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WO2010123039A1 - Composition for immobilizing biological material - Google Patents

Composition for immobilizing biological material Download PDF

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Publication number
WO2010123039A1
WO2010123039A1 PCT/JP2010/057097 JP2010057097W WO2010123039A1 WO 2010123039 A1 WO2010123039 A1 WO 2010123039A1 JP 2010057097 W JP2010057097 W JP 2010057097W WO 2010123039 A1 WO2010123039 A1 WO 2010123039A1
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WIPO (PCT)
Prior art keywords
biological material
amino group
polyallylamine
group
polymer
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PCT/JP2010/057097
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French (fr)
Japanese (ja)
Inventor
悦子 二谷
信二 溝口
寛 窪田
清稔 丸山
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株式会社医学生物学研究所
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Priority to JP2011510349A priority Critical patent/JPWO2010123039A1/en
Publication of WO2010123039A1 publication Critical patent/WO2010123039A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00138—Slides

Definitions

  • the present invention relates to a composition for immobilizing a biological material, specifically, a composition for immobilizing a biological material containing a polyamine polymer, and a solid coated with the composition.
  • the present invention relates to a support and a method for analyzing biological material using the solid support.
  • Cytodiagnosis is widely used as a method for diagnosing the presence or absence of malignant cells by staining and observing under a microscope a biological material containing cells collected from a patient's organ while placed on a microscope slide glass. ing.
  • the biological material needs to be firmly immobilized on the slide so as not to peel off during the staining and washing process. Since the biological material is negatively charged, conventionally, the biological material has been immobilized using a composition containing a positively charged cationic polymer (Patent Document 1).
  • Biological material fixative contains formaldehyde, alcohol, etc. These components have the effect of reducing the affinity between the biological material and a slide glass coated with a conventional cationic polymer. Therefore, when using a composition containing a conventional cationic polymer, the biological material must be thoroughly washed to remove the components of the fixative. However, repeating the process of cleaning the fixative takes time and cost. Therefore, in order to improve the throughput of cytodiagnosis, a composition for immobilizing biological material so that an appropriate specimen can be prepared even with a minimum number of steps of washing the fixing solution, and the composition There is a need to develop a solid support coated with an object, a method for analyzing biological material using the solid support, and an analysis system and kit for performing the method.
  • the present invention provides a composition for immobilizing biological materials.
  • the composition for immobilizing a biological material of the present invention contains a polyallylamine-based polymer, and the amino group of the polymer is a primary amino group that is at least partially modified or a primary amino group. And a secondary amino group, a primary amino group and a tertiary amino group, or a secondary amino group and / or a tertiary amino group.
  • the modified primary amino group may be a secondary amide group or an alkoxycarbonyl group.
  • the modified primary amino group may be 25-75% of the amino group in the same molecule of the polymer.
  • the present invention provides a composition for immobilizing biological materials.
  • the composition for immobilizing a biological material of the present invention contains a polyallylamine-based polymer, and the amino group of the polymer is a primary amino group, or a primary amino group and a secondary amino group. Or a primary amino group and a tertiary amino group, or a secondary amino group and / or a tertiary amino group, and the polymer having only the primary amino group is a secondary amide in the side chain. Group or an alkoxycarbonyl group.
  • the number of secondary amide groups or alkoxycarbonyl groups is 3 minutes of the number of primary amino groups in the same molecule of the polymer. May be 1 to 3 times.
  • the secondary amino group and / or the tertiary amino group are respectively a primary amino group and / or a secondary amino group. It may be denatured.
  • the polymer may be a modified polyallylamine.
  • a primary amino group may be substituted with an acyl group or an alkoxycarbonyl group.
  • the acyl group or alkoxycarbonyl group may have 2 or 3 carbon atoms.
  • the acyl group may be an acetyl group or a propionyl group.
  • composition for immobilizing a biological material of the present invention 25-75% of amino groups in the same molecule of the polymer may be modified.
  • the polyamine polymer may be copolymerized with sulfur dioxide and / or maleic acid.
  • the polymer may be a copolymer of allylamine and diallylamine or diallylmethylamine.
  • the polymer may be polydiallylamine or polydiallylmethylamine.
  • the polymer may have an average molecular weight of 3,000 to 54,000.
  • the concentration of the solution containing the polymer may be 0.05 to 0.2 w / v%.
  • the biological material may be immobilized in the presence of a fixing agent.
  • the fixative may be 0.04 w / v% formaldehyde.
  • the biological material may be a clinical test sample.
  • the biological material may be a cytological examination sample.
  • the biological material may be a cervical cytological examination sample.
  • the present invention provides a solid support for immobilizing biological material coated with a composition for immobilizing biological material of the present invention.
  • the present invention provides a method for producing a solid support for immobilizing a biological material, comprising the step of coating a composition for immobilizing a biological material of the present invention.
  • the present invention provides a microscope slide glass for immobilizing biological material, which is coated with the composition for immobilizing biological material of the present invention.
  • the present invention provides a method for analyzing biological materials using the microscope slide glass of the present invention.
  • the method of the present invention includes (1) a step of immersing and fixing the biological material in a liquid containing a fixing agent, (2) a step of separating the fixed biological material, and (3) Suspending the separated biological material in an aqueous solution; (4) placing the aqueous solution on the microscope slide and immobilizing the biological material on the microscope slide; including.
  • the aqueous solution in step (3) may contain a fixing agent.
  • the aqueous solution in the step (3) may contain 0.04 w / v% formaldehyde.
  • the method for analyzing a biological material of the present invention may include a step of (5) staining a microscope slide glass in which the biological material is immobilized in the step (4).
  • the present invention provides an analysis system for executing the method of analyzing a biological material of the present invention.
  • the analysis system of the present invention includes a holder for holding the microscope slide glass of the present invention, an automatic dispenser unit, and a pipetter unit having a robot arm.
  • the analysis system of the present invention may include a centrifuge unit.
  • the present invention provides a kit for carrying out the method for analyzing a biological material of the present invention.
  • the kit of the present invention includes the microscope slide glass of the present invention.
  • the kit of the present invention may contain a reagent for fixing and storing the collected biological material.
  • the biological material of the present invention includes, but is not limited to, cells, tissues, body fluids and the like collected from subjects.
  • the biological material of the present invention may be a cultured cell derived from a biological species including but not limited to human, mouse, rat and hamster.
  • the biological material of the present invention is used for examining the presence or absence of tumor, inflammation or other pathological changes in the organ by observing with an optical microscope, biochemistry, immunology, molecular biology. It may be used for examination by academic or other methods, but it is not limited to these.
  • the biological material of the present invention may be a cytological examination sample.
  • Samples for cytological examination include gynecological genitals (vulva, vaginal wall, posterior vaginal cap, neck, cervical intima, body, etc.), respiratory organs (eg, sputum, bronchi, alveoli, lavage fluid), body cavity Urinary (urine, lavage fluid, kidney, prostate, testis, etc.), mammary gland (papillary discharge, puncture and aspiration), thyroid gland, parathyroid gland, digestive organ (oral cavity, pharynx, salivary gland, esophagus, stomach, small intestine, large intestine, liver, biliary tract) , Pancreas), brain (brain, cerebrospinal fluid, central nervous system), lymph node, bone, soft organ, hematopoietic organ (blood, bone marrow) .
  • the cytological test sample of the present invention may be collected from the cervix.
  • immobilization of biological material refers to the binding of biological material to the surface of a solid support that includes a microscope slide, which is for subsequent cytochemical techniques. It refers to a bond strong enough to prevent the biological material from detaching from the solid support during processing such as washing, clearing, and staining.
  • the polyallylamine-based polymer refers to a polymer in which at least one monomer selected from the group consisting of allylamine, diallylamine, diallyl (—R) amine, and (—R) 2 allylamine is polymerized.
  • the polyallylamine-based polymer of the present invention includes polyallylamine, polydiallylamine, polydiallyl (—R) amine, and poly (—R) 2 allylamine.
  • Polyallylamine is a polymer in which only primary amine monomer, allylamine is polymerized, and polydiallylamine is a polymer in which only secondary amine monomer, diallylamine, is polymerized, and polydiallyl (—R) amine and poly (—R) 2- Allylamine is a polymer in which only tertiary amine monomers are polymerized.
  • R is an n-alkyl group or iso-alkyl group having 1 to 20 carbon atoms, or an allyl group having 6 to 12 carbon atoms, and includes, but is not limited to, a hydroxyl group and a polyethylene glycol chain. It may have a substituent.
  • the polyallylamine-based polymer includes a copolymer of a primary amine monomer and a secondary amine monomer, and a copolymer of a primary amine monomer and a tertiary amine monomer.
  • the polyallylamine-based polymer of the present invention can be polymerized with sulfur dioxide, maleic acid or other compounds in the main chain, provided that it does not affect the immobilization of the biological material on the solid support of the present invention. It does not matter, and the side chain may be denatured.
  • “modification” means that the hydrogen atom of the amino group in the side chain of the polyallylamine-based polymer of the present invention is covalently modified with another atom or atomic group. That is, the polyallylamine-based polymer of the present invention includes an unmodified polyallylamine-based polymer and a modified polyallylamine-based polymer.
  • modified polyallylamine-based polymer of the present invention refers to a polymer in which hydrogen atoms are substituted with other atoms or atomic groups in at least some amino groups of the unmodified polyallylamine-based polymer molecule. Therefore, the secondary amino group or the tertiary amino group in the polymer of the present invention may be derived from the secondary amine monomer or the tertiary amine monomer, but the primary amine monomer or the second amine group respectively. Although derived from a secondary amine monomer, it may be modified after the polymerization reaction of the monomer.
  • the modified polyallylamine-based polymer of the present invention 1-95% of amino groups in the same molecule are preferably modified, and 10-80% of amino groups in the same molecule may be modified. More preferably, it is even more preferred that 25-75% of the amino groups in the same molecule are modified.
  • the ratio of the modified amino group among the amino groups in the same molecule in the polymer of the present invention can be determined by NMR method, colloid titration method, and other methods well known to those skilled in the art.
  • the atomic group that replaces the hydrogen atom of the amino group is an n-alkyl group or iso-alkyl group having 1 to 20 carbon atoms, or an allyl group having 6 to 12 carbon atoms. And may have substituents including but not limited to hydroxyl groups and polyethylene glycol chains.
  • the atomic group is preferably an acyl group or an alkoxycarbonyl group.
  • the atomic group is more preferably an acyl group having 2-6 carbon atoms or an alkoxycarbonyl group, more preferably an acetyl group, a propionyl group, a methoxycarbonyl group, or an ethoxycarbonyl group, and an acetyl group and a propionyl group. Even more preferably.
  • Methods for substituting hydrogen atoms of amino groups of the polymer of the present invention with these atomic groups are well known to those skilled in the art. For example, when replacing with an acetyl group, the polymer is reacted with acetic anhydride.
  • the amino group of the polyallylamine-based polymer of the present invention may be ionically bonded to one or more other types of cations.
  • the “free base” of the polyallylamine-based polymer means one having no bond with the cation, and is sometimes referred to as a free type.
  • the “salt” of a polyallylamine-based polymer is one having a bond with the cation.
  • Preferred polyallylamine-based polymer salts include, but are not limited to, hydrochloride and acetate.
  • the polyallylamine-based polymer of the present invention is commercially available and its synthesis method is well known to those skilled in the art.
  • the average molecular weight of the polyallylamine-based polymer of the present invention may be in the range of 500 to 1,000,000, provided that it does not affect the immobilization of biological material on the solid support of the present invention. .
  • the average molecular weight of the polyallylamine-based polymer of the present invention is preferably in the range of 2,000 to 100,000, and more preferably in the range of 3,000 to 54,000.
  • the polyallylamine-based polymers in the case of polydiallylamine, it is preferably within the range of 3,000 to 8,000.
  • the preferred molecular weight depends on the proportion of allylamine and diallylamine contained in the polymer.
  • the ratio of allylamine and diallylamine is 1: 1, it is preferably within a range of 3,000 to 54,000, and when 3: 1, it is within a range of 3,000 to 100,000. In the case of 1: 3, it is preferably in the range of 3,000 to 30,000. Since the range of molecular weight changes with the ratio of allylamine and diallylamine, it is not limited to these.
  • the average molecular weight and polydispersity (Mw / Mn) of the polyallylamine-based polymer of the present invention can be determined by gel permeation chromatography or other measurement methods well known to those skilled in the art.
  • the average molecular weight of the polyallylamine-based polymer of the present invention may exhibit polydispersity.
  • a copolymer refers to a polymer compound formed by polymerizing two or more types of monomers, and the polymerization type of the copolymer includes alternating copolymerization, block copolymerization, and random copolymerization. , Graft copolymerization and combinations thereof, but not limited thereto.
  • the copolymer contained in the polyallylamine-based polymer of the present invention has any ratio of two or more types of monomers provided that it does not affect the immobilization of biological material on the solid support of the present invention. It may be copolymerized.
  • the polyallylamine-based polymer of the present invention can be dissolved in deionized water, ethanol or other solvents known to those skilled in the art.
  • the concentration of the polyallylamine-based polymer of the present invention may be any concentration as long as it does not affect the immobilization of biological material on the solid support of the present invention. / V% is preferable, and 0.05 to 0.2 w / v% is more preferable.
  • the solution containing the polyallylamine-based polymer of the present invention can be adjusted to pH using hydrochloric acid, sodium hydroxide, or other acid or base known to those skilled in the art after dissolving the free polyallylamine in the solvent.
  • the pH may be stabilized with an appropriate buffer.
  • the pH of the solution containing the polyallylamine-based polymer may be any pH as long as it does not affect the immobilization of the biological material on the solid support of the present invention. It is preferably within the range, and more preferably within the range of 10 to 11.
  • coating a polyallylamine-based polymer means spraying or applying a polyallylamine-based polymer solution to a solid support, or immersing the solid support in a polyallylamine-based polymer solution.
  • the solid support includes, but is not limited to, a microscope slide glass, a multiwell plate, fine particles for latex aggregation method, and a sensor chip for surface plasmon resonance method.
  • the solid support may be coated with the polyallylamine-based polymer of the present invention only on a specific portion of the surface of the solid support by applying a hydrophobic surface treatment and / or a hydrophobic resin coating. .
  • the solid support coated with the polyallylamine polymer of the present invention may be coated not only with the polyallylamine polymer but also with any other polymer composition together with the polyallylamine polymer. Including.
  • the solid support coated with the polyallylamine polymer of the present invention is preferably subjected to a drying treatment by air drying or heat drying.
  • the solid support coated with the polyallylamine-based polymer of the present invention may be rinsed with deionized water or other aqueous solvent after the drying treatment.
  • the rinsing treatment makes it difficult for co-staining, that is, nonspecific staining in which the dye used for staining reacts with the polyallylamine-based polymer instead of the biological material.
  • the polyallylamine-based polymer of the present invention can immobilize the biological material in a solution containing the components of the fixing solution, even if the co-staining is not caused, incompletely washing the fixing solution.
  • the fixing solution of the present invention refers to an aqueous solution containing a fixing agent.
  • the fixing agent is selected from the group consisting of aldehydes including but not limited to formaldehyde, glutaraldehyde, dialdehyde, alcohols including but not limited to methanol, ethanol, acetone, and picric acid. Or there may be two or more types of compounds.
  • Preferred fixatives are formaldehyde, glutaraldehyde and / or dialdehyde.
  • the biological material suspended in the fixing solution is separated from the fixing solution by centrifugation. After removing the fixing solution, about 0.1 mL of the fixing solution remains in the centrifuge tube together with the biological material, and after washing with a cleaning solution consisting of 2 mL of fresh deionized water, the polyallylamine system of the present invention Placed on a microscope slide coated with polymer. When the fixative is further removed, the biological material is separated by a second centrifugation after washing with 10 mL of fresh deionized water in the centrifuge tube from which the fixative has been removed.
  • the pre-washing solution After the pre-washing solution is removed, it is washed with a washing solution consisting of 2 mL of fresh deionized water and then placed on a microscope slide glass coated with the polyallylamine-based polymer of the present invention. In any centrifugation, as little as 0.1 mL of solution remains in the centrifuge tube with the biological material precipitated. Therefore, it is considered that the fixing solution is diluted 20 times when pre-washing is not performed, and the fixing solution is further diluted 100 times when pre-washing is performed. As shown in the examples of the present specification, the degree of immobilization of the biological material on the slide glass is greatly affected by the presence or absence of pre-washing.
  • the fixative solution used in the following examples contains 0.8 w / v% formaldehyde, so that the biological material is immobilized on a microscope slide coated with the composition of the present invention without pre-washing. When present, 0.04 w / v% formaldehyde is present.
  • the biological material of the present invention is a cytological examination sample
  • the Bethesda system 2001 which is a standard regarding the suitability of specimens used for cytological examinations, mainly cervical cytological examinations, contains at least 5,000 squamous epithelial cells that are well preserved and clearly visible for one specimen. This is because it is stipulated that it must be estimated that In the following examples, the degree of cell immobilization on the microscope slide glass (degree of cell immobilization) was qualitatively evaluated using a 10-point index with a maximum of 10 points. When the degree of cell immobilization is 4 or more, at least 5,000 cells are immobilized on the microscope slide glass, which satisfies the criteria of Bethesda system 2001.
  • the analysis system may include a centrifuge unit and / or a manual describing a method for analyzing the biological material of the present invention.
  • a kit for carrying out the method for analyzing a biological material of the present invention was collected from a microscope slide glass coated with the composition of the present invention, as well as an instrument for collecting the biological material of the present invention. It may include reagents for immobilizing and storing biological materials, manuals describing methods for analyzing biological materials of the present invention, and the like.
  • the reagent contains at least 0.8 w / v% formaldehyde.
  • the degree of acetylation of the acetylated polyallylamine was calculated to be 50% from the area ratio of the NMR waveform corresponding to the hydrogen of the methylene group bonded to the amino group and the hydrogen of the methylene group bonded to the amide group.
  • the average molecular weight polydispersity (Mw / Mn) of the acetylated polyallylamine was 2.637 to 2.730.
  • Microscope slide glass coating The microscope slide glass coated with a water-repellent resin was immersed in the 50% acetylated polyallylamine solution or the unmodified polyallylamine solution for 10 seconds, pulled up, and then air-dried. Thereafter, it was rinsed with deionized water, air dried and stored.
  • Fixing Solution an aqueous solution of 0.8 w / v% formaldehyde, 22 w / v% ethanol and 1.5 w / v% methanol was used.
  • the cells fixed with the fixative were centrifuged at 800 G for 5 minutes to remove the fixative.
  • the precipitated cell pellet contained about 0.1 mL of the fixative.
  • Prewash treatment A prewash solution consisting of 10 mL of deionized water was added to the cell pellet from which the fixative solution had been removed, and the suspended cells were centrifuged at 800 G for 5 minutes to remove the prewash solution.
  • the precipitated cell pellet contained about 0.1 mL of the prewash solution.
  • a washing solution consisting of 2 mL of deionized water is added to the cell pellet from which the fixative solution has been removed or the cell pellet from which the pre-wash solution has been removed. It was placed on a glass slide coated with% acetylated polyallylamine or unmodified polyallylamine and allowed to stand for 10 minutes. Slides with immobilized cells were washed with 100% ethanol or deionized water.
  • Papanicolaou staining was performed to stain the cells.
  • the slide on which the cells were adsorbed was stained with a hematoxylin solution for 1 and a half minutes and with OG-6 for 2 minutes. Thereafter, it was stained with EA-50 for 3 minutes. After dyeing, it was dehydrated and transparently processed and sealed.
  • FIG. 1A is a graph showing the relationship between the concentration of a solution containing 50% acetylated polyallylamine having different average molecular weights and the degree of cell immobilization of a glass slide coated by immersion in the solution.
  • Each of the plot points indicates that the molecular weight of 50% acetylated polyallylamine is 3000 ( ⁇ 5), 5000 ( ⁇ ), 8000 ( ⁇ ), and 15000 ( ⁇ ).
  • the evaluation index for the degree of cell immobilization showed a maximum of 10 at concentrations of 0.03125% (w / v), 0.0625% (w / v) and 0.125% (w / v). .
  • FIG. 1A is a graph showing the relationship between the concentration of a solution containing 50% acetylated polyallylamine having different average molecular weights and the degree of cell immobilization of a glass slide coated by immersion in the solution.
  • Each of the plot points indicates that the molecular weight of 50% acetylated polyallylamine is
  • 1B is a graph showing the relationship between the concentration of a solution containing unmodified polyallylamine having different average molecular weights and the degree of cell immobilization of a glass slide coated by being immersed in the solution.
  • Each of the plotted points indicates that the molecular weight of the unmodified polyallylamine is 3000 ((), 5000 ( ⁇ ), 8000 ( ⁇ ), and 15000 ( ⁇ ).
  • the evaluation index of cell immobilization degree was 9 at the concentrations of 0.03125% (w / v) and 0.0625% (w / v).
  • 50% acetylated polyallylamine with a molecular weight of 15000 has a concentration of 0.125% (w / v) or less
  • unmodified polyallylamine with a molecular weight of 3000 has a concentration of 0.0625% (w / v) or less. It was shown that the degree of cell immobilization was maximal.
  • 50% acetylated polyallylamine had a higher cell immobilization degree than unmodified polyallylamine, and more cells were adsorbed. This suggests that the degree of cell immobilization increases when the native polyallylamine is acylated. This is thought to be because when the amino group is acylated, the hydrophobicity increases, and thus the degree of cell immobilization increases due to the contribution of the hydrophobic bond in addition to the ionic bond.
  • the obtained neutralized solution was subjected to electrodialysis in the same manner as in Example 1 and desalted over 28 hours to produce 25% propionylated polyallylamine free base.
  • the 25% propionylated polyallylamine free base that is, the free base type 25% propionylated polyallylamine is a polymer 25% propionylated with respect to the amino group of the starting polyallylamine.
  • Water in this aqueous solution was substituted in the same manner as in Example 1 and used for NMR measurement.
  • Microscope Slide Glass Coating A microscope slide glass coated with a water-repellent resin was dipped in a 0.1 w / v% aqueous solution of the polymer for 10 seconds, pulled up, and then air-dried. Thereafter, it was rinsed with deionized water, air dried and stored.
  • FIG. 2A shows the relationship between the degree of acylation of an acylated polyallylamine having an average molecular weight of 15,000 and the degree of cell immobilization of a slide glass coated with the acylated polyallylamine for the cells subjected to the prewash treatment. It is a graph which shows. As a control, a slide coated with native polyallylamine was used. The evaluation index of the cell immobilization degree of the acetylated polyallylamine (hatched bar) is 9.5 at 0% acetylation, 9.5 at 25% acetylation, 10 at 50% acetylation, 75% acetylation. It was 9.
  • FIG. 2B shows the relationship between the degree of acylation of an acylated polyallylamine having an average molecular weight of 15,000 and the degree of cell immobilization of a glass slide coated with the acylated polyallylamine for cells not subjected to prewash treatment. It is a graph.
  • the evaluation index of the cell immobilization degree of acetylated polyallylamine was 2 at 0% acetylation, 4 at 25% acetylation, 6 at 50% acetylation, and 5 at 75% acetylation. .
  • the evaluation index (black bar) of the degree of cell immobilization of propionylated polyallylamine was 2 at 0% propionylation, 4 at 25% propionylation, and 6.5 at 50% propionylation. From the above results, when cells not subjected to prewash treatment were used, acetylation and propionylation of native polyallylamine both showed the maximum degree of cell immobilization at 50%.
  • the evaluation index for the degree of cell immobilization decreased compared to the case where cells that had been prewashed were used. This decrease is considered to be due to insufficient removal of the fixative due to the absence of the pre-wash treatment, and some component contained in the fixative inhibited the cell immobilization of the polymer.
  • acylation of unmodified polyallylamine reduces the cation density of polyallylamine and causes a decrease in the degree of cell immobilization.
  • 50% acylated polyallylamine showed a very high degree of cell immobilization compared to native polyallylamine (FIG. 2B). This may be because the hydrophobicity increased with the acylation of polyallylamine, and the cells were more easily adsorbed by hydrophobic bonds.
  • the hydrophobic bond is a component in the remaining fixative solution. It is thought that it is not inhibited by.
  • the decrease in cell immobilization with 75% acylated polyallylamine indicates that cell immobilization in the presence of formaldehyde requires both hydrophobic and ionic bonds, and the degree of acylation is up to 75%. If it goes up, it is thought that it is because the contribution of an ionic bond reduces.
  • FIG. 3 is a graph showing the degree of cell immobilization of glass slides coated with various polyallylamine-based polymers for cells not subjected to pre-washing treatment.
  • Unmodified polydiallylamine and an equimolar copolymer of unmodified allylamine and diallylamine had cell immobility comparable to 50% acetylated polyallylamine with an average molecular weight of 15000.
  • acylation such as acetylation and propionylation
  • cell immobilization of cells not subjected to prewash treatment can be achieved by replacing the primary amino group of native polyallylamine with a secondary amino group.
  • the degree of conversion could be improved. This is probably because the secondary amino group is more basic and more hydrophobic than the primary amino group, so that the cells are easily immobilized.
  • FIG. 4A is a graph showing the relationship between the concentration and average molecular weight of the coated equimolar copolymer and the degree of cell immobilization.
  • the evaluation index of the cell immobilization degree of the 0.05% equimolar copolymer was 4 at an average molecular weight of 3000, 6 at an average molecular weight of 15000, 8 at an average molecular weight of 22000, and 9 at an average molecular weight of 48000.
  • the evaluation index of the cell immobilization degree of the 0.1% equimolar copolymer was 7 at an average molecular weight of 3000, 8 at an average molecular weight of 15000, 7 at an average molecular weight of 22000, and 9 at an average molecular weight of 48000.
  • the evaluation index of the cell immobilization degree of the 0.2% equimolar copolymer was 7 at an average molecular weight of 3000, 7 at an average molecular weight of 15000, 8 at an average molecular weight of 22000, and 7 at an average molecular weight of 48000.
  • the evaluation index of the cell immobilization degree of the equimolar copolymer (PAA-D11-54) having an average molecular weight of 54,000 was almost the same as that of the equimolar copolymer having an average molecular weight of 48,000 at each concentration. (Data not shown). From the above results, it was shown that the cell immobilization degree of the equimolar copolymer increases as the average molecular weight increases. It was also shown that the high molecular weight equimolar copolymer does not reduce the degree of cell immobilization even at a low concentration of 0.05%.
  • FIG. 4B is a graph showing the relationship between the concentration and average molecular weight of the coated polyallylamine and the degree of cell immobilization.
  • the evaluation index of the degree of cell immobilization of 0.05% polyallylamine was 3 with an average molecular weight of 3000 and 4 with an average molecular weight of 15000, 25000 and 65000.
  • the evaluation index of the cell immobilization degree of 0.1% polyallylamine was 3 at an average molecular weight of 3000, 4 at an average molecular weight of 15000, 3 at an average molecular weight of 25000, and 4 at an average molecular weight of 65,000.
  • the evaluation index of the cell immobilization degree of 0.2% polyallylamine was 3 at an average molecular weight of 3000, 4 at an average molecular weight of 15000, 3 at an average molecular weight of 25000, and 5 at an average molecular weight of 65000. From the above results, the maximum value of the cell immobilization degree of polyallylamine was 5 at an average molecular weight of 65000 and a concentration of 0.2%, but it was lower than the maximum value of the cell immobilization degree of an equimolar copolymer. It was shown that.

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Abstract

Developed are: a composition for immobilizing a biological material, whereby an appropriate specimen can be prepared while minimizing procedures for washing away a fixing solution, to thereby improve the throughput of a cytodiagnostic examination; and a method for analyzing a biological material with the use of a solid support having been coated with said composition. Disclosed is a composition for immobilizing a biological material, which comprises a polyallylamine-based polymer, wherein the amino groups in said polyallylamine-based polymer are at least partially denatured primary amino groups, primary amino groups with secondary amino groups, primary amino groups with tertiary amino groups, or secondary amino groups and/or tertiary amino groups. Also disclosed are: a microscopic slide glass for immobilizing a biological material, which has been coated with the aforesaid composition; and a method for analyzing a biological material by using said microscopic slide glass.

Description

生物学的材料の不動化を行うための組成物Composition for immobilizing biological materials
　本発明は、生物学的材料の不動化を行うための組成物、具体的には、ポリアミン系ポリマーを含む生物学的材料の不動化を行うための組成物と、該組成物をコーティングした固体支持体と、該固体支持体を用いて生物学的材料を分析する方法とに関する。 The present invention relates to a composition for immobilizing a biological material, specifically, a composition for immobilizing a biological material containing a polyamine polymer, and a solid coated with the composition. The present invention relates to a support and a method for analyzing biological material using the solid support.
　細胞診検査は、患者の臓器から採取された細胞を含む生物学的材料を顕微鏡スライドガラスに戴置した状態で染色処理し顕微鏡観察することによって悪性細胞の有無等を診断する方法として広く用いられている。 Cytodiagnosis is widely used as a method for diagnosing the presence or absence of malignant cells by staining and observing under a microscope a biological material containing cells collected from a patient's organ while placed on a microscope slide glass. ing.
　前記生物学的材料は染色及び洗浄処理の際に剥離しないようにスライド上にしっかりと不動化される必要がある。前記生物学的材料は負に荷電しているので、従来は、正荷電を有するカチオン性ポリマーを含む組成物を用いて前記生物学的材料を不動化していた（特許文献１）。 The biological material needs to be firmly immobilized on the slide so as not to peel off during the staining and washing process. Since the biological material is negatively charged, conventionally, the biological material has been immobilized using a composition containing a positively charged cationic polymer (Patent Document 1).
特開平１１－１４９０９号公報Japanese Patent Laid-Open No. 11-14909
　生物学的材料の固定液はホルムアルデヒド、アルコール等を含む。これらの成分は、従来のカチオン性ポリマーをコーティングしたスライドガラスと前記生物学的材料との親和性を低下させる作用がある。そのため、従来のカチオン性ポリマーを含む組成物を使用する場合には、生物学的材料を十分洗浄して前記固定液の成分を除去しなければならなかった。しかし、固定液を洗浄する工程を繰り返すことは時間及びコストがかかる。そこで、細胞診検査のスループットを向上させるためには、固定液を洗浄する工程が最小限でも適正な標本の作成ができるように生物学的材料の不動化を行うための組成物と、該組成物をコーティングした固体支持体と、該固体支持体を用いて生物学的材料を分析する方法と、該方法を実行するための分析システム及びキットとを開発する必要がある。 Biological material fixative contains formaldehyde, alcohol, etc. These components have the effect of reducing the affinity between the biological material and a slide glass coated with a conventional cationic polymer. Therefore, when using a composition containing a conventional cationic polymer, the biological material must be thoroughly washed to remove the components of the fixative. However, repeating the process of cleaning the fixative takes time and cost. Therefore, in order to improve the throughput of cytodiagnosis, a composition for immobilizing biological material so that an appropriate specimen can be prepared even with a minimum number of steps of washing the fixing solution, and the composition There is a need to develop a solid support coated with an object, a method for analyzing biological material using the solid support, and an analysis system and kit for performing the method.
　本発明は、生物学的材料の不動化を行うための組成物を提供する。本発明の生物学的材料の不動化を行うための組成物は、ポリアリルアミン系ポリマーを含み、該ポリマーのアミノ基は、少なくとも一部が変性した第１級アミノ基か、第１級アミノ基及び第２級アミノ基か、第１級アミノ基及び第３級アミノ基か、第２級アミノ基及び／又は第３級アミノ基かである。 The present invention provides a composition for immobilizing biological materials. The composition for immobilizing a biological material of the present invention contains a polyallylamine-based polymer, and the amino group of the polymer is a primary amino group that is at least partially modified or a primary amino group. And a secondary amino group, a primary amino group and a tertiary amino group, or a secondary amino group and / or a tertiary amino group.
　本発明の生物学的材料の不動化を行うための組成物において、前記変性した第１級アミノ基は、第２級アミド基又はアルコキシカルボニル基の場合がある。 In the composition for immobilizing a biological material of the present invention, the modified primary amino group may be a secondary amide group or an alkoxycarbonyl group.
　本発明の生物学的材料の不動化を行うための組成物において、前記変性した第１級アミノ基は、前記ポリマーの同一分子中のアミノ基の２５－７５％の場合がある。 In the composition for immobilizing a biological material of the present invention, the modified primary amino group may be 25-75% of the amino group in the same molecule of the polymer.
　本発明は、生物学的材料の不動化を行うための組成物を提供する。本発明の生物学的材料の不動化を行うための組成物は、ポリアリルアミン系ポリマーを含み、該ポリマーのアミノ基は、第１級アミノ基か、第１級アミノ基及び第２級アミノ基か、第１級アミノ基及び第３級アミノ基か、第２級アミノ基及び／又は第３級アミノ基かであって、第１級アミノ基のみのポリマーは、側鎖に第２級アミド基又はアルコキシカルボニル基を含む。 The present invention provides a composition for immobilizing biological materials. The composition for immobilizing a biological material of the present invention contains a polyallylamine-based polymer, and the amino group of the polymer is a primary amino group, or a primary amino group and a secondary amino group. Or a primary amino group and a tertiary amino group, or a secondary amino group and / or a tertiary amino group, and the polymer having only the primary amino group is a secondary amide in the side chain. Group or an alkoxycarbonyl group.
　本発明の生物学的材料の不動化を行うための組成物において、前記第２級アミド基又はアルコキシカルボニル基の数は、前記ポリマーの同一分子中の第１級アミノ基の数の３分の１ないし３倍の場合がある。 In the composition for immobilizing a biological material of the present invention, the number of secondary amide groups or alkoxycarbonyl groups is 3 minutes of the number of primary amino groups in the same molecule of the polymer. May be 1 to 3 times.
　本発明の生物学的材料の不動化を行うための組成物において、前記第２級アミノ基及び／又は第３級アミノ基は、それぞれ、第１級アミノ基及び／又は第２級アミノ基が変性している場合がある。 In the composition for immobilizing a biological material of the present invention, the secondary amino group and / or the tertiary amino group are respectively a primary amino group and / or a secondary amino group. It may be denatured.
　本発明の生物学的材料の不動化を行うための組成物において、前記ポリマーは変性ポリアリルアミンの場合がある。 In the composition for immobilizing a biological material of the present invention, the polymer may be a modified polyallylamine.
　本発明の生物学的材料の不動化を行うための組成物において、前記変性ポリアリルアミンは、第１級アミノ基がアシル基又はアルコキシカルボニル基に置換される場合がある。 In the composition for immobilizing a biological material of the present invention, in the modified polyallylamine, a primary amino group may be substituted with an acyl group or an alkoxycarbonyl group.
　本発明の生物学的材料の不動化を行うための組成物において、前記アシル基又はアルコキシカルボニル基の炭素原子数は２又は３の場合がある。 In the composition for immobilizing the biological material of the present invention, the acyl group or alkoxycarbonyl group may have 2 or 3 carbon atoms.
　本発明の生物学的材料の不動化を行うための組成物において、前記アシル基は、アセチル基又はプロピオニル基の場合がある。 In the composition for immobilizing a biological material of the present invention, the acyl group may be an acetyl group or a propionyl group.
　本発明の生物学的材料の不動化を行うための組成物において、前記ポリマーの同一分子中のアミノ基の２５－７５％が変性される場合がある。 In the composition for immobilizing a biological material of the present invention, 25-75% of amino groups in the same molecule of the polymer may be modified.
　本発明の生物学的材料の不動化を行うための組成物において、前記ポリアミン系ポリマーは、二酸化硫黄及び／又はマレイン酸が共重合される場合がある。 In the composition for immobilizing a biological material of the present invention, the polyamine polymer may be copolymerized with sulfur dioxide and / or maleic acid.
　本発明の生物学的材料の不動化を行うための組成物において、前記ポリマーは、アリルアミンと、ジアリルアミン又はジアリルメチルアミンとの共重合体の場合がある。 In the composition for immobilizing a biological material of the present invention, the polymer may be a copolymer of allylamine and diallylamine or diallylmethylamine.
　本発明の生物学的材料の不動化を行うための組成物において、前記ポリマーは、ポリジアリルアミン又はポリジアリルメチルアミンの場合がある。 In the composition for immobilizing a biological material of the present invention, the polymer may be polydiallylamine or polydiallylmethylamine.
　本発明の生物学的材料の不動化を行うための組成物において、前記ポリマーの平均分子量は３，０００ないし５４，０００の場合がある。 In the composition for immobilizing a biological material of the present invention, the polymer may have an average molecular weight of 3,000 to 54,000.
　本発明の生物学的材料の不動化を行うための組成物において、前記ポリマーを含む溶液の濃度は０．０５ないし０．２ｗ／ｖ％の場合がある。 In the composition for immobilizing the biological material of the present invention, the concentration of the solution containing the polymer may be 0.05 to 0.2 w / v%.
　本発明の生物学的材料の不動化を行うための組成物において、前記生物学的材料の不動化は、固定剤の存在下で行う場合がある。 In the composition for immobilizing a biological material according to the present invention, the biological material may be immobilized in the presence of a fixing agent.
　本発明の生物学的材料の不動化を行うための組成物において、前記固定剤は、０．０４ｗ／ｖ％のホルムアルデヒドの場合がある。 In the composition for immobilizing the biological material of the present invention, the fixative may be 0.04 w / v% formaldehyde.
　本発明の生物学的材料の不動化を行うための組成物において、前記生物学的材料は臨床検査試料の場合がある。 In the composition for immobilizing a biological material of the present invention, the biological material may be a clinical test sample.
　本発明の生物学的材料の不動化を行うための組成物において、前記生物学的材料は細胞診検査試料の場合がある。 In the composition for immobilizing a biological material of the present invention, the biological material may be a cytological examination sample.
　本発明の生物学的材料の不動化を行うための組成物において、前記生物学的材料は子宮頸部細胞診検査試料の場合がある。 In the composition for immobilizing a biological material of the present invention, the biological material may be a cervical cytological examination sample.
　本発明は、本発明の生物学的材料の不動化を行うための組成物をコーティングした、生物学的材料の不動化を行うための固体支持体を提供する。 The present invention provides a solid support for immobilizing biological material coated with a composition for immobilizing biological material of the present invention.
　本発明は、本発明の生物学的材料の不動化を行うための組成物をコーティングするステップを含む、生物学的材料の不動化を行うための固体支持体の製造方法を提供する。 The present invention provides a method for producing a solid support for immobilizing a biological material, comprising the step of coating a composition for immobilizing a biological material of the present invention.
　本発明は、本発明の生物学的材料の不動化を行うための組成物をコーティングした、生物学的材料の不動化を行うための顕微鏡スライドガラスを提供する。 The present invention provides a microscope slide glass for immobilizing biological material, which is coated with the composition for immobilizing biological material of the present invention.
　本発明は、本発明の顕微鏡スライドガラスを用いて生物学的材料を分析する方法を提供する。本発明の方法は、（１）前記生物学的材料を、固定剤を含む液に浸漬して固定するステップと、（２）固定された前記生物学的材料を分離するステップと、（３）分離された前記生物学的材料を水溶液に懸濁するステップと、（４）前記水溶液を前記顕微鏡スライドガラスに戴置して、該顕微鏡スライドガラス上に前記生物学的材料を不動化させるステップとを含む。 The present invention provides a method for analyzing biological materials using the microscope slide glass of the present invention. The method of the present invention includes (1) a step of immersing and fixing the biological material in a liquid containing a fixing agent, (2) a step of separating the fixed biological material, and (3) Suspending the separated biological material in an aqueous solution; (4) placing the aqueous solution on the microscope slide and immobilizing the biological material on the microscope slide; including.
　本発明の生物学的材料を分析する方法において、前記ステップ（３）の水溶液は固定剤を含む場合がある。 In the method for analyzing a biological material of the present invention, the aqueous solution in step (3) may contain a fixing agent.
　本発明の生物学的材料を分析する方法において、前記ステップ（３）の水溶液は０．０４ｗ／ｖ％のホルムアルデヒドを含む場合がある。 In the method for analyzing a biological material of the present invention, the aqueous solution in the step (3) may contain 0.04 w / v% formaldehyde.
　本発明の生物学的材料を分析する方法は、（５）前記ステップ（４）において前記生物学的材料が不動化された顕微鏡スライドガラスを染色するステップを含む場合がある。 The method for analyzing a biological material of the present invention may include a step of (5) staining a microscope slide glass in which the biological material is immobilized in the step (4).
　本発明は、本発明の生物学的材料を分析する方法を実行するための分析システムを提供する。本発明の分析システムは、本発明の顕微鏡スライドガラスを保持するホルダと、自動分注機ユニットと、ロボットアームを備えたピペッタユニットとを含む。 The present invention provides an analysis system for executing the method of analyzing a biological material of the present invention. The analysis system of the present invention includes a holder for holding the microscope slide glass of the present invention, an automatic dispenser unit, and a pipetter unit having a robot arm.
　本発明の分析システムは、遠心機ユニットを含む場合がある。 The analysis system of the present invention may include a centrifuge unit.
　本発明は、本発明の生物学的材料を分析する方法を実行するためのキットを提供する。本発明のキットは本発明の顕微鏡スライドガラスを含む。 The present invention provides a kit for carrying out the method for analyzing a biological material of the present invention. The kit of the present invention includes the microscope slide glass of the present invention.
　本発明のキットは、採取された生物学的材料を固定し保存するための試薬を含む場合がある。 The kit of the present invention may contain a reagent for fixing and storing the collected biological material.
　本発明の生物学的材料は、被験者から採取された細胞、組織、体液等を含むが、これらに限定されない。本発明の生物学的材料は、ヒト、マウス、ラット及びハムスターを含むがこれらに限定されない生物種由来の培養細胞の場合がある。本発明の生物学的材料は、光学顕微鏡で観察することにより、前記臓器に腫瘍、炎症その他の病理学的変化の有無を検査するために利用される場合と、生化学、免疫学、分子生物学その他の手法による検査のために利用される場合とがあるが、これらに限定されない。 The biological material of the present invention includes, but is not limited to, cells, tissues, body fluids and the like collected from subjects. The biological material of the present invention may be a cultured cell derived from a biological species including but not limited to human, mouse, rat and hamster. The biological material of the present invention is used for examining the presence or absence of tumor, inflammation or other pathological changes in the organ by observing with an optical microscope, biochemistry, immunology, molecular biology. It may be used for examination by academic or other methods, but it is not limited to these.
　本発明の生物学的材料は細胞診検査試料の場合がある。前記細胞診検査試料は、婦人科性器（外陰、膣壁、後膣円蓋部、頸部、頸管内膜、体部等）、呼吸器（喀痰、気管支、肺胞、洗浄液等）、体腔液、泌尿器（尿、洗浄液、腎、前立腺、精巣等）、乳腺（乳頭分泌物、穿刺吸引）、甲状腺、副甲状腺、消化器（口腔、咽頭、唾液腺、食道、胃、小腸、大腸、肝臓、胆道、膵臓）、脳（脳、髄液、中枢神経）、リンパ節、骨、軟部臓器、造血臓器（血液、骨髄）を含むが、これらに限定されない被検者の臓器から採取される場合がある。本発明の細胞診検査試料は子宮頸部から採取される場合がある。 The biological material of the present invention may be a cytological examination sample. Samples for cytological examination include gynecological genitals (vulva, vaginal wall, posterior vaginal cap, neck, cervical intima, body, etc.), respiratory organs (eg, sputum, bronchi, alveoli, lavage fluid), body cavity Urinary (urine, lavage fluid, kidney, prostate, testis, etc.), mammary gland (papillary discharge, puncture and aspiration), thyroid gland, parathyroid gland, digestive organ (oral cavity, pharynx, salivary gland, esophagus, stomach, small intestine, large intestine, liver, biliary tract) , Pancreas), brain (brain, cerebrospinal fluid, central nervous system), lymph node, bone, soft organ, hematopoietic organ (blood, bone marrow) . The cytological test sample of the present invention may be collected from the cervix.
　本明細書において、生物学的材料の不動化とは、顕微鏡スライドガラスを含む固体支持体の表面への生物学的材料の結合であって、該結合は、その後の細胞化学的技術のための処理、例えば、洗浄、透徹、染色等の処理の際に前記生物学的材料が前記固体支持体から脱離しない程度に強い結合をいう。 As used herein, immobilization of biological material refers to the binding of biological material to the surface of a solid support that includes a microscope slide, which is for subsequent cytochemical techniques. It refers to a bond strong enough to prevent the biological material from detaching from the solid support during processing such as washing, clearing, and staining.
　本明細書においてポリアリル（ａｌｌｙｌ）アミン系ポリマーとは、アリルアミン、ジアリルアミン、ジアリル（－Ｒ）アミン、（－Ｒ）２アリルアミンからなるグループから選択される少なくとも１種類のモノマーが重合したポリマーをいう。本発明のポリアリルアミン系ポリマーには、ポリアリルアミン、ポリジアリルアミン、ポリジアリル（－Ｒ）アミン、ポリ（－Ｒ）２アリルアミンが含まれる。ポリアリルアミンは第１級アミンモノマーであるアリルアミンだけが重合したポリマーであり、ポリジアリルアミンは第２級アミンモノマーであるジアリルアミンだけが重合したポリマーであり、ポリジアリル（－Ｒ）アミン及びポリ（－Ｒ）２アリルアミンは第３級アミンモノマーだけが重合したポリマーである。ここでＲは、炭素原子数１－２０のｎ－アルキル基又はｉｓｏ－アルキル基か、炭素原子数６－１２のアリル基かであって、ヒドロキシル基、ポリエチレングリコール鎖を含むがこれらに限定されない置換基を有する場合がある。またポリアリルアミン系ポリマーには、第１級アミンモノマーと第２級アミンモノマーとの共重合体と、第１級アミンモノマーと第３級アミンモノマーとの共重合体とが含まれる。 In the present specification, the polyallylamine-based polymer refers to a polymer in which at least one monomer selected from the group consisting of allylamine, diallylamine, diallyl (—R) amine, and (—R) 2 allylamine is polymerized. The polyallylamine-based polymer of the present invention includes polyallylamine, polydiallylamine, polydiallyl (—R) amine, and poly (—R) 2 allylamine. Polyallylamine is a polymer in which only primary amine monomer, allylamine is polymerized, and polydiallylamine is a polymer in which only secondary amine monomer, diallylamine, is polymerized, and polydiallyl (—R) amine and poly (—R) 2- Allylamine is a polymer in which only tertiary amine monomers are polymerized. Here, R is an n-alkyl group or iso-alkyl group having 1 to 20 carbon atoms, or an allyl group having 6 to 12 carbon atoms, and includes, but is not limited to, a hydroxyl group and a polyethylene glycol chain. It may have a substituent. The polyallylamine-based polymer includes a copolymer of a primary amine monomer and a secondary amine monomer, and a copolymer of a primary amine monomer and a tertiary amine monomer.
　本発明の固体支持体への生物学的材料の不動化に影響のないことを条件として、本発明のポリアリルアミン系ポリマーは、主鎖に、二酸化硫黄、マレイン酸その他の化合物が重合してもかまわないし、側鎖が変性されてもかまわない。本発明において変性とは、本発明のポリアリルアミン系ポリマーの側鎖のアミノ基の水素原子が他の原子又は原子団と共有結合による修飾を施されることをいう。すなわち、本発明のポリアリルアミン系ポリマーは、未変性ポリアリルアミン系ポリマーと、変性ポリアリルアミン系ポリマーとを含む。本明細書のポリマーについて、「変性（アシル化、アルコキシカルボニル化等を含む）」又は「未変性」と特に記載されない場合には、「変性ポリマー及び／又は未変性ポリマー」を意味する。本発明の変性ポリアリルアミン系ポリマーとは、未変性ポリアリルアミン系ポリマー分子の少なくとも一部のアミノ基において、水素原子が他の原子又は原子団と置換されたポリマーをいう。したがって、本発明のポリマー中の第２級アミノ基又は第３級アミノ基は、第２級アミンモノマー又は第３級アミンモノマーに由来する場合もあるが、それぞれ、第１級アミンモノマー又は第２級アミンモノマーに由来するが、モノマーの重合反応の後に変性される場合がある。 The polyallylamine-based polymer of the present invention can be polymerized with sulfur dioxide, maleic acid or other compounds in the main chain, provided that it does not affect the immobilization of the biological material on the solid support of the present invention. It does not matter, and the side chain may be denatured. In the present invention, “modification” means that the hydrogen atom of the amino group in the side chain of the polyallylamine-based polymer of the present invention is covalently modified with another atom or atomic group. That is, the polyallylamine-based polymer of the present invention includes an unmodified polyallylamine-based polymer and a modified polyallylamine-based polymer. When the polymer of the present specification is not specifically described as “modified (including acylation, alkoxycarbonylation, etc.)” or “unmodified”, it means “modified polymer and / or unmodified polymer”. The modified polyallylamine-based polymer of the present invention refers to a polymer in which hydrogen atoms are substituted with other atoms or atomic groups in at least some amino groups of the unmodified polyallylamine-based polymer molecule. Therefore, the secondary amino group or the tertiary amino group in the polymer of the present invention may be derived from the secondary amine monomer or the tertiary amine monomer, but the primary amine monomer or the second amine group respectively. Although derived from a secondary amine monomer, it may be modified after the polymerization reaction of the monomer.
　本発明の変性ポリアリルアミン系ポリマーは、同一分子内の中のアミノ基の１－９５％が変性されることが好ましく、同一分子内の中のアミノ基の１０－８０％が変性されることがより好ましく、同一分子内の中のアミノ基の２５－７５％が変性されることがさらにより好ましい。本発明のポリマーにおける同一分子内の中のアミノ基のうち変性アミノ基の割合は、ＮＭＲ法、コロイド滴定法その他の当業者に周知の方法で決定することができる。 In the modified polyallylamine-based polymer of the present invention, 1-95% of amino groups in the same molecule are preferably modified, and 10-80% of amino groups in the same molecule may be modified. More preferably, it is even more preferred that 25-75% of the amino groups in the same molecule are modified. The ratio of the modified amino group among the amino groups in the same molecule in the polymer of the present invention can be determined by NMR method, colloid titration method, and other methods well known to those skilled in the art.
　本発明の変性ポリアリルアミン系ポリマーにおいてアミノ基の水素原子を置換する原子団は、炭素原子数１－２０のｎ－アルキル基又はｉｓｏ－アルキル基か、炭素原子数６－１２のアリル基かであって、ヒドロキシル基、ポリエチレングリコール鎖を含むがこれらに限定されない置換基を有する場合がある。前記原子団は、アシル基又はアルコキシカルボニル基であることが好ましい。前記原子団は、炭素原子数２－６のアシル基又はアルコキシカルボニル基であることがより好ましく、アセチル基、プロピオニル基、メトキシカルボニル基、エトキシカルボニル基であることがさらに好ましく、アセチル基及びプロピオニル基であることがさらにより好ましい。これらの原子団で本発明のポリマーのアミノ基の水素原子を置換する方法は当業者に周知である。例えば、アセチル基で置換する場合には、ポリマーを無水酢酸と反応させる。 In the modified polyallylamine-based polymer of the present invention, the atomic group that replaces the hydrogen atom of the amino group is an n-alkyl group or iso-alkyl group having 1 to 20 carbon atoms, or an allyl group having 6 to 12 carbon atoms. And may have substituents including but not limited to hydroxyl groups and polyethylene glycol chains. The atomic group is preferably an acyl group or an alkoxycarbonyl group. The atomic group is more preferably an acyl group having 2-6 carbon atoms or an alkoxycarbonyl group, more preferably an acetyl group, a propionyl group, a methoxycarbonyl group, or an ethoxycarbonyl group, and an acetyl group and a propionyl group. Even more preferably. Methods for substituting hydrogen atoms of amino groups of the polymer of the present invention with these atomic groups are well known to those skilled in the art. For example, when replacing with an acetyl group, the polymer is reacted with acetic anhydride.
　本発明のポリアリルアミン系ポリマーのアミノ基が他の１種類又は２種類以上のカチオンとのイオン結合を行っていてもかまわない。本明細書において、ポリアリルアミン系ポリマーの「遊離塩基」とは、前記カチオンとの結合がないものをいい、フリータイプともよばれることがある。ポリアリルアミン系ポリマーの「塩」とは、前記カチオンとの結合があるものをいう。好ましいポリアリルアミン系ポリマーの塩には、塩酸塩及び酢酸塩を含むが、これらに限定されない。 The amino group of the polyallylamine-based polymer of the present invention may be ionically bonded to one or more other types of cations. In the present specification, the “free base” of the polyallylamine-based polymer means one having no bond with the cation, and is sometimes referred to as a free type. The “salt” of a polyallylamine-based polymer is one having a bond with the cation. Preferred polyallylamine-based polymer salts include, but are not limited to, hydrochloride and acetate.
　本発明のポリアリルアミン系ポリマーは商業的に入手可能であり、その合成方法は当業者に周知である。本発明のポリアリルアミン系ポリマーの平均分子量は、本発明の固体支持体への生物学的材料の不動化に影響のないことを条件として、５００ないし１，０００，０００の範囲内であればよい。本発明のポリアリルアミン系ポリマーの平均分子量は、２，０００ないし１００，０００の範囲内であることが好ましく、３，０００ないし５４，０００の範囲内であることがより好ましい。ポリアリルアミン系ポリマーのうち、ポリジアリルアミンの場合には、３，０００ないし８０００の範囲内であることが望ましい。アリルアミン及びジアリルアミンの共重合体の場合、好ましい分子量はポリマー中に含まれるアリルアミン及びジアリルアミンの割合に依存する。アリルアミン及びジアリルアミンの割合が１：１の場合には、３，０００ないし５４，０００の範囲内であることが好ましく、３：１の場合には、３，０００ないし１００，０００の範囲内であることが好ましく、１：３の場合には、３，０００ないし３０，０００の範囲内であることが好ましい。分子量の範囲は、アリルアミン及びジアリルアミンの割合によって変化するため、これらに限定されるものではない。本発明のポリアリルアミン系ポリマーの平均分子量及び多分散度（Ｍｗ／Ｍｎ）は、ゲル浸透クロマトグラフィ法その他の当業者に周知の測定法によって決定することができる。本発明のポリアリルアミン系ポリマーの平均分子量は多分散を示す場合がある。 The polyallylamine-based polymer of the present invention is commercially available and its synthesis method is well known to those skilled in the art. The average molecular weight of the polyallylamine-based polymer of the present invention may be in the range of 500 to 1,000,000, provided that it does not affect the immobilization of biological material on the solid support of the present invention. . The average molecular weight of the polyallylamine-based polymer of the present invention is preferably in the range of 2,000 to 100,000, and more preferably in the range of 3,000 to 54,000. Among the polyallylamine-based polymers, in the case of polydiallylamine, it is preferably within the range of 3,000 to 8,000. In the case of allylamine and diallylamine copolymers, the preferred molecular weight depends on the proportion of allylamine and diallylamine contained in the polymer. When the ratio of allylamine and diallylamine is 1: 1, it is preferably within a range of 3,000 to 54,000, and when 3: 1, it is within a range of 3,000 to 100,000. In the case of 1: 3, it is preferably in the range of 3,000 to 30,000. Since the range of molecular weight changes with the ratio of allylamine and diallylamine, it is not limited to these. The average molecular weight and polydispersity (Mw / Mn) of the polyallylamine-based polymer of the present invention can be determined by gel permeation chromatography or other measurement methods well known to those skilled in the art. The average molecular weight of the polyallylamine-based polymer of the present invention may exhibit polydispersity.
　本明細書において、共重合体とは２種類以上の単量体が重合して生成された高分子化合物を指し、前記共重合体の重合形式は、交互共重合、ブロック共重合、ランダム共重合、グラフト共重合及びこれらの組み合わせを含むが、これらに限定されない。本発明のポリアリルアミン系ポリマーに含まれる共重合体は、本発明の固体支持体への生物学的材料の不動化に影響のないことを条件として、２種類以上の単量体がいかなる割合で共重合されてもかまわない。 In the present specification, a copolymer refers to a polymer compound formed by polymerizing two or more types of monomers, and the polymerization type of the copolymer includes alternating copolymerization, block copolymerization, and random copolymerization. , Graft copolymerization and combinations thereof, but not limited thereto. The copolymer contained in the polyallylamine-based polymer of the present invention has any ratio of two or more types of monomers provided that it does not affect the immobilization of biological material on the solid support of the present invention. It may be copolymerized.
　本発明のポリアリルアミン系ポリマーは、脱イオン水、エタノールその他の当業者に周知の溶媒に溶解することができる。本発明のポリアリルアミン系ポリマーの濃度は、本発明の固体支持体への生物学的材料の不動化に影響のないことを条件として、いかなる濃度であってもかまわないが、０．０１ないし１ｗ／ｖ％の範囲内が好ましく、０．０５ないし０．２ｗ／ｖ％の範囲内であることがより好ましい。 The polyallylamine-based polymer of the present invention can be dissolved in deionized water, ethanol or other solvents known to those skilled in the art. The concentration of the polyallylamine-based polymer of the present invention may be any concentration as long as it does not affect the immobilization of biological material on the solid support of the present invention. / V% is preferable, and 0.05 to 0.2 w / v% is more preferable.
　本発明のポリアリルアミン系ポリマーを含む溶液は、遊離型ポリアリルアミンを前記溶媒に溶解した後、塩酸又は水酸化ナトリウムその他当業者に周知の酸又は塩基を用いてｐＨを調製することができる。また前記ｐＨは適切な緩衝液で安定化される場合がある。前記ポリアリルアミン系ポリマーを含む溶液のｐＨは、本発明の固体支持体への生物学的材料の不動化に影響のないことを条件として、いかなるｐＨであってもかまわないが、９ないし１２の範囲内であることが好ましく、１０ないし１１の範囲内であることがより好ましい。 The solution containing the polyallylamine-based polymer of the present invention can be adjusted to pH using hydrochloric acid, sodium hydroxide, or other acid or base known to those skilled in the art after dissolving the free polyallylamine in the solvent. The pH may be stabilized with an appropriate buffer. The pH of the solution containing the polyallylamine-based polymer may be any pH as long as it does not affect the immobilization of the biological material on the solid support of the present invention. It is preferably within the range, and more preferably within the range of 10 to 11.
　本明細書において、ポリアリルアミン系ポリマーをコーティングするとは、ポリアリルアミン系ポリマー溶液を固体支持体に噴霧又は塗布するか、ポリアリルアミン系ポリマー溶液に固体支持体を浸漬することをいう。 In the present specification, coating a polyallylamine-based polymer means spraying or applying a polyallylamine-based polymer solution to a solid support, or immersing the solid support in a polyallylamine-based polymer solution.
　本明細書において固体支持体とは、顕微鏡スライドガラスの他、マルチウェルプレート、ラテックス凝集法用微粒子及び表面プラズモン共鳴法用センサーチップを含むが、これらに限定されない。 In this specification, the solid support includes, but is not limited to, a microscope slide glass, a multiwell plate, fine particles for latex aggregation method, and a sensor chip for surface plasmon resonance method.
　前記固体支持体は、疎水性表面処理及び／又は疎水性樹脂のコーティングを施すことにより、該固体支持体の表面のうち特定の場所だけに本発明のポリアリルアミン系ポリマーがコーティングされる場合がある。 The solid support may be coated with the polyallylamine-based polymer of the present invention only on a specific portion of the surface of the solid support by applying a hydrophobic surface treatment and / or a hydrophobic resin coating. .
　本発明のポリアリルアミン系ポリマーがコーティングされた固体支持体は、該ポリアリルアミン系ポリマーのみがコーティングされる場合の他、前記ポリアリルアミン系ポリマーとともに他のいずれかのポリマー組成物がコーティングされる場合も含む。 The solid support coated with the polyallylamine polymer of the present invention may be coated not only with the polyallylamine polymer but also with any other polymer composition together with the polyallylamine polymer. Including.
　本発明のポリアリルアミン系ポリマーがコーティングされた固体支持体は、風乾又は加熱乾燥によって乾燥処理を施されることが望ましい。 The solid support coated with the polyallylamine polymer of the present invention is preferably subjected to a drying treatment by air drying or heat drying.
　本発明のポリアリルアミン系ポリマーがコーティングされた固体支持体は、前記乾燥処理の後、脱イオン水その他の水性溶媒でリンスされる場合がある。リンス処理により、共染、すなわち、染色に用いる色素が生物学的材料ではなく前記ポリアリルアミン系ポリマーと反応する非特異的染色が起こりにくくなる。本発明のポリアリルアミン系ポリマーは共染が起こらない条件であっても、固定液の洗浄が不完全で固定液の成分を含む溶液中で生物学的材料を不動化することができる。 The solid support coated with the polyallylamine-based polymer of the present invention may be rinsed with deionized water or other aqueous solvent after the drying treatment. The rinsing treatment makes it difficult for co-staining, that is, nonspecific staining in which the dye used for staining reacts with the polyallylamine-based polymer instead of the biological material. The polyallylamine-based polymer of the present invention can immobilize the biological material in a solution containing the components of the fixing solution, even if the co-staining is not caused, incompletely washing the fixing solution.
　本発明の固定液は、固定剤を含む水性溶液をいう。前記固定剤は、ホルムアルデヒド、グルタルアルデヒド、ジアルデヒドを含むがこれらに限定されないアルデヒドと、メタノール、エタノールを含むがこれらに限定されないアルコールと、アセトンと、ピクリン酸とからなるグループから選択される１種類または２種類以上の化合物の場合がある。好ましい固定剤はホルムアルデヒド、グルタルアルデヒド及び／又はジアルデヒドである。 The fixing solution of the present invention refers to an aqueous solution containing a fixing agent. The fixing agent is selected from the group consisting of aldehydes including but not limited to formaldehyde, glutaraldehyde, dialdehyde, alcohols including but not limited to methanol, ethanol, acetone, and picric acid. Or there may be two or more types of compounds. Preferred fixatives are formaldehyde, glutaraldehyde and / or dialdehyde.
　本発明の生物学的材料の処理方法において、固定液に懸濁された生物学的材料は、遠心によって該固定液から分離される。前記固定液を除去した後、０．１ｍＬ程度の固定液が前記生物学的材料とともに遠心管に残っていて、新鮮な脱イオン水２ｍＬからなる洗浄液で洗浄された後、本発明のポリアリルアミン系ポリマーがコーティングされた顕微鏡スライドガラス上に戴置される。固定液をさらに除去する場合には、固定液を除去した遠心管に１０ｍＬの新鮮な脱イオン水からなる予洗液で洗浄された後、２度目の遠心によって前記生物学的材料が分離され、前記予洗液が除去されたのち、新鮮な脱イオン水２ｍＬからなる洗浄液で洗浄された後、本発明のポリアリルアミン系ポリマーがコーティングされた顕微鏡スライドガラス上に戴置される。いずれの遠心においても、０．１ｍＬ程度の溶液が沈殿した生物学的材料とともに遠心管に残る。したがって、予洗を行わない場合には固定液は２０倍に希釈され、予洗を行う場合には固定液はさらに１００倍に希釈されると考えられる。本明細書の実施例に示すとおり、予洗の有無によって生物学的材料のスライドガラスへの不動化の程度は大きな影響を受ける。以下の実施例で用いた固定液は０．８ｗ／ｖ％のホルムアルデヒドを含むので、予洗を行わない場合には、本発明の組成物をコーティングされた顕微鏡スライド上に生物学的材料が不動化される際には０．０４ｗ／ｖ％のホルムアルデヒドが存在する。 In the biological material processing method of the present invention, the biological material suspended in the fixing solution is separated from the fixing solution by centrifugation. After removing the fixing solution, about 0.1 mL of the fixing solution remains in the centrifuge tube together with the biological material, and after washing with a cleaning solution consisting of 2 mL of fresh deionized water, the polyallylamine system of the present invention Placed on a microscope slide coated with polymer. When the fixative is further removed, the biological material is separated by a second centrifugation after washing with 10 mL of fresh deionized water in the centrifuge tube from which the fixative has been removed. After the pre-washing solution is removed, it is washed with a washing solution consisting of 2 mL of fresh deionized water and then placed on a microscope slide glass coated with the polyallylamine-based polymer of the present invention. In any centrifugation, as little as 0.1 mL of solution remains in the centrifuge tube with the biological material precipitated. Therefore, it is considered that the fixing solution is diluted 20 times when pre-washing is not performed, and the fixing solution is further diluted 100 times when pre-washing is performed. As shown in the examples of the present specification, the degree of immobilization of the biological material on the slide glass is greatly affected by the presence or absence of pre-washing. The fixative solution used in the following examples contains 0.8 w / v% formaldehyde, so that the biological material is immobilized on a microscope slide coated with the composition of the present invention without pre-washing. When present, 0.04 w / v% formaldehyde is present.
　本発明の生物学的材料が細胞診検査試料の場合には、顕微鏡スライドガラス上に少なくとも５，０００個の細胞が不動化されることが好ましい。細胞診検査、主に子宮頸部細胞診に用いる標本の適否に関する基準であるベセスダシステム２００１では、１枚の標本について保存状態がよく鮮明に見える扁平上皮細胞が最低でも５，０００個含まれていると推定されなければならないと規定されているからである。以下の実施例で細胞が顕微鏡スライドガラスに不動化する程度（細胞不動化度）は、最大を１０点とする１０段階の指数で定性的に評価された。細胞不動化度が４点以上のとき、顕微鏡スライドガラス上に少なくとも５，０００個の細胞が不動化され、ベセスダシステム２００１の基準を満足する。 When the biological material of the present invention is a cytological examination sample, it is preferable that at least 5,000 cells are immobilized on a microscope slide glass. The Bethesda system 2001, which is a standard regarding the suitability of specimens used for cytological examinations, mainly cervical cytological examinations, contains at least 5,000 squamous epithelial cells that are well preserved and clearly visible for one specimen. This is because it is stipulated that it must be estimated that In the following examples, the degree of cell immobilization on the microscope slide glass (degree of cell immobilization) was qualitatively evaluated using a 10-point index with a maximum of 10 points. When the degree of cell immobilization is 4 or more, at least 5,000 cells are immobilized on the microscope slide glass, which satisfies the criteria of Bethesda system 2001.
　本発明の生物学的材料を分析する方法を実行するための分析システムのうち、本発明の顕微鏡スライドガラスを保持するホルダと、自動分注機ユニットと、ロボットアームを備えたピペッタユニットとは、当業者に周知である。前記分析システムは、遠心機ユニット及び／又は本発明の生物学的材料を分析する方法を説明するマニュアルを含む場合がある。 Among the analysis systems for executing the method for analyzing biological material of the present invention, the holder for holding the microscope slide glass of the present invention, the automatic dispenser unit, and the pipetter unit having a robot arm Are well known to those skilled in the art. The analysis system may include a centrifuge unit and / or a manual describing a method for analyzing the biological material of the present invention.
　本発明の生物学的材料を分析する方法を実行するためのキットは、本発明の組成物をコーティングした顕微鏡スライドガラスの他、本発明の生物学的材料を採取するための器具、採取された生物学的材料を固定し保存するための試薬、本発明の生物学的材料を分析する方法を説明するマニュアル等を含む場合がある。前記試薬は少なくとも０．８ｗ／ｖ％のホルムアルデヒドを含む。 A kit for carrying out the method for analyzing a biological material of the present invention was collected from a microscope slide glass coated with the composition of the present invention, as well as an instrument for collecting the biological material of the present invention. It may include reagents for immobilizing and storing biological materials, manuals describing methods for analyzing biological materials of the present invention, and the like. The reagent contains at least 0.8 w / v% formaldehyde.
異なる平均分子量の５０％アセチル化ポリアリルアミンを含む溶液の濃度と、該溶液に浸漬することによりコーティングされたスライドガラスの細胞不動化度の関係を示すグラフ。The graph which shows the relationship between the density | concentration of the solution containing 50% acetylated polyallylamine of different average molecular weight, and the cell immobilization degree of the glass slide coated by being immersed in this solution. 異なる平均分子量の未変性ポリアリルアミンを含む溶液の濃度と、該溶液に浸漬することによりコーティングされたスライドガラスの細胞不動化度との関係を示すグラフ。The graph which shows the relationship between the density | concentration of the solution containing the native polyallylamine of different average molecular weight, and the cell immobilization degree of the glass slide coated by being immersed in this solution. 予洗処理を施された細胞について、平均分子量１５，０００のアシル化ポリアリルアミンのアシル化の程度と、該アシル化ポリアリルアミンがコーティングされたスライドガラスの細胞不動化度との関係を示すグラフ。The graph which shows the relationship between the degree of acylation of acylated polyallylamine with an average molecular weight of 15,000 and the degree of cell immobilization of a slide glass coated with the acylated polyallylamine for cells that have been subjected to a prewash treatment. 予洗処理を施されない細胞について、平均分子量１５，０００のアシル化ポリアリルアミンのアシル化の程度と、該アシル化ポリアリルアミンがコーティングされたスライドガラスの細胞不動化度との関係を示すグラフ。The graph which shows the relationship between the acylation degree of acylated polyallylamine with an average molecular weight of 15,000, and the cell immobilization degree of the glass slide coated with the acylated polyallylamine for cells not subjected to pre-washing treatment. 予洗処理を施されない細胞について、さまざまなポリアリルアミン系ポリマーでコーティングされたスライドガラスの細胞不動化度を示すグラフ。The graph which shows the cell immobilization degree of the glass slide coated with various polyallylamine-type polymers about the cell which is not pre-washed. コーティングされた等モル共重合体の濃度及び平均分子量と、細胞不動化度との関係を示すグラフ。The graph which shows the relationship between the density | concentration and average molecular weight of a coated equimolar copolymer, and a cell immobilization degree. コーティングされたポリアリルアミンの濃度及び平均分子量と、細胞不動化度との関係を示すグラフ。The graph which shows the relationship between the density | concentration and average molecular weight of the coated polyallylamine, and the degree of cell immobilization.
　以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。 The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the appended claims. Modifications of the present invention, for example, addition, deletion, and replacement of the configuration requirements of the present invention can be made on the condition that the gist of the present invention is not deviated.
　本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。 All documents mentioned in this specification are incorporated herein by reference in their entirety.
　１．ポリアリルアミンのアセチル化の細胞不動化度への影響
　１－１．方法
　未変性ポリアリルアミン
　日東紡績株式会社から購入された未変性ポリアリルアミンは、平均分子量が３０００、５０００、８０００及び１５０００（カタログ番号はそれぞれ、ＰＡＡ－０３、ＰＡＡ－０５、ＰＡＡ－０８及びＰＡＡ－１５Ｃ）であった。 1. 1. Effect of acetylation of polyallylamine on cell immobilization 1-1. Method Unmodified polyallylamine Unmodified polyallylamine purchased from Nittobo Co., Ltd. has an average molecular weight of 3000, 5000, 8000 and 15000 (catalog numbers are PAA-03, PAA-05, PAA-08 and PAA-15C, respectively). )Met.
　５０％アセチル化ポリアリルアミンの合成
　かき混ぜ機、ジムロート還流器、滴下ロート、温度計を備えた４ツ口５ｌ丸底セパラブルフラスコに、分子量１５０００の１５％ポリアリルアミン水溶液を３４４３ｇ（未変性アリルアミンモノマー単位で６．０モル）が仕込まれ、フラスコを氷水で冷やし２００ｒｐｍで撹拌しながら、無水酢酸３１５．７７ｇ（３．００モル）をゆっくり滴下し、４時間かけて全量滴下された。反応時の温度は０～５°Ｃに保持された。引き続きフラスコを冷やしながら撹拌して同温度を維持しながら、１８．４１％の水酸化ナトリウム水溶液６６７．３１ｇ（３．０７モル）が滴下され、副生した酢酸が中和された。中和された反応混合液は、特公平７－６８２８９公報に記載の方法で電気透析に付され、４４時間かけて脱塩され、アセチル化ポリアリルアミン遊離塩基が単離された。該アセチル化ポリアリルアミン遊離塩基の水溶液中の水が重水に置換され、ＮＭＲ測定に供された。アミノ基に結合するメチレン基の水素と、アミド基に結合するメチレン基の水素とに対応するＮＭＲ波形の面積比から、アセチル化ポリアリルアミンのアセチル化度が５０％と算出された。前記アセチル化ポリアリルアミンの平均分子量の多分散度（Ｍｗ／Ｍｎ）は、２．６３７ないし２．７３０であった。 Synthesis of 50% acetylated polyallylamine 3443 g of 15% polyallylamine aqueous solution with a molecular weight of 15000 (unmodified allylamine monomer unit) in a four-necked 5 l round bottom separable flask equipped with a stirrer, Dimroth reflux condenser, dropping funnel and thermometer 6.0 mol) was charged, and 315.77 g (3.00 mol) of acetic anhydride was slowly added dropwise over 4 hours while cooling the flask with ice water and stirring at 200 rpm. The temperature during the reaction was maintained at 0-5 ° C. Subsequently, 667.31 g (3.07 mol) of an 18.41% aqueous sodium hydroxide solution was added dropwise while stirring and maintaining the same temperature while cooling the flask, and the by-produced acetic acid was neutralized. The neutralized reaction mixture was subjected to electrodialysis by the method described in Japanese Patent Publication No. 7-68289 and desalted for 44 hours to isolate acetylated polyallylamine free base. The water in the aqueous solution of the acetylated polyallylamine free base was replaced with heavy water and subjected to NMR measurement. The degree of acetylation of the acetylated polyallylamine was calculated to be 50% from the area ratio of the NMR waveform corresponding to the hydrogen of the methylene group bonded to the amino group and the hydrogen of the methylene group bonded to the amide group. The average molecular weight polydispersity (Mw / Mn) of the acetylated polyallylamine was 2.637 to 2.730.
　顕微鏡スライドガラスのコーティング
　撥水性樹脂を被覆した顕微鏡スライドガラスは、前記５０％アセチル化ポリアリルアミン溶液か、未変性ポリアリルアミン溶液かに１０秒間浸漬され、引き上げた後、風乾された。その後、脱イオン水でリンス処理され、風乾され、保存された。 Microscope slide glass coating The microscope slide glass coated with a water-repellent resin was immersed in the 50% acetylated polyallylamine solution or the unmodified polyallylamine solution for 10 seconds, pulled up, and then air-dried. Thereafter, it was rinsed with deionized water, air dried and stored.
　固定液の調製
　固定液は、０．８ｗ／ｖ％ホルムアルデヒド、２２ｗ／ｖ％エタノール及び１．５ｗ／ｖ％メタノールの水溶液を用いた。 Preparation of Fixing Solution As the fixing solution, an aqueous solution of 0.8 w / v% formaldehyde, 22 w / v% ethanol and 1.5 w / v% methanol was used.
　生物学的材料の調製
　社内ボランティアから採取した細胞診検査試料を前記固定液で固定した。 Preparation of biological material A cytological test sample collected from an in-house volunteer was fixed with the fixative.
　固定液の除去
　前記固定液で固定された細胞は８００Ｇで５分間遠心され、前記固定液が除去された。沈殿した細胞ペレットには前記固定液が約０．１ｍＬ含まれた。 Removal of fixative The cells fixed with the fixative were centrifuged at 800 G for 5 minutes to remove the fixative. The precipitated cell pellet contained about 0.1 mL of the fixative.
　予洗処理
　前記固定液が除去された細胞ペレットに、脱イオン水１０ｍＬからなる予洗液が添加され、懸濁された細胞は８００Ｇで５分間遠心され、前記予洗液が除去された。沈殿した細胞ペレットには前記予洗液が約０．１ｍＬ含まれた。 Prewash treatment A prewash solution consisting of 10 mL of deionized water was added to the cell pellet from which the fixative solution had been removed, and the suspended cells were centrifuged at 800 G for 5 minutes to remove the prewash solution. The precipitated cell pellet contained about 0.1 mL of the prewash solution.
　細胞のコーティング
　前記固定液が除去された細胞ペレットか、前記予洗液が除去された細胞ペレットかに脱イオン水２ｍＬからなる洗浄液が添加され、攪拌及び懸濁の後、０．３ｍＬが、前記５０％アセチル化ポリアリルアミンか、未変性ポリアリルアミンかでコーティングされたスライドガラス上に戴置され、１０分間静置された。細胞が不動化されたスライドは１００％エタノール又は脱イオン水で洗浄された。 Cell Coating A washing solution consisting of 2 mL of deionized water is added to the cell pellet from which the fixative solution has been removed or the cell pellet from which the pre-wash solution has been removed. It was placed on a glass slide coated with% acetylated polyallylamine or unmodified polyallylamine and allowed to stand for 10 minutes. Slides with immobilized cells were washed with 100% ethanol or deionized water.
　細胞の染色
　細胞の吸着後、細胞を染色するために細胞診検査で慣用されるパパニコロウ染色が施された。細胞が吸着されたスライドは、ヘマトキシリン溶液で１分半、ＯＧ－６で２分間染色された。その後、ＥＡ－５０で３分間染色された。染色後は、脱水・透徹処理され、封入された。 Cell Staining After cell adsorption, Papanicolaou staining, commonly used in cytodiagnosis, was performed to stain the cells. The slide on which the cells were adsorbed was stained with a hematoxylin solution for 1 and a half minutes and with OG-6 for 2 minutes. Thereafter, it was stained with EA-50 for 3 minutes. After dyeing, it was dehydrated and transparently processed and sealed.
　スライドの細胞不動化度の測定
　パパニコロウ染色後、顕微鏡の１視野あたりの細胞の数を観察して、５０％アセチル化ポリアリルアミン又は未変性ポリアリルアミンの細胞不動化度は、最大を１０とする１０段階の指数で定性的に評価された。細胞不動化度が４点以上のとき、顕微鏡スライドガラス上に少なくとも５，０００個の細胞が不動化され、ベセスダシステム２００１の基準を満足する。 Measurement of cell immobilization degree of slide After Papanicolaou staining, the number of cells per visual field of a microscope was observed, and the cell immobilization degree of 50% acetylated polyallylamine or unmodified polyallylamine was set to 10 at maximum 10 Qualitatively evaluated with a stage index. When the degree of cell immobilization is 4 or more, at least 5,000 cells are immobilized on the microscope slide glass, which satisfies the criteria of Bethesda system 2001.
　１－２．結果
　図１Ａは、異なる平均分子量の５０％アセチル化ポリアリルアミンを含む溶液の濃度と、該溶液に浸漬することによりコーティングされたスライドガラスの細胞不動化度の関係を示すグラフである。プロット点のそれぞれは、５０％アセチル化ポリアリルアミンの分子量が、３０００（◇）、５０００（■）、８０００（▲）及び１５０００（□）であることを示す。分子量１５０００では、０．０３１２５％（ｗ／ｖ）、０．０６２５％（ｗ／ｖ）及び０．１２５％（ｗ／ｖ）の濃度で細胞不動化度の評価指数が最大の１０を示した。図１Ｂは、異なる平均分子量の未変性ポリアリルアミンを含む溶液の濃度と、該溶液に浸漬することによりコーティングされたスライドガラスの細胞不動化度との関係を示すグラフである。プロット点のそれぞれは、未変性ポリアリルアミンの分子量が、３０００（◇）、５０００（■）、８０００（▲）及び１５０００（□）であることを示す。分子量３０００では、０．０３１２５％（ｗ／ｖ）及び０．０６２５％（ｗ／ｖ）の濃度で細胞不動化度の評価指数が９を示した。以上の結果から、分子量１５０００の５０％アセチル化ポリアリルアミンは０．１２５％（ｗ／ｖ）以下の濃度で、分子量３０００の未変性ポリアリルアミンは０．０６２５％（ｗ／ｖ）以下の濃度で細胞不動化度が最大であることが示された。また、未変性ポリアリルアミンよりも５０％アセチル化ポリアリルアミンの方が細胞不動化度が高く、より多くの細胞が吸着された。これは、未変性ポリアリルアミンがアシル化されると細胞不動化度が増大することを示唆する。これは、アミノ基がアシル化すると疎水性が高まるため、イオン結合に加えて疎水結合の寄与によって細胞不動化度が増大すると考えられる。 1-2. Results FIG. 1A is a graph showing the relationship between the concentration of a solution containing 50% acetylated polyallylamine having different average molecular weights and the degree of cell immobilization of a glass slide coated by immersion in the solution. Each of the plot points indicates that the molecular weight of 50% acetylated polyallylamine is 3000 (、 ５), 5000 (■), 8000 (▲), and 15000 (□). At a molecular weight of 15000, the evaluation index for the degree of cell immobilization showed a maximum of 10 at concentrations of 0.03125% (w / v), 0.0625% (w / v) and 0.125% (w / v). . FIG. 1B is a graph showing the relationship between the concentration of a solution containing unmodified polyallylamine having different average molecular weights and the degree of cell immobilization of a glass slide coated by being immersed in the solution. Each of the plotted points indicates that the molecular weight of the unmodified polyallylamine is 3000 (（), 5000 (■), 8000 (▲), and 15000 (□). When the molecular weight was 3000, the evaluation index of cell immobilization degree was 9 at the concentrations of 0.03125% (w / v) and 0.0625% (w / v). From the above results, 50% acetylated polyallylamine with a molecular weight of 15000 has a concentration of 0.125% (w / v) or less, and unmodified polyallylamine with a molecular weight of 3000 has a concentration of 0.0625% (w / v) or less. It was shown that the degree of cell immobilization was maximal. In addition, 50% acetylated polyallylamine had a higher cell immobilization degree than unmodified polyallylamine, and more cells were adsorbed. This suggests that the degree of cell immobilization increases when the native polyallylamine is acylated. This is thought to be because when the amino group is acylated, the hydrophobicity increases, and thus the degree of cell immobilization increases due to the contribution of the hydrophobic bond in addition to the ionic bond.
　２．アシル化の種類及び割合と、予洗処理の有無との細胞不動化度への影響
　２－１．方法
　２５％アセチル化ポリアリルアミンの合成
　無水酢酸が１５７．８９ｇ（１．５モル）、水酸化ナトリウムが１４．８３％の水酸化ナトリウム水溶液４２５．７７ｇ（１．５８モル）が用いられた以外は、実施例１と同様に操作し、２５％アセチル化ポリアリルアミン遊離塩基が得られた。 2. 2. Effect of kind and ratio of acylation and presence / absence of prewash treatment on cell immobilization degree 2-1. Method Synthesis of 25% acetylated polyallylamine Except for using 157.89 g (1.5 mol) of acetic anhydride and 425.77 g (1.58 mol) of aqueous sodium hydroxide solution with 14.83% sodium hydroxide. By operating in the same manner as in Example 1, 25% acetylated polyallylamine free base was obtained.
　７５％アセチル化ポリアリルアミンの合成
　無水酢酸が４７３．６５ｇ（４．５０モル）、水酸化ナトリウムが１８．４１％の水酸化ナトリウム水溶液１０００．９７ｇ（４．６１モル）が用いられた以外は、実施例１と同様に操作し、７５％アセチル化ポリアリルアミン遊離塩基が得られた。 Synthesis of 75% acetylated polyallylamine Except that 473.65 g (4.50 mol) of acetic anhydride and 100.97 g (4.61 mol) of an aqueous sodium hydroxide solution of 18.41% sodium hydroxide were used, By operating in the same manner as in Example 1, 75% acetylated polyallylamine free base was obtained.
　２５％プロピオニル化ポリアリルアミンの合成
　かき混ぜ機、ジムロート還流器、滴下ロート、温度計を備えた４ツ口３ｌ丸底セパラブルフラスコに、分子量約１５０００の１５％ポリアリルアミン水溶液（ＰＡＡ－１５Ｃ）が１７６３ｇ（未変性アリルアミンモノマー単位で３．０モル）仕込まれ、フラスコを氷水で冷やし２００ｒｐｍで撹拌しながら、無水プロピオン酸１００．６３ｇ（０．７５モル）がゆっくり滴下され、６時間かけて全量滴下された。反応時の温度は０～５°Ｃに保持された。引き続きフラスコを冷やしながら撹拌して同温度に保持しながら、１１．２５％の水酸化ナトリウム水溶液２７９．９８ｇ（０．７９モル）が滴下され、副生したプロピオン酸が中和された。 Synthesis of 25% propionylated polyallylamine 1763 g of 15% polyallylamine aqueous solution (PAA-15C) having a molecular weight of about 15000 was placed in a 4 neck 3 l round bottom separable flask equipped with a stirrer, Dimroth reflux, dropping funnel and thermometer. (3.0 mol in an unmodified allylamine monomer unit) was charged, and while the flask was cooled with ice water and stirred at 200 rpm, 100.63 g (0.75 mol) of propionic anhydride was slowly added dropwise, and the entire amount was added dropwise over 6 hours. It was. The temperature during the reaction was maintained at 0-5 ° C. Subsequently, 279.98 g (0.79 mol) of a 11.25% aqueous sodium hydroxide solution was added dropwise while stirring and maintaining the same temperature while cooling the flask, and the by-produced propionic acid was neutralized.
　得られた中和後の液は、実施例１と同様に電気透析に付され、２８時間かけて脱塩され、２５％プロピオニル化ポリアリルアミン遊離塩基が生成された。ここで、前記２５％プロピオニル化ポリアリルアミン遊離塩基、すなわち遊離塩基型２５％プロピオニル化ポリアリルアミンとは、原料のポリアリルアミンのアミノ基に対し２５％プロピオニル化された重合体である。この水溶液中の水は実施例１と同様に置換され、ＮＭＲの測定に用いられた。 The obtained neutralized solution was subjected to electrodialysis in the same manner as in Example 1 and desalted over 28 hours to produce 25% propionylated polyallylamine free base. Here, the 25% propionylated polyallylamine free base, that is, the free base type 25% propionylated polyallylamine is a polymer 25% propionylated with respect to the amino group of the starting polyallylamine. Water in this aqueous solution was substituted in the same manner as in Example 1 and used for NMR measurement.
　５０％プロピオニル化ポリアリルアミンの合成
　無水プロピオン酸の量を２０１．２５ｇ（１．５０モル）、水酸化ナトリウムを１１．２５％の水酸化ナトリウム水溶液５５９．９６ｇ（１．５８モル）用いた以外は、上記と同様に操作し、遊離塩基型５０％プロピオニル化ポリアリルアミンが得られた。 Synthesis of 50% propionylated polyallylamine Except for using 201.25 g (1.50 mol) of propionic anhydride and 559.96 g (1.58 mol) of 11.25% sodium hydroxide aqueous solution of sodium hydroxide. By operating in the same manner as described above, free base type 50% propionylated polyallylamine was obtained.
　顕微鏡スライドガラスのコーティング
　撥水性樹脂を被覆した顕微鏡スライドガラスは、前記ポリマーの０．１ｗ／ｖ％水溶液中にスライドガラスが１０秒間浸漬され、引き上げた後、風乾された。その後、脱イオン水でリンス処理され、風乾され、保存された。 Microscope Slide Glass Coating A microscope slide glass coated with a water-repellent resin was dipped in a 0.1 w / v% aqueous solution of the polymer for 10 seconds, pulled up, and then air-dried. Thereafter, it was rinsed with deionized water, air dried and stored.
　細胞懸濁液の調製
　細胞は実施例１で説明された方法で調製された。本実施例では、予洗処理を施された後洗浄液に懸濁された細胞と、予洗処理を施されないで固定液が除去された後直接洗浄液に懸濁された細胞とが用いられた。 Preparation of cell suspension Cells were prepared as described in Example 1. In this example, cells suspended in the washing solution after being subjected to the pre-washing treatment and cells suspended directly in the washing solution after the fixing solution was removed without being subjected to the pre-washing treatment were used.
　浸漬細胞のスライドへの吸着、染色及び細胞不動化度の測定は、原則として実施例１で説明された方法に従って実施された。 The adsorption of the immersed cells on the slide, staining, and measurement of the degree of cell immobilization were performed according to the method described in Example 1 in principle.
　２－２．結果
　図２Ａは、予洗処理を施された細胞について、平均分子量１５，０００のアシル化ポリアリルアミンのアシル化の程度と、該アシル化ポリアリルアミンがコーティングされたスライドガラスの細胞不動化度との関係を示すグラフである。対照として、未変性ポリアリルアミンでコーティングされたスライドが用いられた。アセチル化ポリアリルアミンの細胞不動化度の評価指数（ハッチング入りの棒）は、アセチル化０％で９．５、アセチル化２５％で９．５、アセチル化５０％で１０、アセチル化７５％で９であった。また、プロピオニル化ポリアリルアミンの細胞不動化度の評価指数（黒塗りの棒）は、プロピオニル化０％で９．５、プロピオニル化２５％で９、プロピオニル化５０％で１０であった。以上の結果から、予洗処理を施された細胞を用いた場合には、ポリアリルアミンのアシル化の種類及び割合によって、細胞不動化度にほとんど違いはないことが示された。図２Ｂは、予洗処理を施されない細胞について、平均分子量１５，０００のアシル化ポリアリルアミンのアシル化の程度と、該アシル化ポリアリルアミンがコーティングされたスライドガラスの細胞不動化度との関係を示すグラフである。アセチル化ポリアリルアミンの細胞不動化度の評価指数（ハッチング入りの棒）は、アセチル化０％で２、アセチル化２５％で４、アセチル化５０％で６、アセチル化７５％で５であった。また、プロピオニル化ポリアリルアミンの細胞不動化度の評価指数（黒塗りの棒）は、プロピオニル化０％で２、プロピオニル化２５％で４、プロピオニル化５０％で６．５であった。以上の結果から、予洗処理を施されない細胞を用いた場合には、未変性ポリアリルアミンのアセチル化及びプロピオニル化は、ともに５０％で最大の細胞不動化度を示した。しかし、予洗処理を施されない細胞を用いた場合には、予洗処理を施された細胞を用いた場合と比較して、細胞不動化度の評価指数は減少した。この減少は、予洗処理を施されないことによって固定液の除去が不十分となり、固定液中に含まれる何らかの成分がポリマーの細胞不動化を阻害したためであると考えられる。 2-2. Results FIG. 2A shows the relationship between the degree of acylation of an acylated polyallylamine having an average molecular weight of 15,000 and the degree of cell immobilization of a slide glass coated with the acylated polyallylamine for the cells subjected to the prewash treatment. It is a graph which shows. As a control, a slide coated with native polyallylamine was used. The evaluation index of the cell immobilization degree of the acetylated polyallylamine (hatched bar) is 9.5 at 0% acetylation, 9.5 at 25% acetylation, 10 at 50% acetylation, 75% acetylation. It was 9. Moreover, the evaluation index (black bar) of the degree of cell immobilization of propionylated polyallylamine was 9.5 at 0% propionylation, 9 at 25% propionylation, and 10 at 50% propionylation. From the above results, it was shown that when the cells subjected to the prewash treatment were used, there was almost no difference in the degree of cell immobilization depending on the type and ratio of polyallylamine acylation. FIG. 2B shows the relationship between the degree of acylation of an acylated polyallylamine having an average molecular weight of 15,000 and the degree of cell immobilization of a glass slide coated with the acylated polyallylamine for cells not subjected to prewash treatment. It is a graph. The evaluation index of the cell immobilization degree of acetylated polyallylamine (hatched bar) was 2 at 0% acetylation, 4 at 25% acetylation, 6 at 50% acetylation, and 5 at 75% acetylation. . Moreover, the evaluation index (black bar) of the degree of cell immobilization of propionylated polyallylamine was 2 at 0% propionylation, 4 at 25% propionylation, and 6.5 at 50% propionylation. From the above results, when cells not subjected to prewash treatment were used, acetylation and propionylation of native polyallylamine both showed the maximum degree of cell immobilization at 50%. However, when cells that were not subjected to the prewash treatment were used, the evaluation index for the degree of cell immobilization decreased compared to the case where cells that had been prewashed were used. This decrease is considered to be due to insufficient removal of the fixative due to the absence of the pre-wash treatment, and some component contained in the fixative inhibited the cell immobilization of the polymer.
　ここで、未変性ポリアリルアミンのアシル化は、ポリアリルアミンのカチオン密度を低下させ、細胞不動化度の低下を生じさせるとも考えられる。しかし、予洗処理を施されない細胞を用いた場合では、５０％アシル化ポリアリルアミンは、未変性ポリアリルアミンと比較して、非常に高い細胞不動化度を示した（図２Ｂ）。この要因として、ポリアリルアミンのアシル化にともなって疎水性が増大し、細胞が疎水結合によってより吸着されやすくなったことが考えられる。また、予洗処理を施されない細胞を用いた場合において、未変性ポリアリルアミンのアシル化にともなって細胞不動化度の評価指数が増大することを考慮すると、前記疎水結合は残存する固定液中の成分によって阻害されないと考えられる。７５％アシル化ポリアリルアミンで細胞不動化度が減少したことは、ホルムアルデヒド存在下での細胞の不動化には、疎水結合とイオン結合の両方が必要であって、７５％までアシル化の程度が上がってしまうとイオン結合の寄与が低減するためであると考えられる。 Here, it is considered that acylation of unmodified polyallylamine reduces the cation density of polyallylamine and causes a decrease in the degree of cell immobilization. However, in the case of using cells not subjected to prewash treatment, 50% acylated polyallylamine showed a very high degree of cell immobilization compared to native polyallylamine (FIG. 2B). This may be because the hydrophobicity increased with the acylation of polyallylamine, and the cells were more easily adsorbed by hydrophobic bonds. In addition, in the case of using cells that have not been subjected to pre-washing treatment, considering that the evaluation index of the degree of cell immobilization increases with acylation of native polyallylamine, the hydrophobic bond is a component in the remaining fixative solution. It is thought that it is not inhibited by. The decrease in cell immobilization with 75% acylated polyallylamine indicates that cell immobilization in the presence of formaldehyde requires both hydrophobic and ionic bonds, and the degree of acylation is up to 75%. If it goes up, it is thought that it is because the contribution of an ionic bond reduces.
　３．細胞不動化度に対する他のポリアリルアミン系ポリマーの影響
　３－１．方法
　他のポリアリルアミン系ポリマーとして、平均分子量３０００（図３のグラフでは、「ＭＷ３Ｋ」と表記される。）の未変性ポリジアリルアミン（日東紡績、ＰＡＳ－２１、図３のグラフでは、「未変性（２級）」と表記される。）、未変性ポリジアリルメチルアミン（ｐＨ９．０に調製、日東紡績、ＰＡＳ－２２、図３のグラフでは、「未変性（３級）」と表記される。）、未変性アリルアミン及び未変性ジアリルアミンの等モル共重合体（日東紡績、ＰＡＡ－Ｄ１１－３、図３のグラフでは、「未変性（１級／２級）」と表記される。）、未変性アリルアミン及び未変性ジアリルメチルアミンの等モル共重合体（日東紡績、ＰＡＡ－１１１２、図３のグラフでは、「未変性（１級／３級）」と表記される。）を用意した。これらの未変性ポリマーと、実施例１の平均分子量３０００の５０％アセチル化ポリアリルアミン（図３のグラフでは、「アセチル５０％（１級）」と表記される。）と、未変性ポリアリルアミン（図３のグラフでは、「未変性（１級）」と表記される。）と、平均分子量１５０００の５０％アセチル化ポリアリルアミン（図３のグラフでは、「アセチル５０％（１級、ＭＷ１５Ｋ）」と表記される。）とについて、実施例１及び２と同様にして、コーティングされたスライドガラスを調製し、予洗処理を施されない細胞について細胞不動化度を評価した。 3. 3. Effect of other polyallylamine-based polymers on cell immobilization degree 3-1. Method As another polyallylamine-based polymer, an unmodified polydiallylamine (Nittobo, PAS-21, graph of FIG. 3) with an average molecular weight of 3000 (denoted as “MW3K” in the graph of FIG. 3) (Second grade) ”), unmodified polydiallylmethylamine (prepared to pH 9.0, Nittobo, PAS-22, in the graph of FIG. )), An equimolar copolymer of unmodified allylamine and unmodified diallylamine (Nittobo, PAA-D11-3, represented as “unmodified (primary / secondary)” in the graph of FIG. 3). An equimolar copolymer of unmodified allylamine and unmodified diallylmethylamine (Nittobo, PAA-1112, expressed as “unmodified (primary / tertiary)” in the graph of FIG. 3) was prepared. These unmodified polymers, 50% acetylated polyallylamine having an average molecular weight of 3000 of Example 1 (indicated as “acetyl 50% (primary)” in the graph of FIG. 3), unmodified polyallylamine ( In the graph of FIG. 3, “unmodified (primary)”) and 50% acetylated polyallylamine having an average molecular weight of 15000 (in the graph of FIG. 3, “acetyl 50% (primary, MW15K)”). In the same manner as in Examples 1 and 2, coated glass slides were prepared, and the degree of cell immobilization was evaluated for cells that were not subjected to the pre-wash treatment.
　３－２．結果
　図３は、予洗処理を施されない細胞について、さまざまなポリアリルアミン系ポリマーでコーティングされたスライドガラスの細胞不動化度を示すグラフである。未変性ポリジアリルアミンと、未変性アリルアミン及びジアリルアミンの等モル共重合体とは、平均分子量１５０００の５０％アセチル化ポリアリルアミンに匹敵する細胞不動化度であった。この結果、アセチル化、プロピオニル化のようなアシル化の他に、未変性ポリアリルアミンの第１級アミノ基を第２級アミノ基に置換することによっても、予洗処理を施されない細胞についての細胞不動化度を向上させることができた。これは、第２級アミノ基は第１級アミノ基より塩基性が高く、かつ疎水性が高いため、細胞が不動化されやすくなったと考えられる。 3-2. Results FIG. 3 is a graph showing the degree of cell immobilization of glass slides coated with various polyallylamine-based polymers for cells not subjected to pre-washing treatment. Unmodified polydiallylamine and an equimolar copolymer of unmodified allylamine and diallylamine had cell immobility comparable to 50% acetylated polyallylamine with an average molecular weight of 15000. As a result, in addition to acylation such as acetylation and propionylation, cell immobilization of cells not subjected to prewash treatment can be achieved by replacing the primary amino group of native polyallylamine with a secondary amino group. The degree of conversion could be improved. This is probably because the secondary amino group is more basic and more hydrophobic than the primary amino group, so that the cells are easily immobilized.
　４．ポリアリルアミン及び等モル共重合体の濃度及び平均分子量と、細胞不動化度との関係
　４－１．方法
　等モル共重合体及びポリアリルアミン
　日東紡績株式会社から購入された未変性アリルアミン（第１級）及び未変性ジアリルアミン（第２級）の等モル共重合体（以下、「等モル共重合体」という。）は、平均分子量が、３０００、１５０００、２２０００及び４８０００（カタログ番号はそれぞれ、ＰＡＡ－Ｄ１１－３、ＰＡＡ－Ｄ１１－１５、ＰＡＡ－Ｄ１１－２２及びＰＡＡ－Ｄ１１－４８）であった。日東紡績株式会社から購入されたポリアリルアミンは、平均分子量が、３０００、１５０００及び２５０００（カタログ番号はそれぞれ、ＰＡＡ－３、ＰＡＡ－１５Ｃ及びＰＡＡ－２５）であった。また、シグマアルドリッジから購入されたポリアリルアミンは、平均分子量が６５０００（カタログ番号：４７９１４４）であった。 4). 4. Relationship between concentration and average molecular weight of polyallylamine and equimolar copolymer and degree of cell immobilization 4-1. Method Equimolar Copolymer and Polyallylamine Equimolar Copolymer of Unmodified Allylamine (Primary) and Unmodified Diallylamine (Secondary) Purchased from Nittobo Co., Ltd. (hereinafter “Equimolar Copolymer”) The average molecular weights were 3000, 15000, 22000 and 48000 (catalog numbers PAA-D11-3, PAA-D11-15, PAA-D11-22 and PAA-D11-48, respectively). The polyallylamine purchased from Nitto Boseki Co., Ltd. had average molecular weights of 3000, 15000 and 25000 (catalog numbers PAA-3, PAA-15C and PAA-25, respectively). Polyallylamine purchased from Sigma-Aldridge had an average molecular weight of 65000 (catalog number: 479144).
　スライドの細胞不動化度の評価
　等モル共重合体及びポリアリルアミンは、実施例１ないし３と同様にスライドガラスにコーティングされ、予洗処理を施されていない細胞を用いて細胞不動化度が評価された。なお、各実験は２回行われた。 Evaluation of slide cell immobilization degree The equimolar copolymer and polyallylamine were coated on a slide glass in the same manner as in Examples 1 to 3, and the cell immobilization degree was evaluated using cells that had not been prewashed. It was. Each experiment was performed twice.
　４－２．結果
　等モル共重合体のスライドガラスの結果
　図４Ａは、コーティングされた等モル共重合体の濃度及び平均分子量と、細胞不動化度との関係を示すグラフである。０．０５％等モル共重合体の細胞不動化度の評価指数は、平均分子量３０００で４、平均分子量１５０００で６、平均分子量２２０００で８、平均分子量４８０００で９であった。０．１％等モル共重合体の細胞不動化度の評価指数は、平均分子量３０００で７、平均分子量１５０００で８、平均分子量２２０００で７、平均分子量４８０００で９であった。０．２％等モル共重合体の細胞不動化度の評価指数は、平均分子量３０００で７、平均分子量１５０００で７、平均分子量２２０００で８、平均分子量４８０００で７であった。なお、平均分子量が５４０００の等モル共重合体（ＰＡＡ－Ｄ１１－５４）の細胞不動化度の評価指数は、それぞれの濃度で、平均分子量が４８０００の等モル共重合体と同程度であった（データは示されていない。）。以上の結果から、平均分子量が大きくなるにつれて、等モル共重合体の細胞不動化度は高くなることが示された。高分子量の等モル共重合体では、０．０５％の低濃度でも細胞不動化度が減少しないことも示された。 4-2. Results Results of Equimolar Copolymer Slide Glass FIG. 4A is a graph showing the relationship between the concentration and average molecular weight of the coated equimolar copolymer and the degree of cell immobilization. The evaluation index of the cell immobilization degree of the 0.05% equimolar copolymer was 4 at an average molecular weight of 3000, 6 at an average molecular weight of 15000, 8 at an average molecular weight of 22000, and 9 at an average molecular weight of 48000. The evaluation index of the cell immobilization degree of the 0.1% equimolar copolymer was 7 at an average molecular weight of 3000, 8 at an average molecular weight of 15000, 7 at an average molecular weight of 22000, and 9 at an average molecular weight of 48000. The evaluation index of the cell immobilization degree of the 0.2% equimolar copolymer was 7 at an average molecular weight of 3000, 7 at an average molecular weight of 15000, 8 at an average molecular weight of 22000, and 7 at an average molecular weight of 48000. The evaluation index of the cell immobilization degree of the equimolar copolymer (PAA-D11-54) having an average molecular weight of 54,000 was almost the same as that of the equimolar copolymer having an average molecular weight of 48,000 at each concentration. (Data not shown). From the above results, it was shown that the cell immobilization degree of the equimolar copolymer increases as the average molecular weight increases. It was also shown that the high molecular weight equimolar copolymer does not reduce the degree of cell immobilization even at a low concentration of 0.05%.
　ポリアリルアミンのスライドガラスの結果
　図４Ｂは、コーティングされたポリアリルアミンの濃度及び平均分子量と、細胞不動化度との関係を示すグラフである。０．０５％ポリアリルアミンの細胞不動化度の評価指数は、平均分子量３０００で３、平均分子量１５０００、２５０００及び６５０００で４であった。０．１％ポリアリルアミンの細胞不動化度の評価指数は、平均分子量３０００で３、平均分子量１５０００で４、平均分子量２５０００で３、平均分子量６５０００で４であった。０．２％ポリアリルアミンの細胞不動化度の評価指数は、平均分子量３０００で３、平均分子量１５０００で４、平均分子量２５０００で３、平均分子量６５０００で５であった。以上の結果から、ポリアリルアミンの細胞不動化度の最大値は、平均分子量６５０００で、濃度０．２％で５であったが、等モル共重合体の細胞不動化度の最大値よりも低いことが示された。 Results of Polyallylamine Slide Glass FIG. 4B is a graph showing the relationship between the concentration and average molecular weight of the coated polyallylamine and the degree of cell immobilization. The evaluation index of the degree of cell immobilization of 0.05% polyallylamine was 3 with an average molecular weight of 3000 and 4 with an average molecular weight of 15000, 25000 and 65000. The evaluation index of the cell immobilization degree of 0.1% polyallylamine was 3 at an average molecular weight of 3000, 4 at an average molecular weight of 15000, 3 at an average molecular weight of 25000, and 4 at an average molecular weight of 65,000. The evaluation index of the cell immobilization degree of 0.2% polyallylamine was 3 at an average molecular weight of 3000, 4 at an average molecular weight of 15000, 3 at an average molecular weight of 25000, and 5 at an average molecular weight of 65000. From the above results, the maximum value of the cell immobilization degree of polyallylamine was 5 at an average molecular weight of 65000 and a concentration of 0.2%, but it was lower than the maximum value of the cell immobilization degree of an equimolar copolymer. It was shown that.
　結論
　実施例１ないし４の実験結果から、アセチル化、プロピオニル化のようなアシル化と、未変性ポリアリルアミンの第１級アミノ基の第２級アミノ基への置換との他に、第１級アリルアミン及び第２級アリルアミンの共重合体化によっても、細胞不動化度の向上が、予洗処理を施された細胞だけでなく予洗処理を施されない細胞でも認められた。また、共重合体の平均分子量を大きくすることによっても、細胞不動化度の向上が認められた。これは、分子量の高い共重合体では、疎水性が向上すること、一分子あたりの結合点が増大することにより、細胞との接着性が向上するものと考えられる。
 
  Conclusion From the experimental results of Examples 1 to 4, in addition to acylation such as acetylation and propionylation, and substitution of the primary amino group of the unmodified polyallylamine with the secondary amino group, the primary Even by the copolymerization of allylamine and secondary allylamine, an improvement in the degree of cell immobilization was observed not only in the cells subjected to the prewash treatment but also in the cells not subjected to the prewash treatment. In addition, the cell immobilization degree was improved by increasing the average molecular weight of the copolymer. This is presumably because the high molecular weight copolymer improves hydrophobicity and increases the bonding point per molecule, thereby improving the adhesion to cells.

Claims (31)

  1. 　ポリアリルアミン系ポリマーを含み、該ポリマーのアミノ基は、少なくとも一部が変性した第１級アミノ基か、第１級アミノ基及び第２級アミノ基か、第１級アミノ基及び第３級アミノ基か、第２級アミノ基及び／又は第３級アミノ基かであることを特徴とする、生物学的材料の不動化を行うための組成物。 A polyallylamine-based polymer, wherein the amino group of the polymer is at least partially modified primary amino group, primary amino group and secondary amino group, primary amino group and tertiary amino group A composition for immobilizing a biological material, characterized in that it is a group, a secondary amino group and / or a tertiary amino group.
  2. 　前記変性した第１級アミノ基は、第２級アミド基又はアルコキシカルボニル基であることを特徴とする、請求項１に記載の組成物。 The composition according to claim 1, wherein the modified primary amino group is a secondary amide group or an alkoxycarbonyl group.
  3. 　前記変性した第１級アミノ基は、前記ポリマーの同一分子中のアミノ基の２５－７５％であることを特徴とする、請求項１又は２のいずれかに記載の組成物。 3. The composition according to claim 1, wherein the modified primary amino group is 25-75% of the amino group in the same molecule of the polymer.
  4. 　ポリアリルアミン系ポリマーを含み、該ポリマーのアミノ基は、第１級アミノ基か、第１級アミノ基及び第２級アミノ基か、第１級アミノ基及び第３級アミノ基か、第２級アミノ基及び／又は第３級アミノ基かであって、第１級アミノ基のみのポリマーは、側鎖に第２級アミド基又はアルコキシカルボニル基を含むことを特徴とする、生物学的材料の不動化を行うための組成物。 A polyallylamine-based polymer, wherein the amino group of the polymer is a primary amino group, a primary amino group and a secondary amino group, a primary amino group and a tertiary amino group, or a secondary amino group An amino group and / or a tertiary amino group, wherein the polymer containing only the primary amino group contains a secondary amide group or an alkoxycarbonyl group in the side chain. A composition for immobilization.
  5. 　前記第２級アミド基又はアルコキシカルボニル基の数は、前記ポリマーの同一分子中の第１級アミノ基の数の３分の１ないし３倍であることを特徴とする、請求項４に記載の組成物。 The number of the secondary amide group or alkoxycarbonyl group is 1/3 to 3 times the number of primary amino groups in the same molecule of the polymer. Composition.
  6. 　前記第２級アミノ基及び／又は第３級アミノ基は、それぞれ、第１級アミノ基及び／又は第２級アミノ基が変性していることを特徴とする、請求項１又は４に記載の組成物。 5. The secondary amino group and / or the tertiary amino group, respectively, wherein the primary amino group and / or the secondary amino group is modified, respectively. Composition.
  7. 　前記ポリマーは変性ポリアリルアミンであることを特徴とする、請求項１に記載の組成物。 The composition according to claim 1, wherein the polymer is a modified polyallylamine.
  8. 　前記変性ポリアリルアミンは、第１級アミノ基がアシル基又はアルコキシカルボニル基に置換されることを特徴とする、請求項７に記載の組成物。 The composition according to claim 7, wherein the modified polyallylamine has a primary amino group substituted with an acyl group or an alkoxycarbonyl group.
  9. 　前記アシル基又はアルコキシカルボニル基の炭素原子数は２又は３であることを特徴とする、請求項２、４、５及び８のいずれか１つに記載の組成物。 The composition according to any one of claims 2, 4, 5, and 8, wherein the acyl group or alkoxycarbonyl group has 2 or 3 carbon atoms.
  10. 　前記アシル基は、アセチル基又はプロピオニル基であることを特徴とする、請求項９に記載の組成物。 The composition according to claim 9, wherein the acyl group is an acetyl group or a propionyl group.
  11. 　前記ポリマーの同一分子中のアミノ基の２５－７５％が変性されることを特徴とする、請求項７ないし１０のいずれか１つに記載の組成物。 The composition according to any one of claims 7 to 10, wherein 25-75% of amino groups in the same molecule of the polymer are modified.
  12. 　前記ポリアミン系ポリマーは、二酸化硫黄及び／又はマレイン酸が共重合されることを特徴とする、請求項１ないし１１のいずれか１つに記載の組成物。 The composition according to any one of claims 1 to 11, wherein the polyamine polymer is copolymerized with sulfur dioxide and / or maleic acid.
  13. 　前記ポリマーは、アリルアミンと、ジアリルアミン又はジアリルメチルアミンとの共重合体であることを特徴とする、請求項１に記載の組成物。 The composition according to claim 1, wherein the polymer is a copolymer of allylamine and diallylamine or diallylmethylamine.
  14. 　前記ポリマーは、ポリジアリルアミン又はポリジアリルメチルアミンであることを特徴とする、請求項１に記載の組成物。 The composition according to claim 1, wherein the polymer is polydiallylamine or polydiallylmethylamine.
  15. 　前記ポリマーの平均分子量は３，０００ないし５４，０００であることを特徴とする、請求項１ないし１４のいずれか１つに記載の組成物。 The composition according to any one of claims 1 to 14, wherein the polymer has an average molecular weight of 3,000 to 54,000.
  16. 　前記生物学的材料の不動化は、固定剤の存在下で行うことを特徴とする、請求項１ないし１５のいずれか１つに記載の組成物。 The composition according to any one of claims 1 to 15, wherein the biological material is immobilized in the presence of a fixing agent.
  17. 　前記固定剤は、０．０４ｗ／ｖ％のホルムアルデヒドであることを特徴とする、請求項１６のいずれか１つに記載の組成物。 The composition according to claim 16, wherein the fixing agent is 0.04 w / v% formaldehyde.
  18. 　前記生物学的材料は臨床検査試料であることを特徴とする、請求項１ないし１７のいずれか１つに記載の組成物。 18. Composition according to any one of the preceding claims, characterized in that the biological material is a clinical laboratory sample.
  19. 　前記生物学的材料は細胞診検査試料であることを特徴とする、請求項１８に記載の組成物。 The composition according to claim 18, wherein the biological material is a cytodiagnosis sample.
  20. 　前記生物学的材料は子宮頸部細胞診検査試料であることを特徴とする、請求項１９に記載の組成物。 20. The composition of claim 19, wherein the biological material is a cervical cytology sample.
  21. 　請求項１ないし２０のいずれか１つに記載の組成物をコーティングしたことを特徴とする、生物学的材料の不動化を行うための固体支持体。 A solid support for immobilizing a biological material, wherein the composition according to any one of claims 1 to 20 is coated.
  22. 　請求項１ないし２０のいずれか１つに記載の組成物をコーティングするステップを含むことを特徴とする、生物学的材料の不動化を行うための固体支持体の製造方法。 A method for producing a solid support for immobilizing biological material, comprising the step of coating the composition according to any one of claims 1 to 20.
  23. 　請求項１ないし２０のいずれか１つに記載の組成物をコーティングしたことを特徴とする、顕微鏡スライドガラス。 A microscope slide glass, which is coated with the composition according to any one of claims 1 to 20.
  24. 　請求項２３に記載の顕微鏡スライドガラスを用いて生物学的材料を分析する方法であって、
    （１）前記生物学的材料を、固定剤を含む液に浸漬して固定するステップと、
    （２）固定された前記生物学的材料を分離するステップと、
    （３）分離された前記生物学的材料を水溶液に懸濁するステップと、
    （４）前記水溶液を前記顕微鏡スライドガラスに戴置して、該顕微鏡スライドガラス上に前記生物学的材料を不動化させるステップとを含むことを特徴とする、生物学的材料を分析する方法。     A method for analyzing biological material using the microscope slide glass according to claim 23, comprising:
    (1) immersing and fixing the biological material in a liquid containing a fixing agent;
    (2) separating the immobilized biological material;
    (3) suspending the separated biological material in an aqueous solution;
    (4) placing the aqueous solution on the microscope slide glass, and immobilizing the biological material on the microscope slide glass, and a method for analyzing the biological material.
  25. 　前記ステップ（３）の水溶液は固定剤を含むことを特徴とする、請求項２４に記載の方法。 25. The method of claim 24, wherein the aqueous solution of step (3) includes a fixative.
  26. 　前記ステップ（３）の水溶液は０．０４ｗ／ｖ％のホルムアルデヒドを含むことを特徴とする、請求項２５に記載の方法。 The method according to claim 25, characterized in that the aqueous solution of step (3) contains 0.04 w / v% formaldehyde.
  27. 　（５）前記ステップ（４）において前記生物学的材料が不動化された顕微鏡スライドガラスを染色するステップを含むことを特徴とする、請求項２４ないし２６のいずれか１つに記載の方法。 (5) The method according to any one of claims 24 to 26, comprising the step of staining the microscope slide glass in which the biological material is immobilized in the step (4).
  28. 　請求項２３に記載の顕微鏡スライドガラスを保持するホルダと、自動分注機ユニットと、ロボットアームを備えたピペッタユニットとを含むことを特徴とする、請求項２５ないし２７のいずれか１つに記載の方法を実行するための分析システム。 The holder for holding the microscope slide glass according to claim 23, an automatic dispenser unit, and a pipetter unit provided with a robot arm. An analytical system for performing the described method.
  29. 　遠心機ユニットを含むことを特徴とする、請求項２８に記載の分析システム。 The analysis system according to claim 28, further comprising a centrifuge unit.
  30. 　請求項２３に記載の顕微鏡スライドガラスを含むことを特徴とする、請求項２５ないし２７のいずれか１つに記載の方法を実行するためのキット。 A kit for carrying out the method according to any one of claims 25 to 27, comprising the microscope slide glass according to claim 23.
  31. 　採取された生物学的材料を固定し保存するための試薬を含むことを特徴とする、請求項３０に記載のキット。
          31. Kit according to claim 30, characterized in that it contains reagents for fixing and storing the collected biological material.
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