) and the standard deviation of 4 to 8 negative control groups coated with the tissue extract of uninfected mice.

5. Measurement result
(1) Determination of suitable coating conditions In order to realize a high adsorption rate to the microtiter plate, it is necessary to sufficiently dissolve the ^PrPsc- derived protein. Here, the usefulness of GdnSCN and SDS for dissolving PrP ^sc was evaluated (FIG. 5).

In FIG. 5, in order to see the effect of GdnSCN and SDS on the adsorption of PrP ^sc derived protein to microtiter plates, samples from brain tissue (A) and spleen tissue (B) of scrapie infected mice were obtained by Method 1 and Method 5. Each was prepared. At each concentration of SDS and GdnSCN, an extract was collected from an equivalent amount of 1 mg of brain (A) and spleen (B) and coated on 3 wells per microtiter plate.

  Each tissue extract from uninfected mice was prepared, dissolved in 3M GdnSCN-PBS, and used as a negative control group (negative control (Ctrl.); Control). Here, 2 mg brain equivalent (6 wells) or 2.5 mg spleen equivalent (4 wells) was used for each well.

  Moreover, the average value and its standard deviation (SD) were shown in FIG. The cut-off line was determined by adding three standard deviation values (3SD: the same applies hereinafter) to each average value. The indirect method and ABC method were applied to brain tissue and spleen tissue, respectively. The chaotropic ion agent increases the water solubility of hydrophobic molecules, weakens hydrophobic bonds, and is used for extraction of membrane proteins and the like, and examples thereof include GdnSCN.

When dissolving ^PrPsc- derived protein in PBS without a chaotropic agent or detergent, some adsorption to microtiter plates occurs in brain tissue but not in spleen tissue. (FIGS. 5A and 5B: 0 M GdnSCN). Further, even when the SDS concentration was the lowest concentration (0.1%), the adsorption of the ^PrPsc- derived protein did not occur from the brain tissue (FIG. 5 (A)).

  On the other hand, when GdnSCN was added to PBS while increasing the PBS concentration, the coating efficiency was improved, and a peak for the spleen tissue was generated when the GdnSCN concentration was 4M (FIG. 5 (B)). Moreover, the GdnSCN concentration around 3M showed a peak for brain tissue (FIG. 5A). That is, when 1-5M (particularly 3-4M) GdnSCN was used as a solvent, strong and specific adsorption was observed.

  If the GdnSCN concentration in PBS increases, the pH value decreases (≦ 5), so quantitatively replace 0.05M sodium bicarbonate carbonate buffer (pH = 9.6) with PBS. Diluted both GdnSCN and SDS, thereby increasing the final pH to 8 or higher. However, the adsorption rate tended to decrease (data not shown).

(2) Comparison of two different detection methods: indirect method and ABC method The indirect method and the ABC method were compared in terms of sensitivity and specificity. FIG. 6 shows a comparison between the ABC method and the indirect method for detection of antigen-antibody complexes.

  Samples of brain tissue from scrapie-infected mice (A) were prepared by Method 1 and subsequently diluted two-fold sequentially into 3M GdnSCN. For spleen tissue (B), method 3 was applied and 4M GdnSCN was used. In the two measurement methods, the average value in the two detection methods was obtained. A negative control group (control) is also shown. The standard deviation (SD) is too small to be shown. The cut-off line was determined by adding 3SD for each. The cut-off line in the measurement of spleen tissue was determined based on data of a negative control group consisting of 40 mg of spleen equivalent (ie, the amount corresponding to 40 mg of spleen: hereinafter the same).

  According to this measurement result, the ABC method was slightly superior for brain tissue (FIG. 6A), but the ABC method was clearly preferable for spleen tissue (FIG. 6B). . For spleen tissue, the ABC method was at least twice as sensitive as the indirect method. In general, the results obtained by the ABC method were found to be more reproducible than the indirect method (data not shown). Therefore, in the final measurement (comparison between the ELISA method and the WB method in FIG. 8), the ABC method was applied to the spleen tissue and brain tissue.

(3) Establishment of an appropriate sample preparation method (concentration method) Method 3 was used for the ELISA method. This sample preparation (concentration) method is a known sample preparation method for Western blotting (Gracewall et al., 1996). Here, salting out of the PrP ^sc proteins derived by Sakoshiru extraction and NaCl, the method 4, method 5, not shown in method 1.

Then, instead of separating the PrP ^{sc -derived} protein by extraction, PrP ^c (normal prion protein) and other proteins were sufficiently removed from the sample by using 8% (wt / vol) Zwittergent 3-12. It was found that the Twittergent 3-12 is a more effective cleaning agent than Triton X-100. Therefore, the former was used in place of Triton X-100 in the homogenization buffer of Method 1.

  For spleen tissue, collagenase digestion was performed using 4% (wt / vol) Triton X-100 and 0.5% (wt / vol) Circosyl (Method 3, Method 4 and Method 5). In the method 5, the pellet (concentrate) obtained under a centrifugal force of 40,000 rpm is used to remove 8% (wt / vol) Twittergent 3-12 and 0. Further extraction with 5% (wt / vol) circosyl.

(3-1) Brain tissue FIG. 7 shows an appropriate sample preparation method for brain tissue and spleen tissue. Both FIGS. 7A and 7B are measurement results obtained repeatedly.

  The brain tissue extract was prepared (concentrated) by Method 4, Method 3, and Method 1, and dissolved and diluted 2 times in 3M GdnSCN-PBS. Here, the indirect method was used to examine the plate. As a negative control group (control), 8 wells were each coated with an extract obtained in Method 1 from 20 mg brain tissue equivalent of uninfected mice. The cut-off value was an average value obtained by adding 3SD.

  The spleen tissue extract was prepared (concentrated) by Method 4, Method 3, and Method 5, and dissolved and diluted 4 times in 4M GdnSCN-PBS. Here, the ABC method was used to examine the plate. As a negative control group (control), 5 wells in each method were coated with an extract from 40 mg spleen tissue equivalent of uninfected mice. Average values are shown, but SD is too small to show. For the curves represented by ○ and □, the control was close to zero and the cutoff value was 0.1 U on the y-axis, which roughly corresponds to the value obtained repeatedly in the spleen tissue (Fig. 6 and FIG. 8).

According to FIG. 7 (A), it can be seen that PrP ^sc can be detected even in the same amount as that of 7.8 μg of brain tissue by the combination of Twittergent-Circosyl in the homogenization buffer (Method 1).

The sensitivity obtained with the Triton-Sarkosyl combination (Method 4) was compared with that of Method 1. Method 4 is slightly less sensitive than Method 1, but is sufficiently practical. However, in Method 3, PrP ^sc was detected only with a brain equivalent of 125 μg or more. Thus, additional extraction of PrP ^sc from proteins by Sakoshiru and NaCl, it is seen that lead to high sensitivity extraction by ELISA.

  In addition to high sensitivity, Method 1 also revealed a continued weak non-specific reaction of the comparative sample (FIG. 7 (A)). Therefore, Method 1 was considered the most suitable method for preparing brain tissue samples.

(3-2) Regarding Spleen Tissue Wells of the negative control group obtained by Method 4 exhibited a non-specific reaction showing a completely positive signal (FIG. 7B). Therefore, this method was found to be inappropriate for the preparation of spleen tissue. Establishing a sensitive cutoff line in Method 3 and Method 5 based on the negative control group was not possible due to the very weak reaction of the negative control group.

Therefore, the cut-off line was set to 0.1 U on the y-axis, taking into account the values repeatedly established for the spleen tissue, including the results of FIGS. 6 (B) and 8 (C). As a result, an effective reaction for detection of PrP ^sc similar to method 3 and method 5 was realized (FIG. 7B). However, Method 5 did not reach the sensitivity of Method 3 for a large amount of tissue equivalent, and repeated measurements showed sensitivity approximately twice that of Method 3 (data not shown). Therefore, it is necessary to examine the extraction of PrP ^sc derived protein with cercosyl extraction and NaCl.

Comparing the results of all the methods shown in FIG. 7 (B), in order to improve the sensitivity and specificity for spleen tissue, non-specific proteins were combined with a detergent containing Twittergent 3-12. It can be seen that it is an effective and necessary step to sufficiently remove or to separate the ^PrPsc- derived protein by extraction and salting out.

(4) Comparison of sensitivity between Western blot method and ELISA method As a diagnostic method for detecting PrP ^sc extracted from brain tissue or spleen tissue, the usefulness of ELISA method is that of ELISA method and Western blot (WB) method. It was confirmed by sensitivity comparison.

Comparison similar to animal diagnostics in the pre-stage of an infectious disease state, diluted with tissue homogenate from uninfected mice to make comparisons of tissue samples in which only a small amount of PrP ^sc is latent. Tissue homogenate from scrapie infected mice was processed.

  FIG. 8 shows a sensitivity comparison between the ELISA method and the Western blot method. All results are data for each measurement. The results for brain tissue are shown in FIG. 8 (A) (ELISA method) and FIG. 8 (B) (WB method), and the results for spleen tissue are shown in FIG. 8 (C) (ELISA method) and FIG. ) (WB method).

(4-0) Sample preparation for ELISA and WB methods:
Brain tissue was prepared by Method 1 and spleen tissue was prepared by Method 3. Tissue homogenates obtained from scrapie-infected mice were serially diluted 2-fold in homogenate from the 20 mg equivalent of the corresponding tissue of uninfected mice. 2 ^three times (i.e., scrapie proportion of the tissue equivalent for equivalent tissue equivalent total volume of 2.5 mg / 20 mg) shows the result of the dilution process from 2 ^11x to (9.8μg / 20mg).

  ELISA method (FIG. 8 (A), FIG. 8 (C)); for adsorption to microtiter plates, each dilution step sample consists of 20 mg of extract from tissue equivalent, 3M for brain tissue and spleen tissue, respectively. And 4M GdnSCN-PBS. In both tissues, 5 wells of a microtiter plate were coated with 20 mg equivalent of an extract from an uninfected mouse, and used as a negative control group (control) of the ELISA method. Although these are illustrated, the standard deviation (SD) is too small to be illustrated. The cut-off line corresponds to a value obtained by adding 3SD to each average value.

  WB method (FIG. 8 (B), FIG. 8 (D)); For each dilution step, loading was performed per lane using an extract collected from a tissue having a total combined amount of 20 mg. The thin film was exposed to the film for 15 hours.

(4-1) Brain tissue ELISA method, indicated that apparently positive response in the case of 2 ^8-fold dilution step results were found to be cutoff line or even 2 ^9-fold dilution steps. This means that PrP ^sc can be detected even when brain tissue from scrapie-infected mice is only 1/512 of the total homogenate amount (which corresponds to 39 μg in a total amount of 20 mg brain equivalent). (FIG. 8 (A)).

Meanwhile, WB method, after thin film is exposed for 15 hours, 2 ^8-fold dilution steps (which ratio of 1/256, i.e., corresponding to scrapie brain 78μg brain tissue in a total volume of 20 mg.) Very in Only a weak band was shown (FIG. 8 (B)).

  As a result, it can be seen that the ELISA method shows at least the same sensitivity as the WB method.

(4-2) Spleen tissue The ELISA method clearly shows a positive reaction in the case of a ^26- fold dilution step, and the spleen tissue of scrapie-infected mice is only 1/64 of the total amount (ie, Even if it corresponds to 312.5 μg in the total amount of 20 mg spleen equivalent), it shows that PrP ^sc was detected in the extract (FIG. 8 (C)).

WB method, after exposure of 15 hours, (which, 1/32 tissues of scrapie-infected mice, i.e., corresponding to 625μg of splenic tissue equivalent in the total amount of 20 mg.) 2 ⁵ times or less diluted with 24 Bands specific to PrP ^{sc of 5} kDa, 21 kDa, and 17 kDa were shown (FIG. 8 (D)).

  Therefore, it can be seen that the sensitivity of the ELISA method is equivalent to or higher than the sensitivity of the WB method also for the spleen tissue.

  Here, the tissue equivalent required to obtain positive results by ELISA for spleen tissue was only 8 times greater than that required for brain tissue.

6). Evaluation Mouse scrapie was diagnosed by WB method one week after infection (Gracewall et al., 1996), and Reis and Ernst (1992) detected de novo synthesis of PrP ^sc two weeks after infection. Sheep scrapie was also diagnosed at an early stage of infection by the WB method (Ikegami et al., 1991; Murmatsu et al., 1993).

These results are shown in PrP in sheep tonsils by histopathology (Reiss et al., 1992), electron microscopy (Lubenstein et al., 1991), or immunohistochemistry (Schruder et al., 1996). Compared to results obtained with other methods, such as a new report on ^sc preclinicals, the WB method is one of the most sensitive methods in the early stages of scrapie, and is a tedious bioassay. Can be omitted.

Also, one week after infection of the intestinal wall (peritoneum) inner surface, the detection of PrP ^sc in the spleen from the mouse is achieved by sufficient tissue uniform product, made by collagenase digestion of the homogenate (Grace Wall et al., 1996 Year). Thereby, the sensitivity of the WB method by the present inventor is at least consistent with the detection results of PrP ^sc from mouse spleen described in other reports (Lubenstein et al., 1991; Reis and Ernst, 1992). I understood that.

  The inventor applied the ELISA method, which is at least equivalent in sensitivity to the WB method and can be detected easily and in a short time, to brain tissue and spleen tissue.

A major obstacle in developing an ELISA method for scrapie diagnosis was that PrP ^sc easily aggregates into a deposited state (Meyer et al., 1986). Pretreatment of scrapie-infected tissues with formic acid or SDS (Cassack et al., 1987) and further denaturation of pure PrP ^sc with GdnSCN (this was done after adsorption to microtiter plates; Serban et al., 1990) Has been reported, which increases the immunoreactivity of anti-PrP antibodies. It was also reported that the adsorption of BSA fetal bovine serum) to microtiter plates was improved in the presence of guanidine (Zoo et al., 1993). Guanidine probably promotes the development of ^PrPsc , which in turn increases the immunoreactivity of the prion-resistant protein antibody by generating antigenic sites.

Based on these observations and considerations, the present inventors, using GdnSCN of 1M～5M (especially 3M and 4M) to dissolve the PrP ^{sc-containing} materials directly, microtiter plates in the presence of GdnSCN at this concentration Succeeded in adsorbing PrP ^sc to the surface.

  The limit of the tissue equivalent amount that can be analyzed by the ELISA method, that is, the limit value that cannot be expected to improve the sensitivity is around 40 mg (data not shown).

  The advantage of the ELISA method is that many samples can be analyzed at once, thus leading to a wide range of uses to control disease due to infection by diagnosing and selecting large numbers of potentially infected animals.

An obstacle to the method of actually diagnosing TSE (Transmissible Spongiform Encephalopathies) via detection of PrP ^sc is that the blood or its components are still difficult to use for diagnosis. Since PrP ^sc is to be extracted from brain or lymph gland tissue, sample preparation is the most time consuming factor. The method applied to the brain tissue (Method 1) is a fairly simple method and leads to higher sensitivity. However, Method 1 is not sufficient for spleen tissue. In this regard, extraction of PrP ^sc with sarkosyl and subsequent salting out with NaCl (Method 3) improves sensitivity and reduces non-specific signals.

This method is time consuming but useful for examining very small amounts of PrP ^sc . On the other hand, when extraction and salting out of PrP ^sc is omitted and sample preparation (concentration) is performed by Method 5, the sensitivity of the ELISA method is reduced by about 2 times, but it is still equivalent to the WB method (data is Not shown). From this and the fact that Method 5 can be performed in a shorter time than Method 3, Method 5 is considered practical for diagnosis.

The detection limit of PrP ^sc of 0.6 mg of spleen tissue in the WB method found in the present Example was almost equivalent to the detection limit of 0.3 mg (Gracewall et al., 1996). In this case, although the final tissue extract is diluted with SDS-PAGE sample buffer, since using conventional tissue homogenates as a diluent, it is sequentially performed diluted after the first extraction step, PrP ^sc detection condition Was not very advantageous. The extreme detection limits reported for ELISA methods should be found in view of the more difficult conditions.

When the results for brain tissue are compared with those for spleen tissue (FIGS. 6, 7, and 8), the amount of PrP ^sc in the brain tissue is considered to be 8 to 30 times the amount of PrP ^sc in the spleen tissue. Considering that the TSE for different mouse applications varies with PrP ^sc contained in spleen tissue, it is significantly less than 500 times that found in scrapie-infected mice by Rubenstein et al. (1993) and the end of infection It is less than 50 times or more discovered by Sakaguchi et al. (1993) in CJD (Creutfelt Jakob disease) -infected mice. Only Razmezas et al. (1996) recently reported about a 30-fold difference for the mouse model.

The present inventor considers that the efficient recovery of ^PrPsc- derived protein can be realized with improved sample preparation. And the amount of PrP ^sc in the mouse spleen seems to be higher than previously thought.

Paying attention to the potential of lymphoid tissue related to preliminary clinical studies, Van Couren et al. (1996) examined the amount of PrP ^sc in several of the above tissues by immunohistochemistry. According to this study, the tonsils, spleen and intestinal lymph nodes are in this order the most preferred tissues in preliminary clinical practice. The present inventor detected PrP ^sc contained in biopsy surface lymph nodes at the preclinical stage of scrapie for both experimental models (Ikegami et al., 1991) and natural sheep scrapie (Muramatsu et al., 1993). Reported that. However, considering the amount of PrP ^sc and the ease of obtaining each tissue, as proposed in Schruder et al. (1996), biopsy and examination of the tonsils are more suitable as a antemortem test. I think it is.

From the examples described above, it can be concluded that the method of using the ELISA method together with the ABC method for the detection of PrP ^sc from brain and spleen tissue is at least as sensitive as the WB method. In addition, if this ELISA method is applied to the diagnosis of sheep scrapie and cattle BSE, it seems that a more practical and faster diagnosis can be made compared to the current WB method. However, in order to make it suitable as an inspection method, it is necessary to make the labor required for extraction of PrP ^sc simpler.

The ability to easily adsorb PrP ^{sc -derived} protein contained in the raw tissue extract or GdnSCN lysate to the microtiter plate is considered to be a basic matter for enabling detection of PrP ^sc with higher sensitivity. The color development ELISA method is slightly more sensitive than the WB method, but the sensitivity can be further improved by using a reagent such as a fluorescent substance or a chemiluminescent substance.

It is a flowchart which shows the outline | summary of the concentration method of the pathogenic prion protein origin protein in a present Example. It is a flowchart which shows the outline | summary of another concentration method same as the above. It is a flowchart which shows the outline | summary of another concentration method same as the above. It is a flowchart which shows the outline | summary of the enzyme immunosorbent measuring method used for a detection of pathogenic prion protein similarly. 2 is a graph showing changes in the ability to detect pathogenic prion protein depending on the type and concentration of the solvent used in the previous stage of adsorption in the enzyme immunosorbent assay (brain tissue (A), spleen tissue (B)). 3 is a graph showing the detection ability by the color development method (brain tissue (A), spleen tissue (B)). 2 is a graph showing the detection ability of a pathogenic prion protein-derived protein concentration method (brain tissue (A), spleen tissue (B)). In the same figure, a graph showing the detectability in the enzyme immunosorbent assay (brain tissue (A), spleen tissue (C)) and a diagram showing the detectability in the WB method (brain tissue (B), spleen tissue (D)) is there.

Claims (12)

In carrying out the method of detecting pathogenic prion protein from the central nervous system tissue of animals, in the method of concentrating the protein derived from pathogenic prion protein to be detected,
Depending on the type of central nervous system tissue,
(1) n -decyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zugetgent ™ 3-10), n -dodecyl-N, N-dimethyl-3- ammonio -1-propane Sulfonate (Zwittergent ™ 3-12), n -tetradecyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zugetgent ™ 3-14) and n -hexadecyl-N, N -At least one or more surfactants selected from the group consisting of dimethyl-3- ammonio -1-propanesulfonate (Zygentent ™ 3-16);
(2) a first step of homogenizing the central nervous system by using the sub Koshiru (TM) Tona Ru boundary surface active agent,
The homogeneous product obtained in the first step, a second step of decomposing with flop protease,
And a third step of obtaining a concentrate containing a pathogenic prion protein-derived protein from the homogenized product decomposed in the second step.   The concentration method according to claim 1, wherein the central nervous system tissue is brain tissue. Wherein in the second step, using a collagenase and DNA degrading enzymes degrade the uniform product, decomposed with flop protease Furthermore, the method of concentrating claim 1. Before Kipu protease is a proteinase K, method for concentrating according to any one of claims 1 to 3.   The concentration method according to claim 1, further comprising a step of subjecting the concentrate containing the pathogenic prion protein-derived protein obtained in the third step to salting out.   The concentration method according to claim 1, further comprising a washing step of washing the concentrate containing the pathogenic prion protein-derived protein obtained in the third step with a surfactant. In carrying out the method of detecting pathogenic prion protein from the central nervous system tissue of animals, in the method of concentrating the protein derived from pathogenic prion protein to be detected,
Depending on the type of central nervous system tissue in the animal,
(1) n -decyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zugetgent ™ 3-10), n -dodecyl-N, N-dimethyl-3- ammonio -1-propane Sulfonate (Zwittergent ™ 3-12), n -tetradecyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zugetgent ™ 3-14) and n -hexadecyl-N, N -At least one or more surfactants selected from the group consisting of dimethyl-3- ammonio -1-propanesulfonate (Zygentent ™ 3-16);
(2) a first step of homogenizing the central nervous system by using the sub Koshiru (TM) Tona Ru boundary surface active agent,
The homogeneous product obtained in the first step, a second step of decomposing with flop protease,
A third step of obtaining a concentrate containing a pathogenic prion protein-derived protein from the homogenized product decomposed in the second step;
The concentrate containing the pathogenic prion protein-derived protein obtained in the third step was converted into n -decyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zugetgent ™ 3- 10) and a washing step of washing with a surfactant selected from the group consisting of n -dodecyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zugetgent ™ 3-12). Concentration method.   The concentration method according to claim 7, wherein the central nervous system tissue of the animal is brain tissue. Wherein in the second step, using a collagenase and DNA degrading enzymes degrade the uniform product, decomposed with flop protease Furthermore, the method of concentrating claim 7. A reagent kit for concentrating a pathogenic prion protein-derived protein to be detected in carrying out a method for detecting a pathogenic prion protein from an animal's central nervous system tissue,
A first step of homogenizing the central nervous system tissue using a surfactant according to the type of the central nervous system tissue of the animal;
The homogeneous product obtained in the first step, a second step of decomposing with flop protease,
A third step of obtaining a concentrate containing the pathogenic prion protein-derived protein from the homogenized product decomposed in the second step.
(A) used in the first step (1) n -decyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zwittergent ™ 3-10), n -dodecyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zwittergent ™ 3-12), n -tetradecyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zugetgent ™ 3) -14) and at least one surfactant selected from the group consisting of n -hexadecyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zygentent ™ 3-16),
(2) a combination comprising homogenizing reagent central nervous system tissue of Sa Koshiru animals containing a surfactant consisting of (R) and (b) pulp protease be used in the second step, pathogenic Concentration reagent kit for protein-derived prion protein. N washing said third step concentrates of pathogenic prion protein derived from the protein to be used in - decyl -N, N-dimethyl-3-ammonio-1-propane sulfonate (Zui' detergent (TM) 3-10) and 11. The kit according to claim 10, further comprising a surfactant selected from the group consisting of n -dodecyl-N, N-dimethyl-3- ammonio -1-propanesulfonate (Zygentent ™ 3-12). . Before Kipu protease is a proteinase K, kit of claim 10 or 11,.

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