JP2009031121A - Biological sample preserving test tube - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological sample preserving test tube, especially a urine examination test tube hard to cause the adsorption of a very small amount of protein or peptide and usable even in proteome analysis. <P>SOLUTION: The biological sample preserving test tube is constituted, by coating a test tube being a raw material with a copolymer which contains a monomer (a) having a phosphorylcholine group and a hydrophobic monomer (b) as monomers and that the copolymerization ratio a/b of both the monomers (a) and (b) is 35/65-75/25. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a biological sample storage test tube, and more particularly to a biological sample storage test tube, particularly a urine test test tube, which hardly adsorbs a minute amount of protein or peptide and can be used for proteomic analysis.

  Urine contains various substances related to pathological conditions, such as proteins, sugars, electrolytes, blood, and the like, and the presence or state of a disease can be known by measuring the presence or concentration thereof. Therefore, measuring items such as urine glucose, creatinine, NAG, urinary albumin, calcium, magnesium, sodium, potassium, chlor and the like is currently in practical use. In such a case, urine excreted from the human body is transferred to a urine test tube, and these items are measured. In order to examine the amount and type of solid components such as red blood cells, white blood cells, uric acid, cells, and bacteria as urine components, a urine sediment tube is used as a urine test tube (Patent Document 1).

  On the other hand, in recent years, research on proteome analysis has progressed rapidly, and various disease-specific markers have been newly reported. Proteome analysis for urine has been widely pursued, and the search for better biomarkers for renal and urological diseases is being studied. In such disease proteomic research, how to collect and store urine related to the disease is an important issue. If samples collected in daily examinations can be stored as they are, research can be started easily and quickly. Therefore, if a urine test tube handled in daily inspection can be used as it is for sample collection, it is useful without being complicated.

However, according to the study of the present inventor, if a urinalysis test tube used for daily examination is used for proteome analysis research, it was not a problem in normal urinalysis, and it was difficult to store a small amount of protein or peptide in sample storage. Discovered that there was a problem of misinterpreting the results.
JP 7-280800 A

  Under such circumstances, an object of the present invention is to provide a biological sample storage test tube, particularly a urine test test tube, which hardly absorbs a minute amount of protein or peptide and can be used for proteome analysis. .

  The inventors of the present invention have made extensive studies in order to achieve the above object. As a result, surprisingly, when a copolymer with a specific copolymerization ratio is coated on a commercially available urine test tube, even if urine is put into the urine test tube itself, it is difficult for protein to be adsorbed and the urine test items are accurate. The inventors have found that proteomic analysis can be performed accurately and the present invention has been completed based on the findings.

The present invention is as follows.
(1) Copolymer comprising a monomer (a) having a phosphorylcholine group as a monomer and a hydrophobic monomer (b) and having a copolymerization ratio a / b of 35/65 to 75/25 A test tube for storing biological samples, characterized in that the coalescence is coated on a test tube which is a raw material.
(2) The biological sample storage test tube according to (1) above, which is a urine test test tube.
(3) The biological sample storage test tube according to (1) or (2), which also serves as a proteome analysis test tube.
(4) The test tube for biological sample storage according to the above (3), wherein the proteome analysis is a urinary proteome analysis in a low molecular weight region having a molecular weight of 1000 to 6000.
(5) The biological sample storage test tube according to any one of (1) to (4), wherein the biological sample storage test tube is a urine sediment tube.
(6) The biological sample storage test tube according to any one of (1) to (5), wherein the urine test test tube is transparent.
(7) The monomer according to any one of (1) to (6), wherein the monomer having a phosphorylcholine group is 2-methacryloyloxyethyl phosphorylcholine, and the hydrophobic monomer is methacrylic acid-n-butyl ester. Test tube for biological sample storage.
(8) The biological sample storage test tube according to any one of (1) to (7), which is a disposable biological sample storage test tube.
(9) The test tube for biological sample storage according to any one of (1) to (8), wherein the copolymer has a copolymerization ratio a / b of 40/60 to 60/40.
(10) Putting urine into the biological sample storage test tube described in any one of (1) to (9) above, measuring urinalysis items in the urine, and proteomic analysis of the remaining urine Characteristic proteome analysis method.
(11) A test tube which is a raw material contains a monomer (a) having a phosphorylcholine group as a monomer and a hydrophobic monomer (b), and the copolymerization ratio a / b thereof is 35/65 to 75 A method for producing a urinalysis test tube for proteome analysis, which comprises contacting a solution of a copolymer of / 25 and drying the obtained urinalysis test tube.

  The test tube for biological sample storage of the present invention comprises a monomer (a) having a phosphorylcholine group as a monomer and a hydrophobic monomer (b), and the copolymerization ratio a / b thereof is 35/65. A test tube which is a raw material is coated with a copolymer of ~ 75/25.

  The monomer having a phosphorylcholine group in the copolymer used in the present invention has the following formula:

  Although it will not specifically limit if it is a monomer which has the phosphorylcholine group represented by these, Usually, the monomer which has a vinyl group and a phosphorylcholine group can illustrate preferably. Specifically, 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-methacryloyloxypropyl phosphorylcholine, 3-acryloyloxypropyl phosphorylcholine, 4-methacryloyloxybutylphosphorylcholine, 4-acryloyloxybutylphosphorylcholine, Examples include allyl phosphoryl choline and butenyl phosphoryl choline. Among these monomers, the following formula

  The 2-methacryloyloxyethyl phosphorylcholine represented by these is preferable from an acquisition point.

  The hydrophobic monomer in the copolymer used in the present invention is preferably a hydrophobic monomer copolymerizable with a monomer having a phosphorylcholine group. Examples of such hydrophobic monomers include methacrylic acid esters, acrylic acid esters, styrene monomers, vinyl ether monomers, alkene monomers, vinyl monocarboxylate monomers, dialkyl itaconate monomers. Can be illustrated.

  Methacrylic acid ester monomers include: n-butyl methacrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, heptyl methacrylate, octyl methacrylate, methacrylic acid Examples of the monomer include nonyl, tridecyl methacrylate, and 2-hydroxyethyl methacrylate.

  As acrylic acid ester monomers, acrylic acid-n-butyl, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate, octyl acrylate, acrylic acid Examples of the monomer include nonyl, tridecyl acrylate, and 2-hydroxyethyl methacrylate.

  Examples of the styrene monomer include monomers such as styrene, methylstyrene, and chlorostyrene.

  Examples of the vinyl ether monomer include monomers such as ethyl vinyl ether, propyl vinyl ether, and butyl vinyl ether. Examples of alkene monomers include ethylene, propylene, and isobutylene monomers. Examples of the vinyl monocarboxylate monomer include vinyl acetate, vinyl propionate, and vinyl butanoate. Examples of the itaconic acid dialkyl ester monomer include monomers such as dimethyl itaconate, diethyl itaconate, dipropyl itaconate, and di-n-butyl itaconate.

As a hydrophobic monomer, the following formula

  Methacrylic acid-n-butyl ester (CAS No. 97-88-1) represented by the formula is most preferred from the viewpoint of availability.

  In the copolymer used in the present invention, the monomer having a phosphorylcholine group is most preferably 2-methacryloyloxyethyl phosphorylcholine, and the hydrophobic monomer is most preferably methacrylic acid-n-butyl ester.

  The copolymerization ratio a / b of the copolymer used in the present invention is 35/65 to 75/25, more preferably 40/60 to 60/40.

  In the present invention, if the ratio of the copolymer is biased to one of the monomers, the protein adsorption preventing function is weakened.

  The copolymer is preferably used as a coating solution in a state where the copolymer is dissolved in an appropriate solvent. At this time, as the solvent, for example, water, methanol, ethanol, propanol and a mixed solvent thereof can be used, but water is preferable from the viewpoint of safety.

  In the present invention, the raw material test tube for coating is preferably a urine test tube, and most preferably a urine sediment tube.

  The raw material test tube is preferably made of a material that does not change when urine is added. Specifically, the material of the raw material test tube is preferably glass or plastic. In this case, examples of the plastic include polystyrene and polyacrylonitrile styrene. The raw material test tube is preferably manufactured from a transparent substance so that the state of sediment, urine volume and dust can be discriminated. Furthermore, the test tube for raw materials may be a disposable test tube, and the biological sample storage test tube of the present invention may be used as a disposable biological sample storage test tube.

  Examples of the test tube of the raw material to be used include, for example, a centrifugal test tube for urine sediment described in JP-A-7-280800, that is, a cylindrical test tube upper portion and an inner peripheral surface continuously to the lower end of the test tube upper portion. Has a taper-shaped test tube middle part, and a lower part of the test tube whose inner peripheral surface is formed in a small-diameter cylindrical shape continuously from the lower end of the test tube middle part and whose lower end is closed by a bottom wall. A centrifugal test tube for urine sediment (urine sediment tube) characterized by the above can be used.

  In the present invention, in order to produce a test tube for biological sample storage of the present invention from a test tube of raw materials, 1) a method in which the test tube is immersed in a copolymer solution and then dried, and 2) a method in 1) above 3) a method of repeating immersion and drying of a test tube a plurality of times, 3) a method of applying a copolymer solution to the inner surface of the test tube and drying, and 4) a method of 3) above. A method of repeating application and drying a plurality of times can be exemplified. In these cases, after immersing the test tube in the copolymer solution or applying the copolymer solution to the test tube, the resulting test tube may be washed with water and then subjected to a drying operation. Good.

  When producing the biological sample storage test tube of the present invention, the copolymer solution preferably has a copolymer concentration of 0.001 to 5 wt%, more preferably 0.005 to 3 wt%. The immersion time of the test tube in the solution is preferably, for example, 0.5 to 10 minutes. The drying temperature is preferably 20 to 80 ° C, for example. In this case, the drying may be natural drying or vacuum drying.

  If the raw material test tube is transparent, the biological sample storage test tube of the present invention is a transparent biological sample storage test tube even if the coating agent is adsorbed.

  The test tube for biological sample storage of the present invention is useful for urinary proteome analysis, and the molecular weight of peptides and proteins actually analyzed by the urinary proteome is not particularly limited, but urine in a low molecular weight range having a molecular weight of 1000 to 6000. Suitable for proteome analysis. For example, after putting the urine of a patient or a healthy person into the biological sample storage test tube of the present invention and measuring general items as a urine test, the remaining urine is MALDI-TOF-MS, SELDI-TOF-MS, etc. A new marker can be discovered by attaching to a mass spectrometer and comparing the presence and amount of various proteins in urine from patients with a disease state and urine from healthy subjects. In the present specification, a protein includes a peptide. The biological sample storage test tube of the present invention may be a substance in which the target protein is present in a very small amount because protein adsorption hardly occurs. In addition, when measuring a trace amount of protein in urine, the protein can be accurately measured even if urine is stored for a long time because there is almost no adsorption to a biological sample storage test tube.

As a raw material urine sediment tube, a urine sediment centrifuge tube (manufactured by Kunimune Industrial Co., Ltd., made of polyacrylonitrile styrene) described in JP-A-7-280800 was used.
Example 1 and Example 2 Production of urinary sediment tube of the present invention
A 0.05% by weight aqueous solution of a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylic acid-n-butyl ester (copolymerization ratio 50:50) was prepared, and the raw material urine sediment tube was immersed for 1 minute. The urine sediment tube (one coat) of Example 1 was obtained by allowing it to stand in a 50 ° C. incubator overnight and further drying overnight in a vacuum dryer (desiccator). Similarly, the operation of dipping and drying the water-soluble polymer was performed twice to produce the urine sediment tube (twice coat) of Example 2.
Comparative Examples 1 to 3 Production of Comparative Urine Sedimentary Tube As a comparative urinary sedimentary tube, an uncoated raw material urinary sedimentary tube was used as the urinary sedimentary tube of Comparative Example 1.

Further, in Example 1, the copolymerization ratio of 50:50 of the copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylic acid-n-butyl ester was changed to 30:70 and 80:20. The manufactured urinary sediment tubes were used as Comparative Example 2 and 3 urine sediment tubes, respectively.
Example 3 Electrophoretic confirmation test of protein adsorption to urinary sediment tube The urinary protein was applied to the urinary sediment tubes of Examples 1 and 2 and Comparative Examples 1 to 3 prepared by the above method by the urine test paper method, respectively. 1.5 mL of negative urine and urine that was 500 mg / dL or more were dispensed. This was kept at 4 ° C. all day and night, and urine was carefully removed. Next, in order to recover the adsorbed protein, the urine sediment tube is rinsed with PBS (10 mM phosphate buffer, 150 mM NaCl, pH 7.4), and 50 μL of sample buffer (0.5% (v / v)) is added to the sediment tube. Glycerol, 1% (w / v) SDS, 62.5 mM Tris-HCl, 0.2% BPB, pH 6.8) was added. Subsequently, vortex stirring of the urinary sediment tube was performed for 30 seconds to obtain a sample for SDS-PAGE electrophoresis. SDS-PAGE was performed according to the following procedure. 20 μL of the sample was applied to a 10-20% gradient polyacrylamide gel (ATTO), and electrophoresis was performed at a constant current of 20 mA for 75 minutes.

The results are shown in FIG. In urine protein-negative samples 1, 3, 5, and 7, either the uncoated or surface-coated urine sediment tube (Examples 1 and 2 and Comparative Example 2) and the uncoated urine sediment tube (Comparative Example 1) In some cases, no band was confirmed by CBB staining. On the other hand, in the samples 2, 4, 6, and 8 of 500 mg / dL or more, the molecular weight of 66 kDa is around from the urine sediment tube (uncoated) of Comparative Example 1 and the urine sediment tube of Comparative Example 2 (copolymerization ratio 30:70). A band corresponding to albumin was detected. The urine sediment tube of Comparative Example 3 (copolymerization ratio 80:20) also showed similar results (not shown). On the other hand, a urinary sediment tube of Example 1 coated twice and a copolymer of Example 2 which was coated twice with a copolymer of 2-methacryloyloxyethyl phosphorylcholine and methacrylic acid-n-butyl ester (copolymerization ratio 50:50). In the urinary sediment tube, it was revealed from the CBB stained image that there was no band and protein adsorption was suppressed.
Example 4 Confirmation test by SELDI-TOF-MS of protein adsorption to urinary sediment tube The urine protein was applied to the 2-coated urine sediment tube of Example 2 and the uncoated urine sediment tube of Comparative Example 1 by the urine test paper method. 1.5 mL of urine that was negative and 100 mg / dL or more was dispensed. This was held overnight under the following conditions.

(A) After dispensing the urine sample into the urine sediment tube, hold it at room temperature for 2 hours, and then leave it in a 4 ° C cooler (b) After dispensing the urine sample into the urine sediment tube, immediately leave it in a 4 ° C cooler Thereafter, the urine sample was carefully removed, and then the urine sediment tube was rinsed with PBS (10 mM phosphoric acid, 150 mM NaCl, pH 7.4), and 50 μL of 0.1% (v / v) aqueous TFA solution was added. Subsequently, vortex stirring of the urinary sediment tube was performed for 30 seconds to obtain a sample for SELDI-TOF-MS analysis. After this sample was added to NP20 Array (Bio Rad), sine acid (Bio Rad) was applied as a matrix and the spectrum was measured with SELDI-TOF-MS (Bio Rad) using the surface-coated urine sediment tube of Example 2. Comparison was made with the uncoated urine sediment tube of Comparative Example 1.

FIG. 2 shows a SELDI-TOF-MS spectrum of components adsorbed on the urine sediment tube. In the urine protein negative sample, peaks were observed in the uncoated urinary sediment tube of Comparative Example 1 near 2800 m / z and 4700 m / z under the retention conditions (a) and (b) above, and the protein was detected by the urine paper test method. Despite the negative sample, protein / peptide adsorption was revealed. In the urine sediment tube of Example 2 in which the copolymer was coated twice with the same urine protein negative sample, it was found that any peak disappeared and protein / peptide adsorption to the urine sediment tube was avoided. Similarly, in the sample with a urine protein of 100 mg / dL or more, in the uncoated urine sediment tube of Comparative Example 1, peaks were detected at around 2800 m / z, around 3400 m / z, around 4700 m / z, and around 5000 m / z. On the other hand, in the urine sediment tube of Example 2 in which the same urine protein was coated twice with a sample of 100 mg / dL or more, those peaks disappeared or attenuated, and protein / peptide adsorption was suppressed. It has been suggested.
Example 5 Measurement of urine biochemistry items The urine sediment tube of Example 2 and Comparative Example 1 were coated with urine whose protein was negative or 100 mg / dL or more by the urine test paper method, respectively. The sample was transferred to an uncoated urine sediment tube, and glucose, total protein, creatinine, NAG, urinary albumin, calcium, magnesium, Na, K, and Cl were measured with existing clinical laboratory reagents. Here, as a control, urine biochemistry items were also measured for specimens (hereinafter referred to as raw urine) before being transferred to the urinary sediment tube (Tables 1 and 2).

Table 1 shows the measurement results of urine biochemistry items in samples that were negative for protein by the urine test paper method. Table 2 shows the measurement results of the urine biochemical items in the specimen in which the protein was about 100 mg / dL by the urine test paper method. In either case, the measured values of the specimens transferred to the urine sediment tube of Example 2 coated with the copolymer twice and the uncoated urine sediment tube of Comparative Example 1 showed almost the same measured value as that of the original urine. . Thus, it was shown that the urine sediment tube coated with the raw material copolymer can be used for daily examinations like the untreated urine sediment tube that has been conventionally used.

FIG. 1 shows the results of polyacrylamide gel electrophoresis of the components adsorbed on the urine sediment tube. N, A, C, and C2 are urine samples of Comparative Example 1 (uncoated), Comparative Example 2 (copolymerization ratio 30:70), Example 1 (one-time coating), and Example 2 (two-time coating), respectively. The result of a sediment pipe is shown. In addition, 1, 3, 5, and 7 are urine protein negative, and 2, 4, 6, and 8 are the results of using a sample with a urine protein of 500 mg / dl. FIG. 2 shows the SELDI-TOF-MS spectrum of the component adsorbed on the urine sediment tube. The sample is a urine protein negative or 100 mg / dl sample by the urine test paper method, placed in an uncoated urine sediment tube (Comparative Example 1) and a urine sediment tube (Example 2) coated twice with a copolymer at room temperature or It is a substance stored at 4 ° C and adsorbed on the sediment tube.

Claims (11)

  A copolymer comprising a monomer (a) having a phosphorylcholine group as a monomer and a hydrophobic monomer (b) and having a copolymerization ratio a / b of 35/65 to 75/25 as a raw material A test tube for storing a biological sample, characterized in that the test tube is coated.   2. The biological sample storage test tube according to claim 1, which is a urine test test tube.   The test tube for biological sample storage according to claim 1, which also serves as a test tube for proteome analysis.   The test tube for biological sample storage according to claim 3, wherein the proteome analysis is a urinary proteome analysis in a low molecular weight region having a molecular weight of 1000 to 6000.   The biological sample storage test tube according to any one of claims 1 to 4, wherein the biological sample storage test tube is a urine sediment tube.   The biological sample storage test tube according to claim 1, wherein the urine test test tube is transparent.   The test tube for biological sample storage according to any one of claims 1 to 6, wherein the monomer having a phosphorylcholine group is 2-methacryloyloxyethyl phosphorylcholine, and the hydrophobic monomer is methacrylic acid-n-butyl ester. .   The biological sample storage test tube according to claim 1, which is a disposable biological sample storage test tube.   The biological sample storage test tube according to any one of claims 1 to 8, wherein the copolymer has a copolymerization ratio a / b of 40/60 to 60/40.   A proteome analysis method characterized by putting urine into a biological sample storage test tube according to any one of claims 1 to 9, measuring urine test items in the urine, and proteomic analysis of the remaining urine .   A test tube as a raw material contains a monomer (a) having a phosphorylcholine group as a monomer and a hydrophobic monomer (b), and the copolymerization ratio a / b is 35/65 to 75/25. A method for producing a urinalysis test tube for proteome analysis, which comprises contacting a solution of a copolymer and drying the obtained urinalysis test tube.