JP5414975B2 - Method and kit for immunohistochemical detection of AGE-2 in cells or tissues - Google Patents

Description

  The present invention relates to a method for immunohistochemically detecting a glyceraldehyde-derived advanced glycation end product (AGE-2) in a cell or tissue and a kit therefor.

  Terminal glycation products (AGEs) are a general term for structures produced by a non-enzymatic glycation reaction between a reducing sugar such as glucose and a protein. AGEs accumulate in the central nervous system with aging and diabetes, or are considered to be the cause of complications due to diabetes such as neuro / sensory organ disorders and nephropathy. Recently, it has been revealed that it is also involved in neurodegenerative diseases such as Alzheimer's disease, proliferation, metastasis and invasion of malignant tumors.

  AGEs are generated not only from glucose, but also from various aldehyde or carbonyl compounds such as glucose auto-oxides and degradation products. Among these, the terminal glycation product derived from glyceraldehyde (glyceraldehyde-derived terminal glycation product; AGE-2) has a high binding ability to the AGE receptor (RAGE), and through this RAGE, diabetic retinopathy and It is known to be strongly involved in the onset and progression of diabetic vascular complications such as diabetic nephropathy (Non-patent Document 1 and Non-patent Document 2). However, few diagnostic methods and inhibitors have been developed yet. Moreover, although it is useful for the diagnosis of the said disease to detect the AGE-2 in a structure | tissue, and to determine and / or quantify the location, it is difficult at present.

  Various methods for measuring AGEs have been studied so far. The simplest method is to measure fluorescence because AGEs are yellowish brown and emit fluorescence. However, since the specificity and sensitivity to AGEs are low, it is not particularly suitable for biological samples. AGEs with a specific structure can be quantified using HPLC, GC / MS, LC / MS, etc., but are time consuming and suitable for analysis of large numbers of samples, for example, in diagnosis Not.

  At present, an immunological measurement method using an antibody (anti-CML antibody) that recognizes carboxymethyllysine (CML) whose structure is clear among AGEs is mainly performed. However, the sensitivity is low and the antibody itself is expensive. Furthermore, since CML is caused by lipid peroxidation rather than a glycation reaction in vivo, and CML has come to be considered as a marker of oxidative stress, there are problems in using anti-CML antibodies as anti-AGEs antibodies. There is. Thus, an antibody that recognizes the entire AGEs has not been obtained at present.

  In recent years, it has been clarified that single-stranded DNA and single-stranded RNA have various three-dimensional structures and can recognize and bind various compounds from small molecules to proteins and have functions like antibodies. (Non-Patent Document 3 and Non-Patent Document 4). Such a molecule is called an aptamer. Aptamers can be obtained from a random sequence by a selection method called SELEX (Non-Patent Document 4).

  Aptamers have advantages such as being able to be synthesized in large amounts in a test tube, those having a stronger binding force than antibodies, and being able to be stabilized. Therefore, it can be applied to research, detection, medical treatment, and the like, similar to antibodies. Examples of applications that have been examined for medical treatment include RNA aptamers for HIV-1 reverse transcriptase (Non-patent Document 5), RNA aptamers for complement C5 (Non-patent Document 6), and cytomegalovirus (CMV) infection. RNA aptamer (Non-patent document 7), RNA aptamer against vascular endothelial growth factor (Non-patent document 8) being developed as a therapeutic agent for senile macular degeneration, glomerular interstitial proliferative glomerulonephritis model rat An aptamer for platelet-derived growth factor whose symptoms are alleviated when injected intravenously (Non-patent Document 9), an RNA aptamer that restores abnormalities due to overexpression of Drosophila B52 protein (Non-patent Document 10), and the like have been reported.

In view of the above-described problems, the present inventors have tried and acquired an aptamer that recognizes and binds to AGE-2 (Patent Document 1).
International Patent Application Publication No. 2006/080262 Yamagishi S. et al., Biochem. Biophys. Res. Commun., 2002, 290, 973-978 Okamoto T. et al., FASEB J., 2002, 16, 1928-1930 Ellington AD and Szostak JW, Nature, 1990, 346, 818-822 Tuerk C. and Gold L., Science, 1990, 249, 505-510 Kensch O. et al., J. Biol. Chem., 2000, 275, 18271-18278 Biesecker G. et al., Immunopharm., 1999, 42, 219-230 Wang J. et al., RNA, 2000, 6, 571-583 Ruckman J. et al., J. Biol. Chem., 1988, 273, 20556-20567 Floege J. et al., Am. J. Path., 1999, 154, 169-179 Shi H. et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 10031–10038 Tessier et al., Biochem. J., 2003, 369, 705-719

  An object of the present invention is to provide a method and kit suitable for detecting AGE-2 in a cell or tissue and determining and / or quantifying its location.

The present invention provides a method for immunohistochemically detecting a glyceraldehyde-derived terminal glycation product (AGE-2) in a cell or tissue, which specifically binds to the glyceraldehyde-derived terminal glycation product. Contacting the resulting aptamer with a cell or tissue; and detecting the aptamer bound to a glyceraldehyde-derived terminal glycation product in the cell or tissue.

  The present invention further provides a kit for immunohistochemically detecting a glyceraldehyde-derived terminal glycation product in a cell or tissue, which kit can specifically bind to a glyceraldehyde-derived terminal glycation product. Provide aptamer.

  The aptamer is an aptamer that binds to a glyceraldehyde-derived terminal glycation product but does not bind to human serum albumin, the aptamer consists of at least 35 bases, and the cytosine content in the base is at least 35%. Or the guanine content in the base is at least 32%.

  In one embodiment, the aptamer is single stranded DNA.

  In a further embodiment, the single-stranded DNA includes a base sequence described in any one of SEQ ID NOs: 1 to 24 in the sequence listing.

  In a further embodiment, the single-stranded DNA comprises a base sequence set forth in any one of SEQ ID NOs: 25 to 41 in the sequence listing.

  In a further embodiment, the single-stranded DNA includes the base sequence set forth in SEQ ID NO: 1 or 9 in the sequence listing.

  In another embodiment, the aptamer is labeled.

  According to the present invention, a method and a kit capable of immunohistochemically detecting AGE-2 in a cell or tissue are provided.

(AGE-2 aptamer)
An aptamer is a single-stranded DNA or single-stranded RNA that can specifically bind to a specific compound as described above. In the present invention, this particular compound is AGE-2. The AGE-2 aptamer used in the present invention binds to AGE-2 but not to human serum albumin. Either single-stranded DNA or single-stranded RNA may be used.

  The AGE-2 aptamer used in the present invention comprises at least 35 bases, preferably at least 50 bases and at most 120 bases. When it is 34 bases or less, it does not bind to AGE-2.

  The AGE-2 aptamer preferably contains a large amount of either cytosine or guanine in the base constituting the aptamer. If cytosine is high, the cytosine content may be at least 35%, at least 40%, or at least 50%. When guanine is high, the guanine content in the base may be at least 32%, at least 35%, or at least 40%. With such a base content, the aptamer is likely to bind to AGE-2.

  Specific examples include single-stranded DNAs containing the base sequences set forth in SEQ ID NOs: 1 to 24 in the sequence listing. The term “including a base sequence” includes a case where the base sequence is included and a case where a base is further added, deleted or substituted to the base sequence. However, such addition, deletion, or substitution of bases is performed as long as the properties as an aptamer, for example, the binding ability to AGE-2 are not impaired. These single-stranded DNAs comprise a base sequence consisting of 54 to 58 bases and having a cytosine content in the bases of at least 35%. Another specific example is a single-stranded DNA containing the base sequence set forth in any one of SEQ ID NOs: 25 to 41 in the sequence listing. These single-stranded DNAs comprise a base sequence consisting of 61 to 66 bases and having a guanine content in the bases of at least 32%.

The AGE-2 aptamer can be obtained by a SELEX (Systematic Evolution of Ligands by Exponential enrichment) method, which is a general method for obtaining an aptamer. The outline of the SELEX method using single-stranded DNA as a library source will be described below. First, a template DNA containing a random sequence of an appropriate length sandwiched between any two primer sequences is synthesized. In the present invention, the length of the random sequence is suitably 35 to 120 bases. This template DNA is amplified by PCR (Polymerase Chain Reaction) to obtain a random DNA aptamer pool. Next, the random DNA aptamer pool is associated with the target substance, unbound DNA is removed, and bound DNA aptamer is extracted. The obtained DNA aptamer is amplified by PCR using the above primer sequences. At this time, by performing PCR in the presence of 5-8 mM Mg ²⁺ to reduce the replication accuracy and facilitate the introduction of mutations, new DNA that does not exist in the DNA aptamer pool before association with the target substance A DNA aptamer pool containing aptamers is obtained. A new DNA aptamer may have a stronger binding force, i.e., the DNA aptamer may evolve. With respect to this evolved DNA aptamer pool, a DNA aptamer that specifically binds to the target substance is obtained by repeating the above series of operations for 5 to 15 rounds. After the final round, the resulting DNA aptamer pool is cloned and then sequenced by procedures routinely performed by those skilled in the art. In this SELEX method, template DNA synthesis, PCR, and the like, as well as cloning and sequencing are performed by methods commonly used by those skilled in the art. The AGE-2 aptamer of the present invention can be chemically synthesized by a method commonly used by those skilled in the art based on the sequence thus determined.

  In the present invention, the target substance in the SELEX method is AGE-2, preferably a conjugate with human serum albumin (HSA). AGE-2 can be synthesized by any method such as a culture method or a chemical synthesis method. In the culture method, for example, human serum albumin (HSA) is incubated with D-glyceraldehyde for several days. The chemical synthesis method is performed, for example, according to the method of Tessier et al. (Non-patent Document 11). Specifically, acetyl-lysine and glyceraldehyde are mixed in a phosphate buffer solution (pH 7.4), diethylenetriaminepentaacetic acid and 25% (v / v) methanol are added, and then at 37 ° C. for several days. It is obtained by reacting. The obtained AGE-2 is preferably immobilized on an appropriate solid phase (for example, beads) when used in the SELEX method.

  The binding affinity of the obtained aptamer to AGE-2 can be measured by reducing the fluorescence intensity of AGE-2 when an aptamer is added to AGE-2, utilizing the fact that AGE-2 emits fluorescence. Thus, an aptamer with strong affinity among the aptamers of the present invention can be selected.

  The AGE-2 aptamer may be synthesized using a modified nucleotide / nucleotide for the purpose of further stabilization.

(Immunohistochemical detection of AGE-2 in cells or tissues)
According to the present invention, the presence and location of AGE-2 in a cell or tissue can be determined using the AGE-2 aptamer. The AGE-2 aptamer recognizes and binds to AGE-2 present in a cell or tissue when it contacts a target cell or tissue. This AGE-2 aptamer can then be detected to determine the location of AGE-2 to which the AGE-2 aptamer is bound, and thus the presence and location of AGE-2 present in the cell or tissue. Furthermore, the amount of AGE-2 aptamer detected can be calculated to quantify AGE-2 present in cells or tissues.

  The present invention provides a method of detecting AGE-2 immunohistochemically (hereinafter also referred to as an immunostaining method) by using the AGE-2 aptamer. In the method of the present invention, the AGE-2 aptamer is brought into contact with a tissue or cell, and the AGE-2 aptamer is reacted with AGE-2 present in the tissue or cell, whereby the antigen AGE-2 in the tissue or cell is reacted. AGE-2 aptamer bound to is detected. The cell or tissue to be contacted with the AGE-2 aptamer is fixed according to a procedure commonly used by those skilled in the art. In addition, specimens can be prepared by embedding, freezing, and slicing using, for example, paraffin or OCT compounds. If necessary, an antigen activation treatment (eg, proteolytic enzyme treatment or heat treatment (such as a microwave method)) in the prepared specimen can be performed. The immunostaining method can be performed manually or using a commercially available immunostaining apparatus. For example, the Sequenza Immunostaining Center (manufactured by Chandon) can be easily stained by a manual dropping method using a cover plate and can be suitably used.

  As a specific example of the AGE-2 aptamer used for immunohistochemical detection of AGE-2, a single strand comprising the base sequence described in any one of SEQ ID NOs: 1 to 24 in the sequence listing as described above DNA is mentioned. Another specific example is a single-stranded DNA containing the base sequence described in any one of SEQ ID NOs: 25 to 41 in the sequence listing as described above.

  Preferably, an aptamer that is a single-stranded DNA containing the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 9 in the sequence listing is used.

  Single-stranded DNA has a few bases added or deleted to the above base sequence to such an extent that it does not interfere with binding of AGE-2 aptamer to AGE-2 in cells or tissues and immunohistochemical detection of the aptamer. It may have a missing or substituted sequence.

  For detection of the AGE-2 aptamer, the AGE-2 aptamer is preferably labeled in advance. Any nucleic acid labeling method can be used for labeling the aptamer. Such labeling methods include radioisotope labeling, digoxigenin labeling, biotin labeling, dye labeling and the like. Radioisotope labeling can be visualized by autoradiography. The digoxigenin label can be detected using an anti-digoxigenin antibody, and is preferable in view of immunohistochemical visualization. The biotin label can also be detected by an anti-biotin antibody or by streptavidin or avidin. For detection of these labels, the antibody may further bind a dye. Alternatively, the antibody may add a fluorescent or luminescent domain.

  As a sample to be subjected to immunohistochemical detection of AGE-2, typically, various cells or tissues derived from various organisms can be mentioned. For example, kidney, retina, nerve cell, blood vessel and the like can be mentioned.

  The present invention also provides a kit for immunohistochemical detection of AGE-2 in a cell or tissue. This kit includes an AGE-2 aptamer as a reagent for AGE-2 detection. The AGE-2 aptamer provided in the kit is the same as described in the immunohistochemical detection method. Preferably, it is an aptamer that is a single-stranded DNA containing the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 9 in the Sequence Listing. The AGE-2 aptamer can be labeled as described above.

  The kit of the present invention may also be provided with any reagent that can be commonly used in immunostaining. Such reagents include suitable detection reagents that depend on the label of the aptamer. Instructions for AGE-2 detection may further be provided.

  The method and kit of the present invention are used for diseases involving AGE-2, such as diabetic retinopathy, diabetic nephropathy, diabetic complications such as diabetic neuropathy, neurodegenerative diseases such as Alzheimer's disease, and growth of malignant tumors. It can be used to detect or diagnose metastasis, infiltration, etc.

(Example 1: Acquisition of AGE-2 aptamer)
(1-1: Preparation of single-stranded random oligo DNA)
Single-stranded random oligo DNAs containing a random region of 34, 56, or 72 bases and primer sites (SEQ ID NOs: 42 and 43) on both sides thereof were synthesized as follows.

  First, a CPG (controlled pore glass) carrier in which the 3 'terminal nucleotide was bonded via a 3' hydroxyl group was packed in a column. Next, the dimethoxytrityl group, which is a protecting group at the 5'-position of ribose, was removed with trichloroacetic acid, and detritylated. The second nucleotide whose ribose 3 'hydroxyl group is a cyanoethylamidite phosphate derivative is coupled to the 5' hydroxyl group of the detritylated first nucleotide using a base catalyst (tetrazole) and unreacted. The 5 ′ hydroxyl group was acetylated with acetic anhydride. The bond between the two nucleotides was oxidized with iodo to convert trivalent phosphorus to pentavalent phosphate. The above operations from detolylation to conversion to a phosphate ester were repeated until the desired chain length was obtained. The random portion was sequenced using a mixture of four nucleotide amidites (dNTPs) during the coupling reaction. After the reaction, oligo DNA was excised from the column by ammonia treatment and purified by a reverse phase cartridge column. After lyophilization, it was dissolved in an appropriate amount of water, and used as template DNA for the SELEX library.

(1-2: Synthesis of AGE-2)
Human serum albumin (HSA) (Sigma) was incubated with D-glyceraldehyde at 37 ° C. for 7 days under aseptic conditions. Unreacted sugar was first removed through a PD-10 gel filtration column (GE Healthcare Bioscience) and then dialyzed with phosphate buffered saline (PBS). Prior to use, Endospecies ES-20S system (Seikagaku Corporation) was used to confirm the absence of endotoxin.

(1-3: Binding of AGE-2 to beads)
AGE-2 obtained in 1-2 above was immobilized on beads using SIERFOLINK (registered trademark) coupling gel (catalog number 20401) manufactured by PIERCE in accordance with the product instructions as follows.

  First, the coupling gel was placed on a column and equilibrated with a coupling buffer (50 mM Tris-HCl, 5 mM EDTA, pH 8.5). AGE-2 was dissolved in coupling buffer, mixed with the coupling gel and incubated for 1 hour at room temperature. After completion of the reaction, the column was washed several times with coupling buffer. L-cysteine was dissolved in coupling buffer, mixed with the coupling gel and incubated for 30 minutes at room temperature. After completion of the reaction, the column was washed several times with coupling buffer and PBS. The amount of AGE-2 immobilized was calculated by measuring the absorbance before and after the reaction. The gel (beads) after immobilization was aliquoted and stored in a cool dark place until use.

(1-4: Binding of HSA to beads)
HSA was immobilized on the beads using PIERCE's SulfoLink® coupling gel (catalog number 20401) and UltraLink® EDC / DADPA immobilization kit (catalog number 53154).

(1-5: SELEX method)
PCR using random oligo DNA synthesized in 1-1 above as a template and forward primer (SEQ ID NO: 42) and reverse primer (SEQ ID NO: 43) (1 cycle: 94 ° C, 15 seconds; 55 ° C, 15 seconds; 72 ° C) 15 seconds × 12 cycles). After amplification, the positive strand was amplified by asymmetric PCR using only forward primers (1 cycle: 94 ° C., 15 seconds; 55 ° C., 15 seconds; 72 ° C., 15 seconds × 45 cycles). The amplified plus strand was purified by agarose gel electrophoresis and used as a DNA library for SELEX. This DNA library for SELEX was dissolved in PBS, heated at 95 ° C. for 5 minutes, and returned to room temperature. Next, the DNA library for SELEX and the beads immobilized with AGE-2 prepared in the above 1-3 were mixed and incubated at room temperature for 30 minutes. After incubation, the beads were washed several times with PBS. An appropriate amount of water was added to the beads, mixed, and heated at 100 ° C. for 5 minutes to dissociate and collect the DNA bound to the AGE-2 beads. The recovered DNA and beads immobilized with HSA prepared in 1-4 above were mixed and incubated at room temperature for 10 minutes. Passed through DNA (which did not bind to HSA) was collected and concentrated by ethanol precipitation. The above operation was repeated 5 to 15 rounds using the concentrated DNA as a template. At this time, PCR was performed in the presence of 5 to 8 mM Mg ⁺ to introduce mutations.

(1-6: Cloning)
The DNA obtained after 5-15 rounds is amplified by PCR using forward primer (SEQ ID NO: 42) and reverse primer (SEQ ID NO: 43), purified by agarose gel electrophoresis, and AGE-2 specific DNA is obtained. It was. This DNA was introduced into a cloning vector (Invitrogen: Zero Blunt (registered trademark) TOPO (registered trademark) PCR Cloning Kit for Sequencing (catalog number K2875J10)), and the sequence was determined as follows.

  First, AGE-2 specific DNA (PCR product) and a cloning vector (TOPO vector) were mixed and incubated at room temperature for 5 minutes. After completion of the reaction, a part of the reaction solution was added to the competent cell and incubated for 30 minutes under ice cooling. After heat shock at 42 ° C. for 30 seconds, the mixture was cooled on ice for 2 minutes. To the cooled reaction solution, the SOC medium contained in the kit was added and cultured at 37 ° C. for 1 hour. An appropriate amount was inoculated on an agar plate (LB medium containing 50 μg / mL ampicillin) and cultured at 37 ° C. overnight. Several tens of clones were randomly selected and plasmid DNA was prepared by the alkaline method.

(1-7: sequencing)
The sequence of AGE-2 specific DNA in the plasmid DNA obtained in 1-6 above was performed by the BigDye Terminator Cycle sequence method using ABI377 of Applied Biosystem.

  As a result, 34-base single-stranded DNA was not obtained that binds to AGE-2. From the single-stranded DNA of 56 bases, single-stranded DNA of 54 to 58 bases shown in SEQ ID NOs: 1 to 24, that is, AGE-2 aptamer was obtained (Table 1). A 72-base single-stranded DNA was also obtained that binds to AGE-2, but was not sequenced.

  As shown in Table 1, the obtained AGE-2 aptamer had a cytosine content of 35% or more in the base constituting the aptamer.

  Further, an AGE-2 aptamer was prepared in the same manner using a single-stranded random oligo DNA containing a 64-base random region and primer sites (SEQ ID NOs: 42 and 43) on both sides thereof as a template.

  As a result, 61-66 base single-stranded DNAs shown in SEQ ID NOs: 25 to 41, that is, AGE-2 aptamers were obtained (Table 2).

  As shown in Table 2, the obtained AGE-2 aptamer had a guanine content of 32% or more in the base constituting the aptamer.

(Example 2: Staining of rat kidney tissue using AGE-2 aptamer)
(2-1: Preparation of frozen sections of kidney tissue from rats with diabetic nephropathy)
Otsuka Long-Evans Tokushima Fatty RAT (OLETF), a naturally occurring obese type 2 diabetic rat, and non-diabetic control Long-Evans Tokushima Otsuka RAT (LETO) were supplied by Otsuka Pharmaceutical Co., Ltd. Breeding started. Rats were fed with CE-2 for breeding rats (CLEA Japan, Inc.) and were reared with free food and drinking water, and used for experiments.

  During the breeding period, blood glucose was measured at any time to confirm the onset of diabetes in all OLETF rats. Further, during the breeding period, body weight and blood glucose level were measured regularly, and urine was collected at the start of breeding, at the time of confirming the onset of diabetes, and immediately before the termination of breeding, and urine protein was measured. After confirming the onset of diabetes in OLETF rats, they were raised for about 4 weeks and subjected to the final experiment. OLETF and LETO rats were euthanized with anesthetics and kidneys were removed from each rat.

  The removed kidney was fixed according to a method commonly used by those skilled in the art, and then the fixed kidney tissue piece was embedded in an OCT compound, frozen, and sliced with a frozen microtome (cryostat). The prepared frozen section was attached to a silane-coated slide glass to obtain a frozen section slide.

(2-2: Chemical synthesis of digoxigenin-labeled AGE-2 aptamer)
AGE- obtained in Example 1 above by the phosphoramidite method in the same manner as in 1-1 above, except that a substrate for synthesis in which 0.01 mM of digoxigenin (DIG) -dUTP (Rosche) was mixed with dNTP was used. Each AGE-2 aptamer was chemically synthesized based on the sequence of 2 aptamers. The production of digoxigenin-labeled AGE-2 aptamer was confirmed using a kit “Insert Check -Ready-” manufactured by Toyobo.

(2-3: Preparation of Alexa Fluor (registered trademark) 488-labeled anti-digoxigenin mouse monoclonal antibody)
An anti-digoxigenin mouse monoclonal antibody (Rosche; anti-DIG antibody) was dissolved in 100 mM phosphate buffer (pH 7.5, 25 ° C.), and the resulting solution having a concentration of 4 mg / mL (0.0267 μmol / mL) was added to a 1 mL light-shielding bottle. And gently stirred with a silicon stirrer bar.

  1 mg (1.555 μmol) of Alexa Fluor (registered trademark) 488 succinimide ester (Molecular Probes) was weighed in a conical tube and dissolved in 116 μL of dimethyl sulfoxide (DMSO) to prepare a solution having a concentration of 13.405 μmol / mL-DMSO.

  30 μL of this Alexa Fluor (registered trademark) 488 succinimide ester solution was added to the anti-DIG antibody solution all at once and mixed well. The reaction was continued for 2 hours at room temperature with gentle stirring.

  The Alexa Fluor (registered trademark) 488-labeled anti-DIG antibody and unreacted product were fractionated with a HiPrep 26/10 desalting column (GE Healthcare Bioscience) equilibrated with phosphate buffered saline (PBS).

  From the absorbance at 495 nm and 280 nm of the obtained Alexa Fluor (registered trademark) 488-labeled anti-DIG antibody, a labeled antibody in which 3.3 molecules of Alexa Fluor (registered trademark) 488 are bound per molecule of the anti-DIG antibody is obtained. It was.

  A solution of the fraction containing Alexa Fluor (registered trademark) 488-labeled anti-DIG antibody was concentrated by ultrafiltration and filtered through a 0.2 μm membrane filter to obtain a stock solution. When used, it was diluted with PBS to a final concentration of 1% (w / v) bovine serum albumin (BSA) and 0.05% (w / v) Tween®20.

(2-4: Tissue staining of rat kidney tissue frozen section)
The activation process of the frozen section prepared in 2-1 above was performed as follows. As an antigen activation solution, a solution containing Tween (registered trademark) 20 at a final concentration of 0.1% (w / v) in 0.01 M citrate buffer (pH 6.0) is added to a heat-resistant doze (staining bottle). Put in. The frozen section slide was gently immersed in it. The stained doze was treated in an autoclave at 121 ° C. for 10 minutes and then cooled at room temperature for 40 minutes.

  Tissue staining was performed using a Sequenza immunostaining center (manufactured by Chandon). Briefly, frozen section slides prepared from OLETF rats and LETO rat kidneys were each set on a cover plate and pushed into a sequencer cassette to bring the cover plate into close contact with the slide.

  100 μL of digoxigenin-labeled AGE-2 aptamer (prepared in 2-2 above) at a concentration of 1 μg / mL was added to the sequencer cassette and incubated for 2 hours. It was then washed with 2 mL PBS. Next, the Alexa Fluor (registered trademark) 488-labeled anti-DIG antibody solution prepared in 2-3 above was added to PBS at a final concentration of 1% (w / v) bovine serum albumin (BSA) and 0.05% (w / v ) The solution containing Tween (registered trademark) 20 was adjusted to a concentration of 1 μg / mL, and 100 μL of this solution was added to a sequencer cassette and incubated at room temperature for 2 hours. Subsequently, the plate was washed with 2 mL of PBS, and a mounting medium containing 9 volumes of glycerin per 1 volume of PBS was added to the sequencer cassette, and the slide was sealed. As a result, a tissue-stained slide was obtained.

  The tissue-stained slide was observed under a fluorescent stereomicroscope (Nikon SMZ15FLA) and photographed with a digital camera.

  FIG. 1 is a fluorescence micrograph of a frozen kidney section slide stained with AGE-2 aptamer, which is a single-stranded DNA consisting of 56 bases including the base sequence shown in SEQ ID NO: 1 in the sequence listing (100 × magnification). ). In FIG. 1, fluorescence was observed in the mesangial tissue in the glomerulus, clearly showing the accumulation of AGE-2.

  FIG. 2 is a fluorescence micrograph of a frozen kidney section slide stained with AGE-2 aptamer, which is a single-stranded DNA consisting of 56 bases including the base sequence shown in SEQ ID NO: 9 in the Sequence Listing (magnification 200 times). ). In FIG. 2 where the magnification was increased, fluorescence was observed throughout the mesangial tissue.

  Therefore, the accumulation of AGE-2 in mesangial tissue was clearly shown using the AGE-2 aptamer.

  According to the present invention, a method and a kit capable of immunohistochemically detecting AGE-2 in a cell or tissue are provided. The method and kit of the present invention are used for diseases involving AGE-2, such as diabetic retinopathy, diabetic nephropathy, diabetic complications such as diabetic neuropathy, neurodegenerative diseases such as Alzheimer's disease, and growth of malignant tumors. It can be used to detect or diagnose metastasis, infiltration, etc.

It is the fluorescence micrograph of the kidney frozen section slide dye | stained using AGE-2 aptamer which is single strand DNA which consists of 56 bases containing the base sequence of sequence number 1 of a sequence table. It is a fluorescence micrograph of a frozen kidney section slide stained with AGE-2 aptamer, which is a single-stranded DNA consisting of 56 bases including the base sequence shown in SEQ ID NO: 9 in the Sequence Listing.

Claims (2)

A method for immunohistochemically detecting glyceraldehyde-derived advanced glycation end products in renal tissue,
Comprising detecting the aptamers bound to glyceraldehyde-derived advanced glycation end products and the renal tissues; an aptamer capable of specifically binding to the glyceraldehyde-derived advanced glycation end products contacting with the kidney tissue ,
The aptamer binds to the glyceraldehyde-derived advanced glycation end product but does not bind to human serum albumin, the aptamer consists of at least 35 bases, and the cytosine content in the base is at least 35% Or the guanine content in the base is at least 32%,
The aptamer is a single-stranded DNA and contains the base sequence set forth in SEQ ID NO: 9 in the sequence listing; and
The aptamer is digoxigenin-labeled;
Method. A kit for immunohistochemically detecting a glyceraldehyde-derived terminal glycation product in renal tissue, the kit comprising an aptamer that can specifically bind to the glyceraldehyde-derived terminal glycation product, An aptamer that binds to the glyceraldehyde-derived terminal glycation product but does not bind to human serum albumin, the aptamer consists of at least 35 bases, and the cytosine content in the base is at least 35% Or the guanine content in the base is at least 32%;
The aptamer is a single-stranded DNA and contains the base sequence set forth in SEQ ID NO: 9 in the sequence listing; and
The aptamer is digoxigenin-labeled;
kit.

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