WO2015039423A1

WO2015039423A1 - Biomarker preservative solution, reagent and method

Info

Publication number: WO2015039423A1
Application number: PCT/CN2014/074463
Authority: WO
Inventors: 段廷蕊; 雷霆
Original assignee: 深圳迈瑞生物医疗电子股份有限公司; 北京深迈瑞医疗电子技术研究院有限公司
Priority date: 2013-09-18
Filing date: 2014-03-31
Publication date: 2015-03-26
Also published as: CN104459147A

Abstract

Provided is a biomarker preservative solution, comprising a protein stabilizer, alkaline or neutral amino acid, polyhydroxy compound or polymer, and citrate. Also provided are a fluorescent marker reagent prepared by utilizing the preservative solution, preparation method and use of the preservative solution. The preservative solution has good preservation effect on a conjugate of the fluorescent tracers and biomarkers in the fluorescent marker reagent, and improves the activity and stability of the fluorescent tracers, biomarkers and conjugate, so the fluorescent marker reagent can be prepared into commercial liquid reagent for direct and convenient use; and the fluorescent marker reagent prepared from the preservative solution tolerates high-temperature transportation for a short period, reducing costs.

Description

Title: Biomarker Preservation Solution, Reagent, and Method

Technical field

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.

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. Common 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.

The combination of fluorescein or fluorescent protein as a tracer with a monoclonal antibody or a polyclonal antibody to form a fluorescently labeled antibody, and detection of the corresponding antigen expression has become an important detection technique.

There are two main applications for fluorescently labeled antibodies: On the one hand, 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. On the other hand, 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.

With the development of technology, fluorescently labeled antibodies are also used in biochip technology. 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). 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.

With the development of technology, 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.

Due to the high price of raw materials and the complexity of the production process, biomarkers combined with fluorescent tracers, especially fluorescently labeled protein reagents, are generally more expensive. At the same time, due to its high sensitivity and specificity, 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. These characteristics affect the commercial application of fluorescently labeled protein reagents. Therefore, it is an important subject in the field of in vitro diagnostic reagents to effectively maintain protein biological activity, fluorescence stability, stability of fluorescent tracer-protein conjugates in fluorescently labeled protein reagents, facilitate the use of reagents, and effectively reduce the cost of reagents.

In the prior art, there are many preservation solutions for maintaining protein stability and activity, but there is no mention of maintaining fluorescence stability and stability of fluorescently labeled proteins. Moreover, some of these preservation solutions are not suitable for fluorescent labeling protein reagents, and some are not well preserved. There is a need to develop a preservation solution that better maintains the stability of fluorescently labeled protein reagents. Summary of the invention

Based on this, it is necessary to provide a preservation solution capable of effectively maintaining the biological activity of the protein in the fluorescently labeled protein reagent, the stability of fluorescein, and the stability of the fluorescent tracer-protein conjugate.

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,

It is sealed and protected from light at 2 °C ~ 8 °C. It is easy to use and can maintain protein biological activity, fluorescence stability and fluorescence tracer-protein conjugate stability for a long time. The preservation solution can also withstand short-term 25~40°C high temperature transportation conditions and reduce the cost of fluorescent labeling protein reagents. DRAWINGS

Figure 1 is a HPLC analysis of the degradation curve of IgG-PE fluorescent labeled protein reagent in 1#~6# preservation solution by HPLC;

2 is a HPLC method for detecting degradation curves of BSA-PE fluorescent labeled protein reagent in 7#~13# 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;

4 is a comparison result of the CD3-PerCP monochrome reagent preservation solution 15# in the third embodiment and the commercially available preservation solution storage acceleration test;

5 is a comparison test result of the CD8-APC-Cy7 single-color reagent preservation solution 16# in the third embodiment and the commercially available preservation solution preservation accelerated test;

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

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The terms "including", "comprising", etc., are intended to be interpreted in an inclusive sense rather than an exclusive or exhaustive meaning. not limited to". In the Detailed Description section, terms using the singular or plural refer to the plural or singular. In addition, in the context of the present application, the terms "herein," "above," "below," and the like are intended to mean the application as a whole, rather than any particular part of the application. When "or" refers to two or more, this term covers all of the following terms for the term: any item in the sequence, all items in the sequence, and any combination of items in the sequence.

Definition

Unless otherwise stated, the terms used herein have the following meanings.

The term "biomarker" as used herein 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.

The term "fluorescent tracer" as used herein, 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.

The term "fluorescent tracer-biomarker conjugate" as used herein, refers to a compound formed by the attachment of a fluorescent tracer to a biomarker via a covalent bond.

The term "fluorescent labeling protein reagent" as used herein 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.

The term "multicolor fluorescently labeled protein reagent" as used herein refers to a mixture of a plurality of monochromatic fluorescently labeled protein reagents.

The term "antibody" as used herein, 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. We have found that 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. Although not wishing to be bound by theory, 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%.

Although not wishing to be bound by theory, it is believed that 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. .

Further, 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.

Further, 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. There are no special requirements for the type of preservative. Commonly used 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.

In addition to the above main components, 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.

Although not wishing to be bound by theory, it is believed that the above protein stabilizer, alkaline or neutral ammonia The combination of a base acid, a polyhydroxy compound or a polymer and a citrate, in addition to the above-described effects, has synergistic effects between the components. 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. Further, 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.

In the preservation solution of one embodiment, 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.

In the preservation solution of one embodiment, the fluorescent labeling protein reagent type may be a fluorescent protein labeled protein, a fluorescent protein such as PE, APC, PerCP or the like.

In the preservation solution of one embodiment, 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 preservation solution of one embodiment, 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.

Fluorescent label i£» reagent

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. In a preferred embodiment, 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. Preferably, the fluorescent tracer-biomarker conjugate is a fluorescently labeled protein, particularly preferably a fluorescently labeled antibody. Mixing the fluorescent label reagent with the sample to be tested for a period of time, such that the fluorescent tracer-biomarker conjugate in the reagent binds to the cells or cellular components in the sample to be tested, and this process may also be referred to as staining. . After staining is complete, the samples are analyzed on a fluorescence activated flow analyzer. Alternatively, in another embodiment, 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.

Preferably, the fluorescent tracer-biomarker conjugate is a fluorescently labeled protein, particularly preferred,

: Burning the cursor with antibodies.

Hereinafter, a fluorescently labeled protein will be described as an example.

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.

Other

In another aspect, 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.

Example

The invention is described in more detail below by way of examples of fluorescently labeled protein reagents, but the invention is not limited thereto.

1. Equipment:

Shimadzu 20A high performance liquid chromatography (HPLC); BD FACSCanto II flow cytometer; Shanghai Bo Xun Industrial Co., Ltd. digital display electrothermal incubator; its Linbeier instrument factory vortex; METTLER pipette and matching Tips; Shanghai Anting Scientific Instrument Factory low-speed large-capacity centrifuge.

2. Reagents:

Commercially available SurModics StabilGuard® preservation solution;

Commercially available multicolor fluorescently labeled protein reagent A: 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; Commercially available multicolor fluorescently labeled protein reagent B : 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;

Commercially available flow hemolytic agent, BD FACS Lysing Solution (1 Ox concentrate, use deionized water diluted 1: 10 to l x reagent);

Clinical whole blood samples (EDTA-K3 and EDTA-K2 anticoagulation);

Commercially available StatusFlow® Flow Cytometry Control;

Furfural solution, analytically pure;

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 μηι PALL GHP filter.

3. Detection method:

(1) HPLC detection of fluorescent tracer-protein conjugate peak area method: Column GF250 (Agilent, 4μηι, 4.6x250mm) Flow rate 0.5mL / min Detection wavelength 565 匪 mobile phase 0.3M Na ₂ HPO ₄ , pH7.0 Injection volume 2 (^L

(2) Method for detecting samples using a single-color fluorescent labeling protein reagent:

1 Add 20μ fluorescent labeled protein reagent in 12x75mm flow tube, then add ΙΟΟμ whole blood sample, mix at low speed and vortex, incubate for 15min at room temperature (20 °C ~ 25 °C);

2 Add 2mL lx flow hemolytic agent, mix at low speed and vortex, incubate at room temperature in the dark for 10 min; Pour the supernatant after centrifugation for 5 min at 3 OOg;

4 Add 2mLPB S, mix at low speed on the vortex meter, centrifuge at 500g for 5min, then pour the supernatant;

5 Add 500μ PBS fixative containing 1% furfural, vortex at low speed, and measure by flow cytometry.

(3) Method for detecting samples using multicolor fluorescently labeled protein reagents:

1 Add 20 L fluorescent labeled protein reagent in a 12x75mm flow tube, then add 50 L whole blood sample, mix at low speed and vortex at room temperature (20 °C ~ 25 °C) for 15 min;

2 Add 450 μl x flow hemolytic agent, mix at low speed and vortex at room temperature for 15 min;

3 Low-speed vortex mixing hook, upflow cytometry.

Example 1

Prepare 1#~6# preservation solution according to the formula of Table 1, stir and dissolve at room temperature, and set aside. 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 #和6#, the preservation solution works better. It also shows that the preservation solution has a good preservation effect on the fluorescein-antibody conjugate.

1#~6# preservation solution group

Name preservation solution 1# preservation solution 2# preservation solution 3# preservation solution 4# preservation solution 5# preservation solution 6# Weigh

0.05% gelatin 0.08% gelatin 0.08% gelatin 0.24% BSA 0.24% BSA 0.40% BSA

20mM Gan 16mM fine 16mM Gan

4 mM arginine 4 mM glycine I mM glycine

Lysine

0.6% mannose 0.6% mannose group 4% glycerol 4% glycerol 4% glycerol alcohol alcohol 0.6% PEG 1 () () () () 0.6% PEG 20000 0.6% PEG 10000 0.4% seaweed 0.4% seaweed sugar

16 mM citric acid 16m M citric acid 16 mM citric acid 24m M lemon 24m M lemon 24mM lemon trisodium trisodium trisodium citrate trisodium citrate trisodium citrate trisodium

2 mM EDTA-2Na, 0.1% NaN ₃ , 140 mM NaCl, 10 mM phosphate buffer solution, pH 7.0

Example 2

Prepare 7#~13# preservation solution according to the formula of Table 2, stir and dissolve at room temperature, and set aside. 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 μυ 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.

7#~13# preservation solution group

0.12% gelatin 0.12% gelatin 0.12% gelatin 0.12% gelatin 0.16% BSA

8mM refined ammonia 8mM refined ammonia 8mM refined ammonia 8mM refined ammonia 12mM fine 12mM fine 12mM acid acid acid acid lysine

0.1% nectar 1.0% nectar 0.2% nectar

1% glycerin 10% glycerol 6% glycerol 2% glycerol

Alcohol Alcohol Group 1.0% PEG1 0.2% PEG2 0.2% PEG1 0.8% PEG1

0.1% seaweed 1.0% seaweed 0.8% seaweed into 0000 0000 0000 0000

Sugar sugar lOmM lOmM lOmM lOmM 30mM 30mM 30mM

4 citric acid tri-acid three 4 Ning-like acid three 4 Ning-like acid three 4 Ning-like acid three 4 Ning-like acid three 4 Ning-like acid sodium sodium sodium sodium sodium sodium sodium

3mM EDTA-2Na, 0.1% Proclin300, 140mM NaCl, 50mM phosphate buffer solution, pH7.0

Example 3

Prepare 14#~16# preservation solution according to the formula of Table 3, stir and dissolve at room temperature, and set aside. Follow

<

Conventional methods for preparing CD4-FITC (SK3 clone), CD3-PerCP (SK7 clone), CD8-APC-Cy7 (SK8 clone) three monochromatic fluorescently labeled monoclonal antibody reagents, respectively using the above 14#~16# preservation solution The <Three Monochrome Fluorescent Labeled Monoclonal Antibody Reagents were diluted according to Table 4 with commercially available SurModics StabilGuard® Preservatives.

Table 3 14#~16# preservation solution components

Table 4 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 ΙΟΟμυ 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

Take homemade CD45-PerCP (2D1 clone), CD3-FITC (S7 clone), CD4-APC (SK3 clone), CD8-PE (SK8 clone), CD19-APC (SJ25C1 clone), CD56-PE (ACM16) .2 clone;), CD16-PE (B73.1 clone) Monochrome fluorescently labeled monoclonal antibody reagent, prepared by using the preservation solution 17# according to the components and concentrations of the commercially available multicolor fluorescently labeled protein reagent A. 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.

Table 6 Multicolor reagent composition

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 ΙΟΟμυ, 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.

Due to the large difference in clinical samples at different times, 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 results showed that the antibody activity and fluorescence degradation rate of different fluorescently labeled antibody reagents were inconsistent. 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.

Self-made multi-color reagent A1 accelerated test each fluorescence intensity and multi-color fluorescent labeled protein reagent A ratio

Table 8 Self-made multicolor reagents B1 accelerated test The ratio of each fluorescence intensity to the multicolor fluorescent labeling protein reagent B

Note: / indicates no statistics, because the subpopulations of negative and positive cells can not be effectively separated.

Example 5

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. Take the control reagent and the transported reagent separately, and stain the same batch of commercially available StatusFlow® Flow Cytometry Control, hemolysis/washing After the treatment, the flow cytometry was used for flow detection to record the average fluorescence intensity, and each repeated operation was performed three times, and the mean value of the three test results was taken, and the difference between the post-transport reagent and the control test result was compared.

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.

CD4-FITC Monochrome Fluorescent Labeled Monoclonal Antibody Reagent Transport Test Results

Example 6

Take homemade CD45-PerCP (2D1 clone), CD3-FITC (SK7 clone), CD4-APC (SK3 clone), CD8-PE (S 8 clone) monochrome fluorescent labeled monoclonal antibody reagent, use preservation solution 17# 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

Prepare the 19# preservation solution according to the following formula, stir and dissolve at room temperature, and set aside.

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 ΙΟΟμυ 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 method for detecting a sample by the above monochromatic reagent, detecting the fluorescence intensity of the cell binding reagent in the quality control product, each Repeat the operation 3 times and take the average of the three test results. Curves were plotted over time and mean fluorescence intensity of each monoclonal fluorescently labeled monoclonal antibody reagent.

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.

In summary, it can be seen from the above examples that 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.

The present invention has been described above by way of specific embodiments, but the invention is not limited to the specific embodiments. It will be apparent to those skilled in the art that various modifications, equivalents, changes, and the like may be made without departing from the spirit and scope of the invention.

Claims

Claim

A preservation solution for a biomarker, comprising:

Protein stabilizer

Alkaline or neutral amino acid;

a polyhydroxy compound or a polyhydroxy polymer; and

Citrate.

The preservation solution according to claim 1, wherein the protein stabilizer is selected from the group consisting of albumin or gelatin.

3. The preservation solution as claimed in claim 2, wherein the concentration of the protein stabilizing agent is from 0.05% to 0.40%, preferably 0.08% to 0.24% ₀

The preservative solution according to claim 1, wherein the basic or neutral amino acid is selected from the group consisting of arginine, histidine, lysine and glycine, preferably arginine or glycine.

The preservative solution according to claim 4, wherein the concentration of the basic or neutral amino acid is from 1 to 20 mmol/L, preferably from 4 to 16 mmol/L.

The preservative solution according to claim 1, wherein the polyhydroxy compound or polyhydroxy polymer is selected from the group consisting of glycerin, polysaccharide, sugar alcohol, and polyethylene glycol polymer.

The preservation solution according to claim 6, wherein the polysaccharide is selected from trehalose, or the sugar alcohol is selected from mannitol, or the polyethylene glycol polymer is selected from PEG 8000, PEG 10000 or

The preservation solution according to claim 7, wherein the concentration of the glycerin is 1 to 10%, preferably 2 to 6%; or the concentration of the mannitol, trehalose, and polyethylene glycol polymer It is 0.1% to 1.0%, preferably 0.2% to 0.8%.

The preservative solution according to claim 1, wherein the preservative solution further contains at least one antioxidant.

The preservation solution according to claim 9, wherein the antioxidant is selected from the group consisting of sodium ethylenediaminetetraacetate.

The preservation solution according to claim 1, wherein the preservation solution further contains a preservative.

The preservative solution according to claim 1, wherein the preservative solution further contains a buffering agent and maintains a pH of 6 to 8.

1 . A fluorescent label reagent, characterized in that the fluorescent label reagent comprises a fluorescent tracer-biomarker conjugate and the preservation solution according to any one of claims 1 to 12.

14. The reagent according to claim 13 wherein the biomarker in the fluorescent tracer-biomarker conjugate is a protein.

15. The reagent of claim 14, wherein the biomarker in the fluorescent tracer-biomarker conjugate is an antibody or polypeptide.

16. The reagent of claim 13, wherein the fluorescent tracer in the fluorescent tracer-biomarker conjugate is selected from the group consisting of small molecule fluorescein, fluorescent protein, or tandem fluorescent dye.

17. A method of preparing a fluorescent label reagent, comprising

Providing a fluorescent tracer-biomarker conjugate and the biomarker preservation solution of any of claims 1-12;

The fluorescent tracer-biomarker conjugate was diluted to the working concentration with the preservation solution to obtain a fluorescent marker reagent solution that can be used directly.

18. Use of a preservation solution according to any of claims 1-12 in the preparation of a fluorescent label reagent.