JPH02501741A - A core consisting of a pair of helical filaments and its antibodies useful for diagnosing Alzheimer's disease - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Fur consisting of a pair of spiral filaments and its use in diagnosing Alzheimer's disease Nabuki The present invention provides proteins and proteins associated with Alzheimer's type senile dementia. It concerns protein precursors and antibodies against them. This antibody is It can be used to diagnose Zuheimer's disease.

Senile dementia of the Alzheimer type (Alzheimer's disease) affects people aged 65 in the UK. It is an age-related disease that affects about 4% of the elderly. Alzheimer's high Mar's disease causes progressive cognitive impairment and always leads to death. 190 from postmortem brain tissue research Various characteristic lesions discovered by Alzheimer's disease in 2006 are shown. in the brain The main lesions that occur are tangles of nerve wires, nerve bundles I! (plaque) and Granulovacuolar regression. [For an overview, see Tomlinson B, E, “ The aging brain” (The aging brain) Recent Advances in NeuropotholooV, k1.

eds, Sm1th 11. T, and Cavanagh J, B, Ch. See Urchill Livingstone, London 129-159. light]. The tangles consist of a unique superstructure named a pair of helical filaments (PHFs). It was shown to be a dense collection of microstructures. [niidd H, (1964 ), Brain 87.307-32 Q; Wtsniewski H,H , et al. (1976), J. Neurol.

Sci, 27.173-181 (1979), Ann.

The ribbon consists of a double helix overlap of horizontally oriented subunits, and the ribbon is wrapped in a left-handed spiral. It was shown that the overall shape was twisted in a spiral shape. (crowthe r R, ^, et at (1985) EHBOJ, 4 (13B), 36 61-3665: 986), Brlt, Hed, Bull 42 (1 above, 5l-56).

Antibodies raised against nerve 11m11 were isolated from a pair of helical filaments isolated from a tangle. It has been shown to cause cross-reactivity with ment. [Ihara, Y., et al. (1981) , Proc, Jap, ^cad.

57.152-156: ^nderton B, H, et al. (1982), N ature 298.84-86: Ga1betti P, G, et al. (198 3), J, Neuropathol, Exp, Neurol, main 1゜ 69-79], but according to recent data, the neural 1111 miscellaneous of PHF. In fact, the reactivity is due to cross-reactivity between epitopes derived from 1il and tau. It was suggested that [Luca F, C, et al. (1986), PNAS83. 1006-1010; Sternberger N, H, et al. (1985) .

PNAS and 4274-4276Moreover, several research groups have reported the extraction of tau protein from the prepared tangled sections. [niosik K , S et al. (1986) 86), J, Neuropath, Exp, Neur ol', main 5゜647-664: Grundke-rqbal r, et al. (1 986), J, 8io1, CheIm, 261.6084-60891.　 The reactivity of Tau survives under 5DS (sodium dodecyl sulfate) extraction conditions. Although it is clear that (Bionasi A, (1984), Acta , Neuropathol, 64.243-250), Neuropathol 111! As to whether the reactivity can survive the harsher extraction conditions of PHFS. There is some controversy. (RaSOOI C, G et al. (1984), Brain Re5310.249-2601 There is no clinical diagnostic test for Alzheimer's disease in humans. recently, Brain Iri! Presence of a pair of antigenic helical filament proteins in I(C8F) presence was reported. (Hehta P, D, et al., The Lancet, 6 th July 1986) and an enzyme immunoassay containing a so-called anti-PHF antibody. From C8F of Alzheimer's disease patients to C5F of patients with other neurological diseases. It was possible to measure antigens at a higher temperature than that of the sample. However, the test The law was not definitive, and there was considerable overlap in the spread of results. Motsu and heavy The important thing is that the antibody is actually directed against ubiquitin, and the ubiquitin in C3F is It is now clear that it cross-reacts with chitin.

In the present invention, we demonstrate that a pair of isolated helical filaments can be used to form two structural It contains two components that can be differentiated into two components, that is, the entire structural subunits of PHF. Pronase-resistant core and protease account for 100 Kd per unit In electronic microscopic imaging, which is sensitive to They discovered that it consists of a peripheral region. The outer fuzzy coating of PHF is a subunit This corresponds to approximately 20 d per unit, and the fuzzy covering is a real It has an M taste of blocking a qualitative epitope and is immunodominant6. Reacts with PHF, producing microtau protein, and possibly H and cross-react with other proteins such as M neuropil fiber protein and Yupichkin. The antibody is directed against an epitope that exists only in the fuzzy coat of PHF. It is. We are pronase resistant core (hereinafter “PHF core protein”) ) showed no cross-reactivity with such antibodies.

The term "PHF core protein" means that the core is composed of a single protein. Rather, it does not mean that the core structure is It should be understood that it indicates the proteinaceous fur of HF.

According to the present invention, a pair of helical filaments substantially free of proteinaceous material A fur protein consisting of

Pronase has a wide range of action derived from 5treptOtyCeS qriseus. It is a strong protease. Pronase is 70,000 units per g dry IIm It has a specific proteolytic activity of (1 unit is 1 minute at 40℃, pH 7.5. Defined as the amount of enzyme that liberates the products of digestion equivalent to 25 μ9 of tyrosine per It will be done. ) The protein protein of the present invention is a proteinaceous protein that does not have an immunodominant protein (Im) attached. It consists of a PHF core. This fur protein twists in a left-handed ribbon shape. multiple subunits arranged laterally to form a double-helical polymer It was shown to consist of a number of different fragments, which are thought to consist of .

Each subunit seems to be made up of three regions arranged in a 'C' shape. be exposed.

In a third aspect of the invention, a pair of helices containing substantially no other proteinaceous substance are provided. Precursors or fractions of filamentary core proteins are provided. This website Buunits are precursors of paired helical filament core proteins . Such fractions may contain normal brain tissue, and also the brains of Alzheimer's patients, from cerebrospinal fluid. It was found to exist as a soluble protein. Pronase resistant fur The purification and separation of the fur precursor or core fraction will be described in detail hereinafter. It is described in the explanation.

For the PHF core protein or the P)IF core protein precursor, (Such antibodies are included in the present invention.)

) cannot label cytoskeletal proteins found in tissue sections; The anti-W8 cell scaffold antibody that labels the released PHFS does not target the core epitope in vivo. A PHF core stripped of its occluded outer fuzzy region cannot be labeled. child Based on these findings, we selected antibodies based on the reactivity of tissue forehead tangles published in the paper. This strategy forms the PHF core and also takes into account the structure of the PHF subunits. The singular (or This suggests a failure to identify one or more proteins. Also, P Known antibodies against HF cross-react with antigens normally found in C8F. However, no matter how you look at it, it is difficult to interpret the results of tests using them. This makes it difficult to test, and it is easy to get false positive results in any case. However, Therefore, the protein of the present invention can be used to generate antibodies against the protein or its precursor. By making the neural network li! which can be used for PHF measurement. A tangle of miscellaneous things Provides a specifically related antigen. Antibodies can also be used by detecting anti- or using competitive techniques. For example, all The body has specific properties for PHF core protein or PHF core protein precursor. It may be an antibody with isomerism (more preferably a monoclonal antibody), or , or to antibodies specific for the fur protein or precursor. It may also be an antibody that has specificity.

Assays may also be performed using IWgl antigen to detect antibodies.

According to the fourth aspect of the invention, for the protein of the first or second aspect of the invention Antibodies are provided.

The antibody may be a polyclonal antibody, but it is preferable to use a monoclonal antibody. stomach. The monoclonal antibody described in the present invention is clone number 42. It is called by 3.

In yet another aspect of the invention, i) Paired spiral filaments from Alzheimer's neuronal tangles and ii) profiling the isolated paired helical filaments. Digested with Naze, 1ii) P The first aspect of the invention consisting of the step of purifying the HF core protein A process is provided for the preparation of proteins.

Figure 1 shows a synthetic nucleic acid derived from a part of the amino acid sequence of PHF core protein. cDNA prepared by methods involving leotide probes The predicted nucleotide sequence and the predicted amino acid sequence are shown, and FIG. 2 shows the amino acid sequence of the peptide fraction of the PHF core.

The present invention includes PHF core and core fraction regardless of the preparation method. Therefore, preferably PHF fur and core using the method of the present invention Contains fractions. Therefore, the present invention provides products prepared using our method. It includes classes of substances that share the essential characteristics of objects. Furthermore, another aspect of the present invention is the immunological equivalent of the fur and core fractions produced in W4 using the method of the present invention. material with physical characteristics and, where appropriate, electron microscopic structural properties.

The present invention further provides a method for collecting a sample such as brain cerebrospinal fluid (C3F) in a pair of helical Regarding the presence of fur proteins consisting of filaments or their precursors or fractions. A method for diagnosing Alzheimer's disease is provided.

The PHF core protein of the present invention is specifically associated with neuronal II tangles. Therefore, it is possible to identify antigens that can be used to diagnose Alzheimer's disease. provide. of the core protein, i.e. the structure that remains after pronase treatment. can be isolated by the operation described below; This is merely an example of a method.

Death due to Alzheimer's disease confirmed by autopsy, tissue containing tangles of nerve fibers removed from the brain of a patient.

The tissue is homogenized and filtered. The filtered homogenate is prepared in a series (e.g. (e.g. 3 times) density gradient centrifugation. Centrifugation Sucrose density gradient centrifugation It is more preferable. Fractions enriched in tangles and tangled fragments from centrifugation (hereafter “ ifI), i.e., a pair of helical fragments are obtained by centrifugation. is isolated.

The step in the preparation of PHFs is to analyze whole tangles and tangle fragments by fluorescence microscopy. By counting, we use the discriminative rough entanglement morphology observed in It is possible to monitor the (Yen, S-H, C,, F, Ga5k in and R, D, Terry, 1981, Imiunocytocheg +1cal 5 studies of neurofibrillary tang les, ^ta, J, Pathol, 104 Near 7-89>.

The tangles and the tangle fragments of the ifI fraction were separated with pronase and, optionally, in the nuclease. Digest with clease such as Micrococcus nuclease to further remove impurities. . Finally, the tangled fragments are further centrifuged to separate them from pronase and nuclease. Separate from enzyme-resistant fragments. For readers with a lot of experience, “pronase” is generally understood to be a general product for mixtures of multiple proteases. This means that another protease may be used in place of pronase. will understand immediately.

The tangled fragments are removed after digestion with pronase and, optionally, a nuclease. , subjected to density gradient centrifugation. To get good results, actually 1 ．． A sucrose linear density gradient of 18 to 1.05 was used, thereby eliminating unwanted contaminants. The material floats higher up the tube, and is best separated by the sedimentation velocity of the tangled cross section. optimized. Typically, a sucrose gradient is poured onto a cushion of C5Cj and concentrated. The collected fraction is collected at the interface of C5CJ at a density of 1.45.

The PHF core component can be further processed, for example by contacting ifI with formic acid or with succinic acid. The core fraction can be purified and separated by combining with. Ultrasonication The fifj of PHF core protein is further improved by using . the end of the process The end products are the soluble residues of a pair of helical filaments. The residue has a shell electric A large number of PHF core precursors can be separated by pneumophoresis. Coomassie Only the bands seen by staining or mercury staining were confirmed for antibody 423. Corresponds to the Stern plot positive band.

The isolated PHF core protein was prepared using Ohler and Hilstein (Na ture298 (1975)) for the PHF core component. Monochrome antibodies can be used in the preparation. The antibodies we selected are It was named 423 according to its clone number. However, the specific items used Clonal antibodies are of no particular importance to the present invention. The important thing is to operate It is possible to repeatedly obtain monoclonal antibodies against a portion of the PHF core. That is true.

Antibody 423 does not label cytoskeletal nuclear structures. Then, an antibody labeling the isolated PHFs was used. Blast nucleus antibodies do not label PHF cores. Therefore, for the PHF core component Antibodies are divine! useful for providing assays that are sensitive only to fiber entanglements. Therefore, it seems suitable for diagnosing Alzheimer's disease.

The assay for PHF core protein can be performed using any known immunoassay, for example. It can also be carried out according to the technique in A. Immunoassays can be homogeneous or heterogeneous Suitable labels for detecting the presence of antigen-antibody complexes, which may include radiolabels, include There are fluorescent labels, fluorescent labels, and enzyme labels.

For example, the assay may be a radioimmunoassay or an ELISA. Poly Although clonal antibodies can be used instead, monoclonal antibodies are used instead. is more preferable.

The present invention can be used to target PHF core proteins or precursors of core proteins. Probes for the corresponding nucleotide sequences can be prepared: for example, A cDNA library prepared from the patient's frontal cortex or a portion thereof was transferred to PHF. The part of the polynucleotide sequence that encodes the protein or peptide Screening using corresponding oligo or polynucleotide probes can be done. The probe was developed using T4 polynucleotide kinase. can be 1a1 with [γ-32P]ATP. hybridization positive Clones can be plaque purified and sequenced if desired. c. DNA clones or fragments thereof can also be used for research purposes as well as for Alzheimer's disease. It is conceivable that it can also be used for diagnosis.

Therefore, the present invention includes at least a portion of the PHF core or a fraction of the fur. Contains nucleotide probes that This probe has both synthetic and CDNA origins. But that's fine. Derived from the amino acid sequence of a part of the PHF core protein or peptide The cDNA library was screened using the synthetic nucleotide probes obtained. It is more preferable that the The probe must be strong enough to perform hybridization. length, typically consisting of 15 or more contiguous nuclei from the sequence to which the probe corresponds. leotide, e.g. 15 nuclei corresponding to the portion of the peptide sequence shown in Figure 2. Any sequence of leotide may be used.

Using nucleotide probes to characterize the components of the PHF core Related recently published work has shown that some of the fur is tau or tau. It has become clear that it is formed by some similar proteins.

However, monoclonal antibody 423 was isolated in the intact state, It does not react with au protein but reacts weakly with untreated PHFS.

Thus, monoclonal antibody 423 was found in the core protein tau's subsea fence, which until now has been I was not separated from the tea @ (HlGoedert et al., (198, 8), (1988) , PNAS85.4506-4510: C, M, Kaichik et al. (198 8), PNAS Hidama, 4884-4888: BMJ 297. D, 444 (1988). ′) All references cited herein are incorporated herein by reference.

mark Preparation of PHF core protein and monoclonal antibodies against it The brain has been clinically diagnosed with senile dementia of the Alzheimer's type, and sufficient reports have not been made. Obtained postmortem from a patient. In each case, the clinical diagnosis is frontal as well as lateral. Histologically confirmed by the presence of numerous rounded spots and tangles in the head cortex. I was bitten. Tissues obtained from the brains of four humans were used in this study. These people are 65.67.71 and died at the age of 84. The material used to make w4 is the frontal cortex. , taken from the temporal cortex and hippocampus. After removing the pia mater and meninges, create transverse sections. Ta. The gray matter was excised and discarded, leaving 20-40 u of tissue for use in individual preparations. It was. Tissues were stored at -70°C. The tissue was prepared using an equal volume of 0.32 M sucrose, 1 lin HMQCJ2.0.25mM phenylmethylsulfonyl chloride (PMSF> , 1eHEGTA, and 5sH potassium phosphate (p) 16.5). did. The resulting mixture was homogenized in a PTFE-glass manual homogenizer. Genized and filtered through four layers of cotton cloth placed over the syringe barrel, 70-100m was obtained. The filtered homogenate was mixed with 1.5M sucrose, 1*HMa Cj, 0.251M PMSE, 1+1 HEG Dine A and 5iH potassium phosphate ( Becksan SW2 at 15°C. 70-ter (Becksan Instrument, Inc., Pal.

^1 to, California) for 60 minutes in 1ia127. Centrifuge at ooorpm I let go. Discarding the upper slip and pellet, the material at the interface is 1.5M that exists below. ! ! It was harvested together with the sugar liquid layer. This mixture was passed through a PTEF glass homogenizer. Homogenize again inside and 2.0Mj! ! Sugar, 118MQCI, 0.25M A mixture containing PMSF, 1*HEGTA, and 5eH potassium phosphate (pH 6,5) BeCkllanS Centrifuged for 60 minutes in a W40 rotor. The fraction that floated to the top, the upper layer liquid and the bottom I threw away the beret. Harvest the material at the interface and add it to the initial 0.32M sucrose and buffer solution. Resuspend and incubate at 40.0O at 15°C in a Beck+an 3W 4Q oven. Orpm centrifugation was performed for 60 minutes. The final pellet, which is mixed with this ifI material, is Polyaromer centrifuge tubes sealed with plastic film serve as storage wells. It was used and stored at -70°C.

Pronase treatment Further treatment to obtain a PHFs-rich ifI fraction is as follows. Each ifI pellet has a volume of 9-20 Peng H Tris pH 7,1 Homogenize in a buffer consisting of mMCaCj2 and 3 IN M OC12 do. In contrast, 1 in 2011H Tris pH 7 and 1 IN Ca C12 Add 00 μl of Micrococcus nuclease (200 units/μl) and mix the mixture. Digest for 1 hour at 35°C. After 1 hour, add 100 μl of pronase to the 201H birds. 1) In a mixed solution containing H7,118CaC72 and 3*HMQC12, 10ay/ - and digested for an additional hour at 35°C. Digest with 150 μl of 200 By adding iHE GTA and 150μl of 200mHEDTA make it stop. Add 0.5d of 2M NaCl to this mixture to bring the final concentration to 100 Make 5M and add 100 μl of 3M Tris oH8,8 and 100 ay of cholic acid. . After stirring vigorously, the mixture was pipetted with Be1lat made in a Pasteur pipette. Filter through an ini 3 glass bead filter. Next, add 0.7g sucrose to the mixture. In addition, its density is 1.18. 201N Tris pH 7, 218EGTA , 14% J in 2dM EDTA! ! l! The second liquid containing l (specific gravity 1.05) was prepared. Same m* solution (20iH Tris pH 7, 218EGTA, 2*HED A third solution consisting of a density of 68% C5C1,1,45 in TA) was made from 211 It will be done. 13d 5W40 old trac! 2d C5CIFIi solution in ear centrifuge tube Pour the density gradient solution onto the cushion. Slope ranges from 1.18 to 1.05 It is.

This special gradient thereby optimizes the separation of entangled fragments by sinking velocity, Unwanted contaminants float to the top of the tube, and tangled fragments are collected at the C3CI interface. This is very important. The tube is Beckman S W 40 Q-tar. Rotate the container at 150° C. for 40.0 O Orpi + r311.

The pronase-treated PHF-rich layer, called ifI, has a density of 1.45. By inserting the needle of the syringe just below the interface, Therefore, collect it carefully. ifls are all 5W28 centrifuge tubes (3ifl[s/ tube), 5 dM citric acid, 5 mM N a HP 040H5, 1 in 5 Dilute to 0x. These were heated to 28,0 OOrp at 15°C in a 5W 280-tar. Centrifuge at night. The supernatant is discarded and the pellet is harvested for further biochemical extraction.

The PHF core protein is treated with a suitable reagent such as formic acid or a succinylation reagent. By sonicating in the presence, it is possible to further [j. typical The procedure is as follows: N II! Collected together with solution III (18iflrs/2a+e), and E /M CCorp HicroprobeSOniCatOr tuned to 3-4 Ultrasonication was carried out at the bottom of the wall using full power. This material is 5d 5W5 Transfer to a 0 polyaromer tube, make the volume 21 times to 5 d with water, and further sonicate. After that, the material was heated at 15°C and 50.0O in a Becksan S W 50 rotor. Centrifuge for 1H18 in OrD-. After this centrifugation, the collected supernatant was Al75. Call it 5.

Add 1-100% formic acid to the pellet and use the blow sonicator described above. Thoroughly sonicate.

Lai this material! Transfer to Ependorf tube and place on Fpendorf table. Centrifuge for 10 minutes in a top centrifuge. The upper groove of this centrifugation is harvested and freeze-dried. Beret Throw away the pieces. For lyophilizates, oN 5.5 ammonia acetate mv Add Z solution 1111 (30 μm in 50 μm of water distilled in a glass distiller) of glacial acetic acid, Adjust the pH to 5.5 with ammonium hydroxide). Blow out this suspension. Thoroughly sonicate in a centrifuge and centrifuge in an Ependorf tabletop centrifuge. Perform separation. The supernatant obtained from this processing step is the purified (fractionated) alkali Contains Zheimer PI-IF core protein. This internal division is AIZ, F , 5.5.

Aoz, F, 5.5 was subjected to shell electrophoresis and immunoblotting. electricity By electrophoresis, many fragments were separated into different bands. And Coomassie Only the bands seen in staining or silver staining are positive on the western plot with antibody 423. Compatible with various bands. The pellet from this final centrifugation was extracted with 11M , lyophilization, E) re-extraction with H5,5 and recirculation.

Monoclonal antibodies were prepared using the procedure of Ohler and Stein. [Nature 298 (1975)].

Seventy mice were injected over a period of 24 months. Note if ■ for 35 animals. Approximately half of the remaining half were injected with ifI, and the rest were injected with ifI. The collected fraction was injected. Five mice received PI with the fuzzy outer region removed. - Generated in serum that differentially labeled IFS. During this period, the fusion of tiles were prepared from a total of 94 animals. Entanglement reactivity in vivo, isolated antientanglement reactivity, modification of single tPHFs seen in immunoNma, and purified PHF coreta. Various screening techniques, including labeling of protein fractions, are used to detect positive Used to select lawn and serum. Standard immunolabeling protocols and The Eastern blotting protocol was used. 10 positive hybrids in total starting from a strong anti-PHF core reactivity as seen by electron microscopy. 1 clone (derived from the IL injected with if■, hereinafter referred to as 423) The pure antibody survived up to the stage of bulk preparation in autologous medium.

Filament form PHFs (i fII) prepared by a method involving a pronase digestion step are , filaments prepared without proteolysis are defined as PHF boundaries 1i The outer area seen as an area of increasing array of unstained parts along the ME. The difference is that the fuzzy region is lost. Nevertheless, outside this The PHFS from which the side fuzzy-marginal structure has been removed with pronase is the P It retains the ultrastructural features reported to be composed of structural subunits of HF. There is. In fact, every 3 to 4 nl, there are vertical stripes that merge and horizontal stripes spaced approximately 3aI apart. in pronase-treated filaments than in untreated filaments. It's clearly showing up. Therefore, it consists of the three areas previously reported. The subunits survive pronase treatment, indicating that pronase-treated fila Confirmed by image reconstruction based on ment.

Antibodies against fur protein Antiserum, oligoclonal, and monoclonal hybridomas are first Creating a white modification of the PHF core structure as seen by microscopy selected based on their ability to Screening to expose the reactivity of histological tangles ng is a reliable means of predicting clones with PHF core reactivity. It turned out that it wasn't. In fact, the upper groove can produce PHF core labeling. The histological sections were prepared as appropriate until final purification and bulk preparation of the monoclonal. No signs of tangles could be produced in the sections.

All hybridomas that can produce antibodies that strongly modify the PHF core shows a characteristic helical arrangement of gold particles along the filament, and the epitope This suggests that they are regularly and repeatedly arranged in a spiral pattern.

PHFS not treated with pronase (ifI) can be modified with 423. However, at lower dilutions than required for ifl [filament modification] there were.

This suggests that the PHF core epitope required for reactivity with 423 is probably i fI probably partially due to proteins present in Fuzzy II of PHFS means that it is blocked by On the other hand, ifI PHFS is exactly the standard at 423. What is known is that the 423 epitope is abrasive with pronase, but the protein fraction This means that it cannot be created depending on the solution. This interpretation applies at the histological level. This is consistent with the findings of amyloid filament The pronase-treated PHF-enriched fraction used as an immunogen is 7 myloid deposits contained collagen and ribofuscin. PHF core reaction Antibodies from hybridomas selected for It was not possible to modify the roid fibrils. Therefore, the presence of The present protein is distinguished from amyloid by its antigenic properties.

Antibody 423 also targets retracted capillary tubules present in arterioles or in plaque cores. Any histological I! of vascular amyloid precipitates present in! ! also give enlightenment I couldn't do it.

After large-scale production of antibody 423, tangles were prepared by fixing the sections with ethanol. Freshly frozen Alzheimer's brain tissue (frontal cortex) was treated with can be shown to be positive only in frozen sections from the temporal cortex, hippocampus) Met. However, in sections fixed with roseformaldehyde, normal Strong staining of untangled pyramidal cells and smaller neurons was found. child The staining was abolished by pronase treatment. and rose formaldehyde It could not be seen in the ethanol fixative. Staining is limited to the cell body It was There was no staining of dendrites and axons. Staining of these cells However, in that case, staining in the nerve processes was completely absent. It was.

Staining of Burkinje cell bodies and small classes of neurons was seen in the cerebellum. similarly , staining of cell bodies in the pulp was limited to nuclear-bearing it cells. paraformaldehy Staining of tangled neurons in fixed sections may, in some cases, be completely stained. was absent and variable, but in other cases the staining was localized with tangles. was.

Comparison of neurofibril antibody and TAtJ antibody The staining pattern observed with antibody 423 is similar to that observed with anti-1i1a and anti-tau antibodies. A can be compared with the detected pattern. 1 isolated PHFS! be aware of Neural lOIII antibody cannot label Burkinje cell bodies In addition, it is distinctive in that it labels the ectopic cell processes that serve the Burkiniie cysts. Ru. Similarly, the staining pattern observed with antibody 423 is similar to that of phosphorylated or non-vAII. This is distinct from tau, which is distributed in axons or separate parts, respectively.

Anti-tau and anti-neurofibrillar antibodies label isolated PHFs washed with SDS. However, the reactivity of both antibodies is increased after trypsin digestion of PHFs. Main departure In the light, ifI PHFS was weakly labeled with anti-tau antibody. this sign was burned after pronase treatment. Anti-God l1ants Monoclonal (RT97) Weak labeling with was similarly observed for ifI PHFS. This reactivity is Lost after lonase digestion.

Construction of cDNA library and screening RNA were performed using 15-week-old human fetuses; Frontal scalp of a 65-year-old patient with a histologically confirmed diagnosis of Alzheimer's disease. From quality, guanidium inthiocyanate/! 'i! I phenol method [Fera Cisco, J. R. et al. (1982) J. Biol.

Chet257, 11024-11031]. cortical group The texture was obtained in 3 hours. Poly (A) + RNA5 is oligo (6 cells) cell [1-Analysis was performed by affinity chromatography. First helix CDN A synthesis involves oligo(dT) in the presence of actinomycin D (40 μg/-). This was done by using murine reverse transcriptase as a primer. Double screw 1c DNA is produced by RNASeH, DNA polymerase, and Eschcrichia. Gubler and Horfian [G ubler, J.J. and HoNman, B.J. (1983) Gene25, 263-269) by a modification of the procedure described by J.D. 263-269). SJ Nuku Double helix subjected to size selection after treatment with Rease and EC0RI methylase CDNA is the mm434ECORI insertion vector λat with ECORI linker. cloned into lo (25). Fetal poly(A)+RNA (10μg) , generated a library consisting of 4 x 106 clones, and generated a library consisting of 4 x 10 clones, and a 10-ay frontal cortex point. Li(A)+RNA resulted in 6.2×10 6 clones.

Replica filters are made using two types of synthetic oligonucleotides, the sequences of which are described later. leotide Screening was performed using a mixture of probes.

By using T4 polynucleotide kinase, [γ-3 2P]ATP. Clones with positive hybridization are Braaku 11 The 3M Tetramethylammonium chloride 150■H Tris・HCI, pH 8, 0/2 Determined using mHEDTA.

Out of 650,000 clones, a single clone with positive hybridization was obtained. It was. The melting temperature of the hybrid was 56°C, and the oligonucleotide probe 1 This suggests that there is complete correspondence between the cDNA clones and cDNA clones. this is, This clone's 160-base pair hybridization-positive Hael [f/Aft This was confirmed by determining the sequence of the JI fragment. That is, the peptide sequence are found to encode, only a portion of which can be used to design oligonucleotide probes. used to enter.

Approximately 50,000 genes from the fetal brain cDNA library were transferred to Hael [l As a result of screening with /AJuI fragment, 34 additional positives were obtained; Several of them underwent plaque purification. Two of these clones (λPHF5 and and λPHF7), the characteristics were further examined. Unable to disconnect with EC0RI Therefore, the exact insertion size of λPHF5 is unknown. But partial limitations The map shows a length of 2.9kb. CDNA insert into λPHF7 2. The length is f3kb.

Amino acid sequence deduced as nucleotide sequence of cDNA clone Parts determined from clones PHF5 and PHF7 encompassing the complete coding region The minute nucleotide sequence is depicted in Figure 1. It is 37 in the 5′ non-U translated gA region. nucleotides, an open reading frame of 1056 nucleotides, an in-frame stop codon and 3 of 12 'Consists of untranslated sequences. It is the first ATG downstream of the in-frame stop codon. (nucleotides -9 to -7), the translation start site contains nucleotides 1-3. The methionine codon numbered as . The reading frame is 352 am. encodes a protein made of amino acids. The sequence of this protein is the mouse is highly homologous to the sequence of tau and thus represents the human equivalent of mouse tau. It consists of

protein sequence analysis As described above, AIZ, F, 5.5 was used for shell electrophoresis and immunoblotting. I got it. Two major bands at 9.5 and 12 kDa were identified by antibody 423. It was done.

For sequencing, the 9.5 and 12 kDa bands were isolated from polypinylidene difluoride membranes. Transferred and stained with Coomassie blue. Then they are cut out, eluted, and the resulting The diluted peptides were fractionated by Microbore HPLC.

The sequences of the peptides generated by tryptic digestion from the 9.5 and 12-kDa fragments are: Using direct HPLC detection of phenylthiohydantoin-derived amino acids Determined using an Applied Biosystem gas phase sequencer. Ta. By improving the sensitivity of this instrument, in each cycle to be analyzed Can detect 90% of amino acids derived from released phenylthiohydantoin It increased to Amino acid sequences derived from the 9.5 and 12-kDa components (Figure 2) are found to overlap with each other.

G　l　　　-11e-Va　l-'ryr-LVs-Pr.

The protein sequence of (QIVYKP in Figure 2) isolates the cDNA clone. Determination of mixtures of oligonucleotides that serve as hybridization probes for used for zain.

In the present invention, a sample from a breather is used as P)-IF core protein, PHF core fraction. or both. did Therefore, the test (which can be used to diagnose Alzheimer's disease) is P HF core protein or its precursor (or fraction) is added to an antibody that detects human antibodies. It can be used as a source. The specimen may be C3F, and in particular, an antibody. For assays, it may be plasma or serum. If the PHF core fraction is extracted from e.g. serum or It is also conceivable that it may be possible to detect it during dnWi.

The invention described above has been described by way of example only; It is obvious that modifications may be made and fall within the scope of the invention.

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Claims (1)

[Claims] 1. A pair of helical filament cores that are virtually free of other proteinaceous substances protein 2. A pair of helical filaments that are substantially free of immunodominant protein coatings. Core protein 3. A pair of helical filaments that are virtually free of other proteinaceous substances. Precursor of lament core protein and fraction 4.1 pair of helical filament cores -Protein separation fraction 5. (i) A pair of helical filaments from a tangle of nerve fibers in Alzheimer's disease. isolation. (ii) Digesting the isolated pair of helical filaments with pronase. (iii) and purifying the resulting insoluble pronase-resistant residues. consisting of Steps for preparing a pair of helical filament residues that are pronase resistant. 6. Step (i) is (a) homogenizing tissue containing a tangle of nerve fibers, and (b) subjecting the filtered homogenate to a series of concentration gradient centrifugation steps; In claim 5, the method comprises obtaining a fraction enriched in tangles and fragments of tangles. The process being billed. 7. Step (ii) involves quenching the isolated pair of helical filaments with a nuclease. As claimed in claim 5 or 6, further comprising: Process. 8. Step (iii) comprises adding the medium containing the digested filaments to 1.05 and 1. Pronase Claim 1, comprising collecting a fraction of less resistant residues at the base of the gradient. The process claimed in any of paragraphs 5 to 7. 9. Prepare an isolated core fraction by subjecting it to an additional purification step for pronase-resistant residues Claims 5 to 8 further include: The method required. 10. A process claimed in any one of claims 5 to 8 Residues of a pair of pronase-resistant helical filaments prepared using 11. Core fraction prepared using the process claimed in claim 9 12. Core protein claimed in claim 1 or 2 or antibodies to the residues claimed in paragraph 10. 13. The anti-antibody claimed in claim 12 which is a monoclonal antibody body. 14. Core protein award claimed in claim 1 or 2 or of the residues claimed for paragraph 10, by preparing antibodies against them. Use for. 15. The antibody claimed in claim 12 or 13 is involved. , (a) paired helical filaments in cerebrospinal fluid optionally labeled with antibodies. or (b) the presence of the residues claimed under paragraph 10. immunoassay. 16. A pair of helical filament core proteins or from assaying cerebrospinal fluid samples for the presence of residues claimed in How to diagnose Alzheimer's disease 17. By forming a concentrated preparation, Alzheimer's disease can also be cured. A pair of helical filaments (PHFs) are isolated from each pair and concentrated in buffer. The preparation was homogenized and the homogenate was treated with Micrococcus nuclease and and digest with pronase to stop the digestion and adjust the density of the digestion mixture to 1.18; The mixture was passed through a density gradient of 1.18 to 1.05 and a cushion of a solution of CSCl. The pronase-treated PHF fraction was collected at the CsCl interface. A process for removing PHFs "fuzzy" regions. 18. The pronase-treated PHF fraction is further recentrifuged to further purify it. 18. The process as claimed in claim 17, comprising: 19. The pronase-treated PHF fraction was sonicated at pH 5.5 and Centrifuge the treated material, collect the pellet, and sonicate the pellet in the presence of formic acid. The resulting mixture was centrifuged, the supernatant was collected and lyophilized, and the lyophilized product was Ultrasonic treatment was performed at pH 5.5 in an aqueous solvent at H5.5, and the sonicated product was Claim 17 or 18, further comprising centrifuging and collecting the supernatant. The process that is being billed. 20. Claim 9 further comprising subjecting the final supernatant material to an additional separation operation. Step 21. The claimed invention further comprises the preparation of a PHF core-fraction. A process claimed for scope 17 or 18. 22. By the method claimed in claim 17 or 18 Immunological, as well as electronic, characteristics corresponding to the prepared pronase-treated PHF-fraction. Substances with microscopic structural features 23. Claimed in any one of claims 19 to 21 Immunological and, where appropriate, PHF core-fractions prepared by the method is a substance with electron microscopic characteristics. 24. By the method claimed in claim 17 or 18 an antibody against the pronase-treated PHF fraction that has been or can be prepared; Antibodies against the substances claimed in paragraph 22. 25. (a) as claimed in any one of claims 19 to 21; (b) PHF core-fraction prepared or capable of being prepared by the method described in Substances claimed in paragraph 23; (c) before they are claimed in paragraph 3; precursor or fraction; or (d) a fraction as claimed in paragraph 4 or paragraph 11. Monoclonal antibody against min. 26. Claimed in any one of claims 17 to 21 PHF-fraction prepared or capable of being prepared by the method, or paragraph 22 or or the use of the substances claimed in paragraph 23 to prepare those antibodies. . 27. Any one of Claims 12, 13, 24, or 25 Immunoassays involving antibodies claimed in . 28. Specimen from a patient as claimed in claim 3 or 4. assay for the presence of precursors or fractions, or for samples from Alzheimer's disease, which consists of carrying out the immunoassay claimed in paragraph 27. Diagnosis of Immer's disease. 29. Any item claimed in any one of claims 1 to 4 Nucleotide sequence encoding an protein or a precursor or fraction thereof and the substances claimed in paragraph 22 or 23; Prepared by the method claimed in any of paragraphs 17 to 21 Nucleotide probes for pronase-treated PHF fractions.