WO2015039423A1 - Solution de conservation de biomarqueur, réactif et procédé - Google Patents
Solution de conservation de biomarqueur, réactif et procédé Download PDFInfo
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- WO2015039423A1 WO2015039423A1 PCT/CN2014/074463 CN2014074463W WO2015039423A1 WO 2015039423 A1 WO2015039423 A1 WO 2015039423A1 CN 2014074463 W CN2014074463 W CN 2014074463W WO 2015039423 A1 WO2015039423 A1 WO 2015039423A1
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- protein
- preservation solution
- biomarker
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6809—Determination of free amino acids involving fluorescent derivatizing reagents reacting non-specifically with all amino acids
Definitions
- the present invention relates to the field of in vitro diagnostic reagents, and more particularly to a preservation solution, reagent and method for biomarkers. Background technique
- Protein is a macromolecular substance with a complex spatial three-dimensional structure. It is biologically active and easily denatured by external conditions. It is easily degraded and inactivated at low concentrations ( ⁇ 0.1mg/mL), and at low concentrations. Easy to adsorb on the pipe wall and cause damage. For many proteins, which are typically frozen at - 20 ° C or - 80 ° C, repeated freezing/thawing cycles can denature the protein, causing it to form aggregates with reduced activity.
- fluorescein After being irradiated with excitation light, fluorescein absorbs light into an excited state and can immediately retreat and emit light (usually the wavelength of the emitted light is longer than the wavelength of the incident light, ie, the stock shift occurs). It is currently used for cell staining to improve the ability to recognize cells.
- fluoresceins include small molecule compounds such as FITC, Cy5, Cy7 Alexa Fluor, etc., as well as many fluorescent proteins such as PE, APC; and PerCP. Fluorescein is easily quenched, often in solid form (mostly a small molecule compound of fluorescein), ammonium sulfate precipitated form (such as fluorescent protein) or stored in a preservation solution at a high concentration and low temperature, generally not frozen.
- fluorescence microscopy is a technique in which a fluorescently labeled antibody is bound to an antigen on a cell, and the fluorescence intensity of the antibody binding site is observed by a fluorescence microscope to determine the result.
- flow cytometry technology which uses different colors of fluorescein to label different kinds of monoclonal antibodies, can simultaneously analyze multiple antigen molecules on one cell, further enhancing the understanding of cell types and functions. And can fluoresce a lot of cells Quantitative analysis of intensity, combined with computer technology, improves the detection level of immunofluorescence and automates cell analysis. Different fluorescently labeled antibody reagents can detect different antigens, and thus have become a core component of flow cytometry in scientific research or clinical applications.
- Biochip refers to the integration of various biological information that can act as a receptor on a solid substrate, including oligonucleotides, proteins/enzymes, antigens/antibodies, cells, etc., utilizing the reaction between the receptor and the linker (Biological nucleic acid hybridization reactions, antigen/antibody affinity recognition reactions, and the like are included for biological detection. According to different biomolecules and materials fixed by biochips, they can be divided into gene chips, protein chips, chip labs, cell chips and tissue chips.
- a protein chip is a novel biochip consisting of an antigen or antibody microarray immobilized on different kinds of support media.
- the position and composition of the immobilized molecule in the array are known, using a label (fluorescent substance, enzyme or chemiluminescence).
- a label fluorescent substance, enzyme or chemiluminescence.
- the antibody or antigen labeled with a substance or the like is reacted with a probe on the chip, and then detected by a specific scanning device, and the result is analyzed by a computer.
- the molecular spectral fluorescence analysis method of protein has the advantages of high sensitivity, good selectivity, wide dynamic response range and measurement conditions closer to the physiological environment of the living body, and is widely used in protein analysis.
- Fluorescent protein probe technology is a method for analyzing the high sensitivity of proteins in solution at the molecular weight level by utilizing the photophysical and photochemical properties of the substance.
- fluorescent dyes for tracer are not limited to fluorescein and fluorescent protein, trivalent rare earth ions and their chelating agents, or semiconductor nano-grains, which are excited by excitation light, and the wavelength of emitted light is also higher.
- a substance having a long wavelength of excitation light and also having fluorescent properties is also used as a tracer, and is labeled on a biological material such as a protein, a polypeptide, a hormone, a nucleic acid, or a living cell.
- Trivalent rare earth ions and their chelates have longer fluorescence decay times and a larger stock shift for Time-Resolved Fluorescence Immunoassay (TRFIA).
- TRFIA technology has high sensitivity, high specificity, good stability, wide measurement range, easy operation and non-radioactivity. Point, very suitable for biological and medical ultra-micro analysis.
- Semiconductor nano-grains also known as quantum dots (QDs) are semiconductor nanoparticles with a diameter between 1 and 100 nm that can receive excitation light to produce fluorescence. Quantum dots combine with biomolecules, living cells, etc., for DNA detection (DNA chip), protein detection (protein chip) and exploring protein-protein (antigen-antibody, ligand-receptor, enzyme-substrate) reaction principle Provides an advanced method.
- biomarkers combined with fluorescent tracers are generally more expensive.
- the amount of reagents used is generally small and the concentration is low.
- Such reagents require cryopreservation and require high transport and storage conditions, which in turn increases reagent costs.
- a preservation solution for a biomarker comprising: at least one protein stabilizer, at least one basic or neutral amino acid, at least one polyhydroxy compound or polyhydroxy polymer, and citrate.
- a fluorescent label reagent comprising a preservation solution of the above biomarker and a fluorescent tracer-biomarker conjugate.
- a method for preparing a fluorescent label reagent comprising: providing a preservation solution of the above biomarker and a fluorescent tracer-biomarker conjugate; and diluting the fluorescent tracer-biomarker conjugate with a preservation solution to At the working concentration, a fluorescent label reagent solution that can be used directly is obtained.
- the application also discloses the use of the above preservation solution in the preparation of a fluorescent label reagent.
- the above preservation solution is suitable for fluorescent label reagents containing fluorescent tracer-biomarker conjugates, and is particularly suitable for storing small molecule fluorescein labeling proteins, fluorescent protein labeling proteins and tandem dye labeling protein reagents; monochromatic or multicolor fluorescence Labeled protein reagent; fluorescently labeled protein, polyclonal antibody or monoclonal antibody reagent. Directly dilute the fluorescently labeled protein reagent to the working concentration when used,
- Figure 1 is a HPLC analysis of the degradation curve of IgG-PE fluorescent labeled protein reagent in 1# ⁇ 6# preservation solution by HPLC;
- Fig. 3 is a comparison test result of the CD4-FITC monochrome reagent preservation solution 14# and the commercial preservation solution preservation accelerated test in Example 3;
- Figure 6 is a storage solution for CD19-APC monochrome reagent in Example 7 and stored in a commercially available preservation solution. The accelerating test compares the test results. detailed description
- biomarker refers to a biomolecule or living cell derived from an organism.
- the biomolecule is a molecule that specifically binds to a cell or a cellular component, including but not limited to an antibody, an antigen, a receptor, and a ligand. Body, enzyme, substrate, coenzyme, nucleic acid, hormone, etc.
- fluorescent tracer refers to a compound that produces a fluorescent signal and is capable of coupling to a biological material, including but not limited to small molecule fluorescein, fluorescent protein, fluorescent dye, rare earth ion, and chelating agent thereof. , semiconductor nano-micro crystals, etc.
- fluorescent tracer-biomarker conjugate refers to a compound formed by the attachment of a fluorescent tracer to a biomarker via a covalent bond.
- fluorescent labeling protein reagent refers to a fluorescent tracer through a covalent bond to a protein
- the macromolecule is linked to form a fluorescent tracer-protein conjugate, and the conjugate is formulated into a solution for use herein in the term “monochrome fluorescently labeled protein reagent”, which refers to a fluorescent tracer through covalent
- the bond is linked to a biomarker to form a conjugate, which is formulated as a solution.
- multicolor fluorescently labeled protein reagent refers to a mixture of a plurality of monochromatic fluorescently labeled protein reagents.
- antibody includes monoclonal antibodies, polyclonal antibodies, and antibody fragments of various animal origins, including different antibody types, such as immunoglobulin G, immunoglobulin A, immunoglobulin E, immunoglobulin. Protein M, immunoglobulin D and immunoglobulin Y. Preservative solution for biomarkers
- One embodiment of the present invention discloses a preservation solution for a biomarker comprising at least one protein stabilizer, at least one basic or neutral amino acid, at least one polyhydroxy compound or polyhydroxy polymer, and citrate.
- the protein stabilizer in the preservation solution can maintain the biological activity of the protein, can reduce the degradation and inactivation of the low concentration protein, and reduce the loss caused by the adsorption of the low concentration protein on the tube wall.
- protein stabilizers also alleviate protein denaturation caused by adverse environmental factors such as heat, surface tension and chemical factors.
- the protein stabilizer may be selected from gelatin and albumin (e.g., bovine serum albumin BSA and human serum albumin), preferably gelatin and BSA, and the concentration may be 0.05% to 0.40%, preferably 0.08% to 0.24%.
- the alkaline or neutral amino acid in the preservation solution reduces the aggregation of the protein, fluorescent tracer-protein conjugate.
- the amino acid may be selected from the group consisting of arginine, histidine, lysine and glycine, and preferably arginine and glycine, and the concentration may be in the range of 1 to 20 mM, preferably 4 to 16 mM.
- the polyhydroxy compound or polyhydroxy polymer in the preservation solution increases the water solubility and stability of the fluorescently labeled protein reagent.
- it is considered a polyhydroxy compound or polymer It has a tension control function, which can effectively maintain the configuration and activity of the protein, help to maintain the stability of the fluorescent tracer-protein conjugate, and also reduce the fluorescence quenching of fluorescein caused by intermolecular and intramolecular interactions.
- the polyhydroxy compound may be selected from the group consisting of glycerin, sugar alcohols (such as mannitol, xylitol, sorbitol) and sugars (such as trehalose, mannose, xylose) and the like, preferably glycerin, mannitol and trehalose.
- the polyhydroxy polymer may be selected from polyethylene glycol polymers, such as PEG 8000, PEG 10000, PEG 20000, etc., preferably PEG 10000, PEG 20000.
- the glycerol concentration may range from 1 to 10%, preferably from 2 to 6%; the concentration of mannitol, trehalose and PEG may range from 0.1% to 1.0%, preferably from 0.2% to 0.8%.
- the citrate in the preservation solution maintains the pH of the buffer in the preservation solution for a long period of time, reduces protein aggregation, and maintains protein stability.
- the citrate may be selected from citrate-soluble citrate in water, such as sodium citrate, potassium citrate, etc., preferably sodium citrate, and the concentration may range from 5 to 40 mM, preferably from 10 to 30 mM. .
- the preservation solution of one embodiment of the present invention may further comprise at least one antioxidant. While not wishing to be bound by theory, it is believed that antioxidants can be used to reduce oxidative quenching of fluorescein. Sodium ethylenediaminetetraacetate, such as EDTA*2Na, is commonly used in concentrations ranging from 2 to 3 mM.
- the preservation solution of one embodiment of the present invention may further comprise a conventional preservative to facilitate preservation and long-term preservation of the reagent.
- a conventional preservative to facilitate preservation and long-term preservation of the reagent.
- preservatives are NaN 3 , Procline, etc.
- the preservative concentration range is generally 0.05% to 0.10%.
- the concentration and type of preservative are preferably such that they do not affect protein structure and biological activity.
- the preservation solution of one embodiment of the present invention may further comprise a buffer to maintain the pH of the preservation solution at 6-8, preferably about 6.8 ⁇ 7.2, and the buffer is selected so as not to affect the fluorescence intensity. , protein structure and biological activity are appropriate. Common buffer systems such as phosphate buffer solutions, citrate buffer solutions and the like can be used in the present invention.
- the buffer is usually used in an amount of 10 to 50 mM, and more preferably at a pH of 7.0.
- the preservation solution can be directly diluted with the fluorescently labeled protein reagent to a working concentration, and the fluorescently labeled protein reagent diluted by the preservation solution is sealed and stored in the dark at 2 ° C to 8 ° C for more than 1 year. It is easy to understand that the preservation solution can also store the protein reagent as a concentrate and dilute it before use.
- the protein stabilizer in the preservation solution synergizes with the polyhydroxy compound or the polymer to alleviate adverse environmental factors such as heating, surface tension and chemical factors, and can be maintained at a high temperature of 25 ° C to 40 ° C.
- the reagent is stable and non-volatile. It has been proved by experiments that it can withstand high temperature transport conditions of 25 ⁇ 40 °C for a short period of time, which reduces the cost of fluorescent labeled protein reagents and has good commercial value.
- the fluorescent labeling protein reagent may be a small molecule fluorescein labeling protein, and a small molecule fluorescein such as FIT:, Cy5, Cy7, Alexa Fluor or the like.
- the fluorescent labeling protein reagent type may be a fluorescent protein labeled protein, a fluorescent protein such as PE, APC, PerCP or the like.
- the fluorescent labeling protein reagent may be a tandem dye-labeled protein, a tandem dye such as PE-Cy7, APC-Cy7, PE-Cy5, PE-Cy5.5 or the like.
- the fluorescent labeling protein reagent component may be a monochromatic or multicolor fluorescent labeling protein reagent, a monochromatic reagent such as CD4-FITC, CD3-PerCP, etc., a multicolor reagent such as CD3-FITC/CD8-PE /CD45 -PerCP/CD4-APC Four-color reagent and CD3- FITC/CD16 + CD56-PE/CD45-PerCP/CD19-APC four-color reagent.
- CD4-FITC refers to a conjugate of fluorescein FITC formed by covalent bonding to an antibody specific for CD3 cells. Other analogies.
- Another embodiment of the present invention discloses a fluorescent label reagent comprising the above-described preservation solution and fluorescent tracer-biomarker conjugate.
- a biomarker in a fluorescent tracer-biomarker conjugate refers to a biomolecule or living cell from an organism, including but not limited to nucleic acids, polypeptides, proteins, hormones, living cells, protein packs. Including antibodies, enzymes, and the like.
- the biomarker is a monoclonal antibody or a polyclonal antibody.
- the fluorescent tracer in the fluorescent tracer-biomarker conjugate can be any compound capable of generating a fluorescent signal and capable of coupling with a biological material, including but not limited to small molecule fluorescein, fluorescent protein, fluorescent dye, rare earth Ions and their chelating agents, semiconductor nano-microcrystals, preferably small molecule fluorescein, fluorescent protein, tandem fluorescent dye.
- the fluorescent tracer-biomarker conjugate can be a combination of the above, obtained by techniques well known to those skilled in the art.
- the fluorescent tracer-biomarker conjugate is a fluorescently labeled protein, particularly preferably a fluorescently labeled antibody.
- the samples are analyzed on a fluorescence activated flow analyzer.
- the fluorescent tracer-biomarker conjugate can also be used in solid phase immunoassays or biochip assays. Related techniques are well known in the art and are available in standard textbooks. Method for preparing fluorescent label reagent
- a further embodiment of the present invention discloses a method for preparing a fluorescent label reagent, comprising: providing the above preservation solution and a fluorescent tracer-biomarker conjugate;
- the fluorescent tracer-biomarker conjugate was diluted to the working concentration with a preservation solution to obtain a fluorescent marker reagent solution that can be used directly.
- the fluorescent tracer-biomarker conjugate is a fluorescently labeled protein, particularly preferred,
- the fluorescently labeled protein is mixed with other components as needed, dissolved in water, and formulated into a fluorescent label.
- Record protein mother liquor. Weigh the components of the above-mentioned preservation solution, stir and dissolve with water, and dilute with water to a certain concentration, which can be determined according to the concentration of the fluorescent labeled protein mother liquor. If necessary, the prepared preservation solution and the fluorescently labeled protein solution are mixed in proportion so that the concentration of the fluorescently labeled protein is at a working concentration to obtain a fluorescently labeled protein reagent solution.
- the "working concentration" herein means that the prepared fluorescent labeled protein reagent solution can be directly mixed with the sample to be tested without dilution. Since the preservation solution of the present application has a good preservation effect on the fluorescent tracer-biomarker conjugate, the formulated reagent can be lyophilized without using reconstitution, and is convenient to use.
- a further embodiment of the invention discloses the use of the above-described preservation solution for the preparation of a fluorescent label reagent.
- the above preservation solution can be used to prepare a liquid fluorescent labeling reagent which can be stored for a long period of time and can be used directly.
- Multitest CD3- FITC/CD8-PE/CD45-PerCP/CD4-APC (CD3-FITC, SK7 clone; CD8-PE, SKI clone; CD45-PerCP, 2D1 clone; CD4- APC, SK3 clone), no dilution, direct use;
- Multitesl CD3-FITC/CD16 + CD56-PE/CD45 -PerCP/CD19-APC CD3-FITC, SK7 clone; CD16-PE, B73.1 clone; CD56-PE, NACM16 .2 clone; CD45-PerCP, 2D1 clone; CD19-APC, SJ25C1 clone), no need for trouser release, direct use;
- BD FACS Lysing Solution (1 Ox concentrate, use deionized water diluted 1: 10 to l x reagent);
- PBS buffer KC1 0.2 g, KH 2 PO 4 0.2 g, NaCl 8.0 g, Na 2 HP0 4 1.15 g, dissolved in 1 L of deionized water, pH 7.4 ⁇ 0.2, filtered through a 0.2 ⁇ PALL GHP filter.
- a self-made fluorescent protein-labeled polyclonal antibody IgG-PE reagent was prepared according to a conventional method.
- the self-made fluorescent protein-labeled polyclonal antibody IgG-PE reagent was diluted to a solution of 12.5 g/mL with 1 ⁇ 6# preservation solution.
- the diluted fluorescently labeled polyclonal antibody reagent solution is dispensed into 9 small portions (50 L each) at 40 ° C in the dark, and a reagent solution is taken every 3 to 4 days.
- the fluorescent tracer is detected as described above.
- the peak area of the protein-protein conjugate was calculated as a function of increasing the percent degradation of the fluorescent tracer-protein conjugate over time, plotting time and percent degradation.
- the test results are shown in Figure 1.
- the percentage of degradation of the fluorescent tracer-protein conjugate reflects the stability of the fluorescent tracer-protein conjugate and the stability of fluorescein, wherein the protein stabilizer gelatin or BSA concentration is 0.08%. ⁇ 0.24%, when the concentration of arginine or glycine is 4mM ⁇ 16mM (ie, preservation solution 2#, 5#), the degradation percentage of IgG-PE fluorescent tracer-protein conjugate is less than the preservation solution 1#, 3#, 4 # ⁇ 6#, the preservation solution works better. It also shows that the preservation solution has a good preservation effect on the fluorescein-antibody conjugate.
- preservation solution 1 preservation solution 2# preservation solution 3# preservation solution 4# preservation solution 5# preservation solution 6# Weigh
- the fluorescently labeled protein BSA-PE reagent was prepared in accordance with a conventional method.
- the self-made fluorescently labeled protein BSA-PE reagent was diluted to a 12.5 g/mL solution with 7 ⁇ 13# preservation solution.
- the diluted fluorescently labeled protein reagent solution is divided into 8 small portions (each 50 ⁇ is placed at 40 ° C in the dark, and one reagent is taken every 3 to 7 days to detect the fluorescent tracer-protein conjugation as described above. Peak area, calculated as a function of increasing the percentage of degradation of the fluorescent tracer-protein conjugate over time, plotted versus percent degradation.
- the detection results are shown in Fig. 2.
- the percentage of degradation of the fluorescent tracer-protein conjugate reflects the stability of the fluorescent tracer-protein conjugate and the stability of fluorescein, wherein the polyglycerol concentration is 2% ⁇ 6. %, when the concentration of sugar alcohol, saccharide and polyethylene glycol polymer is 0.2% ⁇ 0.8% (ie, preservation solution 9#, 10#, 13#), the percentage of degradation of BSA-PE fluorescent tracer-protein conjugate Less than the preservation solution 7#, 8#, 11#, 12#, the preservation solution is better. It also shows that the preservation solution has a good preservation effect on the fluorescein-protein conjugate.
- CD4-FITC CD3-PerCP CD8- APC-Cy7 Three Monochrome Fluorescent Labeled Monoclonal Antibody Reagent Dilution Method Monochrome Reagent Diluted Preservative Concentration CD4-FITC Preservative 14# and commercially available SurModics StabilGuard® Preservative diluted 5 g/mL
- CD3-PerCP Preservative 15# and commercially available SurModics StabilGuard® Preservatives are diluted separately
- CD8-APC-Cy7 Preservative Solution and commercially available SurModics StabilGuard® Preservation Solution were diluted 5 g/mL, respectively.
- the diluted monochromatic fluorescently labeled monoclonal antibody reagents were each dispensed in 6 aliquots (each ⁇ at 25 °C) Light placement, one reagent per 7 days to determine the same batch of commercially available StatusFlow® Flow Cytometry Control controls, and the fluorescence intensity of the cell binding reagent in the control product is detected by the method of detecting the sample by the above monochromatic reagent. The operation was performed 3 times, and the average value of the test results was taken three times.
- the curve was plotted with time and the average fluorescence intensity of each monochromatic fluorescently labeled monoclonal antibody reagent.
- the detection results are shown in Fig. 3, Fig. 4, and Fig. 5, and the time-fluorescence intensity curve reflects The biological activity and fluorescence stability of the fluorescently labeled monoclonal antibody reagent showed that the preservation solutions 14#, ⁇ 5#, 16# have protective effects on the biological activity of the antibody and the stability of fluorescein or fluorescent protein, compared with the commercially available preservation solution. Save better.
- Example 4 The 17# and 18# preservation solutions were separately prepared according to the formulation of Table 5, and dissolved and filtered at room temperature for use. Table 5 17# ⁇ 18# preservation solution components
- Multicolor fluorescently labeled protein reagent A 1 CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC; Prepared for self-made multicolor fluorescence by preserving the composition and concentration of commercially available multicolor fluorescently labeled protein reagent B with preservation solution 18# Labeled Protein Reagent Bl: CD3 FITC/CD16 + CD56 PE/CD45 PerCP/CD19 APC.
- the composition of the two polychromic reagents is shown in Table 6.
- the diluted two self-made multi-color reagents A1 and B1 and the commercially available multi-color fluorescent labeling protein reagent A and multi-color fluorescent labeling protein reagent B were separately dispensed into 3 small portions (each ⁇ , protected from light at 40 °C) Place for 13 days for accelerated test. Take 1 part of accelerated test multicolor reagents Al, Bl and multicolor fluorescent labeled protein reagent A, multicolor fluorescent labeled protein reagent B on 0 days, 8 days, and 13 days, respectively.
- the method for detecting the sample by the reagent detects the fluorescence intensity of each positive cell group in the clinical sample after the binding reagent, and repeats the operation three times, and takes the average value.
- the concentration of self-made multi-color reagents is different from that of multi-color fluorescent labeling protein reagent A and multi-color fluorescent labeling protein reagent B.
- the average fluorescence intensity of each of the self-made multicolor reagents A1 and B1 is statistically calculated.
- the ratio of the average fluorescence intensity of the fluorescent labeling protein reagent A and the multicolor fluorescent labeling protein reagent B is larger, and the larger the ratio after the accelerated test, the better the preservation effect. If the sub-population negative and positive cells are not effectively separated between the multi-color reagents, the multi-color reagent stops the accelerated test and does not count the data. The results are shown in Table 7 and Table 8.
- the ratio of the multicolor reagents stored in the preservation solution 17# and 18# was greater than the ratio of 0 days, that is, the preservation solution 17#, 18# has a protective effect on the biological activity and fluorescence stability of the multi-color reagent antibody.
- the self-made CD4-FITC (SK3 clone) and CD3-PerCP (SK7 clone) monochromatic fluorescently labeled monoclonal antibody reagents were diluted with the above-mentioned preservation solution 14#, preservation solution 17# and a commercially available SurModics StabilGuard® preservation solution, respectively.
- the concentrations were 5 g/mL and 12 g/mL, respectively.
- Diluted reagents are divided into 2 parts, one is stored at 4 °C in the dark as a control, one is placed in the carton and placed in the trunk of the car on the secondary road for 6 days in the dark, and transported at least 80km per day. , the test time is summer.
- the test results are shown in Table 9, Table 10.
- the preservation solution 14#, 17# can withstand short-term high temperature transport conditions, and the fluorescence labeling protein reagent antibody activity and fluorescence stability change after transport are less than 6%, which has no obvious effect on the reagent performance.
- CD45-PerCP 2D1 clone
- CD3-FITC SK7 clone
- CD4-APC SK3 clone
- CD8-PE S 8 clone monochrome fluorescent labeled monoclonal antibody reagent
- the mixture was mixed into CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC fluorescent labeling protein multicolor reagent, and the multicolor reagent composition was the same as Table 6.
- the diluted reagent is divided into 6 parts (50C ⁇ L per part), stored at 2 ⁇ 8°C in the dark, sealed and stored.
- the commercially available StatusFlow® Flow Cytometry Control was sampled at 0, 3, 6, 9, 12, and 14 months, and the fluorescence intensity of the cell-binding reagent in the control product was detected by the method of detecting the sample by the above-mentioned monochrome reagent. 3 times, take the average of 3 test results, compare the changes of fluorescence intensity with the increase of the time of the sample happening.
- the test results are shown in Table 11. With the increase of time, the fluorescence intensity decreased after the sample was placed.
- the preservation solution 17# was stored at low temperature and protected from light at a low temperature, and the fluorescence intensity decreased by about 10% within 1 year. .
- the preservation solution can protect the biological activity and fluorescence stability of the fluorescent labeling protein reagent, and can maintain the performance of the reagent for one year.
- Table 1 1 CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC multicolor reagent long-term stability, fluorescence intensity test results
- a self-made CD19-APC (SJ25C1 clone) monochromatic fluorescently labeled monoclonal antibody reagent was prepared and diluted to a solution of 5 g/mL with a preservation solution 19# and a commercially available SurModics StabilGuard® preservation solution.
- the diluted monochromatic fluorescently labeled monoclonal antibody reagents are each dispensed in 6 small portions (each ⁇ is placed at 25 ° C in the dark, and one reagent is taken every 7 days to determine the same batch of commercially available StatusFlow® Flow Cytometry Control.
- the detection results are shown in Fig. 6.
- the time and the average fluorescence intensity curve of each monochromatic fluorescently labeled monoclonal antibody reagent reflect the biological activity and fluorescence stability of the fluorescently labeled monoclonal antibody reagent antibody, and the preservation solution 1 9# to the fluorescent protein-antibody
- the conjugate as well as the antibody's biological activity and fluorescence stability have protective effects.
- the above preservation solution has a good preservation effect on the fluorescent tracer-biomarker conjugate, and can preserve the activity of the fluorescent tracer, the biomarker and the conjugate.
- the stability allows the fluorescent label reagent to be prepared into a liquid commercial reagent for direct use, which is convenient to use, and the fluorescent marker reagent prepared by the preservation solution can withstand high-temperature transportation for a short time, which helps to reduce the cost and has a good effect. Business value.
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Abstract
La présente invention concerne une solution de conservation de biomarqueur, comprenant un stabilisateur de protéine, un acide aminé alcalin ou neutre, un composé ou un polymère polyhydroxy, et un citrate. La présente invention concerne également un réactif de marqueur fluorescent préparé par utilisation de la solution de conservation, un procédé de préparation et l'utilisation de la solution de conservation. La solution de conservation présente un bon effet de conservation sur un conjugué des traceurs et des biomarqueurs fluorescents dans le réactif de marqueur fluorescent, et améliore l'activité et la stabilité des traceurs, des biomarqueurs et du conjugué fluorescents, de sorte que le réactif de marqueur fluorescent puisse être préparé sous forme de réactif liquide commercial pour une utilisation directe et pratique; et le réactif de marqueur fluorescent préparé à partir de la solution de conservation supporte un transport à haute température durant une courte période, permettant ainsi de réduire les coûts.
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CN201310430902.4A CN104459147A (zh) | 2013-09-18 | 2013-09-18 | 一种生物标记物的保存液、试剂和方法 |
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CN114324850A (zh) * | 2022-01-26 | 2022-04-12 | 杭州广科安德生物科技有限公司 | 一种化学发光工作试剂保存液及其制备方法 |
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