WO2003054545A1 - Procede permettant de detecter un anticorps anti-telomerase - Google Patents

Procede permettant de detecter un anticorps anti-telomerase Download PDF

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WO2003054545A1
WO2003054545A1 PCT/JP2002/013310 JP0213310W WO03054545A1 WO 2003054545 A1 WO2003054545 A1 WO 2003054545A1 JP 0213310 W JP0213310 W JP 0213310W WO 03054545 A1 WO03054545 A1 WO 03054545A1
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telomerase
antibody
htert
reverse transcriptase
protein
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PCT/JP2002/013310
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English (en)
Japanese (ja)
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Seishi Murakami
Shuichi Kaneko
Kenkichi Masutomi
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Mitsubishi Kagaku Iatron, Inc.
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Priority to JP2003555207A priority Critical patent/JPWO2003054545A1/ja
Priority to AU2002357613A priority patent/AU2002357613A1/en
Publication of WO2003054545A1 publication Critical patent/WO2003054545A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

Definitions

  • the present invention relates to a method for detecting an anti-telomerase antibody.
  • telomeres are special structures at the ends of linear chromosomes in eukaryotic cells and play an essential role in maintaining chromosome length and chromosome stability.
  • the telomere structure is composed of a terminal region of a chromosome in which a TTAGGG sequence called a telomere repeat sequence is repeated, and a specific DNA binding protein.
  • the DNA replication of the telomere sequence portion is performed by an RNA-dependent DNA synthesis reaction, ie, reverse transcription, by an RNA protein complex called telomerase.
  • telomerase reverse transcriptase a rate-limiting factor for telomerase activity and a catalytic component of telomerase, induces immortalization of cells upon introduction into normal cells, and normal epithelial cells upon co-introduction with certain oncogenes. causess malignant transformation of Thus, telomerase reverse transcriptase is strongly involved in cell immortalization and canceration. Clinically, it has been reported that the expression of teguchi-merase reverse transcriptase is significantly increased in hepatocytes during the process from precancerous lesions to cancer. This also suggests the importance of telomerase reverse transcriptase in the carcinogenesis process.
  • telomerase is a specific reverse transcriptase.
  • Human telomerase reverse transcriptase (hereinafter sometimes abbreviated as “hTERT”) has been identified as a catalytically active subunit of telomerase, and its activity has been identified as the type II RNA as the human telomerase RNA subunit (hereinafter referred to as “hTERT”). Is sometimes abbreviated as “hTR”).
  • hTERT human telomerase reverse transcriptase
  • the present inventors first performed expression and purification of soluble recombinant hTERT using an insect cell expression system, fractionated the recombinant purified hTERT using a biochemical method, Purified as a single band of a.
  • Purified hTERT showed a dose-dependent telomerase activity in the presence of in vitro synthesized and purified hTR.
  • Gel shift assay (EMSA) using the hTR probe was shown to form a specific complex with hTERT ⁇ hTR in a dose-dependent manner. From these results, it was confirmed in the in vitro reconstitution system that the two submitters, hT ERT and hTR, were the minimum components of telomerase activity (J. Biol. Chem., 275). 22568-22573 (2000)). Disclosure of the invention
  • the present invention provides a clinical application of a recombinant human telomerase reverse transcriptase having a biological function, such as a method for detecting a blood anti-telomerase antibody using the recombinant human human telomerase reverse transcriptase as an antigen. It was made for the purpose.
  • the present inventors have conducted intensive studies in order to achieve the above object, and as a result, have found that blood-bearing anti-human telomerase antibodies are significantly higher in cancer-bearing patients than in healthy individuals.
  • the inventors have found that blood anti-human telomerase antibodies in patients with chronic liver disease significantly increase as the disease progresses, such as chronic hepatitis, liver cirrhosis, and liver cancer.
  • examination of other diseases revealed high antibody reactivity in patients with autoimmune diseases.
  • the present invention has been achieved based on these findings.
  • (3) The method according to (1) or (2), wherein the detection result of the anti-telomerase antibody is The method according to any one of the above (1) to (3), which is used as a car; (5) the method according to the above (4), wherein the disease marker is a tumor marker; (6) the autoimmune disease The method according to the above (4), which is a marker, is provided.
  • At least one of the above (1) to (6) comprising at least a protein constituting telomerase, type I RNA, or a complex thereof or a partial fragment thereof.
  • a reagent kit for performing any one of the methods is provided.
  • telomere reverse transcriptase the protein constituting telomerase is telomerase reverse transcriptase.
  • FIG. 1 is a diagram showing the results of detection of blood anti-human telomerase antibodies in Example 3.
  • HCC indicates hepatocellular carcinoma
  • LC indicates cirrhosis
  • CH indicates chronic hepatitis
  • Norma 1 indicates healthy persons
  • Others indicates other malignant diseases (cancer)
  • n indicates the number of cases.
  • FIG. 2 is a diagram showing the results of detection of blood anti-human telomerase antibodies in Example 4.
  • ANA posit i Ve indicates an antinuclear antibody positive patient (autoimmune disease patient)
  • Norm a 1 indicates a healthy person.
  • the telomerase used as the antigen in the present invention may be of any origin as long as it can react with the anti-telomerase antibody.
  • the telomerase-constituting protein, ⁇ -type RNA, or a complex thereof or These are partial fragments. Any telomerase can be used as long as it has the ability to replicate a telomere repeat sequence.
  • the protein constituting telomerase include telomerase reverse transcriptase, and it is preferable to use this reverse transcriptase as an antigen.
  • the telomerase reverse transcriptase has, for example, the sequence described in Matthew Meyersonetal., Cell, Vol. 90, 785-795, Au gust22, 1997. 0213310
  • telomerase reverse transcriptase includes natural telomerase reverse transcriptase (ie, type III RNA (TR)) derived from an organism (including an animal, a plant, and a microorganism). Telomerase, which has reverse catalytic activity in the presence of type I RNA (TR) and has the ability to replicate telomere repeats).
  • TR type III RNA
  • those functionally equivalent variants [ie, one or more (preferably one or several) amino acids are deleted, substituted, and / or added in the amino acid sequence of natural telomerase reverse transcriptase]
  • a protein having an amino acid sequence and having the same function as a natural telomerase reverse transcriptase], or a partial fragment of a natural telomerase reverse transcriptase, a partial fragment of a functionally equivalent variant, or a natural telomerase reverse A fusion protein of a transcriptase or a functionally equivalent variant thereof and a fusion partner (for example, a detection protein such as FLAG peptide or glutathione-1S-transferase) is included.
  • Telomerase reverse transcriptase used as an antigen can be prepared by introducing a DNA encoding the same into an appropriate expression vector, culturing a transformant having the vector, and expressing the protein.
  • the DNA is introduced into a vector by a method known per se, for example, the method described in Christopher M. C outereta 1., On cogene (1 998) 16, 1217-1222. Can be.
  • Examples of the vector having the DNA encoding telomerase reverse transcriptase thus prepared include pCI-neo-hTERT-HA (On cogene (1998) 16, 1227-1222), pCI-Ne o—hTERT (J. Biol. Chem., 275; 22568-22573 (2000)). These vectors have the DNA of SEQ ID NO: 1.
  • the method for expressing telomerase reverse transcriptase is not particularly limited, and any method such as an in vitro translation method or a recombinant expression method can be used. Is also good. If the expression product maintains the biological activity of telomerase, it becomes possible to determine the true antibody and the status of the antibody by measuring the telomerase activity. Due to the physiological characteristics of telomerase, it has been difficult to purify the functional telomerase or the telomerase catalytic component hTERT quantitatively by conventional construction—expression in normal animal cells. However, using the expression method using insect cells developed by the present inventors, especially the following construction and preparation method (WO01 / 57227), a substance having a biological function can be obtained. Can be obtained.
  • a step of synthesizing telomerase reverse transcriptase by expressing telomerase reverse transcriptase using insect cells (hereinafter sometimes referred to as a “synthesis step”), and a step of expressing telomerase reverse transcriptase in insect cells
  • synthesis step a step of synthesizing telomerase reverse transcriptase by expressing telomerase reverse transcriptase using insect cells
  • solubilizing step a step of solubilizing telomerase reverse transcriptase using a specific surfactant
  • the baculovirus that can be used in the synthesis step is not particularly limited as long as it is a baculovirus that can be used in a normal baculovirus expression system.
  • Bombyx mori nuclear polyhedrosis A virus nuclear polynedrosis virus; hereinafter, referred to as INPV
  • Au tographaca 1 ifornica NPV Au tographaca 1 ifornica NPV
  • paculovirus expression vector ie, transfer vector
  • transfer vector a transfer vector derived from a paculovirus, for example, p. VL1392 or pVL1393.
  • the host that can be used in the synthesis step is not particularly limited as long as it is an insect cell that can be used in an ordinary paculovirus expression system.
  • a cultured cell derived from Trichoplusiani examples include cultured cells derived from tomato (Spodopterafrugiperda) and cultured cells derived from silkworm (Bombyx mori).
  • culture cells derived from nettle primrose are preferable, and culture cells derived from egg cells of nettle primrose are more preferable.
  • Cultured cells obtained by treatment according to the method described in 1999 are more preferable, and High 5 cells (Biotechnol. Prog., 8, 391-396, 19992; In Vitro Cell. & D) ev., 29 A, 388-390, 19993) are particularly preferred.
  • Igh5 cells are commercially available, for example, from Invitrogen (product number: BTI-TN-5BI-4).
  • the vector and baculovirus are prepared according to a conventional method.
  • a paculovirus containing DNIII encoding telomerase reverse transcriptase can be prepared.
  • the multiplicity of infection (m.o.i.i.) at the time of infection in the synthesis step is not particularly limited, but is, for example, 0: !! to 10, preferably 0.1 to 0.3 or 5 to: 10, more preferably 0.1 to 0.3.
  • the optimal condition is 5 to 10 in a normal Paculouvirus expression system.
  • the culture period after infection is not particularly limited.
  • the culture can be performed for 2 to 8 days, preferably 4 to 6 days. In a normal paculovirus expression system, 2-3 days is considered to be the optimal condition.
  • Telomerase reverse transcriptase expressed in insect cells during the synthesis process accumulates in the cell in an insoluble state.Even if the cell is simply crushed in a buffer, it remains in an insoluble state. Then, the purification operation cannot be performed.
  • the insect cells obtained in the above-mentioned synthesis step or the cell lysate thereof are treated with an aqueous solution containing ND-Darco-N-methylalkaneamide (hereinafter referred to as “solubilization solution”).
  • Solubilized by treatment with N-D-Darco-N-methylalkaneamide used in the solubilization process includes n-Otanoinolay N-methylglucamide (MEGA-8) and n-nonanoyl-N-methyldalcamide (MEGA-9) and n-decanoyl-N-methyldalcamide (MEGA-10).
  • the concentration of N-D-darco-1-N-methylalkaneamide used in the solubilization step is not particularly limited as long as it is a concentration that can solubilize telomerase reverse transcriptase. It can be between 5 and 2% by weight, preferably between 0.5 and 1% by weight.
  • the solubilizing solution may further contain a salt, a protease inhibitor, a reducing agent, and Z or glycerol, if desired.
  • a salt include salts generally used for protein purification, for example, halides of alkali metals or alkaline earth metals.
  • Protease inhibitors include, for example, pheninolemethanesulfur fluoride (PMS F), leptin, pepstatin, antipine, fenanslorin, or benzamide.
  • the reducing agent include dithiothreitol (DTT) and 2-mercaptoethanol.
  • the concentration of salts, protease inhibitors, reducing agents, glycerol, etc. in the solubilization solution is not particularly limited as long as it is a concentration capable of solubilizing telomerase reverse transcriptase.
  • the pH of the solubilization solution is not particularly limited as long as the pH does not lose the biological activity of telomerase reverse transcriptase, but the pH is around neutral (for example, pH 7. It is preferably from 2 to 7.8).
  • the method of treating the insect cells to be solubilized or the cell lysates thereof with the solubilization solution can solubilize the telomerase reverse transcriptase contained in the solubilized object. As long as it is not particularly limited.
  • the solubilization can be carried out by combining the suspension and the solubilizing solution and then thoroughly mixing.
  • the solubilized substance is a solid substance (for example, a precipitate obtained by removing the supernatant after centrifugation)
  • the solid substance and the solubilized solution are combined, and Can be carried out by mixing them.
  • the solution obtained by the solubilization step contains the solubilized telomerase reverse transcriptase.
  • This solution was directly used as a telomerase reverse transcriptase-containing fraction.
  • telomerase reverse transcriptase can be further purified using a known protein purification method (eg, ammonium sulfate salting out, chromatography, dialysis, and / or lyophilization).
  • affinity chromatography using heparin sepharose or poly (U) -sepharose can be used in addition to the above-mentioned protein purification method known per se.
  • the solubilization step is performed on the insect cells obtained in the synthesis step or the cell lysate thereof without performing a preliminary treatment (for example, a partial purification treatment) as it is.
  • a preliminary treatment for example, a partial purification treatment
  • an appropriate partial purification treatment is performed prior to the solubilization step, the purification operation performed on the telomerase reverse transcriptase-containing solution obtained in the solubilization step can be omitted or simplified.
  • the crushed cell is centrifuged to remove the supernatant, and the remaining precipitate can be subjected to a solubilization step.
  • the protein fraction obtained by solubilizing the precipitate including telomerase reverse transcriptase
  • the operation of separating the soluble protein can be omitted or simplified.
  • the crushed cell is centrifuged to remove the first supernatant, and the remaining first precipitate is subjected to N-D-Darco-N-methylalkaneami Treated with a buffer solution containing a surfactant other than amide (for example, Triton X_100, NP_40, or CHAPS (3-[(3-Cholamidopropyl) dimethyl-ammonio] propanesufonic ac id)). Thereafter, centrifugation is again performed to remove the second supernatant, and the remaining second precipitate can be subjected to a solubilization step.
  • a surfactant other than amide for example, Triton X_100, NP_40, or CHAPS (3-[(3-Cholamidopropyl) dimethyl-ammonio] propanesufonic ac id
  • the soluble protein contained in the cell lysate is substantially removed together with the first supernatant, and subsequently, the soluble protein is solubilized by the surfactant remaining in the first precipitate. Since the protein is also almost completely removed together with the second 'supernatant, the second precipitate is removed.
  • the protein fraction obtained by solubilization the fraction containing telomerase reverse transcriptase
  • the operation of separating the soluble protein and the protein solubilized by the surfactant is omitted or simplified. it can.
  • Telomerase can be reconstituted by combining telomerase reverse transcriptase (TERT) obtained in this way with separately prepared type I RNA (RT), for example, in vitro. .
  • RT type I RNA
  • a telomerase reverse transcriptase having a biological function can be prepared.
  • the method for detecting an anti-telomerase antibody of the present invention comprises reacting at least a protein constituting telomerase, type I RNA, or a complex or a partial fragment thereof with an anti-telomerase antibody in a biological sample. At least, the reaction product is analyzed.
  • Biological samples that can be used for detecting antibodies include whole blood, serum, plasma, ascites, pleural effusion, spinal fluid, tissues and cells and their cultures, urine, and the like.
  • the antigen used for detecting the anti-telomerase antibody includes at least a protein constituting telomerase, type I RNA, a complex thereof, or a partial fragment thereof. Among them, telomerase reverse transcriptase or a partial fragment thereof is more preferable. Further, as the telomerase reverse transcriptase used as the antigen, one having the amino acid sequence of SEQ ID NO: 2 is particularly preferred.
  • a method of reacting the anti-telomerase antibody in the biological sample with the anti-telomerase antibody and analyzing the reaction product is a commonly used method known per se An immunological detection or quantification method is used.
  • immunological detection or quantification methods include immunoenzymatic antibody method [Enzyme Linked Immunosorbent Assay (ELISA)], radioactive label immunoassay (RIA), western blotting method, and dot blot method.
  • ELISA Enzyme Linked Immunosorbent Assay
  • RIA radioactive label immunoassay
  • dot blot method examples include a tinting method, an immunoprecipitation method, a hemagglutination method, a latex agglutination method, and a ribosome aggregation method.
  • a method of directly detecting the binding of the antibody to the antigen without labeling using surface plasmon resonance or a crystal oscillator can be used.
  • pretreatment of a biological sample for example, serum or urine can further increase sensitivity.
  • Detection or quantification of antibodies can be accomplished, for example, by enzymatic reactions, It may be performed by detecting or quantifying a signal from light, chemiluminescence, radioactivity, or the like. These methods are based on, for example, the descriptions in the Monoclonal Antibody Experiment Manual (Kodansha Scientific, 1987), Seismic Chemistry Laboratory Course 5 Immunobiochemical Research Method (Tokyo Kagaku Dojin, 1986). It can be easily implemented.
  • the immunoenzymatic antibody method refers to the reaction of the above antigen with an anti-telomerase antibody in a biological sample, and the reaction product is further labeled with an enzyme such as peroxidase or biotin. After reacting the antibody or the binding fragment subjected to the above, the coloring dye is measured with an absorptiometer.
  • Radioactive substance-labeled immunoassay refers to the reaction of the above antigen with an anti-telomerase antibody in a biological sample, followed by the reaction of a reaction product with a radiolabeled antibody or binding fragment. This is a method of measuring with a counter.
  • Western blotting is a method in which the above antigen is plotted on a PVDF membrane or -trocellulose membrane, a biological sample is added to the membrane and reacted with an anti-telomerase antibody, and the reaction product is added to a fluorescent substance such as FITC. This is a method of confirming after reacting an antibody or a binding fragment labeled with an enzyme such as peroxidase or biotin.
  • the level of anti-telomerase antibody in blood was significantly higher in cancer-bearing patients and autoimmune disease patients than in healthy subjects.
  • the blood anti-telomerase antibody can be a marker for various diseases, for example, a tumor marker or an autoimmune disease marker.
  • the anti-telomerase antibody in the biological sample is detected or quantified, and compared with the value of a healthy person. Can be determined.
  • the reagent kit used in the above-described method for detecting an anti-telomerase antibody of the present invention contains at least a protein constituting telomerase, ⁇ -type RNA, a complex thereof, or a partial fragment thereof as an antigen, and utilizes a normal immune reaction. Provided in the same configuration as the kit.
  • As the antigen telomerase reverse transcriptase or a partial fragment thereof is preferable.
  • the telomerase reverse transcriptase used as the antigen has the sequence Those having the amino acid sequence of No. 2 are particularly preferred.
  • the reagent kit of the present invention contains at least the above antigen, and may further contain, as optional components, a sample diluent, a washing solution, a labeled antibody, a dye, a positive control antibody, and the like.
  • Plasmid pCI—Neo—hT ERT (J.Biol.Chem., 275; 22568—22573 (2000)) containing hT ERT cDNA (SEQ ID NO: 1) was replaced with a restriction enzyme. By digestion with EcoRI and restriction enzyme Sa1I, an EcoRI / Sa1I-DNA fragment containing the above hTERT-c0 was obtained.
  • Plasmid pNKFLAG-hTERT was constructed by inserting the EcoRI / Sa1I-DNA fragment into the EcoRI-SalI site of plasmid pNKF LAGZ.
  • the plasmid pNKF LAGZ is composed of the FLAG sequence cDNA of the plasmid pNKF LAG (J. Biol. Cem., 273, 15479—15486, 1998) at the 3 ′ end of the plasmid. ) And modified to a sequence connecting the EcoRI and Sa1I sites.
  • the obtained plasmid pNKF LAG-hTERT contains a DNA encoding a FLAG-hTERT fusion protein (FLAG-labeled hTERT) in which the FLAG peptide is labeled at the N-terminus of hTERT.
  • FLAG-labeled hTERT FLAG-labeled hTERT
  • Not I and B g1 II By cutting the above plasmid pNKF LAG-hTERT with restriction enzymes Not I and B g1 II, Not I / B g 1 II- containing DNA encoding the FLAG-hTERT fusion protein A DNA fragment was obtained. By inserting the Not I / B g1 II—DNA fragment into the Not I—B g1 II site of the paculovirus transfer vector pVL1393 (Pharmingen), plasmid p is obtained. BYK—F LAG—hTERT was constructed.
  • baculovirus For the preparation of the recombinant baculovirus, a commercially available kit (including BaculoGold starter package f9 insect cells and Baccu1oGo1d linearized baculovirus virus DNA); Pharmingen It was carried out in the following procedure.
  • a recombinant baculovirus B VKM-FLAG-h TERT strain expressing the FLAG-hTERT fusion protein was amplified.
  • the measurement of the virus titer using the Sf9 cells was performed according to the instructions attached to the kit.
  • the following experiments used a recombinant Baki Yurowirusu B VKM- F LAG- h TE RT strains of high titer (1. 0 X 1 0 7 pfu / m L or higher).
  • the recombinant baculovirus BV KM-FLAG-h TERT strain was stable for more than about 6 months when stored at 4 ° C, after which the virus titer decreased.
  • Expression levels of the FLAG-hTERT fusion protein were compared using the same Sf9 cells used in Example 1 (2) and High 5 cells (Invitrogen) as insect cells. .
  • Sf9 cells the same 10% fetal serum-containing TNM-FH insect cell culture medium used in Example 1 (2) was used.
  • High 5 cells It was used with the addition of 1 0% ⁇ shea fetal serum; (I nvitrog e n Inc. H IgH F ive serum free medium) commercially available media.
  • the recombinant paculovirus B VKM-FLAG-hTERT strain prepared in Example 1 (2) was infected so that the multiplicity of infection (m.o.i.) was 2, and infection was continued. Thereafter, the cells were cultured at 27 ° C for 3 days.
  • the multiplicity of infection at the time of infection with a recombinant paculovirus is considered to be 5-10 optimal.
  • the expression level of the FLAG-hTERT fusion protein was determined under the condition that the multiplicity of infection at infection was 0.2.
  • the multiplicity of infection was similar to or higher than 5-10.
  • the optimal multiplicity of infection is considered to be about 0.2 because the yield of the FLAG-hTERT fusion protein was reduced.
  • the whole cell extract of each insect cell cultured for 48 hours, 72 hours, 96 hours, 120 hours, or 144 hours is separated by SDS-PAGE. And stained with CBB.
  • the highest expression level of the FLAG-hTE RT fusion protein was reached 4 to 6 days after infection.
  • H igh F ive serum-free medium I n V itrogen Co.
  • I nvitrogen Co. H IgH 5 insect cells
  • I nvitrogen Co. 1. 0 X 1 0 so that the seven 5 X 25 cm
  • the recombinant paculovirus B VKM-FL AG-h TERT strain prepared in Example 1 (2) was tested with a multiplicity of infection (m.o.i.) of 0.2. Infected. After infection, the cells were cultured at 27 ° C for 5 days.
  • PBS (—) phosphate-buffered saline
  • All buffers used were lmmo1 / L phenylinolemethylsulfonyl fluoride (Sigma), 10 mg / mL Pustatin A (Sigma), 1 Omg / mL Leptin (Sigma), 10 mg / mL Aprotune (B OEHR INGER MANNHE IM), 10 mg / mL Fuenanthroline (Sigma), 16 mg / mL Benzami (Sigma) and 1 mmo1 / L dithiothreitol (Nakarai) were added.
  • All buffers used were lmmo1 / L phenylinolemethylsulfonyl fluoride (Sigma), 10 mg / mL Pustatin A (Sigma), 1 Omg / mL Leptin (Sigma), 10 mg / mL Aprotune (B OEHR INGER MANNHE IM), 10 mg / mL Fuenanthroline (Sigma), 16 mg /
  • S2 fraction After centrifuging the above suspension at 10,000 g for 10 minutes, the supernatant (hereinafter referred to as “S2 fraction”) is collected, and the sediment is lysed with lysis buffer B [20 mm o 1 / LT ris -HCl (pH 7.5), 50% glycerol, 0.5% MEGA-9, 500mm o1 / L sodium chloride, 10mm o1 / L—] 3_mercaptoethanol] lmL The cells were suspended and sonicated (10 seconds) three times.
  • lysis buffer B [20 mm o 1 / LT ris -HCl (pH 7.5), 50% glycerol, 0.5% MEGA-9, 500mm o1 / L sodium chloride, 10mm o1 / L—] 3_mercaptoethanol] lmL
  • the obtained suspension was centrifuged again at 10,000 g for 10 minutes, and the supernatant (hereinafter, referred to as “S3 fraction”) was collected.
  • S3 fraction the supernatant
  • the suspension was suspended in lmL of mercaptoethanol and sonicated (10 seconds) three times.
  • This S5 fraction was added to buffer E [2 Omol / L—Tris—HCl (pH 7.5), 50% glycerol, 0.5% MEG A-9, 300 mmol / L sodium chloride , Immol ZL—DTT] and passed through a DNA Sepharose (L KB Pharmacia) column (1. OmL packed amount).
  • the permeate fraction was mixed with a mixture of heparin-1 sepharose CL-6B and sepharose CL-16B (heparin-1 sepharose CL-1B and sepharose CL-6B 1: 1) equilibrated with the buffer E. (Hereinafter simply referred to as "heparin-one-sepharose”), and the mixture was rotated and stirred at 4 ° C. for 3 to 4 hours.
  • the protein bound to heparin-sepharose was collected by low-speed centrifugation, washed with buffer E, and eluted with buffer B (2 mL) to obtain a purified FLAG-hTERT fusion protein.
  • fractionation was performed by SDS-PAGE and staining with CBB.
  • Bovine serum albumin (Sigma) was used as a standard sample for measuring protein concentration.
  • the FLAG-hTERT fusion protein prepared in Example 1 has the catalytic activity of human thermolase
  • the FLAG-hTERT fusion protein was purified with purified human telomeric RNA (hTR ) And force It was reconstituted in vitro to evaluate whether it exhibited telomerase activity.
  • the telomerase activity was measured by two different methods, namely, a telomere repeat sequence amplification protocol using a commercially available kit (Te1oCaser; Toyobo) (Telomere Repeat Amplification Protocol).
  • TRAP assay Assay; hereinafter, referred to as "TRAP assay”
  • TRAPEZE kit ELISA telomerase assay kit
  • a plasmid p GRN 164 containing hTR-cDNA (Science, 269, 123 6-1 24 1, 1995) was used as a type I, and an in vitro transcription system using T7 RNA polymerase was used.
  • h TR was synthesized.
  • the reconstitution buffer A [final concentration: l Ommol / L- HE PES (pH 8. 0), 10 Ommol / L sodium chloride, 25% glycerin, 1 mmo1 / L magnesium chloride, 3 mmo1 ZL potassium chloride, 0.1
  • the TRAP assay was performed using a commercially available kit (TeloChaser; Toyobo). The TRAP assay was performed according to the instructions attached to the kit. The obtained PCR product was fractionated by electrophoresis using a 10% polyacrylamide gel, and then stained with a staining reagent [SYBR Green I; Mo 1 ecu 1ar Probes].
  • telomere synthesis was observed after about 10 minutes, and thereafter, seemed to be linearly maintained for 1 hour. Similar telomere synthesis latencies were observed in the previous 33 ° C treatment without primers and substrate, indicating that a significant structural transformation of the FLAG-hTERT fusion protein after the primer and substrate were added. Suggests that will happen.
  • the catalytic activity of the FLAG-hTERT fusion protein was between 30 ° C. and 37 ° C. and the pH was highest near 8.0, requiring 3 mmo 1 / L of magnesium ions.
  • the reaction of telomerase activity by the FLAG-hTERT fusion protein was also under the same optimal conditions.
  • the TRAP assay performed in (2) above has good sensitivity, but is not suitable for telomerase quantification. Therefore, in vitro in the presence of different ratios of FLAG-hTERT fusion protein and hTR. Quantification of the reconstituted telomerase activity was performed by a TRAP-ELISA method using a commercially available kit (TRAPE ZE kit; Intergen). The TRAP-ELISA method was performed according to the following procedure according to the instructions attached to the kit.
  • reconstitution buffer B [final concentration: l Oinmo1 / L-HEPES (pH 8.0), 100 mm o 1 ZL sodium chloride, 25% glycerin, 1 mm o 1 / L magnesium chloride, 3 mm o 1 / L potassium chloride, 0.1 mm o 1 / L- PMS F, 1 mm o 1 / L— DTT, l OU / ⁇ ⁇ — RN asin, 2 n gZ 1— TS—primer, 1 mm o 1 ZL unlabeled d ATP, lmmo 1 ZL unlabeled dTTP, 0.1 mm o 1 ZL unlabeled d GTP, 0.1 ⁇ C i / n L [a— 32 P] label d GTP (800 C i / mm o 1; Am ersh 0213310
  • telomerase activity was highest when the two components of the FLAG-hTERT fusion protein and hTR were in an equimolar ratio. This suggests efficient complex formation of the two components.
  • Antibodies were detected using serum from a total of 83 patients (aged 40 to 82 years) and serum from healthy subjects. These include 30 cases of hepatocellular carcinoma, 16 cases of cirrhosis, 7 cases of chronic hepatitis, and 12 malignant diseases. Serum from 18 healthy volunteers was used as a control. In addition, at the time of blood collection, informative outlets were obtained in writing. There were no significant differences in age and gender between the patient group and the healthy group.
  • Antibodies were detected using the recombinant purified hTERT (FLAG-hTERT) prepared in Example 1 as an antigen.
  • Western blotting was performed according to a conventional method, and an anti-FLAG M2 monoclonal antibody (Sigma) was used as a positive control.
  • the anti-hTERT antibody of the fractionated protein derived from the serum derived from the patient was detected by Western blotting as follows.
  • RT FLAG-hTERT
  • PB SB blocking buffer
  • the reaction for absorbance measurement was performed by adding 100 ⁇ l of patient serum diluted 1: 100 in PBS to each well and incubating for 1 hour at 37 ° C. 100 ⁇ l of HRP conjugate anti-human IgG diluted 1: 2000 was added to each well D.
  • the ABTS substrate (2, 2'-Azinobis (3 -ethylbenzothiazo line-6-sulfonic Acid; ABTS from Nakarai ELISA POS Substrate ABT S Kit, Code 14351-80) was added, and measurement was performed using a microtiter plate reader and a 405 nm filter.
  • Detection of anti-hTERT antibody in cancer patients was performed using purified hTHER (FLAG-hT ERT).
  • purified proteins were detected by western blotting using patient sera.
  • a reaction specific to the purified protein of about 127 kDa was observed.
  • the reaction in this patient's serum was absorbed by the addition of purified hTERT. This result indicates that the signal of serum from cancer patients by Western blotting is specific.
  • the absorbance (OD) of 30 cases of hepatocellular carcinoma, 16 cases of cirrhosis, 7 cases of chronic hepatitis, 18 cases of healthy subjects and 12 cases of other malignant diseases (cancer) is 0.365 ⁇ 0.021, 0.
  • the values were 3 25 ⁇ 0.019, 0.256 ⁇ 0.020, 0.239 ⁇ 0.013 and 0.319 ⁇ 0.027 ( Figure 1).
  • Differences between liver cancer and healthy subjects and other malignant diseases and healthy subjects were statistically significant (P 0 0.0001 and P 0 0.01, respectively) ( Figure 1).
  • the amount of autoantibodies to hTERT increased with the progression of the disease in chronic liver disease.
  • antibodies to the unrelated protein, serum albumin (BSA) were not found in the sera of patients and healthy individuals.
  • the measured values of anti-hTERT antibodies in 10 patients with autoimmune diseases were compared with the measured values of antibodies in 9 healthy subjects.
  • Example 3 (1) At the time of blood collection, informed consent was obtained in writing in the same manner as in Example 3 (1).
  • the measurement of the anti-hTERT antibody was performed by the ELISA method in the same manner as in Example 3 (5), and the statistical analysis was performed in the same manner.
  • the autoimmune disease patient had a high anti-hTERT antibody titer.
  • the value was 0.286 ⁇ 0.022 for healthy subjects, 0.503 for the autoimmune disease patients, and 0.074 for the autoimmune disease patients, which was significantly higher in the autoimmune disease patients (Fig. 2).
  • This means that anti-hTERT autoantibodies can be measured not only in liver disease and liver cancer, but also in other diseases, and reflect the condition Indicates that information can be obtained.
  • the present invention provides a method for detecting an anti-telomerase antibody in a biological sample.
  • the method of the present invention to detect, for example, an anti-telomerase antibody in the serum of a patient, it becomes possible to detect various diseases, for example, cancer such as liver cancer, and autoimmune diseases.
  • cancer such as liver cancer
  • autoimmune diseases for example, cancer such as liver cancer, and autoimmune diseases.

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Abstract

L'invention concerne une méthode permettant de détecter un anticorps anti-télomérase destiné à être utilisé comme marqueur de maladie. D'une manière plus spécifique, l'invention concerne une méthode permettant de détecter un anticorps anti-télomérase, cette méthode consistant à faire réagir une protéine constituant la télomérase, un ARN messager, un complexe constitué de ces derniers ou des fragments de ces derniers avec l'anticorps anti-télomérase dans un échantillon biologique et à analyser le produit de la réaction. On observe des titres d'anticorps anti-télomérase sensiblement plus élevés dans des échantillons issus de sujets souffrant de diverses maladies que dans des échantillons issus de sujets normaux, ce qui signifie que l'anticorps peut être utilisé comme marqueur de maladie indicateur d'états pathologiques.
PCT/JP2002/013310 2001-12-21 2002-12-19 Procede permettant de detecter un anticorps anti-telomerase WO2003054545A1 (fr)

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JP2003555207A JPWO2003054545A1 (ja) 2001-12-21 2002-12-19 抗テロメラーゼ抗体の検出方法
AU2002357613A AU2002357613A1 (en) 2001-12-21 2002-12-19 Method of detecting antitelomerase antibody

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11127874A (ja) * 1992-05-13 1999-05-18 Univ Texas Syst 検出方法
JPH11253177A (ja) * 1996-10-01 1999-09-21 Geron Corp ヒトテロメラーゼ触媒性サブユニットプロモーター
JPH11276182A (ja) * 1997-12-26 1999-10-12 Chugai Pharmaceut Co Ltd 逆転写酵素モチーフを有する新規遺伝子

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11127874A (ja) * 1992-05-13 1999-05-18 Univ Texas Syst 検出方法
JPH11253177A (ja) * 1996-10-01 1999-09-21 Geron Corp ヒトテロメラーゼ触媒性サブユニットプロモーター
JPH11276182A (ja) * 1997-12-26 1999-10-12 Chugai Pharmaceut Co Ltd 逆転写酵素モチーフを有する新規遺伝子

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MASUTOMI K. ET AL.: "Identification of serum anti-human telome rase reverse transcriptase (hTERT) auto-antibodies during progression to hepatocellular carcinoma", ONCOGENE, vol. 21, no. 38, August 2002 (2002-08-01), pages 5946 - 5950, XP002962916 *

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