WO2019078655A1 - Composition pour infiltrer une biomolécule dans un tissu comprenant un tensioactif zwitterionique à base de sulfobétaïne, et son utilisation - Google Patents

Composition pour infiltrer une biomolécule dans un tissu comprenant un tensioactif zwitterionique à base de sulfobétaïne, et son utilisation Download PDF

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WO2019078655A1
WO2019078655A1 PCT/KR2018/012369 KR2018012369W WO2019078655A1 WO 2019078655 A1 WO2019078655 A1 WO 2019078655A1 KR 2018012369 W KR2018012369 W KR 2018012369W WO 2019078655 A1 WO2019078655 A1 WO 2019078655A1
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biological sample
propanesulfonate
tissue
biological
composition
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PCT/KR2018/012369
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English (en)
Korean (ko)
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최영식
윤종혁
장재명
최승희
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재단법인대구경북과학기술원
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Priority claimed from KR1020170135948A external-priority patent/KR20190043886A/ko
Priority claimed from KR1020180004121A external-priority patent/KR102200618B1/ko
Application filed by 재단법인대구경북과학기술원 filed Critical 재단법인대구경북과학기술원
Publication of WO2019078655A1 publication Critical patent/WO2019078655A1/fr

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    • 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
    • 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
    • G01N1/36—Embedding or analogous mounting of samples
    • 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
    • G01N33/483—Physical analysis of biological material
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • the present invention relates to a composition for penetrating a biological tissue containing a sulfobetaine-zwitterionic surfactant and a use thereof.
  • Biological samples containing biological tissue, organs, or parts thereof are opaque, and it is almost impossible to observe the inside of the tissue under a general optical microscope in a three-dimensional state. Therefore, a confocal microscope or a multiphoton microscope has been used as a means for observing the inside of a biological sample including a living tissue by using an optical imaging technique in a three-dimensional (3D) state, but a light source used can pass through a biological sample The depth is only a few hundred micrometers, so that only very limited information can be obtained. Immunostaining methods have also been used as other methods for observing biological samples of a three-dimensional structure. Recently, a method for effectively observing a biological sample in a three-dimensional state by applying the immuno-staining method and the tissue transparency method has been proposed.
  • Immunostaining is performed by immobilizing the tissue with a fixative and then embedding the tissue with paraffin or polymer. The tissue is then cut to a thickness of several ⁇ m or nm to allow light or electromagnetic waves to pass therethrough, Imaging the sample.
  • this method requires a series of intermittent cuts of several tens of ⁇ thick or less, a series of cumbersome steps of imaging each of them using a microscope and then reconstructing them again .
  • Biological tissue transparency is one of the ways to overcome the limitations of such immuno-staining methods. It modifies the physico-chemical properties of tissues and suppresses absorption, scattering and refraction of light used for imaging, To the surface of the substrate.
  • tissue transparency methods are methods for removing the lipid in the tissue of a biological sample and correcting the refractive index of the sample.
  • information on a biological tissue such as DNA, RNA, or protein is distorted or lost It is important to avoid.
  • a method of staining an antibody or a polymer in a biological sample to observe the three-dimensional structure or distribution of a living tissue or an organ is still under development.
  • particles naturally move themselves through diffusions, mixing themselves with other molecules while doing irregular motions and spreading all around.
  • the diffusion of an antibody or a polymer in a biological tissue composed of various materials and having a complicated structure proceeds slowly, so that it takes a long time to penetrate into a tissue having a certain volume or more. Therefore, in order to solve such a problem, a method of using a small amount of fixing agent for fixing a biological sample such as a biotissue or an organ or a method of widening the structure interval of the hydrogel is also applied.
  • a method of making a tissue porous for rapid diffusion of a polymer has been proposed.
  • this method is mainly troublesome to repeat the process of dissolving and melting the tissues and repeating dehydration and rehydration.
  • Another method has been developed to increase the temperature of the solution containing the polymer or to use microwaves in order to increase the diffusion rate of the antibody or the polymer itself.
  • a method of using the blood vessel for the uniform delivery of the antibody or polymer has also been developed There is a bar.
  • a method of dyeing using an organic solvent a method of applying an external force of an electric field in a rotating solution, a method of using a concentration gradient using a high concentration of a polymer, a method of applying pressure to a polymer using centrifugal force, has been developed.
  • the method of using the electric field can damage the tissue due to the high voltage of 60 to 100 V because the constituents in the tissue or biological sample have electric charge and move in accordance with the electric field.
  • the probabilistic electrotransport method also causes fluid flow along with the external force of the electric field, so that tissue damage may occur depending on the fluid velocity.
  • the method of increasing the moving speed of the polymer by applying the centrifugal force ensures the stability of the tissue, but has a drawback that it flows only in one direction.
  • Concentration gradients with a high concentration of polymer have the advantage of controlling the reaction rate, but they are economically inefficient because a large amount of polymer should be used.
  • these methods have a disadvantage in that a special specification of a mechanical device is required.
  • the method using an organic solvent does not require a special apparatus and shows a higher preservability than an electrical method, but has a disadvantage that only one dyeing can be performed with one tissue.
  • a biological part such as a nucleic acid, a peptide and a polypeptide including an antibody, an antibody fragment, a protein, a polysaccharide,
  • a method capable of rapidly and uniformly penetrating a biological molecule to improve the observation and analysis efficiency of the sample.
  • Another object of the present invention is to provide a composition for tissue infiltration of biological molecules containing a sulfobetaine zwitterionic surfactant.
  • the present invention provides a method for treating a sulfobetaine-zwitterionic surfactant, which comprises treating a biological sample containing tissues, organs or a part thereof, A method for infiltrating a biological molecule into a biological sample using an ionizing surfactant is provided.
  • a method for tissue infiltration of a biological sample using a sulfobetaine zwitterionic surfactant comprises the steps of: (a) immobilizing a biological sample; (b) optionally and optionally washing the biological sample of step (a) and removing the fixative solution; (c) treating the biological sample with a sulfobetaine zwitterionic surfactant; (d) washing the biological sample of step (c) and removing the sulfobetaine zwitterionic surfactant; And (e) adding to the biological sample of step (d) at least one biological molecule selected from the group consisting of nucleic acids, peptides and polypeptides, including antibodies, antibody fragments, proteins, polysaccharides, RNA and DNA do.
  • the biological molecule may be treated with the sulfobetaine zwitterionic surfactant in step (c) or treated prior to step (c) after step (b
  • treating the biological sample with a sulfobetaine zwitterionic surfactant may be by immersing the biological sample in the sulfobetaine zwitterionic surfactant.
  • the present invention also provides a composition for penetrating a biological tissue containing a sulfobetaine-zwitterionic surfactant.
  • the composition for penetrating a living tissue of the present invention and the method for infiltrating tissue using the same can rapidly and uniformly infiltrate biological molecules into the deep part of a tissue or an organ having a thickness of 0.1 mm or more .
  • biological molecules include, but are not limited to, compounds, antibodies, antibody fragments, proteins, polysaccharides, RNA and DNA, nucleic acids, peptides and polypeptides that are intended to penetrate into living tissue , Any polymer and a low molecular weight substance to be transferred into a living tissue.
  • the biological sample includes living tissue, an organ, and cells isolated in vitro, preferably a biological tissue having a thickness of 0.1 mm or more, but is not limited thereto.
  • the sulfobetaine zwitterionic surfactant of the present invention is represented by the following formula (1).
  • R is an alkyl or alkenyl group having 8 to 22 carbon atoms
  • x is an integer of 0 to 3
  • y is an integer of 2 to 4.
  • the surfactant is selected from the group consisting of Dimethylethylammoniumpropane sulfonate; N-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate; Sodium 3,3 '- [(1,2-diphenylethene-1,2-diyl) bis (4,1-phenylene)] bis (oxy) bis (propane-1-sulfonate); 3- (4-Heptyl) phenyl-3-hydroxypropyl) dimethylammoniopropanesulfonate; 3- (Decyldimethylammonio) -propane-sulfonate; 3- [Dimethyl- (2-hydroxyethyl) ammonio] -1-propanesulfonate; 3- (1-Methylpiperidinio) -1-propanesulfonate; 3- (Triphenylphosphonio) propane-1-sulfonate; 3- (Di
  • the concentration of the sulfobetaine zwitterionic surfactant above the critical micelle concentration at which each surfactant has a unique value To the biological sample for a period of time ranging from 4 to 200 hours, preferably, but not exclusively, to the biological sample.
  • the sulfobetaine zwitterionic surfactant may be treated at a temperature ranging from the micelle critical temperature of each of the surfactants to 60 ° C or less, but is not limited thereto.
  • treating the biological sample with the sulfobetaine zwitterionic surfactant is carried out in a rolling bottle incubator at a speed of 10 to 500 rpm, at a speed of 10 to 500 rpm A shaker or a rocker with a speed of 10 to 500 rpm and a slope of 30 to 45 degrees.
  • the biological molecule to be penetrated into the sample may be treated simultaneously with the sulfobetaine zwitterionic surfactant or may be applied before or after the surfactant treatment, depending on its characteristics.
  • the present invention also provides an immunohistochemical staining method using a sulfo-betaine zwitterionic surfactant and a composition for immunohistochemical staining comprising a sulfo-betaine zwitterionic surfactant as an active ingredient.
  • the composition for immunochemical staining of the present invention can be used for immunohistochemically staining the deep part of a living tissue having a thickness of 0.1 mm or more.
  • immunohistochemical staining is an immunohistochemical staining method using a reaction between an antigen and an antibody to identify a substance present in cells or tissues, and used in combination with immunostaining or immunochemical staining.
  • the composition for immunochemical staining of the present invention comprises the sulfobetaine zwitterionic surfactant of Formula 1 as an active ingredient, wherein the sulfobetaine zwitterionic surfactant is selected from the group consisting of dimethylethylammoniumpropane sulfonate; N-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate; Sodium 3,3 '- [(1,2-diphenylethene-1,2-diyl) bis (4,1-phenylene)] bis (oxy) bis (propane-1-sulfonate); 3- (4-Heptyl) phenyl-3-hydroxypropyl) dimethylammoniopropanesulfonate; 3- (Decyldimethylammonio) -propane-sulfonate; 3- [Dimethyl- (2-hydroxyethyl) ammonio] -1-propanesul
  • One embodiment of the present invention provides a method of immunochemically staining a biological sample using the sulfo-betaine zwitterionic surfactant, comprising the steps of: (a) immobilizing a biological sample; (b) optionally and optionally washing the biological sample of step (a) and removing the fixative solution; (c) treating the biological sample with a sulfobetaine zwitterionic surfactant; (d) washing the biological sample of step (c) and removing the sulfobetaine zwitterionic surfactant; (e) immunologically staining the biological sample of step (d) by reacting with the antibody; (f) washing the biological sample of step (e) and removing unreacted antibody; And (g) visualizing the immunologically stained biological sample.
  • the treatment in step (c) is performed by treating the sulfobetaine zwitterionic surfactant with a surfactant, for example, in a solution containing distilled water or PBS (phosphate-buffered saline) But is not limited to, treatment with a solution that is dissolved at a concentration higher than the intrinsic micelle critical concentration possessed by the biological sample, preferably for 4 to 200 hours.
  • a surfactant for example, in a solution containing distilled water or PBS (phosphate-buffered saline)
  • PBS phosphate-buffered saline
  • the treatment is preferably, but not exclusively, a treatment with a sulfobetaine zwitterionic surfactant at a temperature ranging from a micelle critical temperature to 60 ° C.
  • the sulfobetaine zwitterionic surfactant is added to a rolling bottle incubator having a speed of 10 to 500 rpm, a shaker at a speed of 10 to 500 rpm, To a biological sample using a rocker with a speed of ⁇ 500 rpm and a slope of 30 [deg.] To 45 [deg.].
  • the removal of the sulfobetaine zwitterionic surfactant in step (d) may be performed by removing the biological sample treated with the surfactant with, for example, distilled water or PBS (phosphate-buffered saline) To thereby reduce the concentration of the sulfobetaine zwitterionic surfactant present in the sample to a micelle critical concentration or less.
  • PBS phosphate-buffered saline
  • the removal of the sulfobetaine zwitterionic surfactant can be accomplished by the addition of a solvent such as a tilt or a solution having a slope of 30 ° to 45 ° and a speed of 10 to 500 rpm on a stirrer And removing it.
  • a solvent such as a tilt or a solution having a slope of 30 ° to 45 ° and a speed of 10 to 500 rpm on a stirrer And removing it.
  • the step (g) is performed by adding a sulfobetaine zwitterionic surfactant to the vitrification solution at a concentration of the micellar critical concentration or more of the eigenvalues of the respective surfactants, , and the transparentizing step may further include a step of processing after step (d).
  • the composition comprising the sulfobetaine zwitterionic surfactant of the present invention is a composition for pretreatment for optical imaging of biological samples, and is a composition for both immunohistochemical staining and tissue transparency.
  • the present invention relates to a composition for tissue infiltration of a biological sample. According to the present invention, it is possible to quickly and uniformly infiltrate a biological molecule into a deep part of a living tissue having a thick thickness. Therefore, the composition of the present invention can be used for immunochemical staining of a biological sample containing biological tissue.
  • the biological sample is immunochemically stained with the sulfo-betaine zwitterionic surfactant of the present invention, only the membrane penetration protein contained in the biological sample can be selectively removed, and thus the antibody or biopolymer for immuno-staining is analyzed It is possible to effectively and quickly penetrate the desired biotissue, and the sample can be safely preserved without being damaged.
  • the efficiency of immuno-staining analysis can be improved compared to conventional immuno-staining chemical methods.
  • FIG. 1 is a flow chart of a method for immunochemical staining of biological tissues or biological samples by treating a sulfobetaine zwitterionic surfactant.
  • Fig. 2 shows that more antibody was permeated when a sulfobetaine zwitterionic surfactant was applied to a brain tissue slice (100 ⁇ thick).
  • FIG. 3 is a photograph showing the deformation of tissues with time after treating each brain tissue with a phosphate buffer solution, an ionic surfactant, and a zwitterionic surfactant. When the zwitterionic surfactant is treated, Indicating that the tissue was preserved without modification.
  • FIG. 4 shows that when the sulfobetaine-zwitterionic surfactant was treated with brain tissue sections, the biological tissue was preserved and deformation was not observed even at a high concentration when the concentration was above the microcell threshold.
  • FIG. 5 shows the result of confirming that antibody or biopolymer material is well penetrated into tissues as a result of treating the sulfobetaine zwitterionic surfactant by type and staining brain tissue sections.
  • FIG. 6 shows immunochemical staining, including pretreatment with a sulfobetaine zwitterionic surfactant, showing that the antibody penetrated into the deep brain when reacted to whole brain tissue.
  • the present invention relates to a method and a composition for rapidly and evenly infiltrating a biological molecule containing an antibody, an antibody fragment, a peptide, a polypeptide and a protein into the deep part of the sample with respect to the entire biological sample.
  • the composition of the present invention comprises a sulfobetaine zwitterionic surfactant which maintains the protein-protein bond present in the biological sample while it weakens the inter-lipid or lipid-protein bond and inhibits Only the membrane penetration protein can be selectively removed. Therefore, the homeostasis and antigenicity in the tissues to be analyzed are maintained, while the deformation or breakdown of the tissue due to changes in membrane protein or lipid is prevented.
  • the sulfobetaine zwitterionic surfactant of the present invention can be produced by a method in which a nucleic acid, a peptide,
  • a nucleic acid a nucleic acid, a peptide
  • the analysis of the biological sample for example, the efficiency of analysis such as immunochemical staining, And the analysis time can also be effectively shortened.
  • the sulfobetaine zwitterionic surfactant usable in the present invention may be a compound represented by the following formula (1).
  • R is an alkyl or alkenyl group having 8 to 22 carbon atoms
  • x is an integer of 0 to 3
  • y is an integer of 2 to 4.
  • N N- dimethylmyristylammonio propanesulfonate as the sulfobetaine zwitterionic surfactant
  • N-Dodecyl-N N-dimethyl-3-ammonio-1-propanesulfonate
  • N-Dodecyl-N N-dimethyl-3-ammonio-1-propanesulfonate
  • a method of infiltrating a biological sample using a sulfobetaine zwitterionic surfactant comprises the steps of: (a) immobilizing a biological sample; (b) optionally and optionally washing the biological sample of step (a) and removing the fixative solution; (c) treating the biological sample with a sulfobetaine zwitterionic surfactant; (d) washing the biological sample of step (c) and removing the sulfobetaine zwitterionic surfactant; And (e) adding a nucleic acid, a peptide or a polypeptide comprising an antibody, an antibody fragment, a protein, a polysaccharide, RNA and DNA to the biological sample of step (d).
  • the immobilization of the biological sample may be performed by immersing the immobilized tissue or organ in vitro derived from the subject to be analyzed in a solution containing an aldehyde compound.
  • the aldehyde compound may be, but not limited to, paraformaldehyde Or glutaraldehyde may be used.
  • the immobilization of the sample is carried out by immersing the sample in a solution containing an aldehyde-based compound, and then fixing the immobilized sample at 4 ° C to 10 ° C for 4 hours to 12 hours so as to prevent the antigen present in the sample from destroying or leaking do. Subsequently, the solution is placed in a phosphate-buffered saline (PBS) solution for 4 hours to 12 hours to remove unused aldehyde-based compounds from the biological sample.
  • PBS phosphate-buffered saline
  • the surfactant of the present invention weakens the binding between the lipid and the lipid existing in the biological sample or the binding between the lipid and the protein, thereby facilitating the penetration of the analytical sample or the dye sample.
  • treating the sulfobetaine zwitterionic surfactant significantly increases the tissue permeability of the biological molecule to the biological sample.
  • the treatment of the sulfobetaine zwitterionic surfactant according to the present invention is carried out by adding a solution of the sulfobetaine zwitterionic surfactant added at a concentration not lower than the micelle concentration of the sulfobetaine zwitterionic surfactant to be used in distilled water or PBS (phosphate-buffered saline) Can be used.
  • PBS phosphate-buffered saline
  • the sulfobetaine zwitterionic surfactant is used at a concentration of 50% (w / v), which is a critical value of the micelle having an intrinsic value. In this case, if the concentration exceeds 50% (w / v), the antigenicity in the biological sample can not be preserved, and the structure of the sample may be withdrawn and analysis may be difficult.
  • the surfactant is reacted at a micelle critical temperature or more, which is an intrinsic value of each surfactant, more preferably at a micelle critical temperature of 10 Deg.] C to 60 [deg.] C. More preferably, the reaction is carried out at a temperature of from 10 ⁇ to 42 ⁇ . In this case, if the reaction is performed at a temperature exceeding 60 ⁇ , denaturation of the protein may occur. Therefore, it is preferable to perform the reaction within the above temperature range.
  • the treatment of the biological sample with the solution containing the sulfobetaine zwitterionic surfactant can be performed for 4 to 200 hours. If the treatment is carried out for less than 4 hours, the surfactant does not properly remove the lipids present in the biological sample. If the treatment exceeds 200 hours, the biological specimen may be deformed have.
  • the treatment of a biological sample of a sulfobetaine zwitterionic surfactant varies with size and can be processed for 4 to 200 hours, more preferably for 8 hours. Of course, the treatment time may be further extended or reduced depending on the type and size of the biological sample to be treated.
  • a rolling bottle incubator with a speed of 0 to 500 rpm, a shaker incubator with the same speed, and a 30 ° A stirrer such as a rocker having a tilt movement of about 45 [deg.] May be used, but the present invention is not limited thereto.
  • the sulfobetaine-zwitterionic surfactant selectively removes the membrane-penetrating protein present in the biological sample, the effect of homogeneity in the solution in which the micelle critical concentration and temperature distribution of each surfactant are reacted .
  • the biological sample is washed and a step of removing the sulfobetaine zwitterionic surfactant is performed.
  • Removal of the sulfobetaine zwitterionic surfactant means reducing or eliminating the concentration of the surfactant from the biological sample treated with the surfactant, wherein the surfactant-treated biological sample is dissolved in distilled water or PBS -buffered saline buffer to reduce the concentration of the sulfobetaine zwitterionic surfactant present in the sample to below the micelle critical concentration.
  • a sulfobetaine zwitterionic surfactant which was treated on a biological sample through a tilt movement method at a slope of 30 ° to 45 ° and a speed of 10 to 500 rpm on a stirrer, The concentration of the surfactant is lowered to below the critical micelle concentration to stop the activity or action of the surfactant.
  • a biological molecule such as an antibody is added to a biological sample to infiltrate the biological molecule into living tissue.
  • composition containing the sulfobetaine zwitterionic surfactant of the present invention can effectively penetrate biological tissues into biological tissues, it is possible to provide an antibody specific to a component (antigen or substance) to be analyzed in a biological sample containing biological tissue Or by adding a dye sample to the reaction mixture.
  • Methods of performing immunochemical staining of a biological sample using a composition comprising a sulfobetaine zwitterionic surfactant of the present invention include: (a) immobilizing a biological sample; (b) optionally washing the biological sample of step (a) and removing the fixative solution as needed; (c) treating the biological sample with a sulfobetaine zwitterionic surfactant; (d) washing the biological sample of step (c) and removing the sulfobetaine zwitterionic surfactant; (e) adding an antibody, antibody fragment, peptide, polypeptide or protein to the biological sample of step (d); (f) washing the biological sample of step (e) and removing unreacted antibody; And (g) visualizing the immunologically stained biological sample.
  • the above method will be described in more detail.
  • an antigen-specific antibody present in the biological sample may be added, and an antibody conjugated with a probe such as a fluorescent substance may be used for easy detection.
  • the antibody may be used by dilution.
  • a buffer solution containing phosphate, sodium borate and bovine serum albumin may be used, but the present invention is not limited thereto.
  • a sulfobetaine zwitterionic surfactant may be added at a concentration above the micelle critical concentration.
  • the immuno-stained biological sample is made transparent.
  • the transparency process is a process for securing the tissue depth so that the dyed biological sample can be observed.
  • the biological sample that has undergone immunochemical staining can be subjected to a solvent-based inversion process including BABB, 3DISCO, or the like, SeeDB, ClearT, and others.
  • a solvent-based inversion process including BABB, 3DISCO, or the like, SeeDB, ClearT, and others.
  • it is possible to carry out a transparency process by a simple method of putting the tissue into a solution including the solution, SeeDB, ClearT, etc.
  • a hydralization transparency method including Scale or CUBIC can be performed, A hydrogels embedding-based transparency method, etc.
  • a sulfobetaine zwitterionic surfactant may be added at a concentration above the micelle critical concentration of the surfactant.
  • the transparency step according to the present invention may be performed before the immunological staining by reacting the biological sample with the antibody.
  • the present invention provides a composition for immunochemical staining comprising the sulfobetaine zwitterionic surfactant of the present invention as an active ingredient.
  • the composition according to the present invention may further include additional additives for enhancing the integrity of the biological sample or enhancing the permeability of the dye sample.
  • the immunochemical staining method of a biological sample using the sulfobetaine zwitterionic surfactant provided in the present invention can selectively remove the membrane-penetrating protein present in a biological sample.
  • an antibody or a polymer substance And the like can be rapidly diffused into the biological sample, so that the sample can be rapidly dyed and a thick sample can be immunochemically stained.
  • the immunochemical staining method according to the present invention can maintain the antigenicity of the biological sample as it is, not only does not cause deformation of the sample, but also has the effect of stably and for a long time preserving the biological sample.
  • Example One Sulfobetainate Zwitterion
  • the surfactant SB3-14 (3- ( N , N -Dimethylmyristylammonio) propanesulfonate) for staining of brain slices
  • Mouse brain (8.2 mm x 12.5 mm x 6.0 mm, mouse brain (P1), Korea Brain Research Institute) was placed in 4% paraformaldehyde solution for 12 hours at 4 ° C. Next, the purified brain was washed in PBS (phosphate-buffered saline pH 7.4, Thermo Fisher Scientific, USA) for 12 hours to remove paraformaldehyde. Immobilized brains were placed in 30% sucrose (sucrose, Sigma Aldrich, USA) in PBS until they subsided. The sunken brain was frozen and cut to a thickness of 100 ⁇ m.
  • PBS phosphate-buffered saline pH 7.4, Thermo Fisher Scientific, USA
  • the antibody and blocking solution were mixed at 1: 300.
  • the blocking solution was diluted with 10% BSA (Bovine Serum Albumins, Sigma Aldrich, USA), 0.2% Triton X-100 (Triton X-100, Sigma Aldrich, USA), 0.001% Sodium azide ).
  • BSA Bovine Serum Albumins
  • Triton X-100 Triton X-100, Sigma Aldrich, USA
  • 0.001% Sodium azide 0.001% Sodium azide
  • Example 1 The procedure of Example 1 was repeated except that 4% sodium dodecyl sulfate (Sigma Aldrich, USA) was used instead of SB3-14 in Example 1.
  • Example 1 As a result of microscopic observation of the brain tissue sections of the mice stained by the method of Example 1, Comparative Example 1 and Comparative Example 2, the intensity of the immunostaining signal in Example 1 and Comparative Example 2, as shown in FIG. 2, was found to exhibit better coloring as compared with Comparative Example 1. In addition, it was also found that the depth of the tissue showing significant signal intensity was 30 ⁇ for Comparative Example 1 and 70 ⁇ or greater for Example 1. When the depth of the brain tissue segment was taken as the Z axis, It was confirmed that the dyed depth and effect were superior to the examples (see Fig. 5).
  • Example 2 Sulfobetainate Zwitterion
  • the surfactant SB3-14 (3- ( N , N -Dimethylmyristylammonio) propanesulfonate) Treatment of brain tissue by time
  • Mouse brain tissue sections of 100 ⁇ m thickness were treated with SB3-14 at a concentration of 4% at a temperature of 37 ⁇ C. After SB3-14 treatment, the sections of brain tissue were observed with time (1 hour, 2 hours, 4 hours, 8 hours) using a microscope (SMZ745T, Nikon, Japan).
  • the brain tissue staining method using the surfactant treatment was carried out in the same manner as in Example 1, except that the treatment time was different. In order to compare the observed optical characteristics over time, we observed 2.5 mm x 2.5 mm grid paper for comparison, because the same tissues were observed over time in the analysis process.
  • Example 2 The sections of brain tissue stained with the method of Example 2, Comparative Example 3 and Comparative Example 4 were analyzed. As a result, as shown in FIG. 3, the stability of the tissue or biological sample of Example 2 was superior to the other comparative examples as the surfactant treatment time elapsed. In addition, it can be seen that there is almost no change in the surface or thickness of the tissue to be analyzed when it is seen that the scale is not deformed even after the time of the surfactant treatment has elapsed.
  • Example 3 Sulfobetainate Zwitterion
  • the surfactant SB3-14 (3- ( N , N -Dimethylmyristylammonio) propanesulfonate) in brain tissue
  • Example 4 Various kinds of Sulfobetainate Zwitterion Perform brain tissue staining using surfactant
  • Mouse brain tissue sections with a thickness of 100 ⁇ m were treated with various sulfobetaine zwitterionic surfactants at 4% concentration for 4 hours at 37 ⁇ for immunochemical staining.
  • SB3-16 (3-Cholamidopropyl) dimethylammonio] -1-propanesulfonate, Sigma aldrich, Sigma Aldrich, USA
  • CHAPS Sigma Aldrich, USA
  • each of the above surfactants was washed and then washed and reacted with a primary antibody Pan Neuronal Marker (0.5 mg / ml, EMD Millipore, USA) for 8 hours, and then reacted with a secondary antibody Alexa flour 594 mg / mL, Molecular probes, USA) for 2 hours and immunochemically stained by diffusion method.
  • the following confocal microscope (A1, Nikon, Japan) was used to measure in 10 ⁇ m increments.
  • the group treated with the sulfobetaine-based surfactant used in the above experiment exhibited excellent dyeing strength. Also, it was found that the depth of the tissue in which the significant signal intensity is visible is more than 70 ⁇ m.
  • Example 5 Sulfobetainate Zwitterion
  • the surfactant SB3-14 (3- ( N , N -Dimethylmyristylammonio) propanesulfonate) for brain tissue staining
  • Mouse brain (8.2 mm x 12.5 mm x 6.0 mm, mouse brain (P1), Korean Brain Research Institute) was placed in 4% paraformaldehyde for 12 hours at 4 ° C. The immobilized brain was then immersed in PBS solution for 12 hours at room temperature to wash paraformaldehyde. Brain tissues immobilized were immersed in a PBS solution containing 4% SB3-14 (3- ( N , N- dimethylmyristylammonio propanesulfonate, Sigma Aldrich, USA) at a concentration of 4% The brain tissue was treated with a sulfobetaine zwitterionic surfactant using a Rolling Botttle incubator with a speed of rpm and a temperature of 37 ° C.
  • the brain tissue was then immersed in a PBS solution for 12 hours, Tyrosine hydroxylase (Alexa Fluor 488) conjugated anti-tyrosine hydroxylase antibody (0.5 ml / well) was used for immunoblotting of the brain tissues, followed by washing on a rocker with a slope of 100 rpm and a slope of 100 rpm. mg, abcam, USA) was applied for 8 hours through a natural diffusion method to perform immunochemical staining.
  • the antibody was mixed with the blocking solution used in Example 1 at a ratio of 1: 200. After immunostaining, Immunohistochemical staining and washing were carried out in a shaking incubator at a temperature of 100 rpm and 37 ° C.
  • the present invention relates to a method for observing a biological sample containing a biological tissue or organ in a three-dimensional state, wherein a biological molecule such as an antibody, an antibody fragment, a protein, a polysaccharide, a nucleic acid including the RNA and DNA, a peptide and a polypeptide can be used to rapidly and uniformly infiltrate biological molecules.
  • a biological molecule such as an antibody, an antibody fragment, a protein, a polysaccharide, a nucleic acid including the RNA and DNA, a peptide and a polypeptide can be used to rapidly and uniformly infiltrate biological molecules.

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Abstract

La présente invention concerne une composition qui comprend un tensioactif zwitterionique à base de sulfobétaïne et est destinée à l'infiltration d'une biomolécule, telle qu'un acide nucléique, un polysaccharide, un anticorps, un fragment d'anticorps, un peptide, un polypeptide ou une protéine, dans un tissu dans un échantillon biologique. Une composition selon la présente invention peut infiltrer rapidement et uniformément une biomolécule telle qu'un anticorps dans le noyau d'un tissu biologique, et peut donc être utilisée dans la coloration immunohistochimique d'un tissu ou un organe biologique ayant une structure tridimensionnelle. De plus, la composition selon la présente invention peut être utilisée pour rendre un tissu biologique plus transparent.
PCT/KR2018/012369 2017-10-19 2018-10-19 Composition pour infiltrer une biomolécule dans un tissu comprenant un tensioactif zwitterionique à base de sulfobétaïne, et son utilisation WO2019078655A1 (fr)

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KR10-2017-0135948 2017-10-19
KR1020170135948A KR20190043886A (ko) 2017-10-19 2017-10-19 설포베타인계 쯔비터이온성 계면활성제를 유효성분으로 포함하는 생물학적 시료의 면역화학염색용 조성물 및 이를 이용한 면역화학염색 방법
KR1020180004121A KR102200618B1 (ko) 2018-01-12 2018-01-12 설포베타인계 쯔비터이온성 계면활성제를 포함하는 생물학적 분자의 조직침투용 조성물 및 이의 용도
KR10-2018-0004121 2018-01-12

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CN117337827A (zh) * 2023-10-18 2024-01-05 温州市倍可特医疗器械有限公司 一种抑制组织固定液中甲醛挥发的方法及应用

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* Cited by examiner, † Cited by third party
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WO2021095718A1 (fr) * 2019-11-11 2021-05-20 公立大学法人大阪 Procédé de fabrication de tissu biologique transparent et réactif associé
CN117337827A (zh) * 2023-10-18 2024-01-05 温州市倍可特医疗器械有限公司 一种抑制组织固定液中甲醛挥发的方法及应用

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