WO2015039423A1 - 生物标记物的保存液、试剂和方法 - Google Patents

生物标记物的保存液、试剂和方法 Download PDF

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Publication number
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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Prior art keywords
reagent
fluorescent
protein
preservation solution
biomarker
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English (en)
French (fr)
Inventor
段廷蕊
雷霆
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Shenzhen Mindray Bio Medical Electronics Co Ltd
Beijing Shen Mindray Medical Electronics Technology Research Institute Co Ltd
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Shenzhen Mindray Bio Medical Electronics Co Ltd
Beijing Shen Mindray Medical Electronics Technology Research Institute Co Ltd
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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
    • 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
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • 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
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/124—Disinfecting agents, e.g. antimicrobials
    • 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
    • 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

一种生物标记物保存液，包括蛋白稳定剂、碱性或中性氨基酸、多羟基化合物或聚合物和柠檬酸盐，以及使用该保存液配制的荧光标记物试剂、制备方法和该保存液的用途。该保存液对荧光标记物试剂中的荧光示踪物-生物标记物缀合物具有很好的保存效果，能提高荧光示踪物、生物标记物以及缀合物的活性和稳定性，使得荧光标记物试剂可制备成直接使用的液态商品化试剂，方便使用，且该保存液制备的荧光标记物试剂可以耐受短时间的高温运输，降低成本。

Description

发明名称： 生物标记物的保存液、 试剂和方法
技术领域
本发明涉及体外诊断试剂领域， 尤其是一种生物标记物的保存液、 试剂 和方法。 背景技术
蛋白质是一种具有复杂的空间立体结构的大分子物质， 具有生物活性， 易受外界条件的影响发生变性， 在低浓度时 （<0.1mg/mL ) 极易降解而失活， 而且低浓度时易吸附于管壁造成损失。 对于很多蛋白质， 一般冻存于- 20°C或 - 80°C , 重复冷冻 /融化循环可使蛋白变性， 导致其形成活性降低的聚集物。
荧光素经激发光照射后， 吸收光能进入激发态， 并能立即退激发并发出 发射光 （通常发射光的波长比入射光的波长长， 即发生 stock shift )。 目前多 用于细胞染色， 以提高识别细胞的能力。 常见的荧光素有小分子化合物如 FITC、 Cy5、 Cy7 Alexa Fluor等， 还有许多荧光蛋白如 PE、 APC；、 PerCP等。 荧光素易淬灭， 常于固体形态 （多为小分子化合物的荧光素）、 硫酸铵沉淀形 式 （如荧光蛋白） 或于保存液中以高浓度低温避光保存， 一般不可冷冻。
将荧光素或荧光蛋白作为示踪物与单克隆抗体或多克隆抗体结合， 形成 荧光标记抗体， 对相应抗原表达进行检测已成为一项重要的检测技术。
荧光标记抗体应用主要有两个方面： 一方面是荧光显微镜技术， 其将荧 光标记抗体与细胞上的抗原结合后利用荧光显微镜观察抗体结合部位的荧光 强弱从而判断结果。 另一方面就是流式细胞分析技术， 其用不同颜色的荧光 素标记不同种类的单克隆抗体， 可以在一个细胞上同时分析多种抗原分子， 进一步增强了对细胞种类和功能等特性的认识， 并且可以对大量细胞的荧光 强度进行定量分析， 其与计算机技术结合， 提高了免疫荧光的检测水平并实 现了细胞分析的自动化。 不同的荧光标记抗体试剂可检测不同抗原， 因此其 已成为了流式细胞分析在科研或临床应用中的核心组成部分。
随着技术的发展， 荧光标记抗体也应用在生物芯片技术中。 生物芯片 ( biochip ) 是指在固相基质上集成各种可以作为受体的生物信息, 包括寡核 苷酸、 蛋白质 /酶、 抗原 /抗体、 细胞等， 利用受体与连接物间的反应 （包括核 酸杂交反应、 抗原 / 抗体亲和识别反应等）来进行生物学的检测。 根据生物芯 片固定的生物分子及材料不同可分为基因芯片、 蛋白质芯片、 芯片实验室、 细胞芯片及组织芯片。 蛋白质芯片是一种新型的生物芯片， 是由固定于不同 种类支持介质上的抗原或抗体微阵列组成， 阵列中固定分子的位置及组成是 已知的， 用标记 （荧光物质、 酶或化学发光物质等标记） 的抗体或抗原与芯 片上的探针进行反应， 然后通过特定的扫描装置进行检测， 结果由计算机分 析处理。
蛋白质的分子光谱荧光分析法， 具有灵敏度高、 选择性好、 动态响应范 围宽以及测定条件更接近生命体的生理环境的优点而在蛋白质分析中应用广 泛。 荧光蛋白探针技术是利用物质的光物理和光化学特性， 在分子量级上研 究溶液中蛋白质高灵敏度的分析方法。
随着技术的发展， 用于示踪的荧光染料不限于荧光素和荧光蛋白， 三价 稀土离子及其螯合剂， 或半导体纳米微晶粒这些受激发光激发后， 发射光的 波长也会较激发光波长长， 也具有荧光特性的物质也用作示踪物， 标记在蛋 白质、 多肽、 激素、 核酸、 活细胞等生物材料上。 三价稀土离子及其螯合物 具有更长的荧光衰变时间， 更大的 stock shift， 用于时间分辨荧光免疫分析法 (Time-Resolved Fluorescence Immunoassay, 简称 TRFIA)。 TRFIA技术具有灵 敏度高、 特异性强、 稳定性好， 且测定范围更宽， 操作简便和非放射性等特 点， 非常适用于生物学、 医学上的超微量分析。 半导体纳米微晶粒又称量子 点 ( quantum dots, QDs ), 是一种直径在 l〜100nm之间， 能够接收激发光产 生荧光的半导体纳米颗粒。 量子点与生物分子、 活细胞等结合， 为 DNA检测 (DNA芯片）、 蛋白质检测 (蛋白质芯片）和探索蛋白质 -蛋白质之间（抗原-抗体、 配体—受体、 酶—底物)反应原理提供了先进的方法。
由于原料价格高和生产工艺的复杂性等原因， 结合荧光示踪物的生物标 记物， 特别是荧光标记蛋白试剂普遍成本较高。 同时， 因其敏感性和特异性 高， 一般试剂使用量少， 且浓度低。 该类试剂需要低温保存， 对运输和存储 的条件要求偏高， 从而也增加了试剂成本。 这些特点影响了荧光标记蛋白试 剂的商业化应用。 因此， 有效保持荧光标记蛋白试剂中蛋白生物活性、 荧光 稳定性、 荧光示踪物-蛋白缀合物稳定性， 方便试剂使用及有效降低试剂成本 的保存方法成为体外诊断试剂领域重要课题。
现有的技术中， 用于保持蛋白质稳定性和活性的保存液很多， 但均没有 提到对荧光稳定性和荧光标记蛋白稳定性的保持。 而且， 这些保存液有些是 不适合荧光标记蛋白试剂保存， 有些保存效果不佳。 需要开发一种更好地保 持荧光标记蛋白试剂稳定性的保存液。 发明内容
基于此， 有必要提供一种能有效保持荧光标记蛋白试剂中蛋白的生物活 性、 荧光素的稳定性、 荧光示踪物-蛋白缀合物稳定性的保存液。
一种生物标记物的保存液， 包括： 至少一种蛋白稳定剂、 至少一种碱性 或中性氨基酸、 至少一种多羟基化合物或多羟基聚合物和柠檬酸盐。
一种荧光标记物试剂， 包括上述生物标记物的保存液和荧光示踪物 -生物 标记物缀合物。 一种荧光标记物试剂的制备方法， 包括， 提供上述生物标记物的保存液 和荧光示踪物-生物标记物缀合物； 用保存液将荧光示踪物 -生物标记物缀合物 稀释至工作浓度， 获得可以直接使用的荧光标记物试剂溶液。
本申请还公开了上述保存液在制备荧光标记物试剂中的用途。
上述保存液适用于含有荧光示踪物-生物标记物缀合物的荧光标记物试 剂， 特别适合保存小分子荧光素标记蛋白、 荧光蛋白标记蛋白和串联染料标 记蛋白试剂； 单色或多色荧光标记蛋白试剂； 荧光标记蛋白质、 多克隆抗体 或单克隆抗体试剂。 使用时直接稀释荧光标记蛋白试剂至工作浓度， 于
2°C ~8°C密封避光保存， 使用方便， 能长时间保持蛋白生物活性、 荧光稳定性 和荧光示踪物-蛋白缀合物稳定性。 该保存液还可短期耐受 25~40°C高温运输 条件， 降低荧光标记蛋白试剂成本。 附图说明
图 1为实施例 1 中 HPLC检测 IgG-PE荧光标记蛋白试剂于 1#~6#保存液 中降解曲线；
图 2为实施例 2中 HPLC检测 BSA- PE荧光标记蛋白试剂于 7#〜13#保存 液中降解曲线；
图 3为实施例 3中 CD4- FITC单色试剂用保存液 14#保存与市售保存液保 存加速试 ^ 比较检测结果；
图 4为实施例 3 中 CD3- PerCP单色试剂用保存液 15#保存与市售保存液 保存加速试验比较检测结果；
图 5为实施例 3中 CD8- APC- Cy7单色试剂用保存液 16#保存与市售保存 液保存加速试险比较检测结果；
图 6是实施例 7中 CD19- APC单色试剂用保存液 19#保存与市售保存液保 存加速试验比较检测结果。 具体实施方式
为了使本发明的目的、 技术方案及优点更加清楚明白， 以下结合附图及 实施例， 对本发明进行进一步详细说明。 应当理解， 此处所描述的具体实施 例仅仅用以解释本发明， 并不用于限定本发明。
除非上下文清楚地要求， 否则， 贯穿本说明和权利要求， 用语 "包括"、 "包含" 等应以包含性的意义来解释而不是排他性或穷尽性的意义， 即， 其 含义为 "包括， 但不限于"。 在本详细描述部分中， 使用单数或复数的用语也 分别包括复数或单数。 此外， 在用于本申请中时， 用语 "在此"、 "以上，'、 "以 下" 及类似意义的用语指的是作为整体的本申请而不是本申请任何特定部分。 当权利要求使用用语 "或" 来引用两个或更多项时， 此用语覆盖以下所有对 该用语的解释： 序列中的任何项、 序列中的所有项和序列中项的任何组合。
定义
除另有说明外， 本文中使用的术语具有以下含义。
本文中使用的术语 "生物标记物"， 是指来自生物体的生物分子或活细胞， 生物分子为可与细胞或细胞成分特异性结合的分子， 包括但不限于抗体、 抗 原、 受体、 配体、 酶、 底物、 辅酶、 核酸、 激素等。
本文中使用的术语"荧光示踪物"，是指可以产生荧光信号， 并能够和生物 材料偶联的化合物， 包括但不限于小分子荧光素、 荧光蛋白、 荧光染料、 稀 土离子及其螯合剂、 半导体纳米微晶粒等。
本文中使用的术语"荧光示踪物-生物标记物缀合物"， 是指荧光示踪物通 过共价键与生物标记物连接形成的化合物。
本文使用的术语"荧光标记蛋白试剂"，是指荧光示踪物通过共价键与蛋白 质分子连接形成荧光示踪物-蛋白缀合物， 该缀合物配制成的溶液， 用于体外 本文使用的术语"单色荧光标记蛋白试剂 ",是指一种荧光示踪物通过共价 键与一种生物标记物连接形成缀合物， 该缀合物配制成的溶液。
本文使用的术语"多色荧光标记蛋白试剂 "，是指多种单色荧光标记蛋白试 剂的混合物。
本文使用的术语"抗体"， 包括各种动物源的单克隆抗体、 多克隆抗体以及 抗体片段等， 包括不同的抗体种类， 例如免疫球蛋白 G、 免疫球蛋白 A、 免 疫球蛋白 E、 免疫球蛋白 M、 免疫球蛋白 D和免疫球蛋白 Y。 一种生物标记物的保存液
本发明一个实施方式公开了一种生物标记物的保存液， 含有至少一种蛋 白稳定剂、 至少一种碱性或中性氨基酸、 至少一种多羟基化合物或多羟基聚 合物和柠檬酸盐。
保存液中的蛋白稳定剂， 可保持蛋白生物活性， 可降低低浓度蛋白降解 与失活， 减少低浓度蛋白吸附于管壁造成的损失。 我们发现蛋白稳定剂还可 减轻一些不利环境因素如加热、 表面张力及化学因素引起的蛋白变性。 所述 的蛋白稳定剂可选明胶和白蛋白（如牛血清白蛋白 BSA和人血清白蛋白）等， 优选明胶和 BSA, 其浓度可为 0.05%~0.40%, 优选 0.08%〜0.24%。
保存液中的碱性或中性氨基酸， 可降低蛋白、 荧光示踪物-蛋白缀合物的 聚集。 所述氨基酸可选精氨酸、 组氨酸、 赖氨酸和甘氨酸等， 优选精氨酸和 甘氨酸， 其浓度范围可为 l〜20mM, 优选 4〜16mM。
保存液中的多羟基化合物或多羟基聚合物， 可增加荧光标记蛋白试剂的 水溶性和稳定性。 虽然不希望受理论约束， 但是认为多羟基化合物或聚合物 具有张力控制作用， 可有效保持蛋白的构型和活性， 有助于保持荧光示踪物- 蛋白缀合物的稳定， 也可减少荧光素因分子间和分子内作用造成的荧光淬灭。 所述的多羟基化合物可选甘油、糖醇（如甘露醇、木糖醇、 山梨醇）和糖类 (如 海藻糖、 甘露糖、 木糖)等， 优选甘油、 甘露醇和海藻糖。 所述的多羟基聚合 物可选聚乙二醇聚合物， 如 PEG8000、 PEG 10000、 PEG20000 等， 优选 PEG 10000、 PEG20000。 甘油浓度范围可为 1~10%, 优选 2~6%； 甘露醇、 海 藻糖和 PEG浓度范围可为 0.1%〜1.0%, 优选 0.2%〜0.8%。
虽然不希望受理论约束， 但是认为保存液中的柠檬酸盐可以长时间保持 保存液中緩冲液的 pH, 减少蛋白的聚集， 保持蛋白的稳定。 所述柠檬酸盐可 选可与柠檬酸化合的在水中可溶解的柠檬酸盐， 例如柠檬酸钠、 柠檬酸钾等， 优选柠檬酸钠， 其浓度范围可为 5〜40mM， 优选 10〜30mM。
进一步的， 本发明一个实施方式的保存液还可包含至少一种抗氧化剂。 虽然不希望受理论约束， 但是认为抗氧化剂可用于减少荧光素的氧化淬灭。 常用乙二胺四乙酸钠盐， 例如 EDTA*2Na, 其浓度范围可为 2〜3mM。
进一步的， 本发明一个具体实施方式的保存液中还可以包含常规防腐剂， 以有助于试剂的防腐和长期保存。 对于防腐剂的种类没有特殊的要求， 常用 的防腐剂有 NaN3、 Procline等 , 防腐剂浓度范围一般为 0.05%〜0.10%。 防腐 剂的浓度和种类以不影响蛋白质结构和生物活性为宜。
除上述主要组分外， 本发明一个具体实施方式的保存液还可包含缓冲剂， 以将保存液的 pH维持在 6〜8, 优选 6.8〜7.2左右， 对于緩冲剂的选择以不影 响荧光强度、 蛋白质结构和生物活性为宜。 常见的緩冲体系如磷酸盐缓冲溶 液、柠檬酸盐缓沖溶液等均可用于本发明。缓冲剂的使用量一般为 10〜50mM, pH为 7.0时更佳。
虽然不希望受理论约束， 但是认为将上述的蛋白稳定剂、 碱性或中性氨 基酸、 多羟基化合物或聚合物和柠檬酸盐进行组合， 各组分除了上述所述作 用外， 组分间还有协同作用。 该保存液可直接将荧光标记蛋白试剂稀释至工 作浓度进行保存，经验证经该保存液稀释的荧光标记蛋白试剂于 2°C〜8°C密封 避光保存超过 1 年。 容易理解， 该保存液也可以将蛋白试剂保存为浓缩液， 使用前稀释。 更进一步， 该保存液中的蛋白稳定剂和多羟基化合物或聚合物 协同作用， 可减轻不利环境因素如加热、 表面张力及化学因素引起的蛋白变 性， 25°C〜40°C高温下可保持试剂稳定、 不易挥发， 经试验证明可短期耐受 25~40°C高温运输条件，降低了荧光标记蛋白试剂成本，具有^ ί艮好的商业价值。
一实施方式的保存液， 适用荧光标记蛋白试剂种类可为小分子荧光素标 记蛋白， 小分子荧光素如 FIT：、 Cy5、 Cy7、 Alexa Fluor等。
一实施方式的保存液， 适用荧光标记蛋白试剂种类可为荧光蛋白标记蛋 白， 荧光蛋白如 PE、 APC、 PerCP等。
一实施方式的保存液， 适用荧光标记蛋白试剂种类可为串联染料标记蛋 白， 串联染料如 PE- Cy7、 APC- Cy7、 PE- Cy5、 PE- Cy5.5等。
一实施方式的保存液， 适用荧光标记蛋白试剂组分可为单色或多色荧光 标记蛋白试剂， 单色试剂如 CD4- FITC、 CD3 -PerCP 等， 多 色试剂如 CD3-FITC/CD8-PE/CD45 -PerCP/CD4-APC 四色试剂和 CD3- FITC/CD16 + CD56-PE/CD45-PerCP/CD19-APC 四色试剂等。 CD4- FITC 是指荧光素 FITC 通过共价键与对 CD3细胞有特异性的抗体连接形成的缀合物。 其他类推。
荧光标 i£»物试剂
本发明另一个实施方式公开了一种荧光标记物试剂， 包括上述的保存液 和荧光示踪物-生物标记物缀合物。
荧光示踪物 -生物标记物缀合物中的生物标记物是指来自生物体的生物分 子或活细胞， 包括但不限于核酸、 多肽、 蛋白质、 激素、 活细胞， 蛋白质包 括抗体、 酶等。 在一个优选实施方式中， 生物标记物为单克隆抗体或多克隆 抗体。
荧光示踪物-生物标记物缀合物中的荧光示踪物可以是任何能产生荧光信 号并能够和生物材料偶联的化合物， 包括但不限于小分子荧光素、 荧光蛋白、 荧光染料、 稀土离子及其螯合剂、 半导体纳米微晶粒， 优选小分子荧光素、 荧光蛋白、 串联荧光染料。
荧光示踪物-生物标记物缀合物可以是上述的组合， 通过本领域技术人员 熟知的技术得到。 优选的， 荧光示踪物-生物标记物缀合物是荧光标记蛋白， 特别优选的， 是荧光标记抗体。 将荧光标记物试剂与待测样品混合温育一段 时间 , 使得试剂中的荧光示踪物-生物标记物缀合物与待测样品中的细胞或细 胞成分结合， 这个过程也可被称为染色。 染色完成后， 样品在荧光激活的流 式分析仪上进行分析。 或者， 在另一个实施方案中， 荧光示踪物 -生物标记物 缀合物还可以用于固相免疫测试或生物芯片检测。 相关的技术在本领域是公 知的， 可由标准教科书得到。 荧光标记物试剂的制备方法
本发明又一个实施方式公开了一种荧光标记物试剂的制备方法， 包括： 提供上述保存液和荧光示踪物-生物标记物缀合物；
用保存液将荧光示踪物-生物标记物缀合物稀释至工作浓度， 获得可以直 接使用的荧光标记物试剂溶液。
优选的， 荧光示踪物-生物标记物缀合物是荧光标记蛋白， 特别优选的，
：焚光标己抗体。
下面， 以荧光标记蛋白为例进行说明。
将荧光标记蛋白根据需要和其他组分混合， 溶解在水中， 配制成荧光标 记蛋白母液。 称取上述保存液的各组分， 加水搅拌溶解， 用水稀释至一定浓 度， 具体可根据荧光标记蛋白母液浓度确定。 根据需要， 将配好的保存液和 荧光标记蛋白溶液按比例混合， 使得其中荧光标记蛋白的浓度至工作浓度， 获得荧光标记蛋白试剂溶液。这里的"工作浓度"是指配制好的荧光标记蛋白试 剂溶液可无需稀释， 直接与待测样品混合检测。 由于本申请的保存液对荧光 示踪物-生物标记物缀合物具有很好的保存效果， 配制的试剂可以无需冻干， 使用时可以无需复溶配制, 使用方便。
其他
另一方面， 本发明的再一个具体实施方式公开了上述保存液在制备荧光 标记物试剂中的用途。
上述保存液可以用来制备可长期保存且可直接使用的液态荧光标记物试 剂。
实施例
以下通过荧光标记蛋白试剂的实施例来更详细的描述本发明， 但本发明 并非局限于此。
1、 仪器设备：
岛津 20A高效液相色谱仪（ HPLC ); BD FACSCanto II流式细胞仪； 上海 博迅实业有限公司数显电热培养箱； 其林贝尔仪器制造厂涡旋仪； 梅特勒移 液器及配套枪头； 上海安亭科学仪器厂低速大容量离心机。
2、 试剂：
市售 SurModics StabilGuard®保存液；
市售多 色荧光标记蛋白试剂 A： Multitest CD3- FITC/CD8- PE/CD45 -PerCP/CD4-APC ( CD3-FITC, SK7克隆； CD8-PE, SKI克隆； CD45- PerCP, 2D1克隆； CD4-APC, SK3克隆）， 无需稀释， 直接使用； 市售 多 色 荧光标记蛋白 试剂 B ： Multitesl CD3-FITC/CD16 + CD56-PE/CD45 -PerCP/CD19-APC ( CD3-FITC, SK7克隆； CD16- PE， B73.1 克隆； CD56-PE, NACM16.2克隆； CD45- PerCP, 2D1克隆; CD19-APC, SJ25C1 克隆）， 无需裤释， 直接使用；
市售流式溶血剂， BD FACS Lysing Solution ( 1 Ox浓缩液， 使用时需用去 离子水按 1 : 10稀释为 l x试剂 );
临床全血样本 (EDTA- K3和 EDTA- K2抗凝处理）；
市售 StatusFlow® Flow Cytometry Control;
曱醛溶液， 分析纯；
PBS緩冲液， 取 KC1 0.2g, KH2PO4 0.2g, NaCl 8.0g, Na2HP04 1.15g, 溶 于 1L去离子水中， pH为 7.4士0.2, 用 0.2μηι PALL GHP滤膜过滤。
3、 检测方法：
( 1 ) HPLC检测荧光示踪物-蛋白缀合物峰面积方法： 色谱柱 GF250(Agilent, 4μηι, 4.6x250mm) 流速 0.5mL/min 检测波长 565匪 流动相 0.3M Na2HPO4, pH7.0 进样量 2(^L
( 2 ) 使用单色荧光标记蛋白试剂检测样本方法：
① 加 20μ 荧光标记蛋白试剂于 12x75mm流式管中， 之后加入 ΙΟΟμ 全血样本， 低速涡旋混匀， 室温 （20°C〜25°C ) 孵育 15min;
② 加入 2mL l x流式溶血剂， 低速涡旋混匀， 室温避光孵育 lOmin; ③ 5 OOg离心 5 min后倾倒上清；
④ 加入 2mLPB S , 涡旋仪上低速混匀， 500g离心 5min后倾倒上清；
⑤ 加入 500μ 含 1%曱醛的 PBS固定液， 低速涡旋混勾， 上流式细胞 仪检测。
( 3 ) 使用多色荧光标记蛋白试剂检测样本方法：
① 加 20 L荧光标记蛋白试剂于 12x75mm流式管中， 之后加入 50 L 全血样本， 低速涡旋混匀， 室温 （20°C〜25°C ) 孵育 15min;
② 加入 450μ l x流式溶血剂， 低速涡旋混匀， 室温避光孵育 15min;
③ 低速涡旋混勾， 上流式细胞仪检测。
实施例 1
按表 1配方分别配制 1#〜6#保存液， 室温下搅拌溶解过滤， 备用。 按照常 规的方法制备自制的荧光蛋白标记多克隆抗体 IgG- PE试剂。分别用 1〜6#保存 液将自制的荧光蛋白标记多克隆抗体 IgG- PE试剂稀释为 12.5 g/mL的溶液。 稀释后的荧光标记多克隆抗体试剂溶液各分装为 9小份（每一份 50 L )于 40°C 避光放置， 每 3〜4天取一份试剂溶液按前述的方法检测荧光示踪物 -蛋白缀合 物峰面积， 计算随时间增加荧光示踪物 -蛋白缀合物降解百分比， 绘制时间与 降解百分比变化曲线。
检测结果如图 1所示， 荧光示踪物-蛋白缀合物降解百分比反映了荧光示 踪物-蛋白缀合物的稳定性及荧光素稳定性，其中蛋白稳定剂明胶或 BSA浓度 为 0.08%〜0.24%, 精氨酸或甘氨酸浓度为 4mM〜16mM时（即保存液 2#、 5# ), IgG-PE荧光示踪物-蛋白缀合物降解百分比小于保存液 1#、 3#、 4#和 6#， 保 存液效果较佳。 也说明本保存液对荧光素-抗体缀合物有良好的保存效果。
1#〜6#保存液组
名 保存液 1# 保存液 2# 保存液 3# 保存液 4# 保存液 5# 保存液 6# 称
0.05%明胶 0.08%明胶 0.08%明胶 0.24%BSA 0.24%BSA 0.40%BSA
20mM 甘 16mM 精 16mM 甘
4mM精氨酸 4mM甘氨酸 ImM甘氨酸
氨酸 氨酸 氨酸
0.6%甘露 0.6%甘露 组 4%甘油 4%甘油 4%甘油 醇 醇 分 0.6%PEG 1 ()()()() 0.6%PEG20000 0.6%PEG10000 0.4%海藻 0.4%海藻 糖 糖
16mM 柠檬酸 16m M 柠檬酸 16mM 柠檬酸 24m M 柠 24m M 柠 24mM 柠 三钠 三钠 三钠 檬酸三纳 檬酸三纳 檬酸三钠
2mM EDTA-2Na, 0.1% NaN3 , 140mM NaCl, lOmM磷酸盐緩冲溶液， pH7.0
实施例 2
按表 2配方分别配制 7#〜13#保存液， 室温下搅拌溶解过滤， 备用。 按照 常规的方法制备荧光标记蛋白质 BSA- PE试剂。分别用 7〜13#保存液将自制的 荧光标记蛋白质 BSA- PE试剂稀释为 12.5 g/mL的溶液。稀释后的荧光标记蛋 白质试剂溶液各分装为 8小份（每一份 50μυ于 40°C避光放置， 每 3〜7天取 一份试剂按上述的方法检测荧光示踪物-蛋白缀合物峰面积， 计算随时间增加 荧光示踪物-蛋白缀合物降解百分比， 绘制时间与降解百分比变化曲线。
检测结果如图 2所示， 荧光示踪物-蛋白缀合物降解百分比反映了荧光示 踪物-蛋白缀合物的稳定性及荧光素稳定性， 其中多羟基化合物甘油浓度为 2%〜6%, 糖醇、 糖类和聚乙二醇聚合物浓度为 0.2%〜0.8%时 （即保存液 9#、 10#、 13# ), BSA- PE荧光示踪物-蛋白缀合物降解百分比小于保存液 7#、 8#、 11#、 12#, 保存液效果较佳。 也说明本保存液对荧光素-蛋白缀合物有良好的 保存效果。
7#~13#保存液组
Figure imgf000015_0001
0.12%明胶 0.12%明胶 0.12%明胶 0.12%明胶 0.16%BSA
8mM 精氨 8mM 精氨 8mM 精氨 8mM 精氨 12mM 精 12mM 精 12mM 精 酸 酸 酸 酸 氨酸 氨酸 氨酸
0.1%甘露 1.0%甘露 0.2%甘露
1%甘油 10%甘油 6%甘油 2%甘油
醇 醇 醇 组 1.0%PEG1 0.2%PEG2 0.2%PEG1 0.8%PEG1
0.1%海藻 1.0%海藻 0.8%海藻 成 0000 0000 0000 0000
糖 糖 糖 lOmM lOmM lOmM lOmM 30mM 30mM 30mM
4宁檬酸三 樣酸三 4宁樣酸三 4宁樣酸三 4宁樣酸三 4宁樣酸三 4宁樣酸三 钠 钠 钠 钠 钠 钠 钠
3mM EDTA-2Na, 0.1% Proclin300, 140mM NaCl, 50mM磷酸盐緩冲溶液， pH7.0
实施例 3
按表 3配方分別配制 14#〜16#保存液， 室温下搅拌溶解过滤， 备用。 按照
<
常规的方法制备 CD4- FITC ( SK3 克隆）、 CD3- PerCP ( SK7 克隆）、 CD8-APC-Cy7(SK8 克隆）三种单色荧光标记单克隆抗体试剂， 分别用上述 14#〜16#保存液和市售 SurModics StabilGuard®保存液按表 4对这 <三种单色荧 光标记单克隆抗体试剂进行稀释。
表 3 14#〜16#保存液组分
Figure imgf000016_0001
表 4 CD4-FITC：、 CD3-PerCP CD8- APC-Cy7三种单色荧光标记单克隆 抗体试剂稀释方法 单色试剂 稀释保存液 浓度 CD4-FITC 保存液 14#和市售 SurModics StabilGuard®保存液分别稀释 5 g/mL
CD3-PerCP 保存液 15#和市售 SurModics StabilGuard®保存液分别稀释
CD8-APC-Cy7 保存液 16#和市售 SurModics StabilGuard®保存液分别稀释 5 g/mL 稀释后的单色荧光标记单克隆抗体试剂各分装为 6小份（每份 ΙΟΟμυ于 25°C避光放置， 每 7 天各取一份试剂测定同一批市售 StatusFlow® Flow Cytometry Control质控品，按上述单色试剂检测样本的方法，检测质控品中细 胞结合试剂后的荧光强度， 各重复操作 3 次， 取三次检测结果均值。 以时间 和各单色荧光标记单克隆抗体试剂平均荧光强度绘制曲线。 检测结果如图 3、 图 4、 图 5所示， 时间-荧光强度变化曲线反映了荧光标 记单克隆抗体试剂抗体生物活性及荧光稳定性， 结果显示保存液 14#、 \ 5#、 16#对抗体生物活性和荧光素或荧光蛋白的稳定性均有保护作用， 比市售保存 液保存效果更好。
实施例 4 按表 5配方分别配制 17#、 18#保存液， 室温下搅拌溶解过滤， 备用。 表 5 17#〜18#保存液组分
Figure imgf000017_0001
取自制的 CD45- PerCP ( 2D1 克隆）、 CD3-FITC ( S 7克隆）、 CD4-APC ( SK3 克隆）、 CD8-PE ( SK8 克隆）、 CD19-APC ( SJ25C1 克隆）、 CD56-PE ( ACM16.2克隆；)、 CD16-PE ( B73.1克隆） 单色荧光标记单克隆抗体试剂， 用保存液 17#按市售的多色荧光标记蛋白试剂 A的组分和浓度稀释配制自制 的多色荧光标记蛋白试剂 A 1 : CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC; 用 保存液 18#按市售的多色荧光标记蛋白试剂 B 的组分和浓度稀释配制自制的 多色荧光标记蛋白试剂 Bl : CD3 FITC/CD16 + CD56 PE/CD45 PerCP/CD19 APC。 两种多色试剂组成见表 6。
表 6 多色试剂组成
Figure imgf000018_0001
稀释好的两种自制的多色试剂 A1和 B1及市售的多色荧光标记蛋白试剂 A、 多色荧光标记蛋白试剂 B分别分装为 3小份（每份 ΙΟΟμυ, 于 40 °C避光放置 13 天进行加速试验， 分别于 0天、 8天、 13天各取 1份加速试验多色试剂 Al、 Bl 及多色荧光标记蛋白试剂 A、 多色荧光标记蛋白试剂 B, 按上述多色试剂检测样 本的方法， 检测临床样本中各阳性细胞群结合试剂后的荧光强度， 各重复操作 3 次， 取均值。
由于不同时间临床样本差异较大， 自制多色试剂浓度与多色荧光标记蛋白试 剂 A、 多色荧光标记蛋白试剂 B有差异， 数据统计取自制多色试剂 A1和 B1各 平均荧光强度与多色荧光标记蛋白试剂 A、多色荧光标记蛋白试剂 B平均荧光强 度的比值， 加速试验后比值越大， 说明保存效果越好。 若多色试剂检测过程中各 亚群阴性和阳性细胞间不能有效分离， 则该多色试剂停止加速试验和不对该次数 据进行统计。 结果如表 7、 表 8所示， 结果表明不同荧光标记抗体试剂抗体活性和荧光降 解速率不一致， 保存液 17#、 18#保存的多色试剂比值均大于 0天比值， 即保存液 17#、 18#对多色试剂抗体生物活性和荧光稳定性均有保护作用。
自制多色试剂 A1加速试验各荧光强度与多色荧光标记蛋白试剂 A比
Figure imgf000019_0001
表 8 自制多色试剂 B1加速试 各荧光强度与多色荧光标记蛋白试剂 B比值
Figure imgf000019_0002
注： /表示未统计， 因各亚群阴性和阳性细胞间不能有效分离。
实施例 5
取自制的 CD4-FITC ( SK3克隆）、 CD3-PerCP ( SK7克隆）单色荧光标记单 克隆抗体试剂，分别用上述保存液 14#、保存液 17#和市售 SurModics StabilGuard® 保存液稀释， 使其浓度分别为 5 g/mL、 12 g/mL。 稀释好的试剂各分成 2份， 一 份于 4°C避光保存作为对照， 一份不放冰袋直接放入纸盒于车后备箱在二级公路 上避光运输 6天， 每天至少运输 80km, 测试时间为夏季。 分别取对照试剂和运 输后的试剂 , 对同一批市售 StatusFlow® Flow Cytometry Control染色， 溶血 /洗涤 处理后， 用流式细胞仪进行流式检测记录平均荧光强度， 各重复操作 3次， 取三 次检测结果均值， 比较运输后试剂与对照检测结果的差别。
检测结果见表 9、 表 10， 保存液 14#、 17#可短期耐受高温运输条件， 运输后 荧光标记蛋白试剂抗体活性和荧光稳定性变化均小于 6%， 对试剂性能无明显影 响。
CD4-FITC单色荧光标记单克隆抗体试剂运输试验结果
Figure imgf000020_0001
实施例 6
取自制的 CD45- PerCP ( 2D1克隆）、 CD3-FITC ( SK7克隆）、 CD4-APC ( SK3 克隆）、 CD8-PE ( S 8克隆）单色荧光标记单克隆抗体试剂， 用保存液 17# 释 混匀为 CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC荧光标记蛋白多色试剂，多色试 剂组成同表 6。 稀释好的试剂分成 6份（每份 50C^L ), 于 2〜8°C避光、 密封保存。 分别于 0、 3、 6、 9、 12、 14月取样测定市售 StatusFlow® Flow Cytometry Control, 按上述单色试剂检测样本的方法， 检测质控品中细胞结合试剂后的荧光强度， 各 重复操作 3次， 取 3次检测结果均值， 比较随放样时间的增加各荧光强度的变化 情况。
检测结果见表 11 , 随时间增加， 各荧光强度放样后均有降低， 保存液 17#对 荧光标记蛋白试剂进行低温、 避光密封保存， 各荧光强度 1 年内降低幅度约为 10%， 符合要求。 本保存液可对荧光标记蛋白试剂生物活性及荧光稳定性有保护 作用， 可维持试剂性能 1年。
表 1 1 CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC多色试剂长期稳定性各荧 光强度检测结果
Figure imgf000021_0002
Figure imgf000021_0001
按如下配方配制 19#保存液， 室温下搅拌溶解过滤， 备用。
Figure imgf000021_0003
取自制的 CD19-APC ( SJ25C1克隆 )单色荧光标记单克隆抗体试剂， 分别用 保存液 19#和市售 SurModics StabilGuard®保存液稀释为 5 g/mL的溶液。 稀释后 的单色荧光标记单克隆抗体试剂各分装为 6小份（每份 ΙΟΟμυ 于 25°C避光放 置， 每 7天各取一份试剂测定同一批市售 StatusFlow® Flow Cytometry Control, 按上述单色试剂检测样本的方法， 检测质控品中细胞结合试剂后的荧光强度， 各 重复操作 3次， 取三次检测结果均值。 以时间和各单色荧光标记单克隆抗体试剂 平均荧光强度绘制曲线。
检测结果如图 6所示, 时间和各单色荧光标记单克隆抗体试剂平均荧光强度 变化曲线反映了荧光标记单克隆抗体试剂抗体生物活性及荧光稳定性， 保存液 1 9#对荧光蛋白 -抗体缀合物以及抗体生物活性和荧光稳定性均有保护作用。
综上， 从以上的实施例可见， 上述保存液对于荧光示踪物 -生物标记物缀合 物具有 4艮好的保存效果， 能保存荧光示踪物、 生物标记物以及缀合物的活性和稳 定性， 使得荧光标记物试剂可以制备成直接使用的液态商品化试剂， 方便使用， 且该保存液制备的荧光标记物试剂可以耐受短时间的高温运输， 有助于降低成 本， 具有艮好的商业价值。
以上通过具体的实施例对本发明进行了说明， 但本发明并不限于这些具体的 实施例。 本领域技术人员应该明白， 还可以对本发明做各种修改、 等同替换、 变 化等等， 这些变换只要未背离本发明的精神， 都应在本发明的保护范围之内。

Claims

权利要求书
1.一种生物标记物的保存液, 包括：
蛋白稳定剂；
碱性或中性氨基酸；
多羟基化合物或多羟基聚合物； 和
柠檬酸盐。
2.如权利要求 1所述的保存液， 其特征在于， 所述蛋白稳定剂选自白蛋白或 明胶。
3.如权利要求 2 所述的保存液， 其特征在于， 所述蛋白稳定剂的浓度为 0.05%~0.40%, 优选 0.08%~0.24%0
4.如权利要求 1所述的保存液， 其特征在于， 所述碱性或中性氨基酸选自精 氨酸、 组氨酸、 赖氨酸和甘氨酸， 优选精氨酸或甘氨酸。
5.如权利要求 4所述的保存液， 其特征在于， 所述碱性或中性氨基酸的浓度 为 l〜20mmol/L, 优选 4〜16mmol/L。
6.如权利要求 1所述的保存液， 其特征在于， 所述多羟基化合物或多羟基聚 合物选自甘油、 多糖、 糖醇和聚乙二醇聚合物。
7.如权利要求 6所述的保存液， 其特征在于， 所述多糖选自海藻糖， 或者所 述糖醇选自甘露醇， 或者所述聚乙二醇聚合物选自 PEG8000、 PEG10000 或
Figure imgf000023_0001
8.如权利要求 7所述的保存液， 其特征在于， 所述甘油的浓度为 1~10%, 优 选 2-6%; 或者所述甘露醇、 海藻糖和聚乙二醇聚合物的浓度为 0.1%~1.0%, 优 选 0.2%〜0.8%。
9.如权利要求 1所述的保存液， 其特征在于， 所述保存液还含有至少一种抗 氧化剂。
1 0. 如权利要求 9所述的保存液， 其特征在于， 所述抗氧化剂选自乙二胺 四乙酸钠盐。
11. 如权利要求 1所述的保存液， 其特征在于， 所述保存液还含有防腐剂。
12. 如权利要求 1所述的保存液， 其特征在于， 所述保存液还含有缓冲剂， 维持 pH在 6〜8。
1 3. 一种荧光标记物试剂， 其特征在于， 所述荧光标记物试剂包括荧光示 踪物-生物标记物缀合物和权利要求 1 - 1 2任一项所述的保存液。
14. 如权利要求 1 3 所述的试剂， 其特征在于， 所述荧光示踪物-生物标记 物缀合物中的生物标记物为蛋白质。
15. 如权利要求 14 所述的试剂， 其特征在于， 所述荧光示踪物-生物标记 物缀合物中的生物标记物为抗体或多肽。
16. 如权利要求 13 所述的试剂， 其特征在于， 所述荧光示踪物-生物标记 物缀合物中的荧光示踪物选自小分子荧光素、 荧光蛋白或串联荧光染料。
17. 一种荧光标记物试剂的制备方法， 包括，
提供荧光示踪物-生物标记物缀合物和权利要求 1-12任一项所述的生物标记 物保存液；
用所述保存液将所述荧光示踪物-生物标记物缀合物稀释至工作浓度， 获得 可以直接使用的荧光标记物试剂溶液。
18. 权利要求 1-12任一项所述的保存液在制备荧光标记物试剂中的用途。
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