WO2017180160A1 - Methods to reduce evaporation during elevated temperature - Google Patents
Methods to reduce evaporation during elevated temperature Download PDFInfo
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- WO2017180160A1 WO2017180160A1 PCT/US2016/027945 US2016027945W WO2017180160A1 WO 2017180160 A1 WO2017180160 A1 WO 2017180160A1 US 2016027945 W US2016027945 W US 2016027945W WO 2017180160 A1 WO2017180160 A1 WO 2017180160A1
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- Prior art keywords
- reagent
- moieties
- moiety
- evaporation
- reducing agent
- Prior art date
Links
- 238000001704 evaporation Methods 0.000 title claims abstract description 73
- 230000008020 evaporation Effects 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 30
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 57
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 44
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 19
- 230000001413 cellular effect Effects 0.000 claims abstract description 12
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 11
- 239000001257 hydrogen Substances 0.000 claims abstract description 11
- 125000003342 alkenyl group Chemical group 0.000 claims abstract description 8
- 125000000304 alkynyl group Chemical group 0.000 claims abstract description 8
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 8
- 125000003118 aryl group Chemical group 0.000 claims abstract description 7
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 7
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 5
- 239000001301 oxygen Substances 0.000 claims abstract description 4
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims abstract 3
- 239000011593 sulfur Substances 0.000 claims abstract 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 88
- 238000009396 hybridization Methods 0.000 claims description 49
- 125000004429 atom Chemical group 0.000 claims description 9
- 239000000427 antigen Substances 0.000 claims description 5
- 102000036639 antigens Human genes 0.000 claims description 5
- 108091007433 antigens Proteins 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 5
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 150000001412 amines Chemical group 0.000 claims description 4
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims description 4
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 claims description 4
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 3
- 125000001033 ether group Chemical group 0.000 claims description 3
- 125000005843 halogen group Chemical group 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- DNIAPMSPPWPWGF-VKHMYHEASA-N (+)-propylene glycol Chemical compound C[C@H](O)CO DNIAPMSPPWPWGF-VKHMYHEASA-N 0.000 claims description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 2
- YPFDHNVEDLHUCE-UHFFFAOYSA-N 1,3-propanediol Substances OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 230000032677 cell aging Effects 0.000 claims description 2
- 230000003750 conditioning effect Effects 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 229920000166 polytrimethylene carbonate Polymers 0.000 claims description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims 3
- 125000003368 amide group Chemical group 0.000 claims 2
- 125000003827 glycol group Chemical group 0.000 claims 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 40
- 239000000523 sample Substances 0.000 description 31
- 238000003556 assay Methods 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000000203 mixture Substances 0.000 description 8
- 230000009467 reduction Effects 0.000 description 8
- 239000002904 solvent Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 150000001408 amides Chemical group 0.000 description 3
- 125000004430 oxygen atom Chemical group O* 0.000 description 3
- 125000004434 sulfur atom Chemical group 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N Furan Chemical compound C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 239000002608 ionic liquid Substances 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 229940035437 1,3-propanediol Drugs 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000001345 alkine derivatives Chemical class 0.000 description 1
- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000002534 ethynyl group Chemical group [H]C#C* 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000012760 immunocytochemical staining Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 125000000555 isopropenyl group Chemical group [H]\C([H])=C(\*)C([H])([H])[H] 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- -1 isopropynyl Chemical group 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000001749 primary amide group Chemical group 0.000 description 1
- 125000004368 propenyl group Chemical group C(=CC)* 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000002568 propynyl group Chemical group [*]C#CC([H])([H])[H] 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229930192474 thiophene Natural products 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
Definitions
- Immunohistochemical assays and assay techniques based on in situ hybridization are widely used in medical diagnostics such as to diagnose abnormal cells such as those found in cancerous tumors or to diagnose another disease.
- Many assays involve the addition of heat to the sample, such as to a sample in a reagent on a slide. Assay steps at elevated temperatures can cause substantial evaporation of assay reagents. When small volumes are used, sample denaturation, hybridization, wash and aging steps and the resulting assay can be compromised by evaporation.
- a reagent and a method of use of a reagent including an evaporation reducing agent in an aqueous solution operable to reduce evaporation of the reagent in processing of a tissue or cellular sample is disclosed.
- Representative processing for which a reagent as described finds use include, but are not limited to, dewaxing, cell conditioning/antigen retrieval/cell aging, peroxide/phosphatase block, probe denature, probe hybridization, washing, linker hybridization, antibody incubation, probe detection, chromogen precipitation and counterstain involving an elevated temperature step (e.g. an elevated temperature step in an in situ hybridization procedure).
- the solution can representatively be used in
- a suitable reagent may include other components such as a detergent/surfactant which helps with solution spreading in a hybridization solution reagent.
- a suitable amount of an evaporation reducing agent in a reagent operable for tissue or cellular processing is an amount that will limit a loss of the reagent due to evaporation to 20 percent or less under industry acceptable reagent processing conditions such as subjecting a hybridization solution to an elevated temperature between 25°C and 50°C for 10 minutes to 24 hours (e.g., a hybridization or incubation process) or to an elevated temperature of between 60°C and 100°C for two to ninety minutes (e.g., a denaturing or antigen retrieval process).
- a suitable amount of an evaporation reducing agent is in the range of 11 percent to 60 percent by volume of a regent composition (e.g., solution).
- a reagent includes an effective evaporation reducing amount of an evaporation reducing agent comprising the formula of General Formula I:
- R is an alkyl, an alkenyl, an alkynyl or an aromatic moiety of one or more carbon atoms (e.g., one to six carbon atoms in one embodiment and one to four carbon atoms in another embodiment where it is appreciated that if R is a moiety of one carbon, R is an alkyl) that may be substituted with oxygen atom, sulfur atom and/or or nitrogen atom (e.g., -OH, - SH, or - H 2 is substituted for a hydrogen atom of one or more hydrocarbons defining the alkyl, alkenyl, alkynyl or aromatic) or that may be interrupted with an oxygen atom, a sulfur atom and/or a nitrogen atom (e.g., -C-0-C-, -C-SH-C-, -C- H-C-).
- An alkyl moiety is an alkane containing open points of attachment for connection for X 1 and X 2 .
- An alkenyl moiety is an alkene containing open points of attachment for connection for X 1 and X 2 .
- An alkynyl moiety is an alkyne containing open points of attachment for connection to Xi and X 2 .
- Representative alkyls include straight or branched chain alkyls (e.g., methyl, ethyl, propyl, isopropyl) which may or may not be further substituted.
- Representative alkenyls include straight or branched chain alkenyls (e.g., ethenyl, propenyl, isopropenyl).
- alkynyls include straight or branched chain alkynyls which may or may not be further substituted (e.g., ethynyl, propynyl, isopropynyl).
- An aromatic moiety is an unsaturated ring of atoms that is stabilized by an interaction of the bonds forming the ring.
- An example of an aromatic moiety is benzyl.
- interrupted aromatic moieties are pyridine (interrupted with a nitrogen atom); furan (interrupted with an oxygen atom); and thiophene (interrupted with a sulfur atom).
- Xi and X 2 in General Formula I are independently selected to be a moiety where one or both are susceptible to hydrogen bonding and/or contains an electronegative atom.
- Representative moieties for X 1 and X 2 will include functional groups that are capable of hydrogen bonding such as but not limited to hydroxyl moieties (-OH); carbonyl moieties (- CO); amine moieties (- H 2 ); aldehyde moieties (-CHO); halogen moieties (-Y, where Y is CI " , F " , Br “ or ⁇ ); ether moieties (-OR 1 , where R 1 is an alkyl (e.g., an alkyl of one to six carbon atoms) that may be substituted with a moiety that is susceptible or capable of hydrogen bonding and/or contains an electronegative atom); carboxyl moieties (-COOR 2 , where R 2 is a hydrogen atom or an alkyl (e.g., an alkyl of one to six carbon atom
- substitution moieties that are susceptible or capable of hydrogen bonding for the noted alkyls associated with ether, carboxyl and amide moieties for X 1 and X 2 (R 1 , R 2 , R 3 and R 4 ) include hydroxyl, carbonyl, amine, aldehyde, halogen, ether, carboxyl and amide moieties.
- Suitable evaporation reducing agents having General Formula I include ethylene glycol, glycine, serine, isoethionic acid, ethanolamine, polyethylene glycol and 1,3 -propanediol.
- R is not substituted or interrupted.
- Representative of an evaporation reducing agent where R is not substituted or interrupted is ethylene glycol (HO(CH 2 ) 2 OH) where R is ethyl and each of Xi and X 2 is a hydroxyl moiety.
- a suitable reagent includes one or more evaporation reducing agents having the formula of General Formula I (e.g., a reagent includes a combination of two or three evaporation reducing agents and having the formula of General Formula I).
- An evaporation reducing agent may be in a molecular free form (a molecule) or an acceptable salt thereof (a compound) or ionic liquid.
- a salt is a hydrohalide salt of the molecule such as a hydrochloride salt.
- an evaporation reducing agent as a solute reduces the vapor pressure of a solution upon its mixing with one or more other reagents.
- Such solute could be a molecule, an ionic salt, an ionic liquid, a non-ionic agent that may exhibit complete or partial solubility or miscibility with water and is capable of reducing a partial pressure of an aqueous solution to which it is mixed or otherwise introduced.
- an evaporation reducing agent may or may not form an azeotropic mixture with water. If an evaporation reducing agent does form an azeotropic mixture with water, the solute, in one embodiment, forms a negative azeotrope with water.
- a resulting chemical potential of the solvent is lowered so that a partial pressure of the solvent molecules is reduced and, relative to a solvent-only solution, has a reduced tendency to transition into gas phase.
- a partial pressure of water in an aqueous solution where a solute is a nonvolatile evaporation reducing agent will be reduced which would result in elevation of a boiling point of the mixture as well as a reduction in evaporation at the surface.
- Hydrogen bonding can occur when in a molecule, a hydrogen atom is attached to an electronegative atom such as an oxygen, nitrogen or fluorine atom and the molecule comes in proximity with an electropositive atom.
- the electrostatic attraction of this nature has a strength on the order of 5-30kJ/mol which is stronger than Van der Waals attraction but weaker than covalent or ionic bonding.
- an evaporation reducing agent is mixed as a solute with water to reduce a vapor pressure of the water by creating a solution where a partial pressure of the water is reduced such that an assay (e.g., an immunohistologic assay) can be carried out more efficiently than if the partial pressure was not lowered.
- a solute is selected such that there is no or minimum residue left behind which may interfere with an assay known to a person skilled in the art
- a reagent operable for use in processing a cellular or tissue sample is prepared by combining a base reagent and an evaporation reducing agent.
- Additional components may also be combined in a suitable composition.
- the reagent can be added to an aqueous buffer solution to reduce evaporation during elevated temperatures.
- the evaporation reducing agent is combined in an amount to limit any loss of the combination due to evaporation to 20 percent or less and, in another embodiment, to 10 percent or less.
- an evaporation reducing agent or combination of reducing agents is/are present in a composition in an amount between 11 percent and 60 percent by volume or an amount between 1 percent and 60 percent of the reducing agent is a non-glycol- containing compound or a combination of reducing agents.
- the composition may be mixed.
- a method operable in processing of a paraffin-free or deparaffinized tissue or cellular sample for use in an assay at an elevated temperature is disclosed.
- a method is operable to reduce evaporation of any aqueous based processing reagent. The method includes contacting a tissue or cellular sample with a reagent including an evaporation reducing agent (e.g., as a solute) having the formula of General Formula I:
- Such contact includes applying the reagent to a sample on a slide.
- the sample may be processed according to techniques known in the art.
- a method operable in processing of a paraffin-free or deparaffinized tissue or cellular sample for use in an assay at an elevated temperature is disclosed.
- a method is operable to reduce evaporation of any aqueous based processing reagent by adding a solute to a solvent. The resulting chemical potential of the solvent is lowered so that the partial pressure of the solvent molecules is reduced and they have lesser tendency to turn into gas phase. Partial pressure of water in an aqueous solution where solute is a nonvolatile substance is reduced which would result in elevation of boiling point as well as reduction in evaporation at the surface.
- Example 1 Volume loss due to evaporation during oligonucleotide hybridization at different hybridization temperatures.
- hybridization solution 400 ⁇ of hybridization solution (reagent) was applied to each of six sample slides and the slides were individually heated to different temperatures for 30 minutes. It was observed that evaporation occurred on the slide with hybridization solution during the hybridization step.
- Example 2 Reducing evaporation during oligonucleotide hybridization by different reagents.
- hybridization solution 400 ⁇ of hybridization solution (reagent) with different evaporation reducing agents was applied to respective sample slides and heated to 43° C for 30 minutes.
- One sample slide included a hybridization solution with no evaporation reducing agent. It was observed that a significantly greater evaporation of the hybridization solution occurred with the sample slide containing a hybridization solution without an evaporation reducing agent during the 43° C hybridization step but was reduced with the addition of an evaporation reducing agent.
- Example 3 Reducing evaporation using different concentrations of ethylene glycol during hybridization.
- hybridization solution The assay performance for the hybridization solution containing ethylene glycol was found to be similar to hybridization solution without ethylene glycol.
- Example 4 Reproducibility in reducing evaporation volume loss during hybridization.
- Example 5 Reduction in evaporation during denaturation.
- Example 6 Reduction in evaporation during antibody incubation step.
- Example 7 Reducing evaporation during oligonucleotide hybridization by different reagents.
- hybridization solution with different evaporation reducing agents 400 ⁇ was applied to respective sample slides and heated to 43 °C for 30 minutes. A hybridization solution containing no evaporation reducing agent was also applied to a sample slide. It was observed that evaporation occurred in the slide with hybridization solution during the 43°C hybridization step but was reduced with the addition of specific reagent.
- Example 8 Reducing evaporation using ethylene glycol during hybridization at different temperatures.
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
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DK16720619.2T DK3289098T3 (en) | 2015-04-30 | 2016-04-15 | Methods to reduce evaporation at elevated temperature |
BR112017022973A BR112017022973A2 (en) | 2015-04-30 | 2016-04-15 | methods to reduce evaporation during elevated temperature |
CA2983930A CA2983930C (en) | 2016-04-15 | 2016-04-15 | Methods to reduce evaporation during elevated temperature |
AU2016402370A AU2016402370B2 (en) | 2016-04-15 | 2016-04-15 | Methods to reduce evaporation during elevated temperature |
JP2017554865A JP6550471B2 (en) | 2016-04-15 | 2016-04-15 | Evaporation reduction method and reagent used therefor |
CN201680023650.5A CN107614702A (en) | 2016-04-15 | 2016-04-15 | The method for reducing evaporation at elevated temperatures |
EP16720619.2A EP3289098B1 (en) | 2015-04-30 | 2016-04-15 | Methods to reduce evaporation during elevated temperature |
PCT/US2016/027945 WO2017180160A1 (en) | 2016-04-15 | 2016-04-15 | Methods to reduce evaporation during elevated temperature |
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PCT/US2016/027945 WO2017180160A1 (en) | 2016-04-15 | 2016-04-15 | Methods to reduce evaporation during elevated temperature |
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JP (1) | JP6550471B2 (en) |
CN (1) | CN107614702A (en) |
AU (1) | AU2016402370B2 (en) |
CA (1) | CA2983930C (en) |
WO (1) | WO2017180160A1 (en) |
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US20070048770A1 (en) * | 2005-07-15 | 2007-03-01 | Jaekel Robert W | Reagents and methods for processing biological samples |
WO2012055981A1 (en) * | 2010-10-27 | 2012-05-03 | Glaxosmithkline Biologicals S.A. | Immunogenic compositions and methods for treating neurologic disorders |
US20140242595A1 (en) * | 2013-02-22 | 2014-08-28 | Cellular Dynamics International, Inc. | Hepatocyte production via forward programming by combined genetic and chemical engineering |
WO2014144663A1 (en) * | 2013-03-15 | 2014-09-18 | The Johns Hopkins University | Methods and compositions for improving cognitive function |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
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JP6550471B2 (en) | 2019-07-24 |
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