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WO2010123039A1 - Composition d'immobilisation d'une matière biologique - Google Patents

Composition d'immobilisation d'une matière biologique 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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Prior art keywords
biological material
amino group
polyallylamine
group
polymer
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PCT/JP2010/057097
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English (en)
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/ja
Publication of WO2010123039A1 publication Critical patent/WO2010123039A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—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

L'invention porte sur une composition d'immobilisation d'une matière biologique, par laquelle un échantillon approprié peut être préparé tout en rendant minimales les procédures d'élimination par lavage d'une solution de fixage, permettant ainsi d'améliorer le rendement d'un examen cytodiagnostique ; et sur un procédé d'analyse d'une matière biologique à l'aide d'un support solide ayant été revêtu par ladite composition. L'invention porte sur une composition d'immobilisation d'une matière biologique, qui comprend un polymère à base de polyallylamine, les groupes amino dans ledit polymère à base de polyallylamine étant des groupes amino primaires au moins partiellement dénaturés, des groupes amino primaires avec des groupes amino secondaires, des groupes amino primaires avec des groupes amino tertiaires ou des groupes amino secondaires et/ou des groupes amino tertiaires. L'invention porte également sur une lame de verre microscopique pour immobiliser une matière biologique, qui a été revêtue par la composition mentionnée ci-dessus ; et sur un procédé d'analyse d'une matière biologique à l'aide de ladite lame de verre microscopique.
PCT/JP2010/057097 2009-04-21 2010-04-21 Composition d'immobilisation d'une matière biologique WO2010123039A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018197908A (ja) * 2017-05-23 2018-12-13 シスメックス株式会社 形態検査のスキル評価システム
WO2021085526A1 (fr) * 2019-10-31 2021-05-06 東レ株式会社 Biopuce et procédé de détection

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JPH04307366A (ja) * 1991-04-03 1992-10-29 Hitachi Ltd スライドガラス格納機構
JPH1114909A (ja) * 1997-06-20 1999-01-22 Bio Quest:Kk ポリマーコーティングスライドガラス
JP2001521902A (ja) * 1997-11-05 2001-11-13 ジェルテックス ファーマシューティカルズ，インコーポレイテッド 蓚酸塩を低減させるための脂肪族ポリアミン類の使用
JP2008514924A (ja) * 2004-09-23 2008-05-08 トリパス イメージング， インコーポレイテッド 生物学的試料を固定化するためのポリカチオンポリマーコーティング

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JPH04307366A (ja) * 1991-04-03 1992-10-29 Hitachi Ltd スライドガラス格納機構
JPH1114909A (ja) * 1997-06-20 1999-01-22 Bio Quest:Kk ポリマーコーティングスライドガラス
JP2001521902A (ja) * 1997-11-05 2001-11-13 ジェルテックス ファーマシューティカルズ，インコーポレイテッド 蓚酸塩を低減させるための脂肪族ポリアミン類の使用
JP2008514924A (ja) * 2004-09-23 2008-05-08 トリパス イメージング， インコーポレイテッド 生物学的試料を固定化するためのポリカチオンポリマーコーティング

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018197908A (ja) * 2017-05-23 2018-12-13 シスメックス株式会社 形態検査のスキル評価システム
JP7043184B2 (ja) 2017-05-23 2022-03-29 シスメックス株式会社 形態検査のスキル評価システム
WO2021085526A1 (fr) * 2019-10-31 2021-05-06 東レ株式会社 Biopuce et procédé de détection
JP7578907B2 (ja) 2019-10-31 2024-11-07 東レ株式会社 バイオチップおよび検出方法

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