JPH1132795A - Detection of pathogenic prion protein and concentration thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting pathogenic prion protein, capable of rapidly and easily detecting the above protein in high sensitivity even at relatively low concentrations from an animal tissue-derived matter. SOLUTION: This method for detecting pathogenic prion protein comprises the following two consecutive processes: pathogenic prion protein concentration process and enzyme immunoadsorption assay; wherein the former process comprises: 1st process: an animal tissue-derived matter is homogenized using a surfactant and an enzyme according to the kind of the above matter, 2nd process: the homogenized product is decomposed by the use of a catabolic enzyme, and 3rd process: a concentrate containing the above protein is obtained from the homogenized product decomposed in the 2nd process; while the latter process comprises: 4th process: the above concentrated is dissolved in a solvent to obtain a dissolved product, 5th process: the above protein in the dissolved product is made to adsorb to an adsorption surface, and 6th process: the protein adsorbed is subjected to color development.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting a pathogenic prion protein from a substance derived from animal tissues, and a method for detecting the pathogenic prion protein.
Concentrating the pathogenic prion protein to be detected,
The present invention relates to a method for concentrating pathogenic prion proteins.

[0002]

2. Description of the Related Art Sleepy, one of the prion diseases,
It has been known as a serious illness in Western Europe for over 200 years in sheep. In recent years, scrapie-infected sheep have been administered as undigested cattle feed in the UK to produce mad cow disease (bovine spongiform encephalopathy: BSE; Bovine Spongiform Encephal
opathy).

In addition, eating mad cow beef kuzume,
A causal relationship with a new type of human prion disease, Creutfelt Jakob Disease (CJD), has also been pointed out. In other words, it is no exaggeration to say that critical contamination is progressing with respect to lamb and its milk, beef and milk, which are important animal protein resources for humankind.

[0004] However, prion disease is different from infectious bacteria, viral diseases and the like reported so far.
Due to the fact that the pathogenic substance is a protein originally present in living organisms, that its transmission function is new, that it has a relatively long time to onset, and that it is difficult to inactivate pathogenicity,
The development of effective diagnostic and preventive methods is lagging.

[0005] The most sensitive methods for detecting pathogenic prion proteins (abnormal prion proteins) at present are as follows.
Western blotting (WB) for the detection of pathogenic prion proteins at low concentrations before disease on sheep scrapie
Western Blotting) has been developed.

[0006] However, this method is difficult to apply to cattle due to differences in accumulation sites of pathogenic substances, time-consuming implementation of this method, and the number of treated animals. That is, rapid processing is difficult.

[0007] In addition to the above-mentioned methods, at present, methods for detecting infected cattle by immunohistochemical staining and pathological findings using antibodies against pathogenic prion protein are widely practiced.

However, these methods are effective for livestock that exhibit significant neurological symptoms or die after the onset of the disease, and the safety of livestock during the incubation period, in other words, up to the slaughterhouse, The safety of normal cows could not be ensured.

Recently, an enzyme-linked immunosorbent assay (ELISA) has been used from overseas.
There are also reports of diagnostic methods using urine and blood, but their sensitivity and specificity were questionable.

That is, if the pathogenic prion protein contained in the target pathogenic substance is concentrated at the stage of preparing the measurement sample and efficiently adsorbed to a microtiter plate for ELISA, the detection sensitivity by this method can be improved. Can be improved, but the conventional methods have limitations in detection sensitivity.

[0011]

SUMMARY OF THE INVENTION Bovine spongiform encephalopathy (BS)
E: Welsh et al., 1987) occur when cattle feed contains sheep offal contaminated with scraps of meat and bone marrow given to the diet, resulting in newly infected cattle stock. Was clearly recirculated (Virmis et al., 1993).

[0012] Subsequently, in the UK, several captive ungulates (Wyatt et al., 1991), as well as cats, have once again developed spongiform encephalopathy. In all these cases, the BSE is considered to have originated through the contaminated feed.

[0013] A report by Wil et al. (1996) on the specific case of Creutfeld-Jakob disease (CIJ) in the United Kingdom shows that human variants in the group with infectious spongiform encephalopathy (TSE) or prion disease have been identified. One has been reported, but it suggests that BSE may be transmitted to humans (Wyl et al., 1996). For this reason, it is generally considered possible for all TSEs to occur across species (Dillinger, 1995).

[0014] The current top priority for research is to develop methods to detect animals and materials infected with scrapie or BSE and to prevent the spread of infection and entry into the food chain system.

Unfortunately, to date, these preventive measures and control measures, as well as programs to eradicate scrapie and BSE, have not been successful due to difficulties in diagnosis.

The most common diagnostic methods currently used include the histopathological method of diagnosing infection when typical sponge changes in the central nervous system are remarkable (Fraiser, 1976). Partially resistant to the proteinase K treatment method (Bolton et al., 1982; Dillinger et al., 1983) and cannot be extracted with neutral detergents (Meyer et al., 1986; Bolton et al., 1).
Has the characteristics referred to 987) are two scrapie special isoform (PrP ^SC) method for detecting a prion protein can be distinguished from normal prion protein (PrP ^C) in order.

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

On the other hand, histopathology is caused by pathological changes in the central nervous system during the incubation period,
When compared to the PrP ^SC detection method, both experimental scrapie and BSE (Bolton et al., 1991; Gendroska et al., 1991) have reduced their usefulness.

Recently, the importance of prion disease has been increasing, and a more sensitive diagnostic method for screening sheep and cattle at the time of slaughter has been demanded.

Although the ELISA method can be said to be a suitable method for selection, at present, this method is used only in basic research on TSE (Kaskzak et al., 1).
987, Safar et al., 1990; Serban et al., 19
90 years). In these studies, only high-purity PrP ^SC was adsorbed on microtiter plates, but diagnosis also requires the use of original tissue extracts.

As described above, among prion diseases, one of the greatest threats to humans is bovine spongiform encephalopathy (BSE).

This disease is triggered by exogenous pathogenic prion protein, accumulates the pathogenic prion protein in the central nervous system etc. of the livestock, causes neurological symptoms and leads to death. The accumulation level of the prion protein is significantly proportional to the progress of the disease.

For this reason, since the accumulated concentration of the pathogenic substance is low during the incubation period after the disease, a specific and highly sensitive detection method is required. Also, considering the number of cattle treated worldwide, it is not necessary that the method for preparing the measurement sample (that is, the enrichment method) and the detection method be simple, accurate, rapid, economical, and the like. Needless to say.

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

The present invention has been made in view of the above-mentioned conventional circumstances, and has as its object to quickly and simply obtain a relatively low concentration of a substance derived from animal tissue, and to obtain a highly sensitive tissue-specific pathogen. An object of the present invention is to provide a method for detecting a pathogenic prion protein capable of detecting a pathogenic prion protein, and a method for concentrating the pathogenic prion protein to be detected when the detection method is carried out.

[0026]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, in a method for detecting a pathogenic prion protein from a substance derived from animal tissue, a method for detecting a pathogenic prion protein is described. The pathogenic prion protein can be rapidly and easily prepared at a relatively low concentration by appropriately selecting a preparation agent (particularly a surfactant), a preparation method, and a detection method according to the type of the target substance derived from animal tissue. And with high sensitivity.

That is, the present invention relates to a method for detecting a pathogenic prion protein from an animal tissue-derived substance, wherein the method uses a surfactant and an enzyme corresponding to the type of the animal tissue-derived substance. A first step of homogenizing the animal tissue-derived substance, and a second step of decomposing the homogenized product obtained in the first step using a decomposing enzyme;
A step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product degraded in the second step, and a step of concentrating the pathogenic prion protein,
A fourth step of dissolving the concentrate containing the pathogenic prion protein in a solvent to obtain a dissolved substance of the concentrate, and a fifth step of adsorbing the pathogenic prion protein in the dissolved substance on an adsorption surface And detecting the pathogenic prion protein by an enzyme-linked immunosorbent assay having a sixth step of coloring the pathogenic prion protein adsorbed in the fifth step. (Hereinafter, referred to as the detection method of the present invention).

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

Further, since this is detected based on an enzyme-linked immunosorbent assay (ELISA; hereinafter the same) having the fourth to sixth steps, the pathogenic prion protein is specifically and It can be firmly adsorbed (immobilized), and can be detected quickly and easily with high sensitivity.

That is, according to the detection method of the present invention, for example, cows and sheep can be diagnosed and sorted at an early stage of prion disease (scrapie or BSE) infection.
This can be done in large quantities and quickly. In particular, a biopsy using lymph nodes is possible in sheep. According to the present invention, it is considered that a biopsy using lymph nodes can be performed on, for example, cattle.

The present invention also relates to a method for concentrating the pathogenic prion protein to be detected when the method for detecting a pathogenic prion protein is carried out for detecting the pathogenic prion protein from an animal tissue-derived substance. N-dodecyl-N, N-dimethyl- according to the type of the source substance
A first step of homogenizing the animal tissue-derived substance using 3-amino-1-propanesulfonate or t-octylphenoxypolyethoxyethanol and an enzyme, and decomposing the homogenized product obtained in the first step A second step of decomposing using an enzyme; and a third step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product decomposed in the second step. And a method for enriching a pathogenic prion protein (hereinafter, referred to as a first enrichment method of the present invention).

Further, the present invention relates to a method for concentrating the pathogenic prion protein to be detected when the method for detecting a pathogenic prion protein is carried out for detecting the pathogenic prion protein from a substance derived from animal tissue. A first step of homogenizing the animal tissue-derived substance using a surfactant and an enzyme corresponding to the type of the derived substance,
A second step of decomposing the homogenized product obtained in the step (b) using a decomposing enzyme; and a third step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product decomposed in the second step. And the concentrate containing the pathogenic prion protein obtained in the third step and N-dodecyl-
A washing step of washing with a nonionic surfactant comprising N, N-dimethyl-3-amino-1-propanesulfonate, wherein the method comprises the steps of: 2).
Is also provided.

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

Here, the animal tissue-derived substance refers to the animal's central nervous system tissue, reticulum lymphoid tissue and bone, and substances derived from these tissues (for example, food, dura for transplantation, medical treatment, etc.). For collagen) etc.
Similar). Further, the pathogenic prion protein means an abnormal prion protein which is considered to be a cause of prion disease, and examples of the prion disease include the above-mentioned CJD, scrapie, BSE and the like. The detection method of the present invention, the first enrichment method of the present invention, and the second enrichment method of the present invention are not limited to sheep scrapie and BSE, and can deal with various prion diseases.

[0035]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, a detection method according to the present invention will be described.

The first step in the detection method of the present invention includes a homogenizing step of homogenizing the animal tissue-derived substance using a surfactant and an enzyme corresponding to the type of the animal tissue-derived substance. Therefore, the action of the enzyme sufficiently dissolves the animal tissue-derived substance, and also solubilizes a non-specific substance in the presence of the surfactant according to the type, and contains a pathogenic prion protein. The above-mentioned substance derived from animal tissue can be sufficiently homogenized.

Therefore, even if the ratio of the pathogenic prion protein in the animal tissue-derived substance is relatively low,
This can be satisfactorily homogenized, so that a good pathogenic prion protein concentrate can be obtained. That is, it is possible to obtain a homogenate (homogenate) effective for separating and extracting the pathogenic prion protein.

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 replaced with Zwittergent 3-12 Name: N-Dodecyl-N, N-dimethyl-3-amino-1 manufactured by Calbio Chemical Co.
-Propane sulfonate (N-dodecyl-N, N-dimethyl-3-a
mino-1-propanesulfonate): molecular weight 336.6] or Triton X-100 [trade name: manufactured by Sigma: t-octylphenoxypolyethoxyethanol (t-octylpheno)]
xypolyethoxyethanol)]. In addition to the above surfactants, for example, Zwittergent 3-08, 3-10, 3-14, 3-16 manufactured by Calbio Chemical Co., or Nonidet P-40 (octylphenoxypolyethoxyethanol) may be used. .

By using each of the above-mentioned 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, circosyl 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)]
loss less of rP ^SC, sufficiently non-specifically can be extracted proteins, in such contrast lymphoid, spleen tissue, may remove nonspecific contaminating proteins is insufficient, again high concentration of It is considered that it is desirable to selectively extract PrP ^SC with the circosyl.

When the substance derived from animal tissue is a reticulo-lymphoid tissue (for example, spleen, lymph node, bone marrow, etc.), the surfactant is a nonionic surfactant comprising t-octylphenoxypolyethoxyethanol. It is desirable to use an agent.

By using the above surfactant,
Non-specific substances in the spleen tissue can be sufficiently solubilized. In particular, it is more desirable to use spleen tissue as the reticulolymphoid tissue.

Next, since the second step includes a decomposition treatment step of decomposing the homogenized product obtained in the first step using a decomposing enzyme, the pathogenicity in the homogenized product is reduced. Substances containing prion proteins (particularly chromosomes and DNA
) Can be sufficiently decomposed and digested to sufficiently remove the target pathogenic prion protein.

In general, it is considered that the pathogenic prion protein is on a gene in a chromosome. Therefore,
In order to specifically extract this protein, it is necessary to degrade the protein containing it. This second step is an operation for decomposing non-specific substances and decomposing proteins including pathogenic prion proteins.

Here, the homogenized product is decomposed by using a collagenase (collagenase) and a DNAse (DNase: DNase) as the above-mentioned protease, and further, a protease (proteinase: Proteinase) is used.
It is desirable to decompose using nase or protease (Protease).

Further, papain (Papain) is used as the degrading enzyme.
in: SH enzyme) and Bromelain (Bromelain: SH enzyme)
The homogenized product can also be decomposed by using a plant protease (Protease) or a microbial protease.

In particular, bromelin is an enzyme derived from pineapple and mainly acts on proteins, but can also decompose fat. In addition, it can be said that the enzyme has a high stability such that the enzyme temperature is not lost up to about 80 ° C. PrP
^The use of a protease such as bromelin for ^SC detection was first discovered by the present inventors.

When bromelin or the like is used as the degrading enzyme, the degradation of PrP ^SC is at the same level (tolerance) as that of proteinase K at a concentration of 0.08 to 20 Unit / ml and a reaction temperature of about 45 ° C. It is.

Further, proteins and nucleic acids other than PrP ^SC can be sufficiently degraded. Normal protein is 45 ° C
It can be said that bromerin and the like are easily degraded by heat to the extent that bromerin and the like are heat-resistant. (PrP ^SC has a heat resistance of 100 ° C. or more and is therefore subject to thermal decomposition.) Absent).

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

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

The above is the basic configuration of the step of enriching the pathogenic prion protein. In the enrichment step of the detection method of the present invention, for example, in addition to the first to third steps described above, for example, It is desirable to add the following steps.

For example, during the concentration step, a step of decomposing the concentrate containing the pathogenic prion protein obtained in the third step using a decomposing enzyme, and then subjecting the concentrate to a salting-out treatment after separation is further performed. It is desirable to have.

That is, in order to further improve the solubility of the pathogenic prion protein, for example, Sarkosyl (Sarkos
yl, trade name: manufactured by Sigma: C ₁₅ H ₂₅ NO ₃ Na), and then subjected to dissolution treatment and separation treatment using a degrading enzyme, and salting out the obtained separated extract using, for example, NaCl. By performing the separation treatment, a concentrate containing a more concentrated pathogenic prion protein can be obtained.

Preferably, the method further comprises, during the concentration step, a washing step of washing the concentrate containing the pathogenic prion protein obtained in the third step with a surfactant.

That is, as an additional washing step of the concentrate (eg, pellets), by washing the concentrate with a surfactant, an undesired substance (a non-specific substance) in the concentrate can be obtained. ) Can be further removed.

Here, the surfactant used is N
-Dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate (for example, Zittergent 3-12, 3
-08,3-10) are preferred.

Next, the enzyme immunosorbent assay (fourth to sixth steps) based on the detection method of the present invention will be described.

Here, the enzyme immunosorbent assay (ELI)
SA (enzyme-linked immnosorbentassay), also called an enzyme-antibody method, is a method of arranging an antigen on a specific adsorption surface, adsorbing the antigen and the antibody, forming a complex thereof, and detecting this.

First, the fourth step includes a step of dissolving the concentrate containing the pathogenic prion protein in a solvent to obtain a dissolved product of the concentrate (dissolution step). In the adsorption step, a concentrate of the pathogenic prion protein that is easily adsorbed on the adsorption surface can be prepared.

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

Guanidine thiocyanate is considered to have an effect of facilitating the adsorption of the concentrate in the subsequent adsorption step. This is considered to be due to the expression of an antigenic site that increases the immunoreactivity of the anti-prion protein antibody by guanidine thiocyanate,
By lysis treatment with guanidine thiocyanate, a strong and specific reaction of the antigen-antibody complex can be detected.

The guanidine thiocyanate is 1 to 5
It is desirable to use one having a molar concentration (M). This concentration is more preferably 3 to 4 molar. The dissolution of the concentrate in the guanidine thiocyanate described above is desirably performed using a phosphate buffered saline (PBS; pH ≦ 5) or the like as a buffer.

Next, the fifth step includes a step of adsorbing the pathogenic prion protein in the lysate on an adsorption surface (adsorption step). The sex prion protein can be adsorbed on, for example, a microtiter plate. Therefore, a strong and specific reaction for the formation of an antigen-antibody complex can be detected by this method.

Further, the adsorption surface can be formed with a primary antibody, and an antigen-antibody complex of the primary antibody and the pathogenic prion protein can be formed on the adsorption surface. Generally, in the ELISA method, an antigen-antibody complex is formed between an antigen to be measured and an antibody for immobilizing the antigen, and this is detected by a colorimetric method.

Next, as the sixth step, a step of coloring the pathogenic prion protein adsorbed in the fifth step (color developing step) is provided, which is performed by a colorimeter or a spectrophotometer. It can be easily detected by a meter. That is,
The presence or absence of the pathogenic prion protein and the accumulated concentration of the pathogenic prion protein can be examined by coloring the pathogenic prion protein and detecting the coloring concentration.

It is preferable that the color is fluorescent light emission. Furthermore, it is desirable to use a chemiluminescent substance as the fluorescent luminescent agent. For example, as the chemiluminescent substance (coloring reagent), 2,2-azizo-bis (3-
Ethyl-benzthiazoline-6-sulfonate) and the like can be used.

As a method for color development, an avidin-biotin complex for observing color formation based on the formation of a complex of avidin (avidin) binding to the pathogenic prion protein and biotin (biotin) binding to the chemiluminescent substance is used. Body method (ABC method) and horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin (horseradish peroxidase-c)
An indirect method (HRP method) using onjugated donky anti-rabbit IgG) can be used.

The outline of this detection method is, for example, that a secondary antibody (anti-rabbit IgG) -biotin complex in which PrP ^SC adsorbed on the well specifically binds to the polyclonal antibody B103 and recognizes the antibody more specifically. And then bind adivine, then bind horseradish bevel oxidase (HRP) -biotin, and react with 2,2-azizo-bis, which is a substrate of HRP, to develop color.

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

As described above, according to the enzyme-linked immunosorbent assay (ELISA) of the present invention, even if the pathogenic prion protein to be measured has a relatively low concentration, it is strongly and specifically adsorbed. Thus, the pathogenic prion protein can be detected quickly and easily.

The detection by the ELISA method shows at least the same sensitivity as the Western blotting method (WB method) described above, and the measurement is practical and quick. The advantage of ELISA detection is that many samples can be analyzed at one time, potentially infected animals can be diagnosed and sorted in large quantities, and prion diseases (especially BSE) due to infection can be detected. It is possible to use the widespread use of controlling for various purposes.

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

According to the first enrichment method of the present invention, the pathogenic prion protein to be detected is enriched when the method for detecting a pathogenic prion protein is performed for detecting a pathogenic prion protein from a substance derived from animal tissue. In the method,
As the first step, N-dodecyl-N, N-dimethyl-3-amino- according to the type of the animal tissue-derived substance is used.
Since it has a homogenization step of homogenizing the animal tissue-derived material using 1-propanesulfonate or t-octylphenoxypolyethoxyethanol and an enzyme, the animal tissue-derived material can be sufficiently reduced by the action of the enzyme. Dissolve,
Further, in the presence of a nonionic surfactant consisting of N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate or t-octylphenoxypolyethoxyethanol as a surfactant according to the type. The non-specific substance can be solubilized, and the animal tissue-derived substance containing the pathogenic prion protein can be sufficiently homogenized.

Therefore, even if the ratio of the pathogenic prion protein in the animal tissue-derived substance is relatively low,
This can be satisfactorily homogenized, so that a good pathogenic prion protein concentrate can be obtained. That is, it is possible to obtain a homogenate (homogenate) effective for separating and extracting the pathogenic prion protein.

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.

Further, since the second step includes 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 degraded. (Particularly, chromosomes) can be sufficiently decomposed and digested to sufficiently remove the target pathogenic prion protein.

It is desirable to decompose the homogenized product using a collagenase and a DNAse, and further decompose using a protease.

The homogenized product can be decomposed by using the above-mentioned plant protease such as papain or bromelin and / or a microbial protease as the decomposing enzyme.

Next, as a third step, there is a separation step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product degraded in the second step.
The substance containing the pathogenic prion protein sufficiently homogenized and degraded in the first and second steps can be efficiently separated.

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

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

For example, during the concentration step, a step of decomposing the concentrate containing the pathogenic prion protein obtained in the third step using a degrading enzyme, and then subjecting the concentrate to a salting-out treatment after separation is further provided. It is desirable to have.

That is, in order to further improve the solubility of the pathogenic prion protein, dissolution treatment and separation treatment are performed using a degrading enzyme such as circosyl, and the obtained separated extract is, for example, NaCl or the like. After performing salting-out using, a concentrate (for example, a pellet) containing a more concentrated pathogenic prion protein by performing a separation treatment
Can be obtained.

It is preferable that the concentrating step further includes a washing step of washing the concentrate containing the pathogenic prion protein obtained in the third step with a surfactant.

That is, as an additional washing step of the concentrate (eg, pellets), by washing the concentrate with the surfactant, undesired substances (non-specific Substance) can be removed even more. As the surfactant, N-dodecyl-N, N
-Dimethyl-3-amino-1-propanesulfonate (for example, the above-mentioned Zwittergent 3-12, 3-08, 3-10
And the like).

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

According to the second enrichment method of the present invention, the virulence prion protein to be detected is enriched when the pathogenic prion protein detection method for detecting the pathogenic prion protein from animal tissue-derived substances is performed. In the method,
As a first step, the method includes a homogenization step of homogenizing the animal tissue-derived substance using a surfactant and an enzyme corresponding to the type of the animal tissue-derived substance. The animal tissue-derived substance is sufficiently dissolved, and the non-specific substance is solubilized in the presence of the surfactant according to the type thereof, and the animal tissue-derived substance containing the pathogenic prion protein is sufficiently homogenized. Can be

Therefore, even if the ratio of the pathogenic prion protein in the animal tissue-derived substance is relatively low,
This can be satisfactorily homogenized, so that a good pathogenic prion protein concentrate can be obtained. That is, a homogenized product effective for separating and extracting the pathogenic prion protein can be obtained.

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

Next, as a second step, a decomposition treatment step of decomposing the homogenized product obtained in the first step using a degrading enzyme is included, so that the pathogenic prion in the homogenized product is degraded. A substance containing protein (particularly, chromosome) can be sufficiently decomposed and digested to sufficiently extract the target pathogenic prion protein.

Here, it is desirable to decompose the homogenized product using collagenase and DNA degrading enzyme as the decomposing enzyme, and further decompose using a protease.

The homogenized product can be decomposed by using the above-mentioned plant protease such as papain or bromelin and / or a microbial protease as the decomposing enzyme.

Next, as a third step, there is provided a separation step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product decomposed in the second step.
The substance containing the pathogenic prion protein sufficiently homogenized and degraded in the first and second steps can be efficiently separated.

In this separation step, for example, separation and concentration can be carried out by means such as centrifugation (ultracentrifugation).

Next, as a washing step, the concentrate containing the pathogenic prion protein obtained in the third step is made of N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate. The method further comprises a washing step of washing with a nonionic surfactant, so that unwanted substances (non-specific substances; for example, normal prion protein and other proteins) in the concentrate are further removed. can do.

According to the first and second concentration methods of the present invention described above, even if the accumulation concentration of the pathogenic prion protein accumulated in the animal tissue-derived substance is relatively small, it can be sufficiently reduced. Can be concentrated. In addition, the concentrate containing the pathogenic prion protein obtained by each of the above-mentioned enrichment 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.

[0098]

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

In this example, detection of PrP ^sc (pathogenic prion protein: the same applies hereinafter) of PrP ^sc in crude tissue extract from scrapie-infected mice, and Pr to be detected
The concentration of P ^sc is performed.

1. In this example, SIc / ICR mice were used. These mice were infected with Obihiro-species scrapie in the brain (Shinagawa et al., 1985), and brain and spleen tissues were removed from mice that clearly developed scrapie.

2. Enrichment of PrP ^sc from brain and spleen tissues Samples were prepared by eight different enrichment methods as shown in FIGS. All eight methods involved digesting a minced tissue sample with scissors (second step) and removing soluble non-specific material in the presence of a non-ionic detergent. Extract and centrifuge (third step)
This is common in performing the equalization for the purpose of performing (first step).

Hereinafter, the method for concentrating PrP ^sc will be described with reference to Method 1.
Method 8 will be briefly described with reference to FIGS.

Method 1 First, 8% Zwittergent 3-12 (nonionic surfactant), sarkosyl (enzyme), 100 mM sodium chloride (NaCl), 5 mM magnesium chloride (MgCl), 50 mM
M, pH = 7.5 Tris-HCl buffer (Tris-HCl) was added to homogenize the brain tissue and homogenate (homogenate)
Was prepared (first step).

Next, 0.5 mg / 100 mg of collagenase [3,4,24,3] (Collagenase) and
0 μg / 100 mg of DNase [3, 1, 21, 1] (DNas
e) is subjected to a decomposition (digestion) treatment at a temperature of 37 ° C. for 4 to 12 hours, and further using 50 μg / 100 mg of proteinase [3,4,21,14] (Proteinase K).
Decomposition (digestion) was performed at 37 ° C for 0.5 to 2 hours (No. 2).
Process). This decomposition (digestion) treatment is performed for the enzymatic decomposition of the measurement contaminants other than the pathogenic prion protein in the tissue.

Next, after stopping the reaction, the number of rotation was 15,000.
Centrifugation was performed at rpm and room temperature for 20 minutes. This is 5% SDS
(Sodium dodecyl sulfate) was heated and dissolved for 10 minutes (third step) to obtain a concentrate containing PrP ^sc (FIG. 1).

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

Next, after stopping the reaction, the number of rotation was 15,000.
Centrifugation was performed at rpm and room temperature for 20 minutes. This is 5% SDS
(Sodium dodecyl sulfate) was heated and dissolved for 10 minutes (third step) to obtain a concentrate containing PrP ^sc (FIG. 1).

When bromelin is used, the working temperature is preferably 45 to 80 ° C. and the working time is preferably about 1 to 10 hours.
As a result of using bromelin, only one kind of enzyme is used, and the operation is simple (reduction of operation time and cost reduction)
become. Note that the measurement results were the same (same) in sensitivity as in the case of using three types of enzymes (collagenase, DN anase, and proteinase).

Method 3 5-8 times the tissue weight of 4% Triton X-100 (a nonionic surfactant), 0.5% Sarkosyl, 100 mM sodium chloride (NaCl), and 5 mM magnesium chloride (MgCl)
And 50 mM Tris-HCl buffer at pH = 7.5 were added to homogenize the spleen tissue and brain tissue to prepare a homogenized product (first step).

Next, the same decomposition (digestion) treatment as in method 1 was performed (second step). Further, the same separation treatment as in method 1 was performed (third step).

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

Next, this was sonicated, and then centrifuged at 15,000 rpm. HCl was added to the supernatant of the solution obtained by centrifugation at a final concentration of 12%, followed by stirring (salting out). Process). Thereafter, the mixture was ultracentrifuged at a rotation speed of 55,000 rpm, and dissolved by heating using 5% SDS to obtain a PrP ^sc- containing concentrate (FIG. 2).

Method 4 Up to the method 3 and the separation treatment (third step),
After separating the spleen tissue and brain tissue, the obtained precipitate (pellet) was dissolved by heating using 5% SDS,
A concentrate containing rP ^sc was obtained (FIG. 2).

Method 5 Up to the method 3 and the separation treatment (third step),
After the separation of the above spleen tissue, the obtained precipitate (pellet) was mixed with 8% Zwittergent 3-12 (nonionic surfactant), 10 mM Tris-HCl buffer (pH = 7.5). Was added to obtain a uniform product (first step).

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

Method 6 In method 3, in the decomposition treatment step (second step),
Instead of performing the decomposition (digestion) treatment using collagenase, DNase, and proteinase, enzymatic degradation of measurement contaminants other than pathogenic prion protein in tissues is performed using bromelain similar to that in Method 2. A PrP ^sc- containing concentrate was obtained in the same manner as in Method 3 except that the above procedure was performed (FIG. 3).

Method 7 In the method 4, in the decomposition treatment step (second step),
Instead of performing the decomposition (digestion) treatment using collagenase and DNase, and further using proteinase, the same bromelin as in Method 2 was used to enzymatically decompose the contaminants other than the pathogenic prion protein in the tissue. In the same manner as in Method 4, a concentrate containing PrP ^sc was obtained (FIG. 3).

Method 8 In method 5, in the decomposition treatment step (second step),
Instead of performing the decomposition (digestion) treatment using collagenase and DNase, and further using proteinase, the same bromelin as in Method 2 was used to enzymatically decompose the contaminants other than the pathogenic prion protein in the tissue. In the same manner as in Method 5, a concentrate containing PrP ^sc was obtained (FIG. 3).

That is, in methods 3, 4 and 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) of Zwittergent 3-12 is used instead of the Triton X-100. Was added.

[0120] Zittergent 3-12 used in Method 5
Centrifugation in the third step (15,000 rpm) to remove more undesired materials (non-specific substances)
(TLA 100.3 rotor, Optima TLX desktop ultracentrifuge, manufactured by Beckman) was used as an additional washing step of the pellets.

[0121] In method 3, for increasing the solubility of PrP ^sc, the PrP ^sc 6.25% by (wt / vol) Sakoshiru p
Extracted at H = 9.2, which is higher than the conventionally used pH. After the circosyl extraction, the pH was returned to neutral with 10% (vol / vol) HCl to reduce the solubility of PrP ^sc again, and then Pr with 12% (wt / vol) NaCl.
^{Salting out} of P ^sc and final centrifugation at 55,000 rpm were performed to obtain a more concentrated pellet containing PrP ^sc . This circosyl extraction and salting out of PrP ^sc with NaCl was omitted in Methods 4, 5 and 1 to simplify the sample preparation process.

3. Western blot analysis The brain and spleen of mice infected with scrapie were used as samples. The brain was prepared by Method 1 and the spleen 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. For blocking, 5% skim milk was used, and for detection, a B103 polyclonal antibody was used. ELC-Western blot detection sys
tem (Amersham).

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

Next, after the centrifugation treatment (3), the obtained pellet-like precipitate was subjected to 100 μl of different concentrations (1 to 5 M) of guanidine thiocyanate (guanidine thiocyanate: GdnSCN), PBS (phosphate buffered saline). : Last p
Ultrasonic dissolution was performed in H ≦ 5) (4). The steps (1) to (4) so far correspond to the above-described fourth step.

Further, as shown in FIG.
In order to perform the measurement with the use of GdnSCN, pellets were prepared at 100 μl different concentrations (0.1-4% (vol / v
ol)) in SDS, PBS (final pH ≦ 5).

Next, each solution was added to a 96-well round bottom maxisorp immunoplate (trade name: Maxisorp immunoplatform).
e: Nunc) and cultured under shaking at room temperature overnight (5) (for example, Corning ELISA plate high binding type 430452 may be used).

Next, the plate was washed three times with PBS (6) and blocked (closed) at 37 ° C. for 1 hour in PBS-5% skim milk (7).

Next, the plate was washed three times with PBS containing 0.05% Tween 20 (hereinafter referred to as PBST) (8).

Next, rabbit antiserum B-103 (Horiuchi et al.,
1995) as the primary antibody, using PBST-0.5 after precipitation with 33% ammonium sulfate.
% After diluting 2,000 times in skim milk, the wells (the above holes:
The same applies hereinafter). The plate was incubated at room temperature with shaking for 1 hour to form an antigen-antibody reaction complex (9). Here, the steps (5) to (5)
(9) corresponds to a fifth step.

After the plate was washed three times with PBST,
Avidin-Biotin complex (ABC)
When the antigen-antibody complex was visualized by the method as described below, it became visible. However, ABC
VectorStain Elite ABC as a kit for law
Kit, Vector Laboratory (Vectastain Elite ABC ki
t, Vector Laboratories).

Biotin (vitamin H) -modified anti-rabbit immunoglobulin (anti-rabbit IgG) was diluted 1,500-fold in PBST-0.5% skim milk, and the wells were cultured at room temperature for 1 hour with shaking at room temperature. (10).

Next, the plate was washed four times with PBST and once with PBS (11). Then, the components A (avidin) and B (biotin) of the kit were each diluted with PBS at a 200-fold dilution rate.
And distributed in the wells (12). here,
The kit includes a set of tools and preparations for performing the above-mentioned ABC method.

[0133] Culture and washing were then performed on the biotinylated antibody (ie, adsorbed antibody) (1
3).

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

Then, a microplate reader [Mode
l 2550, Bio-Red Laboratorie
s)], color development at a wavelength of 405 nm was confirmed (15).

Instead of the color development method based on the ABC method, horseradish peroxidase conjugated donkey anti-rabbit IgG (ho
rse radish peroxidase-conjugated donky annti-rabbi
Wells were cultured in PBST-0.5% skim milk at a dilution of 800 times with 100 μl of t IgG (manufactured by Amersham) (16). Thereafter, the plate was washed four times with PBST and once with PBS (1.
7). Hereinafter, this method is referred to as an indirect method.

In addition, culturing 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 a tissue extract of an uninfected mouse.

[0138] 5. Measurement Results (1) Determination of Appropriate Coating Conditions In order to achieve a high adsorption rate to a microtiter plate, it is necessary to sufficiently dissolve PrP ^sc . Here, the usefulness of GdnSCN and SDS for dissolving PrP ^sc was evaluated (FIG. 5).

In FIG. 5, samples from the brain tissue (A) and the spleen tissue (B) of the scrapie-infected mouse were used to examine the effects of GdnSCN and SDS on the adsorption of PrP ^sc to the microtiter plate. 5 respectively. At each concentration of each SDS and GdnSCN, extracts were taken from brain (A) and spleen (B) equivalents of 1 mg and coated on three wells per microtiter plate.

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

FIG. 5 shows the average value and its standard deviation (S.
D.). The cut-off line was determined by adding three standard deviation values (3 SD: hereinafter the same) to each average value. The indirect method and ABC method were applied to brain tissue and spleen tissue, respectively. The chaotropic ionic agent increases the water solubility of hydrophobic molecules, weakens hydrophobic bonds, and is used for extracting membrane proteins and the like, and examples include GdnSCN.

Dissolution of PrP ^sc in PBS, without chaotropic agents or detergents, causes some adsorption to microtiter plates in brain tissue but not in spleen tissue. None (Fig. 5 (A), (B): GdnSCN of OM). Even when the SDS concentration was the lowest (0.1%), no PrP ^sc was adsorbed from brain tissue (FIG. 5 (A)).

On the other hand, while increasing the PBS concentration, G
Addition of dnSCN to PBS improved coating efficiency and produced a peak for spleen tissue at the GdnSCN concentration of 4M (FIG. 4 (B)). G near 3M
At the dnSCN concentration, the brain tissue showed a peak (FIG. 4 (A)). That is, 1 to 5 M (particularly 3 to 4 M)
Strong specific adsorption was observed when using GdnSCN of M).

The higher the GdnSCN concentration in PBS, the higher the pH
As the values decrease (≦ 5), both GdnSCN and SDS were diluted by quantitatively replacing 0.05 M sodium bicarbonate carbonate buffer (pH = 9.6) with PBS, thereby bringing the final pH to 8 Increased above. However, the adsorption rates tended to decrease (data not shown).

(2) Two different detection methods: indirect and
Comparison of ABC method The indirect method and the ABC method were compared for sensitivity and specificity.
FIG. 6 shows a comparison between the ABC method and the indirect method for detecting an antigen-antibody complex.

A sample (A) of brain tissue of a scrapie-infected mouse was prepared according to Method 1, followed by 3M GdnSCN
The solution was serially diluted two-fold. For spleen tissue (B),
Method 3 was applied and 4M GdnSCN was used. For the two measurement methods, the average value of the two detection methods was obtained. The negative control group (control) is also shown. The standard deviation (SD) is too small to show. The cutoff line was determined by adding 3 SD to each. The cut-off line in the measurement of spleen tissue was determined based on the data of a negative control group consisting of 40 mg of spleen equivalent (that is, an amount corresponding to 40 mg of spleen; hereinafter the same).

According to the measurement results, the brain tissue
The ABC method is slightly better (Fig. 5 (A)).
The method was found to be clearly preferred for spleen tissue (FIG. 5 (B)). For spleen tissue, the ABC method was at least twice as sensitive as the indirect method. Also, 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 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 sample preparation method for a known Western blot method (Gracewall et al., 1996). Here, circosyl extraction and PrP ^{sc with} NaCl
The salting out of the methods 4, 5, and 1 was omitted.

Then, instead of the separation of PrP ^sc by extraction, PrP ^c (normal prion protein) and other proteins were sufficiently removed from the sample using 8% (wt / vol) of Zwittergent 3-12. . Said Twittergent 3-12
Was found to be a more effective detergent than Triton X-100. Therefore, the former was used in the homogenization buffer of Method 1 instead of Triton X-100.

For spleen tissue, digestion with collagenase was performed using 4% (wt / vol) Triton X-100 and 0.5% (wt / vol) Sarkosyl (methods 3, 4 and 5). Then, in method 5, in order to remove non-specific substances, the pellet (concentrate) obtained under the centrifugal force of 40,000 rpm was subjected to 8% (wt / vol) of Zwittergent 3-1.
Further extraction with 2 and 0.5% (wt / vol) Sarkosyl.

(3-1) About Brain Tissue FIG. 7 shows an appropriate sample preparation method for brain tissue and spleen tissue. 7 (A) and 7 (B) are the results of repeated measurements.

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

The spleen tissue extract was prepared (concentrated) by Method 4, Method 3, or Method 5, dissolved and diluted 4-fold in 4M GdnSCN-PBS. Here, ABC to check the plate
Method was used. As a negative control group (control), 5 wells were used for each method, and 40 wells of uninfected mice were used.
g of the extract from spleen tissue equivalent.
Mean values are shown but SD is too small to show. ○
And for the curve represented by □, the control is close to zero,
The cut-off value was 0.1 U on the y-axis, which roughly corresponds to the value obtained repeatedly in spleen tissue (FIGS. 6 and 8).

According to FIG. 7 (A), the combination of Zwittergent-Sarcosyl in the homogenization buffer (method 1) resulted in Pr at the same amount as 7.8 μg of brain tissue.
It can be seen that the detection of P ^sc was enabled.

The sensitivity obtained with the Triton-Sarcosyl combination (Method 4) was compared with that of Method 1. Method 4 is less practical than Method 1, but is sufficiently practical. However, in method 3, PrP ^sc was detected only in brain equivalents of 125 μg or more. Thus, additional extraction of PrP ^sc with sarkosyl and NaCl results in EL
It can be seen that high-sensitivity extraction by the ISA method is not achieved.

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

(3-2) Spleen Tissue The well of the negative control group obtained by Method 4 exhibited a nonspecific reaction showing a completely positive signal (FIG. 7 (B)).
). Therefore, this method was found to be unsuitable for preparing spleen tissue. Establishing a sensitive cut-off in methods 3 and 5 based on the negative control group was not possible due to the very weak response of the negative control group.

Therefore, the cutoff line was set to 0.1 U on the y-axis in consideration of 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 detecting PrP ^sc similar to that of Method 3 and Method 1 was realized (FIG. 7 (B)). However, Method 5 did not reach the sensitivity of Method 3 for large amounts of tissue equivalents and showed twice the sensitivity of Method 3 by repeated measurements (data not shown).
This requires consideration of circosyl extraction and salting out of PrP ^sc with NaCl.

A comparison of the results of all the methods shown in FIG. 7 (B) shows that in order to improve the sensitivity and specificity of spleen tissue, non-specific It can be seen that sufficient removal of the target protein or separation of PrP ^sc by extraction and salting-out is an effective and necessary step.

(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 the ELISA method is EL.
It was confirmed by sensitivity comparison between the ISA method and the Western blot (WB) method.

A comparison similar to the method of diagnosing animals at the pre-onset stage of infectious disease, in which a tissue homogenate from uninfected mice was used to compare tissue samples in which only a small amount of PrP ^sc was buried. Tissue homogenates from diluted scrapie infected mice were processed.

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

(4-0) Sample preparation by ELISA method and WB method: 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 a homogenate from a 20 mg equivalent of the corresponding tissue in 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) 2 ¹¹ times (9.8μg / 20mg)
3 shows the results of the dilution step into the sample.

ELISA method (FIGS. 8A and 8C);
For adsorption to microtiter plates, samples from each dilution step consisted of extracts from 20 mg tissue equivalents, 3M and 4M for brain and spleen tissues, respectively.
In GdnSCN-PBS. For both tissues, 5 wells of the microtiter plate were
A negative control group (control) for ELISA was coated with g equivalent of the extract. These are shown, but the standard deviation (SD) is too small to show.
The cutoff line corresponds to the value obtained by adding 3 SD 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 combined total amount of 20 mg. The film was exposed to the film for 15 hours.

[0166] (4-1) Brain tissue ELISA method indicates its possible clearly positive response in the case of 2 ^8-fold dilution step occurs, but also the cut-off line or in two ^9-fold dilution steps Do you get it. This is the case when brain tissue from scrapie-infected mice is only 1/512 of the total homogenate (this is the brain equivalent 2
Equivalent to 39 μg of the total amount of 0 mg. ) But PrP ^sc
Can be detected (FIG. 8 (A)).

[0167] On the other hand, WB method, after exposing 15 hours a thin film, 2 ^8-fold dilution steps (which ratio of 1/256, i.e., 2
Equivalent to 78 μg scrapie brain in 0 mg total brain tissue. ) Showed only a very weak band (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 ELISA showed that a positive reaction was clearly observed in the case of a ^26- fold dilution step, and that the amount of spleen tissue from scrapie-infected mice was only 1/64 of the total amount (I.e., equivalent to 312.5 μg in the total amount of 20 mg spleen equivalent.) Even in this case, PrP ^sc was detected in the extract (FIG. 8).
(C)).

The WB method shows that after 15 hours of exposure, a dilution of less than ^25- fold (this is 1/32 of the tissue of scrapie-infected mice)
Ie, 625 μg in a total of 20 mg spleen tissue equivalent. ) Showed 24.5 kDa, 21 kDa and 17 kDa bands specific to PrP ^sc (FIG. 8 (D)).

Accordingly, the spleen tissue was also analyzed by ELISA.
It can be seen that the sensitivity of the method is equivalent to or higher than the sensitivity of the WB method.

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

6. Evaluation Mouse scrapes were diagnosed by the WB method one week after infection (Gracewall et al., 1996), and Reis and Ernst (1992b) 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; Muramatsu et al., 19993).

The results were compared with histopathology (Histopatholo
gy) (Reis et al., 1992a), PrP ^{sc in} tonsils of sheep by electron microscopy (Rubenstein et al., 1991) or immunohistochemistry (Schruder et al., 1996).
The WB method is one of the most sensitive methods in the early stages of scrapie, compared to the results obtained by other methods, such as a new report on preclinical studies of it can.

One week after infection of the inner surface of the intestinal wall (peritoneum), the detection of PrP ^{sc in} the spleen from the mouse was achieved by sufficient tissue homogenization and performed by collagenase digestion of the homogenate (Gracewall). Other 199
6 years), which makes the sensitivity of the WB method by the present inventor at least in agreement with the detection result of PrP ^sc from mouse spleen described in another report (Rubenstein et al., 1991; Reis and Ernst 1992b). I knew it was there.

The present inventor applied an 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 the easy aggregation of PrP ^sc into a sedimented state (Meyer et al., 1986). Pretreatment of scrapie-infected tissues with formic acid or SDS (Casksack et al., 1987) Furthermore, pure PrP ^sc GdnSC
Denaturation with N (which was performed after adsorption to microtiter plates; Serban et al., 1990) has been reported, which increases the immunoreactivity of anti-PrP antibodies.
Also, BSA fetal bovine serum on microtiter plate)
Adsorption was reported (Zhu et al., 1993) can be improved in the presence of guanidine guanidine probably development of PrP ^sc to sequentially increase the immunoreactivity of anti-prion protein antibody by the antigenic sites generated It is thought that it promotes.

[0178] Based on these observations and considerations, the present inventors have, 1M ~ to dissolve the PrP ^{sc-containing} substances directly
Using 5M (especially 3M and 4M) GdnSCN,
PrP on microtiter plates in the presence of dnSCN
^Sc was successfully adsorbed.

The limit of the tissue equivalent which can be analyzed by the ELISA method, that is, the limit at which no improvement in sensitivity can be expected is around 40 mg (data not shown).

The advantage of the ELISA method is that many samples can be used.
Because it can be analyzed in one run, the diagnosis and selection of potentially infected animals in large numbers can lead to a wide range of uses for controlling diseases caused by infection.

An obstacle to the method of actually diagnosing TSE (Transmissible Spongiform Encephalopathies) via detection of PrP ^sc is that it is still difficult to use blood or its components for diagnosis. Since PrP ^sc is to be extracted from brain or lymphatic tissue, sample preparation is the most time-consuming factor. The method applied to brain tissue (method 1) is a fairly simple method, leading to higher sensitivity. However, method 1
Not enough for spleen tissue. In this regard, extraction of PrP ^sc with Sarksyl followed by salting out with NaCl (method 3) increases sensitivity and reduces non-specific signals.

This method is time-consuming, but requires only a small amount.
Useful for testing PrP ^sc . On the other hand, when the extraction and salting out of PrP ^sc were omitted and the sample preparation (concentration) was performed by the method 5, the sensitivity of the ELISA method was reduced by about 2 times, but was still equivalent to that of the WB method (data: Not shown). This and the fact that Method 5 can be performed in a shorter time than Method 3 suggest that Method 5 is practical for diagnosis.

The detection limit of PrP ^sc of 0.6 mg of spleen tissue in the WB method, which was found during the execution of this example, is known.
Detection limit of 0.3 mg (Gracewall et al., 1996)
Was almost equivalent to In this case, the final tissue extract is
Although diluted with SDS-PAGE sample buffer,
Since normal tissue homogenate was used as a diluent and dilution was performed after the first extraction step, the PrP ^sc detection conditions were not very advantageous. The extreme detection limits reported for the ELISA method should be found in view of the more difficult conditions.

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

The present inventor has considered that high efficiency of PrP ^sc recovery can be achieved with improved sample preparation. And the amount of PrP ^sc in the mouse spleen appears to be higher than previously thought.

Focusing on the potential of lymphoid tissue for preclinical studies, Van Coolen et al. (1996) examined the PrP ^sc levels of some of these tissues by immunohistochemistry. According to this study, the tonsils, spleen, and mesenteric lymph nodes, in this order, are the most preferred tissues for preclinical studies. The present inventors have detected PrP ^sc in biopsy surface lymph nodes at the preliminary clinical stage of scrapie in 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 easiness of obtaining each tissue, biopsy and examination of the tonsils were carried out in a prenatal test (antemortemt) as proposed in Schruder et al. (1996).
It seems to be more suitable as est).

From the above examples, it can be concluded that the method using the ELISA method together with the ABC method for the detection of PrP ^sc from brain and spleen tissues has at least the same sensitivity as the WB method. Also, if this ELISA method is applied to the diagnosis of sheep scrapie and cattle BSE, the current W
It seems that a more practical and faster diagnosis can be performed as compared with the method B. However, in order to make it appropriate as a research method, it is necessary to further simplify the labor required for extracting PrP ^sc .

It is considered that the ability to easily adsorb PrP ^sc contained in the original tissue extract or the lysate of GdnSCN to the microtiter plate is a basic matter in enabling the detection of PrP ^sc with higher sensitivity. Although the color development ELISA method has a slightly higher sensitivity than the WB method, the sensitivity can be further improved by using a coloring reagent such as a fluorescent substance or a chemiluminescent substance.

[0189]

According to the detection method of the present invention, a method for detecting a pathogenic prion protein from an animal tissue-derived substance, comprising the step of: A first step of homogenizing the animal tissue-derived substance using an enzyme, a second step of decomposing the homogenized product obtained in the first step using a decomposing enzyme, and And a third step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product decomposed in the step, and a step of concentrating the pathogenic prion protein comprising: Dissolving in a solvent to obtain a solution of the concentrate;
And a fifth step of adsorbing the pathogenic prion protein in the lysate on an adsorption surface, and a sixth step of coloring the pathogenic prion protein adsorbed in the fifth step. It is characterized by detecting the pathogenic prion protein by enzyme-linked immunosorbent assay, first, in the step of enriching the pathogenic prion protein,
It has the above-described first to third steps, and in particular,
Since this is homogenized by using a surfactant corresponding to the type of the animal tissue-derived substance, even if the accumulation concentration of the pathogenic prion protein accumulated in the animal tissue-derived substance is relatively small, it can be sufficiently reduced. It can be concentrated.

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

That is, according to the detection method of the present invention, for example, cows and sheep can be diagnosed and sorted at an early stage of prion disease (scrapie or BSE) infection.
This can be done in large quantities and quickly.

Further, according to the first and second concentration methods of the present invention, when the method for detecting a pathogenic prion protein for detecting a pathogenic prion protein from an animal tissue-derived substance is to be carried out, it should be detected. In the method for concentrating the pathogenic prion protein, it is possible to provide an effective enrichment method capable of sufficiently concentrating the accumulated concentration even if the accumulated concentration is relatively small.

[Brief description of the drawings]

FIG. 1 is a flow chart showing an outline of a method for concentrating a pathogenic prion protein in this example.

FIG. 2 is a flowchart showing an outline of another enrichment method.

FIG. 3 is a flowchart showing an outline of another enrichment method.

FIG. 4 is a flow chart showing an outline of an enzyme-linked immunosorbent assay used for detecting a pathogenic prion protein.

FIG. 5 is a graph showing the change in the ability to detect a pathogenic prion protein depending on the type and concentration of a solvent used before the adsorption in the enzyme immunosorbent assay (brain tissue (A),
Spleen tissue (B)).

FIG. 6 is a graph showing the detection ability by the colorimetric method (brain tissue (A), spleen tissue (B)).

FIG. 7 is a graph showing the detection ability of the pathogenic prion protein by the enrichment method (brain tissue (A), spleen tissue (B))
).

FIG. 8 is a graph showing the detection ability in the enzyme immunosorbent assay (brain tissue (A), spleen tissue (C)), and a diagram showing the detection ability in the WB method (brain tissue (B), spleen tissue ( D)).

Claims (28)

[Claims] 1. A method for detecting a pathogenic prion protein from a substance derived from an animal tissue, the method comprising: detecting a pathogenic prion protein from the substance derived from an animal tissue; A first step of homogenizing, a second step of decomposing the homogenized substance obtained in the first step using a decomposing enzyme, and a step of decomposing the homogenized substance obtained in the second step. And a third step of obtaining a concentrate containing the pathogenic prion protein. The step of concentrating the pathogenic prion protein comprises: dissolving the concentrate containing the pathogenic prion protein in a solvent to dissolve the concentrate A fourth step of obtaining a product, a fifth step of adsorbing the pathogenic prion protein in the lysate on an adsorption surface, and coloring the pathogenic prion protein adsorbed in the fifth step. A method for detecting a pathogenic prion protein, comprising detecting the pathogenic prion protein by an enzyme-linked immunosorbent assay having a sixth step. 2. The animal tissue-derived substance is a central nervous system tissue, and the surfactant is N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate or t-octylphenoxypolyethoxyethanol. The method for detecting a pathogenic prion protein according to claim 1, wherein the method is a nonionic surfactant. 3. The method for detecting a pathogenic prion protein according to claim 2, wherein the central nervous system tissue is a brain tissue. 4. The pathogenic prion according to claim 1, wherein the animal tissue-derived substance is a reticulo-lymphoid tissue, and the surfactant is a nonionic surfactant composed of t-octylphenoxypolyethoxyethanol. Method for detecting proteins. 5. The method for detecting a pathogenic prion protein according to claim 4, wherein the reticulolymphatic tissue is a spleen tissue. 6. The pathogen according to claim 1, wherein, in the second step, the homogenized product is decomposed by using a collagenase and a DNAse as the protease, and further decomposed by using a protease. Method for detecting soluble prion protein. 7. The method for detecting a pathogenic prion protein according to claim 1, wherein in the second step, the homogenized product is decomposed using a plant protease and / or a microbial protease as the decomposing enzyme. 8. The method according to claim 8, wherein during the concentration step, the concentrate containing the pathogenic prion protein obtained in the third step is decomposed using a decomposing enzyme, and then subjected to a salting-out treatment after separation. The method for detecting a pathogenic prion protein according to claim 1, which has 9. The pathogen according to claim 1, further comprising a washing step of washing the concentrate containing the pathogenic prion protein obtained in the third step with a surfactant during the concentration step. Method for detecting soluble prion protein. 10. The method according to claim 10, wherein the surfactant is N-dodecyl-N,
The method for detecting a pathogenic prion protein according to claim 9, wherein the method is a nonionic surfactant comprising N-dimethyl-3-amino-1-propanesulfonate. 11. The method for detecting a pathogenic prion protein according to claim 1, wherein guanidine thiocyanate is used as the solvent in the fourth step. 12. The method for detecting a pathogenic prion protein according to claim 11, wherein the guanidine thiocyanate is used in a concentration of 1 to 5 molar. 13. The pathogenic prion protein according to claim 1, wherein the adsorption surface is formed with a primary antibody, and an antigen-antibody complex of the primary antibody and the pathogenic prion protein is formed on the adsorption surface. Detection method. 14. In the sixth step, the pathogenic prion protein is colored, and its color density is detected.
A method for detecting a pathogenic prion protein according to claim 1. 15. The method according to claim 1, wherein the coloring is performed by fluorescence emission.
4. The method for detecting a pathogenic prion protein according to 4 above. 16. The method for detecting a pathogenic prion protein according to claim 15, wherein a chemiluminescent substance is used as the fluorescent luminescent agent. 17. A method for concentrating the pathogenic prion protein to be detected in carrying out a method for detecting a pathogenic prion protein for detecting a pathogenic prion protein from a substance derived from an animal tissue, the method comprising: N-dodecyl according to
A first step of homogenizing the animal tissue-derived material using N, N-dimethyl-3-amino-1-propanesulfonate or t-octylphenoxypolyethoxyethanol and an enzyme; A second step of decomposing the obtained homogenized product using a decomposing enzyme, and a third step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product decomposed in the second step. A method for concentrating a pathogenic prion protein, comprising: 18. The method for enriching a pathogenic prion protein according to claim 17, wherein the animal tissue-derived substance is a central nervous system tissue. 19. The central nervous system tissue is a brain tissue.
A method for concentrating a pathogenic prion protein according to claim 18. 20. The pathogen according to claim 17, wherein in the second step, the homogenized product is decomposed by using a collagenase and a DNAse as the decomposing enzyme, and further decomposed by using a protease. Method for concentrating soluble prion protein. 21. The method for concentrating a pathogenic prion protein according to claim 17, wherein in the second step, the homogenized product is decomposed using a plant protease and / or a microbial protease as the decomposing enzyme. 22. The method according to claim 17, further comprising a step of decomposing the concentrate containing the pathogenic prion protein obtained in the third step using a decomposing enzyme, and then subjecting the concentrate to a salting-out treatment after separation. A method for enriching the described pathogenic prion protein. 23. The method for concentrating a pathogenic prion protein according to claim 17, further comprising a washing step of washing the concentrate containing the pathogenic prion protein obtained in the third step with a surfactant. . 24. A method for concentrating the pathogenic prion protein to be detected in carrying out a method for detecting a pathogenic prion protein which detects a pathogenic prion protein from a substance derived from an animal tissue, the method comprising: A first step of homogenizing the animal tissue-derived substance using a surfactant and an enzyme corresponding to the above, and a second step of decomposing the homogenized product obtained in the first step using a decomposing enzyme. A step of obtaining a concentrate containing the pathogenic prion protein from the homogenized product degraded in the second step, and containing the pathogenic prion protein obtained in the third step The concentrate is treated with N-dodecyl-N, N-dimethyl-3.
A washing step of washing with a nonionic surfactant comprising amino-1-propanesulfonate. 25. The method for concentrating pathogenic prion protein according to claim 24, wherein the animal tissue-derived substance is a reticulum lymphoid tissue. 26. The method for concentrating pathogenic prion protein according to claim 25, wherein the reticulolymphoid tissue is a spleen tissue. 27. The pathogen according to claim 24, wherein in the second step, the homogenized product is decomposed by using a collagenase and a DNAse as the decomposing enzyme, and further decomposed by using a protease. Method for concentrating soluble prion protein. 28. The method for enriching a pathogenic prion protein according to claim 24, wherein, in the second step, the homogenized product is decomposed using a plant protease and / or a microbial protease as the decomposing enzyme.