CN116539896A - Magnetic microsphere electrochemiluminescence immunoassay kit for detecting serum galectin-3 and preparation of kit - Google Patents
Magnetic microsphere electrochemiluminescence immunoassay kit for detecting serum galectin-3 and preparation of kit Download PDFInfo
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- CN116539896A CN116539896A CN202310521908.6A CN202310521908A CN116539896A CN 116539896 A CN116539896 A CN 116539896A CN 202310521908 A CN202310521908 A CN 202310521908A CN 116539896 A CN116539896 A CN 116539896A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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- G01N2800/00—Detection or diagnosis of diseases
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the field of electrochemical detection, in particular to a magnetic microsphere electrochemiluminescence immunoassay kit for detecting serum galectin-3 and preparation of the kit. The invention provides a composition and application thereof in preparing Gal-3 detection kit. Gal-3 has small molecular weight, and is difficult to meet the requirement of repeatability and stability when being made into commercial diagnosis products. The invention combines the high specificity of the antibody-antigen reaction with the high sensitivity of the terpyridyl ruthenium to emit light, utilizes photons generated by the terpyridyl ruthenium under DBAE to detect the concentration of the product, improves the sensitivity, the stability and the repeatability, shortens the reaction time, has high anti-interference performance and is simple to operate.
Description
Technical Field
The invention relates to the field of electrochemical detection, in particular to a magnetic microsphere electrochemiluminescence immunoassay kit for detecting serum galectin-3 and preparation of the kit.
Background
Chronic Heart Failure (CHF) is a clinical syndrome characterized by severely impaired ventricular ejection function and abnormal peripheral blood flow distribution, and its pathological processes are mainly represented by cardiac remodeling and myocardial fibrosis, which are caused by high mortality due to high morbidity and poor prognosis.
Serum Gal-3 is a soluble β -galactoside binding lectin, a protein of relative molecular mass 30kDa that binds G-galactoside and is involved in a variety of biological activities, mainly known to mediate tumor growth, invasion and metastasis, while it is also associated with increased age, diabetic nephropathy, fibrotic diseases such as liver fibrosis, kidney fibrosis, idiopathic pulmonary fibrosis and chronic pancreatitis. Is associated with cardiac fibrosis and myocardial remodeling, and can promote proliferation of cardiac fibroblasts, collagen deposition and ventricular dysfunction.
Gal-3 is an important mediator of inflammatory response, and plays an important role in myocardial remodeling and pathological development of heart failure, and the higher the plasma Gal-3 level expression of heart failure patients is, the more easily the heart failure worsens, and the higher the hospitalization rate and the death rate are. Serum Gal-3 promotes activation and migration of cardiac macrophages, accelerates fibroblast proliferation and collagen deposition, and leads to cardiac remodeling and cardiac fibrosis, which are important determinant factors affecting chronic heart failure. The ubiquity of the expression of serum Gal-3 in human tissue organs allows the production of serum Gal-3 without being limited by specific conditions. In recent years, serum Gal-3 has been increasingly used as an important diagnostic index for heart failure at home and abroad. At present, gal-3 has become an important index for clinical diagnosis and assessment of recent prognosis of heart failure patients in European and American countries. In addition, gal-3 can be used for fibrosis of various organs, including but not limited to: heart, kidney, liver, etc.
To date, gal-3 has rarely been used in routine clinical patient examinations, although Gal-3 is believed to be useful for additional risk stratification in patients with dynamic or acute heart failure. The method for detecting serum galectin-3 (Gal-3) in human serum mainly comprises the following steps: enzyme-linked immunosorbent assay (ELISA) and enzymatic magnetic particle chemiluminescence. Gal-3 has smaller molecular weight and is also present in all tissues and organs, so that the basic data of the detection data is higher, and the repeatability, sensitivity, specificity, stability, detection time and the like of the prior art and the detection of products cannot meet the requirements of commercial clinical diagnosis.
Disclosure of Invention
In view of the above, the invention provides a magnetic microsphere electrochemiluminescence immunoassay kit for serum galectin-3 detection and a preparation method thereof. The invention provides a composition and application thereof in preparing a serum galectin-3 (Gal-3) magnetic particle electrochemiluminescence detection kit. The invention combines the high specificity of the antibody-antigen reaction with the high sensitivity of the terpyridyl ruthenium to emit light through experiments, utilizes photons generated by the terpyridyl ruthenium under DBAE to detect the concentration of the product, and has the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a composition comprising an antibody containing a biotin label which can specifically bind Gal-3, an antibody containing a terpyridyl ruthenium label which can specifically bind Gal-3, and streptavidin superparamagnetic microspheres;
the particle size of the streptavidin superparamagnetic microsphere is 1.0-6.0 mu m.
In some embodiments of the invention, the antibody that specifically binds Gal-3 comprises an anti-Gal-3 monoclonal antibody.
In some embodiments of the invention, the amount of biotin molecular label on the surface of an antibody molecule that specifically binds Gal-3 comprises 2 to 5.
In some embodiments of the invention, the antibody that specifically binds Gal-3 comprises an anti-Gal-3 monoclonal antibody.
In some embodiments of the invention, the amount of ruthenium molecular label on the surface of the antibody molecule that specifically binds Gal-3 comprises 2 to 10.
In some embodiments of the invention, the particle size of the streptavidin superparamagnetic microspheres comprises 3 μm.
In some embodiments of the invention, the composition further comprises a buffer;
the buffer solution comprises:
20-200 mmol/L phosphate buffer, pH=7.4; and/or
20mmol/L to 200mmol/L of tris buffer solution, pH=7.4.
In some embodiments of the invention, the composition further comprises a first cleaning fluid;
the first cleaning solution comprises 2-N-dibutylamino ethanol;
the first cleaning solution further comprises phosphate buffer solution.
In some embodiments of the invention, the first cleaning solution comprises dibutyl ethanolamine at a concentration of 90mmol/L and comprises phosphate buffer at a concentration of 300 mmol/L.
In some embodiments of the invention, the composition further comprises a stabilizer;
the stabilizer comprises one or more of glycine, methionine, PEG20000, dextran 200000, pullulan or polyvinylpyrrolidone 360.
In some embodiments of the invention, the stabilizer comprises:
in some embodiments of the invention, the volume ratio of the stabilizing agent to the buffer is 5:100.
in some embodiments of the invention, the composition further comprises a second cleaning solution;
the second cleaning liquid comprises a cleaning liquid 1, a cleaning liquid 2, a cleaning liquid 3 and/or a cleaning liquid 4;
the cleaning liquid 1 comprises isooctanol polyoxyvinyl ether with the polymerization degree of 14, lauryl alcohol polyoxyethylene ether with the polymerization degree of 9 and/or Proclin950;
the cleaning liquid 2 comprises sodium hydroxide, sodium chloride and/or sodium hypochlorite solution;
the cleaning liquid 3 comprises isooctyl alcohol polyoxyvinyl ether with the polymerization degree of 14, laurinol polyoxyethylene ether with the polymerization degree of 9 and/or potassium hydroxide;
the cleaning liquid 4 comprises 85 weight percent of phosphoric acid, dichloroacetamide, isooctyl alcohol polyoxyvinyl ether with the polymerization degree of 14, laurinol polyoxyethylene ether with the polymerization degree of 9, monopotassium phosphate and/or 2-N-dibutylamino ethanol.
In some embodiments of the invention, the cleaning liquid 1 comprises:
isooctanol polyoxyvinyl ether having a degree of polymerization of 14 1.0g/L
Laurinol polyoxyethylene ether with polymerization degree of 9 of 10g/L
Proclin950 75g/L
In some embodiments of the invention, the cleaning liquid 2 comprises:
sodium hydroxide 120g/L
Sodium chloride 87.75g/L
25g/L sodium hypochlorite solution
In some embodiments of the invention, the cleaning liquid 3 comprises:
isooctanol polyoxyvinyl ether having a degree of polymerization of 14 1.0g/L
Laurinol polyoxyethylene ether with polymerization degree of 9 of 10g/L
Potassium hydroxide 9.856g/L
In some embodiments of the invention, the cleaning solution 4 comprises:
the invention also provides a preparation method of the composition, which comprises the steps of: mixing an antibody capable of specifically binding Gal-3 with biotin, and removing unlabeled biotin after the reaction is finished to prepare the antibody containing biotin label capable of specifically binding Gal-3;
the preparation method of the terpyridyl ruthenium labeled antibody capable of specifically binding Gal-3 comprises the following steps: mixing an antibody capable of specifically binding Gal-3 with terpyridyl ruthenium uniformly, and removing unlabeled terpyridyl ruthenium after the reaction is finished to prepare the antibody containing terpyridyl ruthenium label capable of specifically binding Gal-3;
the preparation method of the streptavidin superparamagnetic microsphere comprises the following steps: and coating the super paramagnetic microsphere with streptavidin to obtain the super paramagnetic microsphere of the streptavidin.
In some embodiments of the invention, the method for preparing an antibody containing a biotin label that specifically binds Gal-3 comprises: 2.0mg of antibody for labeling biotin Gal-3 was used, the buffer was changed to phosphate buffer (pH=7.8) using desalting column PD10, the concentration was adjusted to 2.0mg/mL after concentration using ultrafiltration tube, 80. Mu.g of biotin (dissolved using DMF) was added, and the reaction was carried out for 30 minutes after mixing, and unlabeled biotin was removed using desalting column PD10 to prepare the antibody containing biotin-labeled Gal-3.
In some embodiments of the invention, the preparation method of the terpyridyl ruthenium labeled antibody capable of specifically binding Gal-3 comprises the following steps: 2.0mg of Gal-3 antibody for labeling ruthenium terpyridyl was used, the buffer solution was changed to phosphate buffer solution (pH=7.8) using a desalting column PD10, the concentration was adjusted to 2.0mg/mL after concentration using an ultrafiltration tube, 80. Mu.g of ruthenium terpyridyl (dissolved using DMF) was added, the mixture was uniformly reacted for 30 minutes, and unlabeled ruthenium was removed using a desalting column PD10 to prepare the antibody containing ruthenium terpyridyl labeled specifically binding Gal-3.
In some specific embodiments of the present invention, the streptavidin superparamagnetic microsphere is a superparamagnetic microsphere with streptavidin coated on the surface, the particle size of the magnetic microsphere is 1.0-6.0 microns, the magnetic particle coating buffer is 20 mmol/L-200 mmol/L phosphate buffer, the PH=7.4 or 20 mmol/L-200 mmol/L tris buffer, and the PH=7.4.
The invention also provides application of the composition in preparation of Gal-3 detection kit.
The invention also provides a kit comprising the composition.
In some embodiments of the invention, the kit comprises: gal-3 reagent Ra, gal-3 reagent Rb, streptavidin superparamagnetic microsphere, calibration product, dibutyl ethanolamine cleaning solution and pipeline cleaning solution.
In some embodiments of the invention, the Gal-3 reagent Ra is an anti-Gal-3 monoclonal antibody containing biotin labels, the molecular label weight of the biotin on the surface of each antibody molecule is 2-5, the buffer solution is 20-200 mmol/L phosphate buffer solution, the PH=7.4 or 20-200 mmol/L tris buffer solution, and the PH=7.4.
In some embodiments of the invention, the Gal-3 reagent Rb is an anti-Gal-3 monoclonal antibody containing terpyridyl ruthenium label, the molecular weight of ruthenium on the surface of each antibody molecule is 2-10, the buffer solution is 20-200 mmol/L phosphate buffer solution, the PH=7.4 or 20-200 mmol/L tris buffer solution, and the PH=7.4.
In some specific embodiments of the present invention, the streptavidin superparamagnetic microsphere is a superparamagnetic microsphere with streptavidin coated on the surface, the particle size of the magnetic microsphere is 1.0-6.0 microns, the magnetic particle coating buffer is 20 mmol/L-200 mmol/L phosphate buffer, the PH=7.4 or 20 mmol/L-200 mmol/L tris buffer, and the PH=7.4.
The invention also provides the use of any of the following in Gal-3 detection:
(I) The composition; and/or
(II) the kit.
The invention also provides a Gal-3 magnetic microsphere electrochemiluminescence detection method, which is used for detecting Gal-3 based on any of the following items:
(I) The composition; and/or
(II) the kit.
The invention also provides a detection method of the Gal-3 magnetic microsphere electrochemiluminescence kit, which comprises the following steps:
step 1, taking 15 mu L of a sample to be detected, adding 15 mu L of a Gal-3 reagent Ra70 mu L and a Gal-3 reagent Rb70 mu L into a reaction tube, incubating for 9min at 37 ℃, and finally adding 40 mu L of streptavidin superparamagnetic microspheres, and incubating for 9min at 37 ℃;
step 2, sucking the reaction tube after incubation into an electrochemical flow cell through a liquid suction steel needle, and adsorbing the reaction tube by a magnet of the flow cell;
and 3, sucking a second cleaning solution by a liquid suction steel needle, cleaning an antibody and a sample of the labeled ruthenium which are not combined with the superparamagnetic microspheres, powering up a flow cell, and emitting light by terpyridyl ruthenium under the condition of DBAE.
And 4, recording a luminescence value by the photomultiplier, and calculating the concentration of Gal-3 in the sample according to the established standard curve.
The invention includes, but is not limited to, achieving the following benefits:
the invention combines the high specificity of the antibody-antigen reaction with the high sensitivity of the terpyridyl ruthenium to emit light, and utilizes photons generated by the terpyridyl ruthenium under DBAE to detect the concentration of the product, thereby having the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the linear range of the reaction process employed in example 7;
FIG. 2 shows a reagent bottle configuration;
FIG. 3 shows a device structure;
fig. 4 shows the signal test results.
Detailed Description
The invention discloses a magnetic microsphere electrochemiluminescence immunoassay kit for serum galectin-3 detection and a preparation method of the kit, and a person skilled in the art can refer to the content of the kit and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The method adopted by the invention is an electrochemiluminescence method, and the ruthenium pyridine is adopted as a chemiluminescent marker, which has obvious advantages and is mainly represented by: the stability is better, ruthenium is a metal ion, the molecular weight is small, and the steric hindrance of the antibody is not influenced. The production process is short and the repeatability is good. The detection range is wide, and the repeatability is good. The electrochemiluminescence reaction is controllable, and the difficulty of signal acquisition is reduced.
The magnetic particles can be used as carriers of biological macromolecules, the magnetic particles coated by the antibodies are called immunomagnetic particles, and the magnetic particles have the characteristics of combining antigens and magnetism, so the magnetic particles have more advantages in the aspects of separating, purifying and concentrating target microorganisms, cells, biological macromolecules and the like from complex samples, and have the advantages of rapidness, strong specificity, simple and convenient operation, wide application range and the like. The nanometer material is a new material which is developed rapidly after the 90 th century of the 20 th century, and the nanometer magnetic particles (the particle diameter is smaller than 10 nm-100 nm) are greatly different from the common magnetic particles in the aspects of magnetic structure and magnetism: the nano magnetic particles have more particles per unit volume and larger specific surface area; has superparamagnetism and weak magnetic interaction; it can directionally move under the action of external magnetic field to make some special components be separated, concentrated or purified, etc.. The magnetic particle chemiluminescence method established by the invention has the advantages of high sensitivity, strong specificity, accuracy, rapidness, short detection time and higher accuracy and repeatability of detection results.
The invention provides a Gal-3 magnetic microsphere electrochemiluminescence kit, which comprises the following components: gal-3 reagent Ra, gal-3 reagent Rb, streptavidin superparamagnetic microsphere, calibration product, dibutyl ethanolamine cleaning solution and pipeline cleaning solution.
The Gal-3 magnetic microsphere electrochemiluminescence kit comprises a Gal-3 reagent Ra, a biotin-containing labeled anti-Gal-3 monoclonal antibody, wherein the molecular label weight of biotin on the surface of each antibody molecule is 2-5, the buffer solution is 20 mmol/L-200 mmol/L phosphate buffer solution, the pH=7.4 or 20 mmol/L-200 mmol/L tris buffer solution, and the pH=7.4. Gal-3 reagent Rb is an anti-Gal-3 monoclonal antibody containing terpyridyl ruthenium label, the molecular label weight of ruthenium on the surface of each antibody molecule is 2-10, the buffer solution is 20 mmol/L-200 mmol/L phosphate buffer solution, and the pH=7.4 or 20 mmol/L-200 mmol/L tris buffer solution, and the pH=7.4.
The Gal-3 magnetic microsphere electrochemiluminescence kit, wherein the streptavidin superparamagnetic microsphere is a superparamagnetic microsphere with streptavidin coated on the surface, the particle size of the magnetic microsphere is 1.0-6.0 microns, the magnetic particle coating buffer solution is 20-200 mmol/L phosphate buffer solution, the pH=7.4 or 20-200 mmol/L tris buffer solution, and the pH=7.4.
The Gal-3 magnetic microsphere electrochemiluminescence kit, wherein the biotin-labeled Gal-3 antibody is a monoclonal antibody;
the detection method of the Gal-3 magnetic microsphere electrochemiluminescence kit comprises the steps of preparing a cleaning solution, wherein the cleaning solution is dibutyl ethanolamine with the concentration of 90mmol/L, and the cleaning solution contains phosphate buffer with the concentration of 300 mmol/L.
A detection method of a Gal-3 magnetic microsphere electrochemiluminescence kit comprises the following steps:
1) Adding 15 mu L of a sample into a reaction tube, sequentially adding 70 mu L of Gal-3 reagent Ra and 70 mu L of Gal-3 reagent Rb, incubating at 37 ℃ for 9min, and finally adding 40 mu L of streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 9min;
2) Sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid suction steel needle, and adsorbing by a magnet of the flow cell;
3) The liquid-absorbing steel needle absorbs the second cleaning liquid, after the antibody of the marked ruthenium which is not combined with the superparamagnetic microsphere and the sample are cleaned, the flow cell is electrified, and the terpyridyl ruthenium emits light under the condition of DBAE.
4) The photomultiplier tube records the luminescence value, and calculates the concentration of Gal-3 in the sample according to the established standard curve.
2. Statistics and calculation model
Such an assessment will not generally be correct for all (i.e., 100%) of the subjects to be identified. However, subjects of statistically significant significance (e.g., a group in a population study) can be identified. Whether there is statistical significance can be determined by those skilled in the art using a variety of known statistical evaluation tools, such as confidence intervals, P-values, the Stokes t-test, the Mann-Whitney test, etc., and details can be found in Dowdy and Werden, statistics for research (John Wiley & Sons, new York, 1983). The confidence interval may be set to 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value may be set to 0.1, 0.05, 0.01, 0.005 or 0.000l. The population may be set to at least 60%, at least 70%, at least 80% or at least 90% of the subjects that can be correctly identified by the methods of the invention.
Advantages and positive effects
1) The electrochemiluminescence immunoassay technology has the advantages of high sensitivity, rapidness, accuracy, good repeatability, safety, no toxicity, no pollution and the like. Luminol, isoluminol and derivatives thereof are the earliest class of chemiluminescent substances used but are applied to chemistryLuminescent immunoassays require the use of catalysts and enhancers, which will result in enhanced background luminescence, limiting the sensitivity of this technique and its application and development. The acridinium ester luminous system is simple, no catalyst is needed, and the acridinium ester luminous system is placed in H 2 O 2 The solution can emit light without a catalytic process and an enhancer, so that background light emission is reduced, sensitivity is improved, interference effect is small, but the acridinium ester is easy to hydrolyze, and meanwhile, the emission of the acridinium ester is released rapidly. The luminescence peak value is 0.4s, so that in-situ sample injection is needed, and the requirement on equipment is high. The terpyridyl ruthenium is easy to realize protein connection, has small molecular weight and small influence on the conformation of the antibody after connection, and the marker is metal ion, so that the stability is good, and the luminescence is controllable under the condition of power-on. Therefore, the electrochemical method applied to Gal-3 detection can improve the sensitivity of the product, shorten the process marking time, improve the linear range and shorten the test time, thereby providing basis for timely coping with brain trauma treatment clinically.
The electrochemical luminescent marker pyridine ruthenium has very stable property, and the luminous efficiency is not influenced by factors such as temperature, pH, ionic strength and the like. The electrochemiluminescent reagent signal value is reduced by less than 3% compared to the fresh reagent. The bottle opening period is three months, and the bottle can be stabilized for more than 15 months at the temperature of 2-8 ℃.
| Luminous system | Horseradish enzyme-luminol | Alkaline phosphatase | Electrochemiluminescence |
| Time-stamping | For more than 24 hours | For more than 24 hours | 60 minutes |
| Test time | 60 minutes | 30 minutes | 18 minutes |
| Expiration date of reagent | For 12 months | For 12 months | For greater than 15 months |
The electrochemical labelling reaction is rapid and only half an hour is needed for the whole reaction. The marking efficiency reaches 70%. The proportion of the marks can be controlled by the feeding ratio, and the productivity is improved by more than 50 percent. Ruthenium has small molecular weight (800D), small steric hindrance and good antibody activity. The absorption peak at 455nm can be controlled to control the batch-to-batch difference.
Streptavidin and biotin have high specific binding capacity, streptavidin and biotin-labeled high-purity antibody are subjected to non-covalent bond specific binding, and have cascade amplification, and the reaction is highly specific. Therefore, the sensitivity is improved, the nonspecific interference is not increased, and the binding characteristic is not affected by the high dilution of the reactant, so that the nonspecific effect of the reactant can be reduced to the greatest extent in practical application.
The invention combines the high specificity of the antibody-antigen reaction with the high sensitivity of the terpyridyl ruthenium to emit light, and utilizes photons generated by the terpyridyl ruthenium under DBAE to detect the concentration of the product, thereby having the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.
2) The magnetic particles are selected, and the sensitivity is high.
3) The portable pollution rate is low, and the sensitivity and the accuracy are high.
4) Long effective period and long bottle opening period.
5) The special reagent bottle bending opening arrangement (figures 3 and 4) can effectively prevent liquid leakage and reagent evaporation, and improve the reagent effective period and the bottle opening effective period.
The equipment used was a full-automatic chemiluminescence immunoassay (model UD90 DT) manufactured by tokyo-co-tek technologies limited.
If not specified, the magnetic microsphere electrochemiluminescence immunoassay kit for serum galectin-3 detection and the raw materials and reagents used in the preparation of the kit can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 selection of magnetic microparticles
(1) Experimental materials
| Name of the name | Particle size (mum) | Lot number |
| Alternative magnetic bead 1 | 3.0 | ---- |
| Alternative magnetic bead 2 | 1.0 | ---- |
| Alternative magnetic bead 3 | 6.0 | ---- |
(2) Instrument for measuring and controlling the intensity of light
(3) Experiment site:
location: laboratory of company research and development
(4) The experimental process comprises the following steps:
the experimental method comprises the following steps:
specific experimental procedure for TSH (sandwich method), T4 (competition method):
TSH (sandwich method):
step 1: samples of 50 μl were incubated with Ra60 μl containing biotinylated TSH-specific monoclonal antibody and Ra50 μl containing ruthenium (Ru) complex labeled TSH-specific monoclonal antibody to form antigen-antibody immune complexes.
Step 2: the magnetic separation reagent containing streptavidin-coated magnetic bead particles was added at 40. Mu.L, and the immunocomplexes formed above were allowed to bind to the magnetic bead particles by interaction between biotin and streptavidin.
Step 3: after incubation, the reaction mixture is sucked into a measuring pool, magnetic bead particles are adsorbed onto an electrode through a magnet, unbound substances are washed away by a cleaning solution, chemiluminescence is generated after the electrode is electrified, and a luminescence value is obtained through measurement by a photomultiplier tube.
After the main curve information is scanned into the instrument through the reagent bar code, scanning the calibration product information card, scanning the information and the concentration of the calibration product into the instrument, correcting the main curve to obtain a calibration curve, and automatically calculating by software through the calibration curve to obtain a detection result.
T4 (competition method):
the total thyroxine (TT 4) assay was competition and the detection steps were as follows:
step 1: samples 15 μl, ra75 μl of ruthenium (Ru) -containing complex-labeled T4 antibody were incubated together; t4 bound to the binding protein in the sample is released by ANS action.
Step 2: 30. Mu.L of a magnetic separation reagent containing Rb 75. Mu.L of biotinylated T4 and magnetic bead particles coated with streptavidin was added, and the biotinylated T4 was bound to unbound labeled antibody to form an antibody-hapten complex, and the immunocomplex formed above was bound to the magnetic bead particles by the interaction between biotin and streptavidin.
Step 3: after incubation, the reaction mixture is sucked into a measuring pool, magnetic bead particles are adsorbed onto an electrode through a magnet, unbound substances are washed away by a cleaning solution, chemiluminescence is generated after the electrode is electrified, and a luminescence value is obtained through measurement by a photomultiplier tube.
After the main curve information is scanned into the instrument through the reagent bar code, scanning the calibration product information card, scanning the information and the concentration of the calibration product into the instrument, correcting the main curve to obtain a calibration curve, and automatically calculating by software through the calibration curve to obtain a detection result.
The signal values of low-value serum and high-value serum of TSH (sandwich method) and T4 (competition method) were detected by streptavidin magnetic beads (1, thermoFisher scientific; 2, JSRLIFe Sciences), and the background signal values and the signal to noise ratio were compared. The concentration value of serum is defined by cobase411 and matched reagent of Roche diagnosis, and the magnetic bead diluent adopts Roche magnetic bead diluent.
(5) Experimental results
TABLE 1TSH (Sandwich method)
Table 2T4 (Competition method)
(6) Conclusion of experiment:
from the results (tables 1 and 2), it can be seen that: the difference of signal to noise ratios of the streptavidin magnetic beads with different particle sizes is obvious, and the larger the signal to noise ratio is, the higher the sensitivity is, and the streptavidin magnetic beads with the particle sizes of 3.0 mu m are determined. The statistical P value is larger than 0.05, and the normal distribution is met.
Example 2 detection of Portable contamination Rate
Cleaning liquid 1 formula: (1L)
Isooctanol polyoxyvinyl ether having a degree of polymerization of 14 1.0g/L
Laurinol polyoxyethylene ether with polymerization degree of 9 of 10g/L
Proclin950 75g/L
The formula of the cleaning liquid 2 is as follows: (1L)
Sodium hydroxide 120g/L
Sodium chloride 87.75g/L
25g/L sodium hypochlorite solution
The formula of the cleaning liquid 3 is as follows: (1L)
Isooctanol polyoxyvinyl ether having a degree of polymerization of 14 1.0g/L
Laurinol polyoxyethylene ether with polymerization degree of 9 of 10g/L
Potassium hydroxide 9.856g/L
Cleaning liquid 4 formula: (1L)
The test flow of the carrying pollution rate:
three signal values are needed to be obtained in the analysis of the portable pollution rate, namely, a background measurement value (ProCellRef for short) is a background luminescence value which is cleaned and tested by using cleaning liquid, a high-concentration test value (ProCellSAP for short) of the portable pollution rate is a luminescence value for testing high-dose ruthenium (the concentration is 1 nmol/L), and a low-concentration test value (ProCellCo for short) of the portable pollution rate is a luminescence value which is cleaned by using the cleaning liquid after the high-dose ruthenium is used, and the background luminescence value is tested again). In the test, the cleaning solution (5 times), the high-dose ruthenium (3 times) and the cleaning solution (3 times) are placed at the test position of a full-automatic chemiluminescence immunoassay analyzer (model UD90 DT), and a photometry program is started. The resulting 5 procallref, 3 procallsap, 3 procallco values, respectively. The luminescence values measured were used to calculate carryover, and the smaller the calculated values, the better the cleaning effect, at the same high dose of ruthenium.
The use of these four cleaning fluids (second cleaning fluid) can reduce the cross contamination rate of the measuring cell to improve the accuracy and sensitivity of Gal-3 detection.
The calculation formula of the carrying pollution rate is as follows:
before improvement:
carryover=93.06 calculated according to the above formula
After improvement:
carryover=30.96 was calculated according to the above formula
From the above carryover contamination detection data, it can be seen that the combined use of these four cleaning fluids significantly (P < 0.05) reduces the carryover contamination rate of the test, which means higher sensitivity and accuracy.
EXAMPLE 3 expiration date and bottle opening expiration date
The compound stabilizer is added into the reagent components, and can obviously prolong the storage period and the bottle opening period of the Gal-3 reagent.
The formula of the stabilizer is as follows:
and (5) qualification standard: the blank limit is less than or equal to 60pg/mL; the linear correlation coefficient r is required to be more than or equal to 0.99; the accuracy is required to meet the deviation within +/-10%; the precision needs to satisfy: CV is less than or equal to 10 percent.
TABLE 3 real-time stability data for reagents without stabilizer added to Gal-3 dilutions
TABLE 4 real-time stability data for reagents after the addition of stabilizers (5% v/v) to Gal-3 dilutions
From the above data, it can be seen that: before the stabilizer is not added, the Gal-3 reagent has a linear correlation coefficient of less than 0.99 in the 15 th month, and the accuracy deviation is more than 10%, which indicates that the reagent is unstable at the moment and the correlation performance is lower than the established index. After the stabilizer is added, the performance of the Gal-3 reagent meets the set index no matter in the 15 th month or the 18 th month, and the addition of the stabilizer has obvious effect on improving the stability of the reagent.
TABLE 5 data on stability to opening of reagents without stabilizer for Gal-3 dilutions
TABLE 6 data on the stability of the reagent in open bottle after the addition of stabilizer (5% v/v) to Gal-3 dilutions
The above data can be seen: before the stabilizer is not added, the Gal-3 reagent has a linear correlation coefficient of less than 0.99 at week 12, and the accuracy deviation is more than 10%, which indicates that the reagent is unstable at this time and the correlation performance is lower than the set index. After the stabilizer is added, the performance of the Gal-3 reagent meets the set index no matter in week 12 or week 24, so that the addition of the stabilizer has obvious effect on improving the uncapping stability of the reagent.
EXAMPLE 4 preparation of biotinylated Gal-3 antibody and reagent Ra
Gal-3 antibody for labeling biotin was purchased from Beijing edge Tian Xin wild technology Co., ltd., product number YT-Gal-3-002, clone number 6E9.
2.0mg of antibody for labeling biotin Gal-3 was used, the buffer was changed to phosphate buffer (pH=7.8) using desalting column PD10, the concentration was adjusted to 2.0mg/mL using ultrafiltration tube, 80. Mu.g of biotin (dissolved using DMF) was added, the reaction was carried out for 30 minutes after mixing, unlabeled biotin was removed using desalting column PD10, the protein concentration of the collected antibody was measured using BCA method, and the kit used was commercial kit of Thermo, accession number 23225. The biotin-labeled Gal-3 antibody was diluted to 1mg/L with a phosphate buffer (pH=7.4) containing 1% of bovine serum albumin as Gal-3 reagent Ra.
EXAMPLE 5 preparation of ruthenium-labeled Gal-3 antibody and reagent Rb
Gal-3 antibody for labeling terpyridyl ruthenium was purchased from Beijing edge Tian Xin wild technology Co., ltd., product number YT-Gal-3-003, clone number 5H4.
2.0mg of Gal-3 antibody for labeling ruthenium terpyridyl was used, the buffer was changed to phosphate buffer (pH=7.8) using desalting column PD10, the concentration was adjusted to 2.0mg/mL using ultrafiltration tube, 80. Mu.g of ruthenium terpyridyl (dissolved using DMF) was added, the reaction was carried out for 30 minutes after mixing, unlabeled ruthenium was removed using desalting column PD10, the protein concentration of the collected antibody was measured using BCA method, and the kit used was commercial kit of Thermo, accession number 23225.. The Gal-3 antibody labeled ruthenium was diluted to 1mg/L with a phosphate buffer solution (pH=7.4) containing 1% bovine serum albumin as Gal-3 reagent Rb.
Example 6 sandwich assay for Gal-3
Gal-3 is measured by a sandwich method, and the detection principle is as follows:
1) Taking 15 mu L of a sample, adding the sample into a reaction tube, sequentially adding 70 mu L of the Gal-3 reagent Ra prepared in the example 4 and 70 mu L of the Gal-3 reagent Rb prepared in the example 5, incubating at 37 ℃ for 9min, and finally adding 40 mu L of streptavidin magnetic microspheres, and incubating at 37 ℃ for 9min;
2) Sucking the reaction mixed liquid pipe after the incubation reaction into an electrochemical flow cell through a liquid suction steel needle, and adsorbing by a magnet of the flow cell;
3) The liquid-absorbing steel needle absorbs the second cleaning liquid, after the antibody of the marked ruthenium which is not combined with the superparamagnetic microsphere and the sample are cleaned, the flow cell is electrified, and the terpyridyl ruthenium emits light under the condition of DBAE.
4) The photomultiplier records the luminescence value, and calculates the concentration of Gal-3 in the sample according to a standard curve established after the curve provided by the enterprise is corrected by using the luminescence value of the calibration product.
Step 1: a sample of 15. Mu.L was added to the reaction tube, followed by the addition of 70. Mu.L of Gal-3 reagent Ra prepared in example 4 and 70. Mu.L of Gal-3 reagent Rb prepared in example 5, incubation at 37℃for 9min, and formation of antigen-antibody immunocomplexes.
Step 2: and adding magnetic bead particles coated with streptavidin for incubation, and allowing the immune complex formed above to be combined to the magnetic bead particles through interaction between biotin and streptavidin to form a reaction mixture.
Step 3: and sucking the formed reaction mixed solution into a measuring pool, adding a second cleaning solution, adsorbing the combined compound on an electrode through magnetic bead particles in the magnet adsorption compound, washing unbound substances by the cleaning solution, applying voltage on the electrode to generate chemiluminescence, and measuring by a photomultiplier to obtain a luminescence value.
After the main curve information is scanned into the instrument through the reagent bar code, scanning the calibration product information card, scanning the information and the concentration of the calibration product into the instrument, and correcting the main curve to obtain a calibration curve. The measured luminescence value is automatically calculated through calibration curve software to obtain a detection result.
Effect example 1 blank test
In the reaction method of example 7, the RLU value (relative luminescence value) of 20 measurement results was obtained by using the zero concentration diluent as a sample, the average value (M) and Standard Deviation (SD) thereof were calculated, and m+2sd was obtained, while samples of adjacent concentrations were repeatedly tested 2 times, and a two-point regression fit was performed according to the concentration-RLU between the zero concentration diluent and the samples of adjacent low concentrations, to obtain a primary equation, and the RLU value of m+2sd was substituted into the above equation, and the corresponding concentration value was obtained as a blank (table 7, table 10).
TABLE 7 blank for the reaction method in example 7
Effect example 2 verification of Linear Range
High value samples near the upper limit of the linear range (114 ng/mL) were diluted in a proportion to at least 5 concentrations in the reaction method of example 7, where low value samples would be near 2ng/mL. And repeatedly detecting the sample with each concentration for 2 times, calculating the average value of the samples to obtain the measured concentration, performing linear fitting on the diluted concentration and the measured concentration by using a least square method, and calculating a linear correlation coefficient r, wherein r is not less than 0.99. The results of the straight line fitting of the diluted concentration to the measured concentration are shown in fig. 1 and table 8.
TABLE 8 blank for the reaction method in example 7
| Project | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 |
| Dilution concentration (ng/mL) | 2 | 20 | 50 | 80 | 114 |
| Measured concentration 1 | 1.92 | 19.56 | 51.26 | 79.88 | 113.56 |
| Measured concentration 2 | 1.96 | 19.74 | 50.48 | 78.24 | 113.48 |
| Measured concentration | 1.94 | 19.65 | 50.87 | 79.06 | 113.52 |
| X-axis | 4 | 20 | 50 | 80 | 114 |
| y-axis | 1.94 | 19.65 | 50.87 | 79.06 | 113.52 |
R 2 ＝0.9995
Comparative example 1 comparison of reaction systems
The electrochemical luminescence immunoassay method in the market at present mostly adopts a reaction system of a pyridine ruthenium compound and Tripropylamine (TPA), such as Roche diagnosis, and the electrochemical luminescence immunoassay method selects the reaction system of the pyridine ruthenium compound and 2-N-dibutylamino ethanol (DBAE). The advantages are as follows:
1. DBAE has better water solubility, and the solubility is about 4g/L at 20 ℃; tripropylamine (TPA) has a solubility of about 2.6g/L at 20 ℃; if a Tripropylamine (TPA) reaction system is adopted, the working concentration of tripropylamine is about 26g/L and is 10 times of the solubility of tripropylamine, so that in order to increase the solubility of Tripropylamine (TPA), a surfactant and phosphoric acid are required to be added, and in addition, a certain stirring time and stirring speed are required to be met, so that the difficulty of a production process is greatly increased.
2. Tripropylamine (TPA) toxicity is classified as high toxic, DBAE toxicity is classified as toxic, and the use of DBAE can reduce the toxicity of the substrate solution, which is more friendly to the health of the inspection workers and the environment.
3. The reaction system of DBAE and ruthenium pyridine has higher luminous efficiency, and the signal test results are shown in FIG. 4 by using 2g/L DBAE buffer solution and 2g/L Tripropylamine (TPA) respectively:
the luminescence intensity of the equivalent concentration of DBAE is about 6 times that of Tripropylamine (TPA).
4. In addition, in the research, we also found that the reaction system using ruthenium pyridine and DBAE can improve the sensitivity of the test, and we respectively detect the sensitivity of serum galectin-3 (Gal-3) by using the two systems, and the test method is as follows: and (3) taking the zero-concentration diluent as a sample to obtain an RLU value (relative luminescence value) of 20 measurement results, calculating an average value (M) and a Standard Deviation (SD) of the RLU value to obtain M+2SD, repeating the test for 2 times by using samples with adjacent concentrations, performing two-point regression fitting according to the concentration-RLU between the zero-concentration diluent and the samples with low concentrations to obtain a primary equation, substituting the RLU value of the M+2SD into the equation, and obtaining the corresponding concentration value, namely the blank limit. The sensitivities of the ruthenium pyridine complex and Tripropylamine (TPA) reaction systems are shown in table 9, and the sensitivities of the ruthenium pyridine complex and Dibutylethanolamine (DBAE) reaction systems are shown in tables 10 and 7.
TABLE 9 sensitivity of ruthenium pyridine complexes and Tripropylamine (TPA) reaction systems
TABLE 10 sensitivity of ruthenium pyridine complex and dibutyl ethanolamine (DBAE) reaction systems
The method claimed in the patent can thus be seen to have a higher sensitivity than conventional methods.
5. Since a reaction system of DBAE and ruthenium pyridine is used, the signal generated by the reaction of 26g/L DBAE and ruthenium pyridine is 3 times that generated by the reaction of 26g/LTPA and ruthenium pyridine, so that a smaller amount of antibody is required to detect the signal.
Protein concentration was determined using BCA method based on antibodies collected in the method of example 5 using a commercial kit from Thermo under the accession number 23225.
Thus, it can be seen that the method claimed in the patent has a more economical antibody than the conventional method.
Comparative example 2 case comparison of kit
The detection method of the Gal-3 magnetic microsphere electrochemiluminescence kit as described in the embodiment 7 comprises the following steps:
1) Adding 15 mu L of a sample into a reaction tube, sequentially adding 70 mu L of Gal-3 reagent Ra and 70 mu L of Gal-3 reagent Rb, incubating at 37 ℃ for 9min, and finally adding 40 mu L of streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 9min;
2) Sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid suction steel needle, and adsorbing by a magnet of the flow cell;
3) The liquid-absorbing steel needle absorbs the second cleaning liquid, after the antibody of the marked ruthenium which is not combined with the superparamagnetic microsphere and the sample are cleaned, the flow cell is electrified, and the terpyridyl ruthenium emits light in the presence of tripropylamine or DBAE.
4) The photomultiplier tube records the luminescence value, and calculates the concentration of Gal-3 in the sample according to the established standard curve.
ELISA method (kit is HumanGalectin-3Immunoassay (catalog number DGAL 30) from R & Dsystem Co.):
1) The required reaction wells were taken, 100 μl of diluent was added, and then serum was added (using diluent 1:20 dilution), 50 μl of standard, and incubating for 120min at room temperature;
2) Removing the incubated liquid, washing for 4 times by using a washing liquid containing a surfactant, adding 200 mu L of Gal-3 horseradish enzyme antibody reagent, and incubating for 120min at room temperature;
3) After removing the incubated liquid and washing 4 times with a washing liquid containing a surfactant, 200. Mu.L of a substrate was added, and after incubation at room temperature for 30 minutes in the absence of light, 50. Mu.L of a stop solution was added.
4) Using an enzyme-labeled instrument, a 450nm filter was selected and the OD value of each sample well was tested.
The concentration of Gal-3 in the sample was calculated from a standard curve established for the standard.
Table 11 summarizes the case of the comparative kits
| This example | Control group 1 | |
| Methodology of | Electrochemical luminescence sandwich method | ELISA test |
| Sensitivity of | 0.29-1.18ng/ml | 2.5ng/ml |
| Linear range | 2-114ng/ml | 3.13-100ng/ml |
| Detection time | 18 minutes | 240 minutes |
| Antibody treatment | 60 minutes | For more than 10 hours |
The steps show that the sandwich method reaction mode adopted by the invention utilizes the principle of magnetic microsphere electrochemistry to quantitatively detect Gal-3 content in human serum or plasma samples, thereby ensuring the detection sensitivity. And is suitable for use in fully automated equipment. The detection speed and the detection flux are increased, the detection efficiency is improved, and meanwhile, errors caused by manual operation are avoided.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A composition comprising an antibody that specifically binds to Gal-3 comprising a biotin label, an antibody that specifically binds to Gal-3 comprising a ruthenium terpyridyl label, and a streptavidin superparamagnetic microsphere;
the particle size of the streptavidin superparamagnetic microsphere is 1.0-6.0 mu m.
2. The composition of claim 1, wherein the particle size of the streptavidin superparamagnetic microspheres comprises 3 μm.
3. The composition of claim 1 or 2, further comprising a buffer;
the buffer solution comprises:
20-200 mmol/L phosphate buffer, pH=7.4; and/or
20mmol/L to 200mmol/L of tris buffer solution, pH=7.4.
4. A composition according to any one of claims 1 to 3, further comprising a first cleaning fluid;
the first cleaning solution comprises 2-N-dibutylamino ethanol;
the first cleaning solution further comprises phosphate buffer solution.
5. The composition of any one of claims 1 to 4, further comprising a stabilizer;
the stabilizer comprises one or more of glycine, methionine, PEG20000, dextran 200000, pullulan or polyvinylpyrrolidone 360.
6. The composition of claim 5, wherein the stabilizer comprises:
7. the composition of claim 5 or 6, wherein the volume ratio of the stabilizer to the buffer is 5:100.
8. the composition of any one of claims 1 to 7, further comprising a second cleaning fluid;
the second cleaning liquid comprises a cleaning liquid 1, a cleaning liquid 2, a cleaning liquid 3 and/or a cleaning liquid 4;
the cleaning liquid 1 comprises isooctanol polyoxyvinyl ether with the polymerization degree of 14, lauryl alcohol polyoxyethylene ether with the polymerization degree of 9 and/or Proclin950;
the cleaning liquid 2 comprises sodium hydroxide, sodium chloride and/or sodium hypochlorite solution;
the cleaning liquid 3 comprises isooctyl alcohol polyoxyvinyl ether with the polymerization degree of 14, laurinol polyoxyethylene ether with the polymerization degree of 9 and/or potassium hydroxide;
the cleaning liquid 4 comprises 85 weight percent of phosphoric acid, dichloroacetamide, isooctyl alcohol polyoxyvinyl ether with the polymerization degree of 14, laurinol polyoxyethylene ether with the polymerization degree of 9, monopotassium phosphate and/or 2-N-dibutylamino ethanol.
9. Use of a composition according to any one of claims 1 to 8 for the preparation of a Gal-3 assay kit.
10. Kit comprising a composition according to any one of claims 1 to 8.
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