WO2024096042A1 - METHOD FOR MEASURING AMOUNT OF Vanin-1 PROTEIN IN SAMPLE BY MEANS OF IMMUNOCHROMATOGRAPHY AND TESTER - Google Patents
METHOD FOR MEASURING AMOUNT OF Vanin-1 PROTEIN IN SAMPLE BY MEANS OF IMMUNOCHROMATOGRAPHY AND TESTER Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Definitions
- This specification discloses a method for measuring Vanin-1 protein in a sample by immunochromatography, and a test device.
- Vanin-1 in urine is a marker of renal function that has been reported to increase with cisplatin-induced renal damage (Non-Patent Document 1) and with decreased renal function in hypertensive patients (Non-Patent Document 2).
- Non-Patent Documents 1 and 2 Vanin-1 protein is measured using the ELISA method.
- An objective of the present invention is to provide a measurement system using immunochromatography that enables measurement of Vanin-1 protein in a shorter period of time.
- a method for measuring Vanin-1 protein in a sample by immunochromatography comprising: contacting the sample with an antibody; measuring a complex between the antibody and Vanin-1 protein on the test strip; an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen; anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 229 to alanine 379 as an antigen, or anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 389 to arginine 497 as an antigen, either alone or in combination with said antibodies; Use NanoAct® or gold colloids as detection particles; Method.
- Item 2 As a capture antibody, Using an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 229th valine to the 379th alanine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen, As a detection antibody, The method according to claim 1, which uses an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 389th valine to the 497th arginine of the Vanin-1 polypeptide
- Item 3 The method according to item 1, wherein the measurement is a qualitative measurement, a semi-quantitative measurement, or a quantitative measurement.
- Item 4 The method according to Item 1, wherein the sample is urine.
- Item 5. The method according to Item 4, wherein a measured value obtained based on the measurement is equal to or greater than a reference value, suggests that the subject from whom the urine was collected has a renal disease.
- a test device for measuring Vanin-1 protein in a sample by immunochromatography comprising a test strip having a first portion through which a measurement sample containing a sample and a detection antibody labeled with a detection particle is permeated, or through which a sample or a dilution of the sample is permeated, and a second portion on which a capture antibody is immobilized, the second portion does not overlap the first portion;
- the capture antibody is an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen;
- a testing device comprising an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 229th valine to the 379th alanine as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 389th valine to the 497th arginine
- Vanin-1 protein can be measured in a shorter time.
- Example 1 shows the appearance of the test device
- B shows the appearance of another form of the test device.
- the following is a list of the antibodies used.
- the results of Example 1 are shown.
- the results of Example 2 are shown below.
- Measurement Method This invention relates to a method for measuring Vanin-1 protein by immunochromatography (hereinafter sometimes referred to as the "measurement method").
- the measurement method includes contacting a sample with an antibody and measuring a complex between the antibody and Vanin-1 protein on a test strip.
- Vanin-1 protein (hereinafter sometimes simply referred to as "Vanin-1") is a protein registered in humans as National Center for Biotechnology Information (NCBI) Reference Sequence: NP_004657.2, and is a polypeptide represented by sequence number 1. Immunochromatography is a method for measuring antigens and antibodies on a test strip.
- a measurement sample is prepared by mixing the sample with a detection antibody.
- the sample and the detection antibody can be mixed in a buffer with a pH of about 6.0 to 8.5.
- the buffer include Tris-buffer.
- the buffer may also contain a surfactant such as Triton (registered trademark)-X or Tween (registered trademark)-20 at a final concentration of about 0.5% to 2.0%, or bovine serum albumin (BSA) at a final concentration of about 0.05% to 2.0%.
- the detection antibody is labeled with a detection particle for visualizing the presence of the antigen-antibody complex.
- the measurement sample is permeated into the portion of the test strip where the capture antibody is not immobilized. If a complex of antigen and detection antibody is present in the measurement sample, the complex of antigen and detection antibody is immobilized in the portion where the capture antibody is immobilized. The complex of antigen and detection antibody becomes visible as the detection particles are concentrated due to immobilization.
- a test strip is prepared by applying or permeating a detection antibody labeled with detection particles in advance, and immobilizing a capture antibody in a portion of the liquid downstream of the portion where the detection antibody was applied/permeated.
- the specimen, or a mixture of the specimen and buffer is permeated in a portion of the liquid upstream of the portion where the detection antibody was applied/permeated. If the specimen contains an antigen, it comes into contact with the detection antibody as it permeates the test strip, and a complex between the antigen and the detection antibody is formed.
- the complex flows over the test strip in accordance with the permeating flow of the specimen, or the mixture of the specimen and buffer, and is immobilized on the immobilized capture antibody.
- the complex between the antigen and the detection antibody becomes visible as the detection particles become concentrated as the detection particles are immobilized.
- the antibody used in immunochromatography is not limited as long as it is capable of detecting Vanin-1 protein. It may be a polyclonal or monoclonal antibody.
- an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the first methionine to the 350th valine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen e.g., Anti-VNN1/Vanin-1 antibody: ab231056: abcam, etc.
- an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 22nd glutamine to the 192nd lysine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen e.g., Anti-VNN1 Antibody: PB10106: Boster Biological Technology, etc.
- anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO:1 Recombinant Vanin-1 (VNN1): RPC598Hu01: CLOUD-CLONE CO
- Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 229th valine to the 379th alanine of the Vanin-1 polypeptide represented by SEQ ID NO:1 e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu02: CLOUD-CLONE CORP., etc.
- SEQ ID NO:1 e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu02: CLOUD-CLONE CORP., etc.
- Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 232nd isoleucine to the 427th leucine of the Vanin-1 polypeptide represented by SEQ ID NO:1 e.g., Anti-VNN1/Vanin-1 antibody: ab96171: abcam, etc.
- an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 300th leucine to the 415th alanine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen e.g., Anti-VNN1 Polyclonal Antibody: LS-B15409: LifeSpan Biosciences, Inc., etc.
- An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 300th leucine to the 500th methionine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen e.g., Anti-VNN1/Vanin-1 antibody: ab205912: abcam, etc.
- An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 302nd serine to the 513th tryptophan of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen e.g.,
- mice There are no limitations on the animals that can be inoculated with the antigen to obtain the above-mentioned antibodies. Examples include rabbits, mice, rats, guinea pigs, goats, pigs, camels, etc.
- an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having the sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 is preferable to use as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having the sequence from the 229th valine to the 379th alanine as an antigen.
- the detection antibody is composed of: It is preferable to use an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 36th leucine to the 223rd leucine, or an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 389th valine to the 497th arginine.
- an anti-Vanin-1 antibody obtained from animals administered a polypeptide having the sequence from the 36th leucine to the 223rd leucine as an antigen are used in combination, a polyclonal antibody can be used as the antibody constituting the capture antibody, and the monoclonal antibody clone C4 antibody can be used as the detection antibody.
- NanoAct registered trademark particles for in vitro diagnostic drugs (Asahi Kasei Corporation, hereinafter sometimes simply referred to as "NanoAct"
- gold colloid particles can be used.
- the particle diameter of the gold colloid particles is about 5 to 400 nm, with 20 nm to 60 nm being preferred.
- Subjects may or may not have renal disease, such as impaired renal function or renal impairment.
- Samples include urine, blood samples (whole blood, serum, plasma), ascites, etc.
- Urine may be natural urine, pooled urine, catheter urine, urine obtained by bladder or kidney puncture, etc.
- the measurement may be a qualitative measurement that determines whether Vanin-1 is present in the sample.
- the presence or absence of Vanin-1 in the sample can be determined by visually determining whether or not the detection particles on the test strip are concentrated.
- the presence or absence of the detection particles may be determined using an immunochromatography reader (C10066-10, Hamamatsu Photonics).
- the measurement may also be a semi-quantitative measurement of the Vanin-1 concentration in the sample.
- a semi-quantitative measurement is a method of determining the Vanin-1 concentration in the sample as “none,” “low,” “moderate,” or “high” depending on the color intensity caused by the concentration of detection particles on the test strip. The determination may be made visually or using a reader.
- the measurement may be a quantitative measurement of the Vanin-1 concentration in the sample.
- Quantitative measurement is a method in which the color intensity caused by the concentration of detection particles on the test strip is measured by a reader or the like, and the concentration of Vanin-1 is quantified based on the measured value.
- the measured value of the color intensity at the portion on the test strip where the detection antibody is immobilized (actual value) and the measured value of the color intensity at the portion where the detection antibody is not immobilized (background value) are obtained, and the value obtained by subtracting the background value from the actual value or the value obtained by dividing the actual value by the background value is obtained as a normalized value.
- a standard sample with a known concentration of Vanin-1 is similarly measured to obtain a normalized value, and a regression equation (calibration curve) is created between the standard sample normalized value and the concentration of Vanin-1.
- the normalized value of the subject sample can be applied to the regression equation to obtain the measured value of the Vanin-1 concentration in the sample.
- the measured value of the Vanin-1 concentration in urine may be corrected by the blood creatinine concentration or the creatinine clearance value, etc., but this is not necessary.
- the osmolality of the urine may be measured at the same time. Urine osmolality can be measured by the test strip method, the freezing point depression method, etc. If these tests show that the urine osmolality is outside the standard range, the measured value of the Vanin-1 concentration can be corrected based on the urine osmolality, and if the urine osmolality is within the standard range, the measured value of the Vanin-1 concentration can be left uncorrected.
- Kidney diseases can include decreased renal function or renal impairment.
- urinary Vanin-1 concentration tends to increase faster than estimated glomerular filtration rate (eGFR) changes, so even in subjects with normal estimated glomerular filtration rate (eGFR), measuring urinary Vanin-1 concentration can detect kidney disease earlier.
- eGFR estimated glomerular filtration rate
- the reference value of Vanin-1 is a value for distinguishing between subjects with and without renal disease, and can be set appropriately based on indices such as sensitivity, specificity, positive predictive value, and negative predictive value obtained by ROC (Receiver Operating Characteristic curve) curve analysis obtained from the measured values of Vanin-1 in samples from subjects with and without renal disease.
- the reference value may also be the mean, median, maximum, third quartile, etc. of the measured values of Vanin-1 in subjects without renal disease.
- the reference value may also be the minimum or first quartile of the measured values of Vanin-1 in subjects with renal disease.
- test instrument 1 for measuring Vanin-1 protein in a specimen by immunochromatography will be described with reference to Fig. 1 (A).
- a measurement sample containing a specimen and a detection antibody is prepared in advance, and the measurement sample is applied to a test strip 11.
- the terms used in this section and in 1. above are incorporated herein by reference.
- the test device 1 includes a test strip 11 .
- Test strip 11 comprises at least a first portion 13 and a second portion 14 .
- the first portion 13 is a portion through which a measurement sample containing a specimen and a detection antibody that labels a detection particle is permeated.
- the second portion 14 is a portion through which a capture antibody is immobilized.
- the first portion 13 and the second portion 14 do not overlap.
- it is preferable that the first portion 13 and the second portion 14 are separated by about 0.5 cm to 2 cm.
- the test strip 11 is made of a membrane material such as nitrocellulose, mixed cellulose esters, cellulose acetate, cellulose nitrate, glass fiber, nylon, polyester, polyethylene, polyvinyl chloride, or polyvinylidene fluoride.
- a membrane material such as nitrocellulose, mixed cellulose esters, cellulose acetate, cellulose nitrate, glass fiber, nylon, polyester, polyethylene, polyvinyl chloride, or polyvinylidene fluoride.
- the measurement sample that has permeated the first portion 13 permeates the membranous material in the direction of the arrow shown in Figure 1 (A) and creates a flow. If a complex between Vanin-1 and the detection antibody is present in the measurement sample, a further complex is formed with the capture antibody immobilized in the second portion 14, and the complex between Vanin-1 and the detection antibody is immobilized in the second portion 14. As the detection particles are concentrated due to immobilization, the complex between the antigen and the detection antibody becomes visible.
- the test strip 11 may have a portion 16 on which a positive control antibody is immobilized, which indicates that the test is being performed correctly.
- the positive control antibody is an anti-immunoglobulin antibody or the like that can capture the antibody that constitutes the detection antibody.
- the positive control antibody will be an anti-rabbit immunoglobulin antibody or an anti-rabbit IgG antibody.
- the portion 16 on which the positive control antibody is immobilized does not overlap with the first portion 13 and the second portion 14.
- test device 1 may also include a case 12 for carrying test strip 11 .
- test device 2 for measuring Vanin-1 protein in a sample by immunochromatography will be described with reference to Fig. 1 (B) .
- a detection antibody is applied to or impregnated into a test strip 21.
- the terms used in this section and in the above 1. are defined by reference in their explanation in the above 1.
- the test device 2 includes a test strip 21 .
- Test strip 21 comprises at least a first portion 23 and a second portion 24. First portion 23 and second portion 24 do not overlap.
- the first portion 23 is permeated with urine or a diluted solution of urine.
- the second portion 24 corresponds to the second portion 14 of the testing device 1 .
- the optional parts 26 where the positive control antibody is immobilized and the case 22 correspond to the optional parts 16 where the positive control antibody is immobilized and the case 12 in the test device 1, respectively.
- the third portion 25 of the test device 2 is coated or impregnated with a detection antibody. Since the detection antibody must move with the flow of the liquid, it is not immobilized like the capture antibody or positive control antibody.
- Example 1 Antibodies Four types of commercially available antibody solutions were used, as shown in Figure 2. An Amicon centrifugal ultrafiltration filter (UFC510024; Merck; molecular weight cutoff 100 kD) was set in a tube, and each antibody solution was added to the Amicon filter unit in three portions, and centrifuged once each. The flow-through was discarded, and 1x TBS buffer was added to the Amicon filter unit to elute the antibodies. The antibody concentration was measured using the Pierce BCA Protein Assay kit, and the concentration of each antibody solution was adjusted to 1 mg/mL.
- UDC510024 Amicon centrifugal ultrafiltration filter
- Merck molecular weight cutoff 100 kD
- antibody solution PAC598Hu01 shown in Figure 2 will be abbreviated as “01”, antibody solution PAC598Hu02 as “02”, antibody solution PAC598Hu03 as “03”, and antibody solution MAC598Hu22 as “M”.
- NanoAct labeling (1) Reagents Detection particles: NanoAct Blue (Asahi Kasei) BL2CAA001 Lot; BL2QFLRCV Labeling buffer: 100mM Tris-HCl (pH7.0-9.0) Blocking solution: 1% Casein, 50 mM Tris-HCl (pH 9.0) Washing solution: 50 mM Tris-HCl (pH 9.0) Drying solution (storage solution): 0.2% Casein, 15% Sucrose, 50 mM Tris-HCl (pH 9.0) Sample diluent: 0.9% TritonX100, 0.1% BSA, 100mM Tris-HCl (pH 8.5)
- the mixture was centrifuged at 15,000 g for 15 minutes at 20° C., and the supernatant was removed.
- vi. 4.8 mL of washing buffer was added to the sediment from v. above, and the mixture was subjected to ultrasonic irradiation to disperse the sediment.
- the dispersion liquid of vi above was centrifuged at 15,000 g for 15 minutes at 20° C., and the supernatant was removed.
- viii. 82 ⁇ L of storage buffer was added to the sediment of vii. above, and the sediment was dispersed by ultrasonic irradiation.
- the dispersion liquid of vii above was stored in a refrigerator as a NanoAct-labeled antibody liquid.
- Colloidal gold labeling (1) Reagents Particles: Colloidal gold (40 nm) GOLD COLLOID, BBI Solutions, EM.GC40, BATCH 20040072 50 ⁇ L of 10 mM Tris-HCl (pH 8.0) was added to 100-150 ⁇ L of the colloidal gold stock solution.
- Antibody The antibody solution was diluted with 10 mM Tris-HCl (pH 8.0) to adjust the concentration to 0.2 mg/mL.
- 1% BSA (pH 8.0) solution BSA was added to 10 mM Tris-HCl (pH 8.0) to make the concentration 1% w/v.
- 1% PEG 100 mg of PEG (20000) was added to 9.9 g of water.
- BSA-PEG mixed solution Prepared by mixing 1% BSA (pH 8.0) solution and 1% PEG solution in a ratio of 9:1.
- Colloidal gold storage solution 50 mg of trehalose dihydrate in 1 mL of 1% BSA (pH 8.0) solution was added and prepared.
- the mixture was centrifuged at 8,000 g (7,000 rpm) for 10 minutes, and the supernatant was removed.
- iv. 1 mL of the BSA-PEG mixture was added to the sediment from iii above, and the sediment was dispersed using a Vortex mixer. It was then left to stand for 15 minutes.
- the gold colloid preservative was added to the dispersion liquid of iv. above, and the mixture was redispersed. 200 ⁇ L of the gold colloid preservative was added to the dispersion liquid containing antibodies 01 and 02, and 100 ⁇ L of the gold colloid preservative was added to the dispersion liquid containing antibodies 03 and M. vi.
- the redispersed solution obtained in v. above was stored in a refrigerator as a gold colloid-labeled antibody solution.
- Reagents Antigen solution Recombinant Vanin-1 (VNN1) RPC598Hu04 as antigen (vanin-1 Gln22 to Gly491 with N-terminal His Tag) (CLOUD-CLONE CORP) was used. The antigen was diluted using the antibody diluent described below.
- Antibody solution The antibody solutions prepared in the above 1. at 1 mg/mL were used.
- Sample diluent Tris-buffer (containing 0.9% TritonX-100 and 0.1% BSA) Labeled antibody solution: The NanoAct labeled antibody solution prepared in 2-1. (2) above, or the gold colloid labeled antibody solution prepared in 2-2. (2) above was used.
- Figure 3 shows the visual judgment results for each combination of capture antibody and detection antibody when NanoAct was labeled and when gold colloid was labeled. Spots that were visible to the naked eye were marked with "+", and those that were not visible were marked with "-”.
- the combination of capture antibody 01 and detection antibody M, or the combination of capture antibody M and detection antibody 01 allowed visual determination down to 1 ng/mL depending on the antigen concentration.
- Example 2 The same method as in Example 1 was used, with 01 used as the capture antibody and NanoAct-labeled and gold colloid-labeled antibody M used as the detection antibody.
- 20 ⁇ L of urine sample not spiked with antigen was added to the wells to which the labeled antibodies had been dropped, and mixed.
- the strip was inserted, and the absorbance was measured 10 minutes later using a reader (C10066-10, Hamamatsu Photonics).
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Abstract
The present invention addresses the problem of providing a measurement system employing immunochromatography, the measurement system being capable of measuring the amount of Vanin-1 protein in a shorter time. The problem is solved by a method for measuring the amount of Vanin-1 protein in a sample by means of immunochromatography, the method including: bringing the sample into contact with an antibody and measuring the amount of a complex of the antibody and the Vanin-1 protein on a test strip, wherein NanoAct (registered trademark) or a gold colloid is used as detection particles.
Description
本明細書には、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定する方法、及び検査器具が開示される。
This specification discloses a method for measuring Vanin-1 protein in a sample by immunochromatography, and a test device.
尿中のVanin-1は、シスプラチンによる腎障害（非特許文献１）、高血圧患者における腎機能低下に伴って上昇すること（非特許文献２）が報告されている腎機能マーカーである。
Vanin-1 in urine is a marker of renal function that has been reported to increase with cisplatin-induced renal damage (Non-Patent Document 1) and with decreased renal function in hypertensive patients (Non-Patent Document 2).
非特許文献１及び２では、ELISA法を用いてVanin-1タンパク質を測定している。
本発明は、より短時間でVanin-1タンパク質を測定可能な、イムノクロマトグラフィーを用いた測定系を提供することを課題とする。 In Non-Patent Documents 1 and 2, Vanin-1 protein is measured using the ELISA method.
An objective of the present invention is to provide a measurement system using immunochromatography that enables measurement of Vanin-1 protein in a shorter period of time.
本発明は、より短時間でVanin-1タンパク質を測定可能な、イムノクロマトグラフィーを用いた測定系を提供することを課題とする。 In
An objective of the present invention is to provide a measurement system using immunochromatography that enables measurement of Vanin-1 protein in a shorter period of time.
項１．検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定する方法であって、
検体と抗体とを接触させることと、
検査ストリップ上で抗体とVanin-1タンパク質との複合体を測定することを含み、
抗体として、配列番号１で表されるVanin-1ポリペプチドの
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体を単独、又は前記抗体を組み合わせて使用し、
検出粒子として、NanoAct(登録商標)、又は金コロイドを使用する、
方法。
項２．捕捉抗体として、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
配列番号１で表されるVanin-1ポリペプチドの第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体を使用し、
検出抗体として、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
配列番号１で表されるVanin-1ポリペプチドの第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体
を使用する、項１に記載の方法。
項３．測定が、定性的測定、半定量的測定又は定量的測定である、項１に記載の方法。
項４．検体が尿である、項１に記載の方法。
項５．測定に基づいて取得された測定値が基準値以上であるとき、尿を採取した被検者が腎疾患を有することを示唆する、項４に記載の方法。
項６．検体と検出粒子を標識した検出抗体を含む測定試料を浸透させる、又は検体もしくは検体の希釈液を浸透させる第１の部分と、捕捉抗体が固相化された第２の部分を有する検査ストリップを備えた、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定するための検査器具であって、
第２の部分は第１の部分と重ならず、
捕捉抗体は、
抗体として、配列番号１で表されるVanin-1ポリペプチドの
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、である
検査器具。Item 1. A method for measuring Vanin-1 protein in a sample by immunochromatography, comprising:
contacting the sample with an antibody;
measuring a complex between the antibody and Vanin-1 protein on the test strip;
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen;
anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 229 to alanine 379 as an antigen, or anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 389 to arginine 497 as an antigen, either alone or in combination with said antibodies;
Use NanoAct® or gold colloids as detection particles;
Method.
Item 2. As a capture antibody,
Using an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 229th valine to the 379th alanine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen,
As a detection antibody,
The method according toclaim 1, which uses an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 389th valine to the 497th arginine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen.
Item 3. The method according toitem 1, wherein the measurement is a qualitative measurement, a semi-quantitative measurement, or a quantitative measurement.
Item 4. The method according toItem 1, wherein the sample is urine.
Item 5. The method according to Item 4, wherein a measured value obtained based on the measurement is equal to or greater than a reference value, suggests that the subject from whom the urine was collected has a renal disease.
Item 6. A test device for measuring Vanin-1 protein in a sample by immunochromatography, comprising a test strip having a first portion through which a measurement sample containing a sample and a detection antibody labeled with a detection particle is permeated, or through which a sample or a dilution of the sample is permeated, and a second portion on which a capture antibody is immobilized,
the second portion does not overlap the first portion;
The capture antibody is
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen;
A testing device comprising an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 229th valine to the 379th alanine as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 389th valine to the 497th arginine as an antigen.
検体と抗体とを接触させることと、
検査ストリップ上で抗体とVanin-1タンパク質との複合体を測定することを含み、
抗体として、配列番号１で表されるVanin-1ポリペプチドの
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体を単独、又は前記抗体を組み合わせて使用し、
検出粒子として、NanoAct(登録商標)、又は金コロイドを使用する、
方法。
項２．捕捉抗体として、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
配列番号１で表されるVanin-1ポリペプチドの第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体を使用し、
検出抗体として、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
配列番号１で表されるVanin-1ポリペプチドの第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体
を使用する、項１に記載の方法。
項３．測定が、定性的測定、半定量的測定又は定量的測定である、項１に記載の方法。
項４．検体が尿である、項１に記載の方法。
項５．測定に基づいて取得された測定値が基準値以上であるとき、尿を採取した被検者が腎疾患を有することを示唆する、項４に記載の方法。
項６．検体と検出粒子を標識した検出抗体を含む測定試料を浸透させる、又は検体もしくは検体の希釈液を浸透させる第１の部分と、捕捉抗体が固相化された第２の部分を有する検査ストリップを備えた、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定するための検査器具であって、
第２の部分は第１の部分と重ならず、
捕捉抗体は、
抗体として、配列番号１で表されるVanin-1ポリペプチドの
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、である
検査器具。
contacting the sample with an antibody;
measuring a complex between the antibody and Vanin-1 protein on the test strip;
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen;
anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 229 to alanine 379 as an antigen, or anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 389 to arginine 497 as an antigen, either alone or in combination with said antibodies;
Use NanoAct® or gold colloids as detection particles;
Method.
Using an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 229th valine to the 379th alanine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen,
As a detection antibody,
The method according to
Item 3. The method according to
Item 4. The method according to
Item 5. The method according to Item 4, wherein a measured value obtained based on the measurement is equal to or greater than a reference value, suggests that the subject from whom the urine was collected has a renal disease.
Item 6. A test device for measuring Vanin-1 protein in a sample by immunochromatography, comprising a test strip having a first portion through which a measurement sample containing a sample and a detection antibody labeled with a detection particle is permeated, or through which a sample or a dilution of the sample is permeated, and a second portion on which a capture antibody is immobilized,
the second portion does not overlap the first portion;
The capture antibody is
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen;
A testing device comprising an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 229th valine to the 379th alanine as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 389th valine to the 497th arginine as an antigen.
より短時間でVanin-1タンパク質を測定できる。
Vanin-1 protein can be measured in a shorter time.
１．測定方法
Vanin-1タンパク質をイムノクロマトグラフィーにより測定する方法（以下、「測定方法」と称することがある）に関する。
測定方法は、検体と抗体とを接触させることと、検査ストリップ上で抗体とVanin-1タンパク質との複合体を測定することを含む。 1. Measurement Method This invention relates to a method for measuring Vanin-1 protein by immunochromatography (hereinafter sometimes referred to as the "measurement method").
The measurement method includes contacting a sample with an antibody and measuring a complex between the antibody and Vanin-1 protein on a test strip.
Vanin-1タンパク質をイムノクロマトグラフィーにより測定する方法（以下、「測定方法」と称することがある）に関する。
測定方法は、検体と抗体とを接触させることと、検査ストリップ上で抗体とVanin-1タンパク質との複合体を測定することを含む。 1. Measurement Method This invention relates to a method for measuring Vanin-1 protein by immunochromatography (hereinafter sometimes referred to as the "measurement method").
The measurement method includes contacting a sample with an antibody and measuring a complex between the antibody and Vanin-1 protein on a test strip.
Vanin-1タンパク質（以下、単に「Vanin-1」と称することがある）は、ヒトの場合、National Center for Biotechnology Information (NCBI) Reference Sequence: NP_004657.2として登録されているタンパク質であり、配列番号１で表されるポリペプチドである。 イムノクロマトグラフィーは、検査ストリップ上で、抗原や抗体を測定する方法である。
Vanin-1 protein (hereinafter sometimes simply referred to as "Vanin-1") is a protein registered in humans as National Center for Biotechnology Information (NCBI) Reference Sequence: NP_004657.2, and is a polypeptide represented by sequence number 1. Immunochromatography is a method for measuring antigens and antibodies on a test strip.
イムノクロマトグラフィー法により検体中の抗原を測定する場合、はじめに検体と検出抗体を混合した測定試料を調製する。検体と検出抗体の混合は、pH6.0～8.5程度のバッファー内で行うことができる。バッファーは、Tris-buffer等を挙げることができる。また、バッファーは、終濃度で0.5～2.0％程度のTriton（登録商標）-X、Tween（登録商標）-20等の界面活性剤、終濃度で0.05％～2.0％程度のウシ血清アルブミン（BSA）等を含んでいてもよい。
検出抗体には、抗原抗体の複合体の存在を可視化するための検出粒子が標識されている。 When measuring an antigen in a sample by immunochromatography, a measurement sample is prepared by mixing the sample with a detection antibody. The sample and the detection antibody can be mixed in a buffer with a pH of about 6.0 to 8.5. Examples of the buffer include Tris-buffer. The buffer may also contain a surfactant such as Triton (registered trademark)-X or Tween (registered trademark)-20 at a final concentration of about 0.5% to 2.0%, or bovine serum albumin (BSA) at a final concentration of about 0.05% to 2.0%.
The detection antibody is labeled with a detection particle for visualizing the presence of the antigen-antibody complex.
検出抗体には、抗原抗体の複合体の存在を可視化するための検出粒子が標識されている。 When measuring an antigen in a sample by immunochromatography, a measurement sample is prepared by mixing the sample with a detection antibody. The sample and the detection antibody can be mixed in a buffer with a pH of about 6.0 to 8.5. Examples of the buffer include Tris-buffer. The buffer may also contain a surfactant such as Triton (registered trademark)-X or Tween (registered trademark)-20 at a final concentration of about 0.5% to 2.0%, or bovine serum albumin (BSA) at a final concentration of about 0.05% to 2.0%.
The detection antibody is labeled with a detection particle for visualizing the presence of the antigen-antibody complex.
続いて、捕捉抗体が固相化されている検査ストリップの捕捉抗体が固相化されていない部分に測定試料を浸透させる。測定試料に抗原と検出抗体の複合体が存在していれば、捕捉抗体を固相化した部分に抗原と検出抗体の複合体が固定化される。固定化に伴う検出粒子の濃縮に伴って抗原と検出抗体の複合体が可視化される。
Then, the measurement sample is permeated into the portion of the test strip where the capture antibody is not immobilized. If a complex of antigen and detection antibody is present in the measurement sample, the complex of antigen and detection antibody is immobilized in the portion where the capture antibody is immobilized. The complex of antigen and detection antibody becomes visible as the detection particles are concentrated due to immobilization.
あるいは、あらかじめ検出粒子を標識した検出抗体を塗布するか又は浸透させ、検出抗体が塗布／浸透した部分よりも液体の流れの下流となる部分に捕捉抗体を固相化した検査ストリップを準備する。検体、又は検体とバッファーの混合液を検出抗体が塗布／浸透した部分よりも液体の流れの上流となる部分に浸透させる。検体に抗原が含まれる場合、検査ストリップに浸透していく過程において、検出抗体と接触し、抗原と検出抗体の複合体が形成される。複合体は、検体、又は検体とバッファーの混合液の浸透していく流れにしたがって検査ストリップ上を流れ、固相化した捕捉抗体に固定化される。固定化に伴う検出粒子の濃縮に伴って抗原と検出抗体の複合体が可視化される。
Alternatively, a test strip is prepared by applying or permeating a detection antibody labeled with detection particles in advance, and immobilizing a capture antibody in a portion of the liquid downstream of the portion where the detection antibody was applied/permeated. The specimen, or a mixture of the specimen and buffer, is permeated in a portion of the liquid upstream of the portion where the detection antibody was applied/permeated. If the specimen contains an antigen, it comes into contact with the detection antibody as it permeates the test strip, and a complex between the antigen and the detection antibody is formed. The complex flows over the test strip in accordance with the permeating flow of the specimen, or the mixture of the specimen and buffer, and is immobilized on the immobilized capture antibody. The complex between the antigen and the detection antibody becomes visible as the detection particles become concentrated as the detection particles are immobilized.
イムノクロマトグラフィーに用いる抗体は、Vanin-1タンパク質を検出できる抗体である限り制限されない。ポリクローナル抗体であってもモノクローナル抗体であってもよい。
The antibody used in immunochromatography is not limited as long as it is capable of detecting Vanin-1 protein. It may be a polyclonal or monoclonal antibody.
抗体として、例えば、
配列番号１で表されるVanin-1ポリペプチドの第１番目のメチオニンから第３５０番目のバリンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1/Vanin-1 抗体：ab231056：abcam等）、
配列番号１で表されるVanin-1ポリペプチドの第２２番目のグルタミンから第１９２番目のリシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1 Antibody抗体：PB10106：Boster Biological Technology等）、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチド（Recombinant Vanin-1 (VNN1)：RPC598Hu01：CLOUD-CLONE CORP.等）を抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Polyclonal antibody to Vanin1 (VNN1)：PAC598Hu01：CLOUD-CLONE CORP.；Monoclonal antibody to Vanin1 (VNN1) ：MAC598Hu22（Clone No. C4）：CLOUD-CLONE CORP.等）、
配列番号１で表されるVanin-1ポリペプチドの第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチド（Recombinant Vanin-1 (VNN1)：RPC598Hu02：CLOUD-CLONE CORP.等）を抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Polyclonal antibody to Vanin1 (VNN1)：PAC598Hu02：CLOUD-CLONE CORP.等）、 配列番号１で表されるVanin-1ポリペプチドの第２３２番目のイソロイシンから第４２７番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1/Vanin-1 抗体：ab96171：abcam等）、
配列番号１で表されるVanin-1ポリペプチドの第２３２番目のイソロイシンから第４５７番目のアスパラギン酸までの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、VNN1 Polyclonal Antibody：PA5-22335：Thermo Fischer等）、又は
配列番号１で表されるVanin-1ポリペプチドの第２９８番目のリシンから第３９７番目のイソロイシンまでの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、VNN1 Monoclonal Antibody：H00008876-M05（Clone No. 2B10）：Thermo Fischer等）、
配列番号１で表されるVanin-1ポリペプチドの第３００番目のロイシンから第４１５番目のアラニンまでの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、Anti VNN1 Polyclonal Antibody：LS-B15409：LifeSpan Biosciences, Inc.等）、
配列番号１で表されるVanin-1ポリペプチドの第３００番目のロイシンから第５００番目のメチオニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1/Vanin-1 抗体：ab205912：abcam等）、
配列番号１で表されるVanin-1ポリペプチドの第３０２番目のセリンから第５１３番目のトリプトファンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、VNN1 Polyclonal antibody抗体：21745-1-AP：Proteintech Group, Inc.等）、
配列番号１で表されるVanin-1ポリペプチドの第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチド（Recombinant Vanin-1 (VNN1)：RPC598Hu03：CLOUD-CLONE CORP.等）を抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Polyclonal antibody to Vanin1 (VNN1)：PAC598Hu03：CLOUD-CLONE CORP.等）、及び
配列番号１で表されるVanin-1ポリペプチドの第１番目のメチオニンから第５１３番目のトリプトファンまでの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、VNN1 Polyclonal Antibody：PA5-75160：Thermo Fischer等）、
を単独、又は前記抗体を組み合わせて使用することができる。
上記抗体は、捕捉抗体としても、検出抗体を構成する抗体としても使用することができる。 As an antibody, for example,
An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the first methionine to the 350th valine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1/Vanin-1 antibody: ab231056: abcam, etc.);
an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 22nd glutamine to the 192nd lysine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1 Antibody: PB10106: Boster Biological Technology, etc.);
anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (Recombinant Vanin-1 (VNN1): RPC598Hu01: CLOUD-CLONE CORP., etc.) as an antigen (e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu01: CLOUD-CLONE CORP.; Monoclonal antibody to Vanin1 (VNN1): MAC598Hu22 (Clone No. C4): CLOUD-CLONE CORP., etc.);
Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 229th valine to the 379th alanine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu02: CLOUD-CLONE CORP., etc.) as an antigen; Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 232nd isoleucine to the 427th leucine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (e.g., Anti-VNN1/Vanin-1 antibody: ab96171: abcam, etc.);
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 232nd isoleucine to the 457th aspartic acid of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., VNN1 Polyclonal Antibody: PA5-22335: Thermo Fischer, etc.); or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 298th lysine to the 397th isoleucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., VNN1 Monoclonal Antibody: H00008876-M05 (Clone No. 2B10): Thermo Fischer, etc.);
an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 300th leucine to the 415th alanine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1 Polyclonal Antibody: LS-B15409: LifeSpan Biosciences, Inc., etc.);
An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 300th leucine to the 500th methionine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1/Vanin-1 antibody: ab205912: abcam, etc.);
An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 302nd serine to the 513th tryptophan of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., VNN1 Polyclonal antibody: 21745-1-AP: Proteintech Group, Inc., etc.);
Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 389th valine to the 497th arginine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu03: CLOUD-CLONE CORP., etc.) as an antigen; and anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 1st methionine to the 513th tryptophan of the Vanin-1 polypeptide represented by SEQ ID NO:1 as an antigen (e.g., VNN1 Polyclonal Antibody: PA5-75160: Thermo Fischer, etc.),
The antibodies can be used alone or in combination.
The above-mentioned antibody can be used as a capture antibody or as an antibody constituting a detection antibody.
配列番号１で表されるVanin-1ポリペプチドの第１番目のメチオニンから第３５０番目のバリンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1/Vanin-1 抗体：ab231056：abcam等）、
配列番号１で表されるVanin-1ポリペプチドの第２２番目のグルタミンから第１９２番目のリシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1 Antibody抗体：PB10106：Boster Biological Technology等）、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチド（Recombinant Vanin-1 (VNN1)：RPC598Hu01：CLOUD-CLONE CORP.等）を抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Polyclonal antibody to Vanin1 (VNN1)：PAC598Hu01：CLOUD-CLONE CORP.；Monoclonal antibody to Vanin1 (VNN1) ：MAC598Hu22（Clone No. C4）：CLOUD-CLONE CORP.等）、
配列番号１で表されるVanin-1ポリペプチドの第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチド（Recombinant Vanin-1 (VNN1)：RPC598Hu02：CLOUD-CLONE CORP.等）を抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Polyclonal antibody to Vanin1 (VNN1)：PAC598Hu02：CLOUD-CLONE CORP.等）、 配列番号１で表されるVanin-1ポリペプチドの第２３２番目のイソロイシンから第４２７番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1/Vanin-1 抗体：ab96171：abcam等）、
配列番号１で表されるVanin-1ポリペプチドの第２３２番目のイソロイシンから第４５７番目のアスパラギン酸までの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、VNN1 Polyclonal Antibody：PA5-22335：Thermo Fischer等）、又は
配列番号１で表されるVanin-1ポリペプチドの第２９８番目のリシンから第３９７番目のイソロイシンまでの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、VNN1 Monoclonal Antibody：H00008876-M05（Clone No. 2B10）：Thermo Fischer等）、
配列番号１で表されるVanin-1ポリペプチドの第３００番目のロイシンから第４１５番目のアラニンまでの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、Anti VNN1 Polyclonal Antibody：LS-B15409：LifeSpan Biosciences, Inc.等）、
配列番号１で表されるVanin-1ポリペプチドの第３００番目のロイシンから第５００番目のメチオニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Anti-VNN1/Vanin-1 抗体：ab205912：abcam等）、
配列番号１で表されるVanin-1ポリペプチドの第３０２番目のセリンから第５１３番目のトリプトファンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体（例えば、VNN1 Polyclonal antibody抗体：21745-1-AP：Proteintech Group, Inc.等）、
配列番号１で表されるVanin-1ポリペプチドの第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチド（Recombinant Vanin-1 (VNN1)：RPC598Hu03：CLOUD-CLONE CORP.等）を抗原として投与した動物から取得された抗Vanin-1抗体（例えば、Polyclonal antibody to Vanin1 (VNN1)：PAC598Hu03：CLOUD-CLONE CORP.等）、及び
配列番号１で表されるVanin-1ポリペプチドの第１番目のメチオニンから第５１３番目のトリプトファンまでの配列を有するポリペプチドを抗原として投与された動物から取得された抗Vanin-1抗体（例えば、VNN1 Polyclonal Antibody：PA5-75160：Thermo Fischer等）、
を単独、又は前記抗体を組み合わせて使用することができる。
上記抗体は、捕捉抗体としても、検出抗体を構成する抗体としても使用することができる。 As an antibody, for example,
An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the first methionine to the 350th valine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1/Vanin-1 antibody: ab231056: abcam, etc.);
an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 22nd glutamine to the 192nd lysine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1 Antibody: PB10106: Boster Biological Technology, etc.);
anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (Recombinant Vanin-1 (VNN1): RPC598Hu01: CLOUD-CLONE CORP., etc.) as an antigen (e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu01: CLOUD-CLONE CORP.; Monoclonal antibody to Vanin1 (VNN1): MAC598Hu22 (Clone No. C4): CLOUD-CLONE CORP., etc.);
Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 229th valine to the 379th alanine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu02: CLOUD-CLONE CORP., etc.) as an antigen; Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 232nd isoleucine to the 427th leucine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (e.g., Anti-VNN1/Vanin-1 antibody: ab96171: abcam, etc.);
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 232nd isoleucine to the 457th aspartic acid of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., VNN1 Polyclonal Antibody: PA5-22335: Thermo Fischer, etc.); or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 298th lysine to the 397th isoleucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., VNN1 Monoclonal Antibody: H00008876-M05 (Clone No. 2B10): Thermo Fischer, etc.);
an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 300th leucine to the 415th alanine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1 Polyclonal Antibody: LS-B15409: LifeSpan Biosciences, Inc., etc.);
An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 300th leucine to the 500th methionine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., Anti-VNN1/Vanin-1 antibody: ab205912: abcam, etc.);
An anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 302nd serine to the 513th tryptophan of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen (e.g., VNN1 Polyclonal antibody: 21745-1-AP: Proteintech Group, Inc., etc.);
Anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 389th valine to the 497th arginine of the Vanin-1 polypeptide represented by SEQ ID NO:1 (e.g., Polyclonal antibody to Vanin1 (VNN1): PAC598Hu03: CLOUD-CLONE CORP., etc.) as an antigen; and anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from the 1st methionine to the 513th tryptophan of the Vanin-1 polypeptide represented by SEQ ID NO:1 as an antigen (e.g., VNN1 Polyclonal Antibody: PA5-75160: Thermo Fischer, etc.),
The antibodies can be used alone or in combination.
The above-mentioned antibody can be used as a capture antibody or as an antibody constituting a detection antibody.
上記抗体を取得するために抗原を接種される動物は制限されない。例えば、ウサギ、マウス、ラット、モルモット（ギニアピッグ）、ヤギ、ブタ、ラクダ等を挙げることができる。
There are no limitations on the animals that can be inoculated with the antigen to obtain the above-mentioned antibodies. Examples include rabbits, mice, rats, guinea pigs, goats, pigs, camels, etc.
捕捉抗体として、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体
を使用することが好ましい。 As a capture antibody,
It is preferable to use an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having the sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having the sequence from the 229th valine to the 379th alanine as an antigen.
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体
を使用することが好ましい。 As a capture antibody,
It is preferable to use an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having the sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having the sequence from the 229th valine to the 379th alanine as an antigen.
検出抗体を構成する抗体として、
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体
を使用することが好ましい。
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体同士を組み合わせて使用する場合、捕捉抗体を構成する抗体としてポリクローナル抗体を使用し、検出抗体としてモノクローナル抗体クローンC4抗体を使用することができる。 The detection antibody is composed of:
It is preferable to use an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 36th leucine to the 223rd leucine, or an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 389th valine to the 497th arginine.
When anti-Vanin-1 antibodies obtained from animals administered a polypeptide having the sequence from the 36th leucine to the 223rd leucine as an antigen are used in combination, a polyclonal antibody can be used as the antibody constituting the capture antibody, and the monoclonal antibody clone C4 antibody can be used as the detection antibody.
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体
を使用することが好ましい。
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体同士を組み合わせて使用する場合、捕捉抗体を構成する抗体としてポリクローナル抗体を使用し、検出抗体としてモノクローナル抗体クローンC4抗体を使用することができる。 The detection antibody is composed of:
It is preferable to use an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 36th leucine to the 223rd leucine, or an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 389th valine to the 497th arginine.
When anti-Vanin-1 antibodies obtained from animals administered a polypeptide having the sequence from the 36th leucine to the 223rd leucine as an antigen are used in combination, a polyclonal antibody can be used as the antibody constituting the capture antibody, and the monoclonal antibody clone C4 antibody can be used as the detection antibody.
検出粒子は、イムノクロマトグラフィーで、抗原と検出抗体の複合体を検出できる限り制限されない。例えば、体外診断薬用標識粒子NanoAct（登録商標）（旭化成株式会社、以下、単に「NanoAct」と称することがある）、金コロイド粒子を使用することができる。NanoActの色は特に制限されない。金コロイド粒子の粒子径は5～400nm程度であるが、中でも20nm～60nmが好ましい。
There are no limitations on the detection particles, so long as they can detect a complex of antigen and detection antibody by immunochromatography. For example, NanoAct (registered trademark) particles for in vitro diagnostic drugs (Asahi Kasei Corporation, hereinafter sometimes simply referred to as "NanoAct") and gold colloid particles can be used. There are no particular limitations on the color of NanoAct. The particle diameter of the gold colloid particles is about 5 to 400 nm, with 20 nm to 60 nm being preferred.
検査ストリップについては、次項で詳細に説明する。
Test strips are explained in more detail in the next section.
被検者は、腎機能低下や腎障害等の腎疾患を有する者であっても有さない者であってもよい。
Subjects may or may not have renal disease, such as impaired renal function or renal impairment.
検体として、尿、血液試料（全血、血清、血漿）、腹水等を挙げることができる。尿は自然尿、蓄尿、カテーテル尿、膀胱や腎臓の穿刺尿等であってもよい。
Samples include urine, blood samples (whole blood, serum, plasma), ascites, etc. Urine may be natural urine, pooled urine, catheter urine, urine obtained by bladder or kidney puncture, etc.
前記測定は、Vanin-1が検体中に存在するかいなかを判定する定性的な測定であってもよい。定性的な測定の場合、検査ストリップ上の検出粒子の濃縮の有無を目視で判定し、検体中のVanin-1の有無を判定することができる。また、イムノクロマトリーダー（C10066-10、浜松ホトニクス）を使って、検出粒子の濃縮の有無を判定してもよい。
The measurement may be a qualitative measurement that determines whether Vanin-1 is present in the sample. In the case of a qualitative measurement, the presence or absence of Vanin-1 in the sample can be determined by visually determining whether or not the detection particles on the test strip are concentrated. Alternatively, the presence or absence of the detection particles may be determined using an immunochromatography reader (C10066-10, Hamamatsu Photonics).
また、前記測定は、検体中のVanin-1濃度の半定量的な測定であってもよい。半定量的な測定とは、検査ストリップ上の検出粒子の濃縮によって生じる色の濃さに応じて検体中のVanin-1の濃度を「無」、「低度」、「中等度」、「高度」のように判定する方法である。判定は、目視で行ってもよく、リーダーを使用してもよい。
The measurement may also be a semi-quantitative measurement of the Vanin-1 concentration in the sample. A semi-quantitative measurement is a method of determining the Vanin-1 concentration in the sample as "none," "low," "moderate," or "high" depending on the color intensity caused by the concentration of detection particles on the test strip. The determination may be made visually or using a reader.
また、前記測定は、検体中のVanin-1濃度の定量的な測定であってもよい。定量的な測定とは、検査ストリップ上において検出粒子の濃縮によって生じる色の濃さをリーダー等によって計測し、計測値に基づいて、Vanin-1の濃度を定量化する方法である。定量の場合、例えば、検査ストリップ上で検出抗体を固相化している箇所の色の濃さの計測値（実測値）と検出抗体を固相化していない箇所の色の濃さの計測値（バックグラウンド値）を取得し、実測値からバックグラウンド値を差し引いた値、もしくは実測値をバックグラウンド値で除した値を正規化値として取得する。また、別途Vanin-1の濃度が既知の標準試料を用いて同様に測定し正規化値を得て、標準試料正規化値とVanin-1の濃度との間で回帰式（検量線）を作成する。被検者検体の正規化値を回帰式に当てはめ検体中のVanin-1濃度の測定値とすることができる。
The measurement may be a quantitative measurement of the Vanin-1 concentration in the sample. Quantitative measurement is a method in which the color intensity caused by the concentration of detection particles on the test strip is measured by a reader or the like, and the concentration of Vanin-1 is quantified based on the measured value. In the case of quantification, for example, the measured value of the color intensity at the portion on the test strip where the detection antibody is immobilized (actual value) and the measured value of the color intensity at the portion where the detection antibody is not immobilized (background value) are obtained, and the value obtained by subtracting the background value from the actual value or the value obtained by dividing the actual value by the background value is obtained as a normalized value. In addition, a standard sample with a known concentration of Vanin-1 is similarly measured to obtain a normalized value, and a regression equation (calibration curve) is created between the standard sample normalized value and the concentration of Vanin-1. The normalized value of the subject sample can be applied to the regression equation to obtain the measured value of the Vanin-1 concentration in the sample.
また、尿中のVanin-1濃度の測定値は、血中クレアチニン濃度や、クレアチニンクリアランス値等で補正を行ってもよいが、行わなくてもよい。尿が必要以上に薄くないかを確認するため、尿の浸透圧等を同時に測定してもよい。尿浸透圧は、試験紙法、氷点降下法等で測定が可能である。これらの試験により、尿浸透圧が基準範囲外の時には、尿浸透圧に基づいてVanin-1濃度の測定値を補正し、尿浸透圧が基準範囲内の時には、Vanin-1濃度の測定値を補正しないとしてもよい。
The measured value of the Vanin-1 concentration in urine may be corrected by the blood creatinine concentration or the creatinine clearance value, etc., but this is not necessary. To check that the urine is not unnecessarily dilute, the osmolality of the urine may be measured at the same time. Urine osmolality can be measured by the test strip method, the freezing point depression method, etc. If these tests show that the urine osmolality is outside the standard range, the measured value of the Vanin-1 concentration can be corrected based on the urine osmolality, and if the urine osmolality is within the standard range, the measured value of the Vanin-1 concentration can be left uncorrected.
尿を検体とする場合であって、前記検体中のVanin-1濃度の測定値が基準値以上であるとき、尿を採取した被検者が腎疾患を有することを示唆する。
When urine is used as a sample, if the measured concentration of Vanin-1 in the sample is equal to or higher than the reference value, it suggests that the subject from whom the urine was collected has renal disease.
腎疾患には、腎機能低下又は腎障害等が含まれ得る。特に尿中Vanin-1濃度は、推定糸球体濾過率（eGFR）が変動するよりも早く増加する傾向があるため、推定糸球体濾過率（eGFR）が正常な被検者であっても、尿中Vanin-1濃度を測定することによって、より早く腎疾患を検出可能となる。
Kidney diseases can include decreased renal function or renal impairment. In particular, urinary Vanin-1 concentration tends to increase faster than estimated glomerular filtration rate (eGFR) changes, so even in subjects with normal estimated glomerular filtration rate (eGFR), measuring urinary Vanin-1 concentration can detect kidney disease earlier.
Vanin-1の基準値は、腎疾患を有する被検者と有さない被検者を判別するための値であり、例えば、腎疾患を有する被検者と有さない被検者の検体中のVanin-1の測定値から得られるROC(Receiver Operatorating Characteristic curve、受信者動作特性曲線)曲線解析によって求められる感度、特異度、陽性的中率、陰性的中率などの指標に基づいて適宜設定することができる。また、基準値は、腎疾患を有さない被検者のVanin-1の測定値の平均値、中央値、最大値、第３四分位等としてもよい。基準値は、腎疾患を有する被検者のVanin-1の測定値の最小値、第１四分位としてもよい。
The reference value of Vanin-1 is a value for distinguishing between subjects with and without renal disease, and can be set appropriately based on indices such as sensitivity, specificity, positive predictive value, and negative predictive value obtained by ROC (Receiver Operating Characteristic curve) curve analysis obtained from the measured values of Vanin-1 in samples from subjects with and without renal disease. The reference value may also be the mean, median, maximum, third quartile, etc. of the measured values of Vanin-1 in subjects without renal disease. The reference value may also be the minimum or first quartile of the measured values of Vanin-1 in subjects with renal disease.
２．検査器具
（１）第１の実施形態
図１（Ａ）を用いて、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定するための検査器具１について説明する。この実施形態は、あらかじめ検体と検出抗体を含む測定試料を調製し、測定試料を検査ストリップ１１にアプライする形態である。 本項で使用する用語であって、上記１．において使用されている用語は、上記１．の説明をここに援用する。
検査器具１は、検査ストリップ１１を備える。
検査ストリップ１１は、少なくとも第１の部分１３と第２の部分１４を備える。
第１の部分１３は、検体と検出粒子を標識した検出抗体を含む測定試料を浸透させる部分である。第２の部分１４は捕捉抗体を固相化する部分である。第１の部分１３と第２の部分１４は重ならない。また、第１の部分１３と第２の部分１４は0.5 cm～2 cm程度離れていることが好ましい。 2. Test Instrument (1) First EmbodimentA test instrument 1 for measuring Vanin-1 protein in a specimen by immunochromatography will be described with reference to Fig. 1 (A). In this embodiment, a measurement sample containing a specimen and a detection antibody is prepared in advance, and the measurement sample is applied to a test strip 11. The terms used in this section and in 1. above are incorporated herein by reference.
Thetest device 1 includes a test strip 11 .
Test strip 11 comprises at least a first portion 13 and a second portion 14 .
Thefirst portion 13 is a portion through which a measurement sample containing a specimen and a detection antibody that labels a detection particle is permeated. The second portion 14 is a portion through which a capture antibody is immobilized. The first portion 13 and the second portion 14 do not overlap. In addition, it is preferable that the first portion 13 and the second portion 14 are separated by about 0.5 cm to 2 cm.
（１）第１の実施形態
図１（Ａ）を用いて、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定するための検査器具１について説明する。この実施形態は、あらかじめ検体と検出抗体を含む測定試料を調製し、測定試料を検査ストリップ１１にアプライする形態である。 本項で使用する用語であって、上記１．において使用されている用語は、上記１．の説明をここに援用する。
検査器具１は、検査ストリップ１１を備える。
検査ストリップ１１は、少なくとも第１の部分１３と第２の部分１４を備える。
第１の部分１３は、検体と検出粒子を標識した検出抗体を含む測定試料を浸透させる部分である。第２の部分１４は捕捉抗体を固相化する部分である。第１の部分１３と第２の部分１４は重ならない。また、第１の部分１３と第２の部分１４は0.5 cm～2 cm程度離れていることが好ましい。 2. Test Instrument (1) First Embodiment
The
The
検査ストリップ１１は、ニトロセルロース、セルロース混合エステル、酢酸セルロース、硝酸セルロース、ガラス繊維、ナイロン、ポリエステル、ポリエチレン、ポリ塩化ビニル、ポリフッ化ビニリデン等の膜性素材から構成される。
The test strip 11 is made of a membrane material such as nitrocellulose, mixed cellulose esters, cellulose acetate, cellulose nitrate, glass fiber, nylon, polyester, polyethylene, polyvinyl chloride, or polyvinylidene fluoride.
第１の部分１３に浸透した測定試料は、図１（A）に示す矢印の方向に向かって膜性素材に浸透し流れを作る。測定試料中にVanin-1と検出抗体の複合体が存在する場合、第２の部分１４に固相化された捕捉抗体とさらなる複合体を形成し、第２の部分１４にVanin-1と検出抗体の複合体が固定化される。固定化に伴う検出粒子の濃縮に伴って抗原と検出抗体の複合体が可視化される。
The measurement sample that has permeated the first portion 13 permeates the membranous material in the direction of the arrow shown in Figure 1 (A) and creates a flow. If a complex between Vanin-1 and the detection antibody is present in the measurement sample, a further complex is formed with the capture antibody immobilized in the second portion 14, and the complex between Vanin-1 and the detection antibody is immobilized in the second portion 14. As the detection particles are concentrated due to immobilization, the complex between the antigen and the detection antibody becomes visible.
任意ではあるが、図１（Ａ）に示すように、検査ストリップ１１は、検査が正しく行われていることを示す、陽性コントロール抗体を固相化した部分１６を備えていてもよい。陽性コントロール抗体は、検出抗体を構成する抗体を捕捉することができる、抗イムノグロブリン抗体等である。例えば、検出抗体を構成する抗体がウサギで作製されたものである場合、陽性コントロール抗体は、抗ウサギイムノグロブリン抗体、抗ウサギＩｇＧ抗体となる。陽性コントロール抗体を固相化した部分１６は、第１の部分１３と第２の部分１４とは重ならない。
Optionally, as shown in FIG. 1(A), the test strip 11 may have a portion 16 on which a positive control antibody is immobilized, which indicates that the test is being performed correctly. The positive control antibody is an anti-immunoglobulin antibody or the like that can capture the antibody that constitutes the detection antibody. For example, if the antibody that constitutes the detection antibody is produced in a rabbit, the positive control antibody will be an anti-rabbit immunoglobulin antibody or an anti-rabbit IgG antibody. The portion 16 on which the positive control antibody is immobilized does not overlap with the first portion 13 and the second portion 14.
このような陽性コントロール抗体を固相化した部分１６を設けることにより、Vanin-1と検出抗体の複合体が存在せず第２の部分１４に検出粒子の濃縮が認められなかったとしても、陽性コントロール抗体固相化部分１６に検出粒子の濃縮が認められれば、少なくとも検出抗体は第２の部分１４を通過しており、検査自体は成立していたと判定することができる。すなわち、第２の部分１４に検出粒子の濃縮が認められず、陽性コントロール抗体を固相化した部分１６に検出粒子の濃縮が認められれば、測定試料に含まれるVanin-1は検出限界以下であった、もしくはVanin-1は存在していなかったと判定することができる。
また、任意ではあるが、検査器具１は、検査ストリップ１１を担持するケース１２を備えていてもよい。 By providing such aportion 16 on which the positive control antibody is immobilized, even if no complex between Vanin-1 and the detection antibody is present and no concentration of detection particles is observed in the second portion 14, if concentration of detection particles is observed in the positive control antibody immobilized portion 16, it can be determined that at least the detection antibody has passed through the second portion 14 and the test itself has been established. In other words, if no concentration of detection particles is observed in the second portion 14 but concentration of detection particles is observed in the portion 16 on which the positive control antibody is immobilized, it can be determined that Vanin-1 contained in the measurement sample was below the detection limit or that Vanin-1 was not present.
Optionally,test device 1 may also include a case 12 for carrying test strip 11 .
また、任意ではあるが、検査器具１は、検査ストリップ１１を担持するケース１２を備えていてもよい。 By providing such a
Optionally,
（２）第２の実施形態
図１（Ｂ）を用いて、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定するための検査器具２について説明する。この実施形態は、検査ストリップ２１に検出抗体が塗布、又は浸透されている。
本項で使用する用語であって、上記１．において使用されている用語は、上記１．の説明をここに援用する。
検査器具２は、検査ストリップ２１を備える。
検査ストリップ２１は、少なくとも第１の部分２３と第２の部分２４を備える。第１の部分２３と第２の部分２４は重ならない。
第１の部分２３には、尿、又は尿の希釈液を浸透させる。
第２の部分２４は、検査器具１における第２の部分１４に対応する。 (2) Second embodiment Atest device 2 for measuring Vanin-1 protein in a sample by immunochromatography will be described with reference to Fig. 1 (B) . In this embodiment, a detection antibody is applied to or impregnated into a test strip 21.
The terms used in this section and in the above 1. are defined by reference in their explanation in the above 1.
Thetest device 2 includes a test strip 21 .
Test strip 21 comprises at least a first portion 23 and a second portion 24. First portion 23 and second portion 24 do not overlap.
Thefirst portion 23 is permeated with urine or a diluted solution of urine.
Thesecond portion 24 corresponds to the second portion 14 of the testing device 1 .
図１（Ｂ）を用いて、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定するための検査器具２について説明する。この実施形態は、検査ストリップ２１に検出抗体が塗布、又は浸透されている。
本項で使用する用語であって、上記１．において使用されている用語は、上記１．の説明をここに援用する。
検査器具２は、検査ストリップ２１を備える。
検査ストリップ２１は、少なくとも第１の部分２３と第２の部分２４を備える。第１の部分２３と第２の部分２４は重ならない。
第１の部分２３には、尿、又は尿の希釈液を浸透させる。
第２の部分２４は、検査器具１における第２の部分１４に対応する。 (2) Second embodiment A
The terms used in this section and in the above 1. are defined by reference in their explanation in the above 1.
The
The
The
また、任意の部分である陽性コントロール抗体を固相化した部分２６及びケース２２は、それぞれ、検査器具１における陽性コントロール抗体を固相化した部分１６及びケース１２に対応する。
Furthermore, the optional parts 26 where the positive control antibody is immobilized and the case 22 correspond to the optional parts 16 where the positive control antibody is immobilized and the case 12 in the test device 1, respectively.
検査器具２における第３の部分２５には、検出抗体が塗布または浸透されている。ここで、検出抗体は液体の流れにしたがって移動しなければならないため、捕捉抗体や陽性コントロール抗体のように固相化はされていない。
The third portion 25 of the test device 2 is coated or impregnated with a detection antibody. Since the detection antibody must move with the flow of the liquid, it is not immobilized like the capture antibody or positive control antibody.
第１の部分２３に尿、又は尿の希釈液を浸透させると、これらは検査ストリップ２３に浸透し、はじめに検出抗体と接触する。尿中にVanin-1が存在していれば、第３の部分２５に存在している検出抗体と反応し、第２の部分２４に固相化された捕捉抗体に捕捉され、固定化される。
When urine or a diluted urine solution is permeated into the first portion 23, it permeates the test strip 23 and first comes into contact with the detection antibody. If Vanin-1 is present in the urine, it reacts with the detection antibody present in the third portion 25, and is captured by the capture antibody solid-phased in the second portion 24 and immobilized.
以下に実施例を示して本発明についてより詳細に説明する。しかし、本発明は、実施例に限定して解釈されるものではない。
The present invention will be explained in more detail below with reference to examples. However, the present invention should not be interpreted as being limited to these examples.
Ｉ．実施例１
１．抗体
図２に示す４種類の市販の抗体液を使用した。アミコン遠心式限外濾過フィルター（UFC510024; メルク; 分画分子量100kD）をチューブにセットし、各抗体液を３回に分けてアミコンフィルターユニットに添加し、１回ずつの遠心を行った。フロースルー液を捨て、１×TBSバッファーをアミコンフィルターユニットに添加し、抗体を溶出した。
抗体濃度をPierce BCA Protein Assay kitを使用して測定し、各抗体液の濃度を1 mg/mLに調整した。 I. Example 1
1. Antibodies Four types of commercially available antibody solutions were used, as shown in Figure 2. An Amicon centrifugal ultrafiltration filter (UFC510024; Merck;molecular weight cutoff 100 kD) was set in a tube, and each antibody solution was added to the Amicon filter unit in three portions, and centrifuged once each. The flow-through was discarded, and 1x TBS buffer was added to the Amicon filter unit to elute the antibodies.
The antibody concentration was measured using the Pierce BCA Protein Assay kit, and the concentration of each antibody solution was adjusted to 1 mg/mL.
１．抗体
図２に示す４種類の市販の抗体液を使用した。アミコン遠心式限外濾過フィルター（UFC510024; メルク; 分画分子量100kD）をチューブにセットし、各抗体液を３回に分けてアミコンフィルターユニットに添加し、１回ずつの遠心を行った。フロースルー液を捨て、１×TBSバッファーをアミコンフィルターユニットに添加し、抗体を溶出した。
抗体濃度をPierce BCA Protein Assay kitを使用して測定し、各抗体液の濃度を1 mg/mLに調整した。 I. Example 1
1. Antibodies Four types of commercially available antibody solutions were used, as shown in Figure 2. An Amicon centrifugal ultrafiltration filter (UFC510024; Merck;
The antibody concentration was measured using the Pierce BCA Protein Assay kit, and the concentration of each antibody solution was adjusted to 1 mg/mL.
以下、図２に示す抗体液PAC598Hu01を「01」と、抗体液PAC598Hu02を「02」と、抗体液PAC598Hu03を「03」と、抗体液MAC598Hu22を「M」と略記する。
Hereinafter, antibody solution PAC598Hu01 shown in Figure 2 will be abbreviated as "01", antibody solution PAC598Hu02 as "02", antibody solution PAC598Hu03 as "03", and antibody solution MAC598Hu22 as "M".
２．抗体の標識
２－１．NanoAct標識
（１）試薬
検出粒子：NanoAct 青（旭化成社）BL2CAA001 Lot; BL2QFLRCV
標識バッファー：100mM Tris-HCl (pH7.0-9.0)
ブロッキング液：1% Casein, 50 mM Tris-HCl (pH9.0)
洗浄液：50 mM Tris-HCl(pH9.0)
乾燥液（保存液）：0.2% Casein, 15% Sucrose, 50 mM Tris-HCl（pH9.0）
検体希釈液：0.9% TritonX100, 0.1% BSA, 100mM Tris-HCl (pH 8.5) 2. Antibody labeling 2-1. NanoAct labeling (1) Reagents Detection particles: NanoAct Blue (Asahi Kasei) BL2CAA001 Lot; BL2QFLRCV
Labeling buffer: 100mM Tris-HCl (pH7.0-9.0)
Blocking solution: 1% Casein, 50 mM Tris-HCl (pH 9.0)
Washing solution: 50 mM Tris-HCl (pH 9.0)
Drying solution (storage solution): 0.2% Casein, 15% Sucrose, 50 mM Tris-HCl (pH 9.0)
Sample diluent: 0.9% TritonX100, 0.1% BSA, 100mM Tris-HCl (pH 8.5)
２－１．NanoAct標識
（１）試薬
検出粒子：NanoAct 青（旭化成社）BL2CAA001 Lot; BL2QFLRCV
標識バッファー：100mM Tris-HCl (pH7.0-9.0)
ブロッキング液：1% Casein, 50 mM Tris-HCl (pH9.0)
洗浄液：50 mM Tris-HCl(pH9.0)
乾燥液（保存液）：0.2% Casein, 15% Sucrose, 50 mM Tris-HCl（pH9.0）
検体希釈液：0.9% TritonX100, 0.1% BSA, 100mM Tris-HCl (pH 8.5) 2. Antibody labeling 2-1. NanoAct labeling (1) Reagents Detection particles: NanoAct Blue (Asahi Kasei) BL2CAA001 Lot; BL2QFLRCV
Labeling buffer: 100mM Tris-HCl (pH7.0-9.0)
Blocking solution: 1% Casein, 50 mM Tris-HCl (pH 9.0)
Washing solution: 50 mM Tris-HCl (pH 9.0)
Drying solution (storage solution): 0.2% Casein, 15% Sucrose, 50 mM Tris-HCl (pH 9.0)
Sample diluent: 0.9% TritonX100, 0.1% BSA, 100mM Tris-HCl (pH 8.5)
（２）方法
i. 5mLのProtein LoBind（登録商標）チューブに、１％ NanoAct 青 40μLと標識バッファー 360μLを添加し混合した。その混合液に、1 mg/mLに調整した抗体液40μLを添加し、Vortexミキサーで攪拌した。
ii. 前記i．で調製した混合液を37℃にて、120分間インキュベーターでインキュベートした。
iii. インキュベーションが終了した混合液にブロッキング液を4.8 mL添加し、Vortexミキサーで攪拌した。
iv. 前記iii．で調製した混合液を37℃にて120分間インキュベーターでインキュベートした。
v. 上記ivにおいてインキュベーションが終了した混合液を、15000g, 15分間、 20℃の条件で遠心し、上清を除去した。
vi. 上記v.の沈渣に洗浄バッファーを4.8 mLを添加し、超音波を照射し、沈渣を分散させた。
vii. 上記viの分散液を、15000g, 15分間、 20℃の条件で遠心し、上清を除去した。
viii. 上記vii．の沈渣に保存バッファー82μLを添加し、超音波を照射し、沈渣を分散させた。
ix. 上記viii．の分散液をNanoAct標識抗体液として冷蔵保存した。 (2) Method
i. 40 μL of 1% NanoAct Blue and 360 μL of labeling buffer were added to a 5 mL Protein LoBind® tube and mixed. 40 μL of antibody solution adjusted to 1 mg/mL was added to the mixture and stirred with a Vortex mixer.
ii. The mixture prepared in i. above was incubated in an incubator at 37° C. for 120 minutes.
iii. After incubation, 4.8 mL of blocking solution was added to the mixture and stirred using a vortex mixer.
iv. The mixture prepared in iii. above was incubated in an incubator at 37° C. for 120 minutes.
v. After the incubation in iv above, the mixture was centrifuged at 15,000 g for 15 minutes at 20° C., and the supernatant was removed.
vi. 4.8 mL of washing buffer was added to the sediment from v. above, and the mixture was subjected to ultrasonic irradiation to disperse the sediment.
vii. The dispersion liquid of vi above was centrifuged at 15,000 g for 15 minutes at 20° C., and the supernatant was removed.
viii. 82 μL of storage buffer was added to the sediment of vii. above, and the sediment was dispersed by ultrasonic irradiation.
ix. The dispersion liquid of vii above was stored in a refrigerator as a NanoAct-labeled antibody liquid.
i. 5mLのProtein LoBind（登録商標）チューブに、１％ NanoAct 青 40μLと標識バッファー 360μLを添加し混合した。その混合液に、1 mg/mLに調整した抗体液40μLを添加し、Vortexミキサーで攪拌した。
ii. 前記i．で調製した混合液を37℃にて、120分間インキュベーターでインキュベートした。
iii. インキュベーションが終了した混合液にブロッキング液を4.8 mL添加し、Vortexミキサーで攪拌した。
iv. 前記iii．で調製した混合液を37℃にて120分間インキュベーターでインキュベートした。
v. 上記ivにおいてインキュベーションが終了した混合液を、15000g, 15分間、 20℃の条件で遠心し、上清を除去した。
vi. 上記v.の沈渣に洗浄バッファーを4.8 mLを添加し、超音波を照射し、沈渣を分散させた。
vii. 上記viの分散液を、15000g, 15分間、 20℃の条件で遠心し、上清を除去した。
viii. 上記vii．の沈渣に保存バッファー82μLを添加し、超音波を照射し、沈渣を分散させた。
ix. 上記viii．の分散液をNanoAct標識抗体液として冷蔵保存した。 (2) Method
i. 40 μL of 1% NanoAct Blue and 360 μL of labeling buffer were added to a 5 mL Protein LoBind® tube and mixed. 40 μL of antibody solution adjusted to 1 mg/mL was added to the mixture and stirred with a Vortex mixer.
ii. The mixture prepared in i. above was incubated in an incubator at 37° C. for 120 minutes.
iii. After incubation, 4.8 mL of blocking solution was added to the mixture and stirred using a vortex mixer.
iv. The mixture prepared in iii. above was incubated in an incubator at 37° C. for 120 minutes.
v. After the incubation in iv above, the mixture was centrifuged at 15,000 g for 15 minutes at 20° C., and the supernatant was removed.
vi. 4.8 mL of washing buffer was added to the sediment from v. above, and the mixture was subjected to ultrasonic irradiation to disperse the sediment.
vii. The dispersion liquid of vi above was centrifuged at 15,000 g for 15 minutes at 20° C., and the supernatant was removed.
viii. 82 μL of storage buffer was added to the sediment of vii. above, and the sediment was dispersed by ultrasonic irradiation.
ix. The dispersion liquid of vii above was stored in a refrigerator as a NanoAct-labeled antibody liquid.
２－２．金コロイド標識
（１）試薬
粒子：金コロイド (40 nm) GOLD COLLOID, BBI Solutions, EM.GC40,
BATCH 20040072
100-150μLの金コロイド原液に50μLの10 mM Tris-HCl (pH 8.0)を加えた。
抗体：10 mM Tris-HCl (pH 8.0)で抗体液を希釈し0.2 mg/mLに調整した。
1%BSA(pH8.0)溶液：10 mM Tris-HCl (pH 8.0)に1%w/vとなるようにBSAを添加した。
1%PEG：PEG(20000) 100 mgに9.9 gの水を添加した。
BSA-PEG混合液：1%BSA(pH8.0)溶液と1%PEG溶液を9:1の割合で混合し、調製した。
金コロイド保存液：50 mgのトレハロース二水和物に1%BSA(pH8.0)溶液1 mL
を添加し、調製した。 2-2. Colloidal gold labeling (1) Reagents Particles: Colloidal gold (40 nm) GOLD COLLOID, BBI Solutions, EM.GC40,
BATCH 20040072
50 μL of 10 mM Tris-HCl (pH 8.0) was added to 100-150 μL of the colloidal gold stock solution.
Antibody: The antibody solution was diluted with 10 mM Tris-HCl (pH 8.0) to adjust the concentration to 0.2 mg/mL.
1% BSA (pH 8.0) solution: BSA was added to 10 mM Tris-HCl (pH 8.0) to make theconcentration 1% w/v.
1% PEG: 100 mg of PEG (20000) was added to 9.9 g of water.
BSA-PEG mixed solution: Prepared by mixing 1% BSA (pH 8.0) solution and 1% PEG solution in a ratio of 9:1.
Colloidal gold storage solution: 50 mg of trehalose dihydrate in 1 mL of 1% BSA (pH 8.0) solution
was added and prepared.
（１）試薬
粒子：金コロイド (40 nm) GOLD COLLOID, BBI Solutions, EM.GC40,
BATCH 20040072
100-150μLの金コロイド原液に50μLの10 mM Tris-HCl (pH 8.0)を加えた。
抗体：10 mM Tris-HCl (pH 8.0)で抗体液を希釈し0.2 mg/mLに調整した。
1%BSA(pH8.0)溶液：10 mM Tris-HCl (pH 8.0)に1%w/vとなるようにBSAを添加した。
1%PEG：PEG(20000) 100 mgに9.9 gの水を添加した。
BSA-PEG混合液：1%BSA(pH8.0)溶液と1%PEG溶液を9:1の割合で混合し、調製した。
金コロイド保存液：50 mgのトレハロース二水和物に1%BSA(pH8.0)溶液1 mL
を添加し、調製した。 2-2. Colloidal gold labeling (1) Reagents Particles: Colloidal gold (40 nm) GOLD COLLOID, BBI Solutions, EM.GC40,
BATCH 20040072
50 μL of 10 mM Tris-HCl (pH 8.0) was added to 100-150 μL of the colloidal gold stock solution.
Antibody: The antibody solution was diluted with 10 mM Tris-HCl (pH 8.0) to adjust the concentration to 0.2 mg/mL.
1% BSA (pH 8.0) solution: BSA was added to 10 mM Tris-HCl (pH 8.0) to make the
1% PEG: 100 mg of PEG (20000) was added to 9.9 g of water.
BSA-PEG mixed solution: Prepared by mixing 1% BSA (pH 8.0) solution and 1% PEG solution in a ratio of 9:1.
Colloidal gold storage solution: 50 mg of trehalose dihydrate in 1 mL of 1% BSA (pH 8.0) solution
was added and prepared.
（２）方法
i. 1.5mLチューブに金コロイド溶液と抗体液（0.2 mg/mL)を容積比で1.5:1となるように加え、Vortexミキサーで攪拌した。01、及び02は200 μLの抗体液使用し、03、及びMは100 μLの抗体液を使用した。攪拌後の金コロイドと抗体の混合液を室温で15分間静置した。
ii. 上記i. にて静置が終了した後の金コロイドと抗体の混合液に1%BSA-PEG混合液を0.4 mL加え、Vortexミキサーで攪拌した後、室温で15分間静置した。
iii. 上記ii. にて静置が終了した後の混合液を、8,000g (7,000 rpm）で10分間遠心し、上清を除去した。
iv. 上記iiiの沈渣にBSA-PEG混合液を1 mL加え、Vortexミキサーで沈渣を分散させた。その後15分間静置した。
v. 上記iv.の分散液に、金コロイド保存液を加えて、再分散させた。01、及び02の抗体を含む分散液には金コロイド保存液を200μL添加し、03及びMの抗体を含む分散液には金コロイド保存液を100μL添加した。
vi. 上記v．の再分散液を金コロイド標識抗体液として冷蔵保存した。 (2) Method
i. A gold colloid solution and an antibody solution (0.2 mg/mL) were added to a 1.5 mL tube at a volume ratio of 1.5:1 and mixed with a Vortex mixer. 200 μL of antibody solution was used for 01 and 02, and 100 μL of antibody solution was used for 03 and M. After mixing, the mixture of gold colloid and antibody was left to stand at room temperature for 15 minutes.
ii. After leaving the mixture of gold colloid and antibody in step i above, 0.4 mL of a 1% BSA-PEG mixture was added, stirred with a vortex mixer, and then left to stand at room temperature for 15 minutes.
iii. After leaving the mixture in step ii. above, the mixture was centrifuged at 8,000 g (7,000 rpm) for 10 minutes, and the supernatant was removed.
iv. 1 mL of the BSA-PEG mixture was added to the sediment from iii above, and the sediment was dispersed using a Vortex mixer. It was then left to stand for 15 minutes.
v. The gold colloid preservative was added to the dispersion liquid of iv. above, and the mixture was redispersed. 200 μL of the gold colloid preservative was added to the dispersion liquid containing antibodies 01 and 02, and 100 μL of the gold colloid preservative was added to the dispersion liquid containing antibodies 03 and M.
vi. The redispersed solution obtained in v. above was stored in a refrigerator as a gold colloid-labeled antibody solution.
i. 1.5mLチューブに金コロイド溶液と抗体液（0.2 mg/mL)を容積比で1.5:1となるように加え、Vortexミキサーで攪拌した。01、及び02は200 μLの抗体液使用し、03、及びMは100 μLの抗体液を使用した。攪拌後の金コロイドと抗体の混合液を室温で15分間静置した。
ii. 上記i. にて静置が終了した後の金コロイドと抗体の混合液に1%BSA-PEG混合液を0.4 mL加え、Vortexミキサーで攪拌した後、室温で15分間静置した。
iii. 上記ii. にて静置が終了した後の混合液を、8,000g (7,000 rpm）で10分間遠心し、上清を除去した。
iv. 上記iiiの沈渣にBSA-PEG混合液を1 mL加え、Vortexミキサーで沈渣を分散させた。その後15分間静置した。
v. 上記iv.の分散液に、金コロイド保存液を加えて、再分散させた。01、及び02の抗体を含む分散液には金コロイド保存液を200μL添加し、03及びMの抗体を含む分散液には金コロイド保存液を100μL添加した。
vi. 上記v．の再分散液を金コロイド標識抗体液として冷蔵保存した。 (2) Method
i. A gold colloid solution and an antibody solution (0.2 mg/mL) were added to a 1.5 mL tube at a volume ratio of 1.5:1 and mixed with a Vortex mixer. 200 μL of antibody solution was used for 01 and 02, and 100 μL of antibody solution was used for 03 and M. After mixing, the mixture of gold colloid and antibody was left to stand at room temperature for 15 minutes.
ii. After leaving the mixture of gold colloid and antibody in step i above, 0.4 mL of a 1% BSA-PEG mixture was added, stirred with a vortex mixer, and then left to stand at room temperature for 15 minutes.
iii. After leaving the mixture in step ii. above, the mixture was centrifuged at 8,000 g (7,000 rpm) for 10 minutes, and the supernatant was removed.
iv. 1 mL of the BSA-PEG mixture was added to the sediment from iii above, and the sediment was dispersed using a Vortex mixer. It was then left to stand for 15 minutes.
v. The gold colloid preservative was added to the dispersion liquid of iv. above, and the mixture was redispersed. 200 μL of the gold colloid preservative was added to the dispersion
vi. The redispersed solution obtained in v. above was stored in a refrigerator as a gold colloid-labeled antibody solution.
３．スポット法（簡易イムノクロマト法）での測定
（１）試薬
抗原液：抗原としてRecombinant Vanin-1 (VNN1) RPC598Hu04
（vanin-1 Gln22～Gly491 with N-terminal His Tag）（CLOUD-CLONE CORP社）を使用した。
抗原は、後述する抗体希釈液を用いて希釈した。
抗体液：上記１．において1 mg/mLに調整した各抗体液を使用した。
検体希釈液：Tris-buffer（TritonX-100 0.9%、BSA 0.1%含有)
標識抗体液：上記２－１．（２）において作成したNanoAct標識抗体液、又は上記２－２．（２）において作製した金コロイド標識抗体液を使用した。 3. Measurement by spot method (simple immunochromatography method) (1) Reagents Antigen solution: Recombinant Vanin-1 (VNN1) RPC598Hu04 as antigen
(vanin-1 Gln22 to Gly491 with N-terminal His Tag) (CLOUD-CLONE CORP) was used.
The antigen was diluted using the antibody diluent described below.
Antibody solution: The antibody solutions prepared in the above 1. at 1 mg/mL were used.
Sample diluent: Tris-buffer (containing 0.9% TritonX-100 and 0.1% BSA)
Labeled antibody solution: The NanoAct labeled antibody solution prepared in 2-1. (2) above, or the gold colloid labeled antibody solution prepared in 2-2. (2) above was used.
（１）試薬
抗原液：抗原としてRecombinant Vanin-1 (VNN1) RPC598Hu04
（vanin-1 Gln22～Gly491 with N-terminal His Tag）（CLOUD-CLONE CORP社）を使用した。
抗原は、後述する抗体希釈液を用いて希釈した。
抗体液：上記１．において1 mg/mLに調整した各抗体液を使用した。
検体希釈液：Tris-buffer（TritonX-100 0.9%、BSA 0.1%含有)
標識抗体液：上記２－１．（２）において作成したNanoAct標識抗体液、又は上記２－２．（２）において作製した金コロイド標識抗体液を使用した。 3. Measurement by spot method (simple immunochromatography method) (1) Reagents Antigen solution: Recombinant Vanin-1 (VNN1) RPC598Hu04 as antigen
(vanin-1 Gln22 to Gly491 with N-terminal His Tag) (CLOUD-CLONE CORP) was used.
The antigen was diluted using the antibody diluent described below.
Antibody solution: The antibody solutions prepared in the above 1. at 1 mg/mL were used.
Sample diluent: Tris-buffer (containing 0.9% TritonX-100 and 0.1% BSA)
Labeled antibody solution: The NanoAct labeled antibody solution prepared in 2-1. (2) above, or the gold colloid labeled antibody solution prepared in 2-2. (2) above was used.
（２）スポット法による抗原の測定
以下の方法により、スポット法（簡易イムノクロマト法）に基づく測定系を構築した。測定は、抗体液と各標識抗体の組み合わせについて独立して3回行った。
i. ニトロセルロースメンブレン製の無担持ストリップに0.5 μLの01、02、03、Mの抗体液をそれぞれ滴下し、45℃にて30分間インキュベートし抗体を固相化した。
ii. 96穴プレートの各ウェルに、各標識抗体を3 μLずつ滴下した。 (2) Antigen measurement by spot method A measurement system based on the spot method (simple immunochromatography) was constructed as follows: Measurements were performed three times independently for each combination of antibody solution and each labeled antibody.
i. 0.5 μL of antibody solutions 01, 02, 03, and M were dropped onto unloaded nitrocellulose membrane strips and incubated at 45° C. for 30 minutes to immobilize the antibodies.
ii. 3 μL of each labeled antibody was dropped into each well of a 96-well plate.
以下の方法により、スポット法（簡易イムノクロマト法）に基づく測定系を構築した。測定は、抗体液と各標識抗体の組み合わせについて独立して3回行った。
i. ニトロセルロースメンブレン製の無担持ストリップに0.5 μLの01、02、03、Mの抗体液をそれぞれ滴下し、45℃にて30分間インキュベートし抗体を固相化した。
ii. 96穴プレートの各ウェルに、各標識抗体を3 μLずつ滴下した。 (2) Antigen measurement by spot method A measurement system based on the spot method (simple immunochromatography) was constructed as follows: Measurements were performed three times independently for each combination of antibody solution and each labeled antibody.
i. 0.5 μL of
ii. 3 μL of each labeled antibody was dropped into each well of a 96-well plate.
iii. 検体希釈液で各濃度（100 ng/mL、10 ng/mL、1 ng/mLに希釈した抗原希釈液20 μLを標識抗体を滴下したウェルに添加した後、ピペッティングにより混和した。またblankとして、標識抗体を滴下したウェルに検体希釈液を20μL加えて混和した。
iv. ストリップを入れ、10分後に目視判定にてスポットの有無を確認した。 iii. 20 μL of antigen dilution solution diluted with specimen dilution solution to each concentration (100 ng/mL, 10 ng/mL, 1 ng/mL) was added to the wells containing the labeled antibody, and then mixed by pipetting. As a blank, 20 μL of specimen dilution solution was added to the wells containing the labeled antibody, and mixed.
iv. The strip was placed and 10 minutes later, the presence or absence of spots was confirmed by visual inspection.
iv. ストリップを入れ、10分後に目視判定にてスポットの有無を確認した。 iii. 20 μL of antigen dilution solution diluted with specimen dilution solution to each concentration (100 ng/mL, 10 ng/mL, 1 ng/mL) was added to the wells containing the labeled antibody, and then mixed by pipetting. As a blank, 20 μL of specimen dilution solution was added to the wells containing the labeled antibody, and mixed.
iv. The strip was placed and 10 minutes later, the presence or absence of spots was confirmed by visual inspection.
４．結果
測定結果を図３に示す。図３には、NanoActを標識した場合、金コロイドを標識した場合における各捕捉抗体と検出抗体の組み合わせにおける目視判定結果を示す。目視にてスポットが視認できたものを「＋」、視認できなかったものを「－」とした。 4. Results The measurement results are shown in Figure 3. Figure 3 shows the visual judgment results for each combination of capture antibody and detection antibody when NanoAct was labeled and when gold colloid was labeled. Spots that were visible to the naked eye were marked with "+", and those that were not visible were marked with "-".
測定結果を図３に示す。図３には、NanoActを標識した場合、金コロイドを標識した場合における各捕捉抗体と検出抗体の組み合わせにおける目視判定結果を示す。目視にてスポットが視認できたものを「＋」、視認できなかったものを「－」とした。 4. Results The measurement results are shown in Figure 3. Figure 3 shows the visual judgment results for each combination of capture antibody and detection antibody when NanoAct was labeled and when gold colloid was labeled. Spots that were visible to the naked eye were marked with "+", and those that were not visible were marked with "-".
NanoActの場合、抗原の濃度に応じて1ng/mLまで目視判定可能であるのは、捕捉抗体01と検出抗体03またはMの組み合わせ、または捕捉抗体02と検出抗体01の組み合わせであった。
In the case of NanoAct, the combinations that allowed visual determination down to 1ng/mL depending on the antigen concentration were capture antibody 01 and detection antibody 03 or M, and capture antibody 02 and detection antibody 01.
金コロイドの場合、抗原の濃度に応じて1ng/mLまで目視判定可能であるのは、捕捉抗体01と検出抗体Mの組み合わせ、または捕捉抗体Mと検出抗体01の組み合わせであった。
In the case of gold colloid, the combination of capture antibody 01 and detection antibody M, or the combination of capture antibody M and detection antibody 01, allowed visual determination down to 1 ng/mL depending on the antigen concentration.
ＩＩ．実施例２
実施例１と同様の方法で、捕捉抗体に01を用い、検出抗体にNanoAct標識および金コロイド標識した抗体Mを用いた。尿試料に各濃度（100 ng/mL、10 ng/mL、1 ng/mL）となるように抗原をスパイクした検体液20 μLを、標識抗体を滴下したウェルに添加した後、ピペッティングにより混和した。またblankとして、標識抗体を滴下したウェルに抗原をスパイクしていない尿試料を20μL加えて混和した。ストリップを入れ、10分後にリーダー（C10066-10、浜松ホトニクス）で吸光度を測定した。 II. Example 2
The same method as in Example 1 was used, with 01 used as the capture antibody and NanoAct-labeled and gold colloid-labeled antibody M used as the detection antibody. 20 μL of sample solution in which antigens were spiked into urine samples to give various concentrations (100 ng/mL, 10 ng/mL, 1 ng/mL) was added to the wells to which the labeled antibodies had been dropped, and then mixed by pipetting. As a blank, 20 μL of urine sample not spiked with antigen was added to the wells to which the labeled antibodies had been dropped, and mixed. The strip was inserted, and the absorbance was measured 10 minutes later using a reader (C10066-10, Hamamatsu Photonics).
実施例１と同様の方法で、捕捉抗体に01を用い、検出抗体にNanoAct標識および金コロイド標識した抗体Mを用いた。尿試料に各濃度（100 ng/mL、10 ng/mL、1 ng/mL）となるように抗原をスパイクした検体液20 μLを、標識抗体を滴下したウェルに添加した後、ピペッティングにより混和した。またblankとして、標識抗体を滴下したウェルに抗原をスパイクしていない尿試料を20μL加えて混和した。ストリップを入れ、10分後にリーダー（C10066-10、浜松ホトニクス）で吸光度を測定した。 II. Example 2
The same method as in Example 1 was used, with 01 used as the capture antibody and NanoAct-labeled and gold colloid-labeled antibody M used as the detection antibody. 20 μL of sample solution in which antigens were spiked into urine samples to give various concentrations (100 ng/mL, 10 ng/mL, 1 ng/mL) was added to the wells to which the labeled antibodies had been dropped, and then mixed by pipetting. As a blank, 20 μL of urine sample not spiked with antigen was added to the wells to which the labeled antibodies had been dropped, and mixed. The strip was inserted, and the absorbance was measured 10 minutes later using a reader (C10066-10, Hamamatsu Photonics).
測定結果を図４に示す。
いずれの条件も尿試料からの検出が可能であった。金コロイドを用いた場合は、1ng/mLまでの測定が可能であった。 The measurement results are shown in FIG.
Detection from urine samples was possible under all conditions. When colloidal gold was used, measurements were possible down to 1 ng/mL.
いずれの条件も尿試料からの検出が可能であった。金コロイドを用いた場合は、1ng/mLまでの測定が可能であった。 The measurement results are shown in FIG.
Detection from urine samples was possible under all conditions. When colloidal gold was used, measurements were possible down to 1 ng/mL.
Claims (6)
- 検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定する方法であって、
検体と抗体とを接触させることと、
検査ストリップ上で抗体とVanin-1タンパク質との複合体を測定することを含み、
抗体として、配列番号１で表されるVanin-1ポリペプチドの
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体を単独、又は前記抗体を組み合わせて使用し、
検出粒子として、NanoAct(登録商標)、又は金コロイドを使用する、
方法。 A method for measuring Vanin-1 protein in a sample by immunochromatography, comprising:
contacting the sample with an antibody;
measuring a complex between the antibody and Vanin-1 protein on the test strip;
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen;
anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 229 to alanine 379 as an antigen, or anti-Vanin-1 antibodies obtained from animals administered with a polypeptide having a sequence from valine 389 to arginine 497 as an antigen, either alone or in combination with said antibodies;
Use NanoAct® or gold colloids as detection particles;
Method. - 捕捉抗体として、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
配列番号１で表されるVanin-1ポリペプチドの第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体を使用し、
検出抗体として、
配列番号１で表されるVanin-1ポリペプチドの第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
配列番号１で表されるVanin-1ポリペプチドの第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体
を使用する、請求項１に記載の方法。 As a capture antibody,
Using an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from the 229th valine to the 379th alanine of the Vanin-1 polypeptide shown in SEQ ID NO: 1 as an antigen,
As a detection antibody,
The method described in claim 1 uses an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 36th leucine to the 223rd leucine of the Vanin-1 polypeptide represented by SEQ ID NO: 1, or an anti-Vanin-1 antibody obtained from an animal administered as an antigen a polypeptide having a sequence from the 389th valine to the 497th arginine of the Vanin-1 polypeptide represented by SEQ ID NO: 1. - 測定が、定性的測定、半定量的測定又は定量的測定である、請求項１に記載の方法。 The method according to claim 1, wherein the measurement is a qualitative measurement, a semi-quantitative measurement, or a quantitative measurement.
- 検体が尿である、請求項１に記載の方法。 The method of claim 1, wherein the sample is urine.
- 測定に基づいて取得された測定値が基準値以上であるとき、尿を採取した被検者が腎疾患を有することを示唆する、請求項４に記載の方法。 The method according to claim 4, wherein, when the measured value obtained based on the measurement is equal to or greater than the reference value, it is suggested that the subject from whom the urine was collected has a renal disease.
- 検体と検出粒子を標識した検出抗体を含む測定試料を浸透させる、又は検体もしくは検体の希釈液を浸透させる第１の部分と、捕捉抗体が固相化された第２の部分を有する検査ストリップを備えた、検体中のVanin-1タンパク質をイムノクロマトグラフィーにより測定するための検査器具であって、
第２の部分は第１の部分と重ならず、
捕捉抗体は、
抗体として、配列番号１で表されるVanin-1ポリペプチドの
第３６番目のロイシンから第２２３番目のロイシンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、
第２２９番目のバリンから第３７９番目のアラニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、又は
第３８９番目のバリンから第４９７番目のアルギニンまでの配列を有するポリペプチドを抗原として投与した動物から取得された抗Vanin-1抗体、である
検査器具。 A test device for measuring Vanin-1 protein in a sample by immunochromatography, comprising a test strip having a first portion through which a measurement sample containing a sample and a detection antibody labeled with a detection particle is permeated, or through which a sample or a dilution of the sample is permeated, and a second portion on which a capture antibody is immobilized,
the second portion does not overlap the first portion;
The capture antibody is
an anti-Vanin-1 antibody obtained from an animal administered with a polypeptide having a sequence from leucine 36 to leucine 223 of the Vanin-1 polypeptide represented by SEQ ID NO: 1 as an antigen;
A testing device comprising an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 229th valine to the 379th alanine as an antigen, or an anti-Vanin-1 antibody obtained from an animal administered a polypeptide having a sequence from the 389th valine to the 497th arginine as an antigen.
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