CN113125752A - Composition for N-MID osteocalcin detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method - Google Patents

Composition for N-MID osteocalcin detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method Download PDF

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CN113125752A
CN113125752A CN202110407515.3A CN202110407515A CN113125752A CN 113125752 A CN113125752 A CN 113125752A CN 202110407515 A CN202110407515 A CN 202110407515A CN 113125752 A CN113125752 A CN 113125752A
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mid
osteocalcin
reagent
mid osteocalcin
buffer solution
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秦军
谢元东
谢良思
马东钰
王晓宁
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Jiangsu Youni Taike Biotechnology Co ltd
Beijing Unidiag Tech Inc
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Jiangsu Youni Taike Biotechnology Co ltd
Beijing Unidiag Tech Inc
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531Production of immunochemical test materials
    • G01N33/532Production of labelled immunochemicals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326Magnetic particles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577Immunoassay; 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/04Endocrine or metabolic disorders
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/10Musculoskeletal or connective tissue disorders
    • G01N2800/108Osteoporosis

Abstract

The invention relates to the field of electrochemical detection, in particular to a composition for detecting N-MID osteocalcin (N-MIDOsteocalcin), application thereof, a magnetic microsphere electrochemical luminescence immunoassay kit and a detection method. The method adopted by the invention is an electrochemical luminescence method, and the ruthenium pyridine adopted as the chemiluminescent marker has obvious advantages, which are mainly shown in that: the sensitivity is better, the stability is better, ruthenium is metal ion, the molecular weight is small, and the steric hindrance of the antibody is not influenced. Short production process, good repeatability and wide detection range. The electrochemical luminescence reaction is controllable, and the signal acquisition difficulty is reduced.

Description

Composition for N-MID osteocalcin detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method
Technical Field
The invention relates to the field of electrochemical detection, in particular to a composition for detecting N-MID Osteocalcin (N-MID osteopalcin), application of the composition, a magnetic microsphere electrochemiluminescence immunoassay kit containing the composition, and a magnetic microsphere electrochemiluminescence immunoassay method based on the composition or the kit.
Background
N-MID Osteocalcin (N-MID Osteocalcin) is a non-collagen bone matrix protein secreted by mature osteoblasts, and accounts for 25% of non-collagen components in a bone matrix and 2% of total bone protein. Intact osteocalcin consists of 49 amino acids and has a relative molecular mass of 5800. Osteocalcin in serum has diversity, about 1/3 is intact osteocalcin (whole fragment osteocalcin), 1/3 is osteocalcin N-terminal middle molecule (N-MID) fragment, and 1/3 is amino acid short peptide. Osteocalcin is a specific marker of osteoblast function and bone mineralization, and is increasingly used clinically as a marker of bone turnover. N-MID is a specific non-collagen protein important in bone matrix, the binding effect of bone calcium depends on vitamin K, and the content of the protein is related to the bone conversion rate in various bone metabolic disorders. Abnormal N-MID content is often found in osteoporosis, primary or secondary hyperparathyroidism, etc.
Up to now, the methods for detecting the content of N-MID Osteocalcin (N-MID osteoalcin) in human serum samples mainly include: enzyme linked immunosorbent assay (ELISA) and enzymatic magnetic particle chemiluminescence. The method adopted by the invention is an electrochemical luminescence method, and the ruthenium pyridine adopted as the chemiluminescent marker has obvious advantages, which are mainly shown in that: the sensitivity is better, the stability is better, ruthenium is metal ion, the molecular weight is small, and the steric hindrance of the antibody is not influenced. Short production process, wide detection range and good repeatability. The electrochemical luminescence reaction is controllable, and the signal acquisition difficulty is reduced.
Disclosure of Invention
The invention provides a composition for detecting N-MID Osteocalcin (N-MID Osteocalcin), application thereof, a magnetic microsphere electrochemiluminescence immunoassay kit and a detection method, and has the advantages of high production efficiency, short detection time, suitability for full-automatic detection, higher sensitivity, wide linear range and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a composition for detecting N-MID Osteocalcin (N-MID Osteocalcin), which comprises an N-MID Osteocalcin reagent Ra, an N-MID Osteocalcin reagent Rb and streptavidin superparamagnetic microspheres;
the N-MID Osteocalcin reagent Ra comprises an anti-N-MID Osteocalcin monoclonal antibody containing a biotin label;
the N-MID Osteocalcin reagent Rb comprises an anti-N-MID Osteocalcin monoclonal antibody containing a terpyridyl ruthenium marker;
the streptavidin superparamagnetic microspheres comprise superparamagnetic microspheres with streptavidin coated on the surfaces.
In some embodiments of the invention, the superparamagnetic microspheres have a particle size of 1.5 to 5.0 μm.
In some embodiments of the invention, the amount of the biotin molecular marker on the surface of each antibody molecule in the N-MID osteopecalin reagent Ra is 2-5; in the N-MID Osteocalcin reagent Rb, the number of ruthenium molecular markers on the surface of each antibody molecule is 2-10.
In some embodiments of the present invention, the N-MID Osteocalcin reagent Ra is prepared by the following steps: mixing an anti-N-MID Osteocalcin monoclonal antibody with biotin in the presence of a buffer solution to prepare an N-MID Osteocalcin reagent Ra; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.
In some embodiments of the present invention, the N-MID Osteocalcin reagent Ra is prepared by the following steps: 2.0mg of an antibody for labeling biotin N-MID Osteocalcin (N-MID Osteocalcin) was taken, the buffer was changed to a phosphate buffer (pH 7.8) using a desalting column PD10, the mixture was concentrated using an ultrafiltration tube and then adjusted to a concentration of 2.0mg/mL, 80 μ g of biotin (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled biotin was removed using a desalting column PD 10. An N-MID Osteocalcin (N-MID Osteocalcin) antibody labeled with biotin was diluted to 1mg/L with a phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as an N-MID Osteocalcin reagent Ra.
In some embodiments of the invention, the N-MID Osteocalcin reagent Rb is prepared by the following method: mixing an anti-N-MID Osteocalcin monoclonal antibody with terpyridyl ruthenium in the presence of a buffer solution to prepare an N-MID Osteocalcin reagent Rb; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.
In some embodiments of the invention, the N-MID Osteocalcin reagent Rb is prepared by the following method: 2.0mg of an N-MID Osteocalcin (N-MID osteopalcin) antibody for labeling ruthenium terpyridyl was taken, the buffer was changed to a phosphate buffer (pH 7.8) using a desalting column PD10, the mixture was concentrated using an ultrafiltration tube and then adjusted to a concentration of 2.0mg/mL, 30 to 80. mu.g of succinamide ruthenium terpyridyl (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled ruthenium was removed using a desalting column PD 10. Ruthenium-labeled N-MID Osteocalcin (N-MID Osteocalcin) antibody was diluted to 1mg/L with 1% bovine serum albumin-containing phosphate buffer (pH 7.4) as N-MID Osteocalcin reagent Rb.
In some embodiments of the invention, the compositions provided herein further comprise a taggant and/or a cleaning solution; the cleaning solution comprises tripropylamine with the concentration of 150-200 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L; or dibutylethanolamine with the concentration of 80-100 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L.
In some embodiments of the present invention, the cleaning solution includes, but is not limited to, a tripropylamine cleaning solution, a dibutylethanolamine cleaning solution, a pipeline cleaning solution.
In some more specific embodiments of the present invention, the cleaning solution comprises tripropylamine at a concentration of 180mmol/L and a phosphate buffer at a concentration of 300 mmol/L; or dibutylethanolamine at a concentration of 90mmol/L and phosphate buffer at a concentration of 300 mmol/L.
In some embodiments of the invention, the volume ratio of the N-MID Osteocalcin reagent Ra, the N-MID Osteocalcin reagent Rb to the streptavidin superparamagnetic microspheres is (50-80): (50-80): (20-40).
On the basis of the research, the invention also provides the application of the composition in preparing a magnetic microsphere electrochemiluminescence immunoassay kit of N-MID Osteocalcin (N-MID Osteocalcin).
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay kit of N-MID Osteocalcin (N-MID osteopalcin), which comprises the composition and a reagent acceptable for detection.
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay method of N-MID Osteocalcin (N-MID osteopalcin), which is based on the composition or the kit and comprises the following steps:
step 1: taking a sample, sequentially adding an N-MID Osteocalcin reagent Ra and an N-MID Osteocalcin reagent Rb, incubating at 37 ℃ for 8-12 min, finally adding streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 8-12 min to obtain a reaction solution; wherein the volume ratio of the sample, the N-MID Osteocalcin reagent Ra, the N-MID Osteocalcin reagent Rb to the streptavidin superparamagnetic microspheres is 15: (50-80):
(50~80):(20~40);
step 2: adsorbing the reaction solution by using a magnet;
and step 3: taking a cleaning solution, cleaning the ruthenium-labeled antibody and the sample which are not bonded to the superparamagnetic microspheres, electrifying, and enabling the terpyridyl ruthenium to emit light under the condition of the presence of the cleaning solution;
and 4, step 4: and recording the luminous value, establishing a standard curve, and obtaining the concentration of the N-MID Osteocalcin in the sample according to the established standard curve.
In some embodiments of the invention, the incubation is at 37 ℃ for 9 min.
In some embodiments of the present invention, the detection method specifically comprises:
step 1: adding 20 μ l of sample into reaction tube, sequentially adding N-MID Osteocalcin reagent Ra and N-MID Osteocalcin reagent Rb, incubating at 37 deg.C for 9min, adding streptavidin superparamagnetic microsphere, and incubating at 37 deg.C for 9 min;
step 2: sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid absorption steel needle, and adsorbing the reaction tube by a magnet of the flow cell;
and step 3: and (3) sucking a cleaning solution (tripropylamine or DBAE) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium under the condition that the tripropylamine or DBAE exists.
And 4, step 4: recording the luminous value by the photomultiplier, establishing a standard curve, and calculating the concentration of the N-MID Osteocalcin in the sample according to the established standard curve.
The magnetic particles can be used as carriers of biological macromolecules, the antibody-coated magnetic particles are called immune magnetic particles, and the immune magnetic particles have the characteristics of antigen combination and magnetism, so that the immune 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 comprise rapidness, strong specificity, simple and convenient operation, wide application range and the like. The nanometer material is a new material which is rapidly developed after 90 years in the 20 th century, and the nanometer magnetic particles (the particle size is less 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; the magnetic material has superparamagnetism, and the magnetic interaction is weak; it can move directionally under the action of external magnetic field to separate, concentrate or purify some special components. 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 a detection result.
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 results of a straight line fit of the diluted concentration to the measured concentration in the reaction method of example 8- (1);
FIG. 2 shows the results of straight line fitting of the diluted concentration to the measured concentration in the reaction method of example 8- (2).
Detailed Description
The invention discloses a magnetic microsphere electrochemiluminescence immunoassay kit for detecting N-MID Osteocalcin (N-MID Osteocalcin), which can be realized by appropriately improving process parameters by referring to the content of the kit by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides the following technical scheme: an N-MID Osteocalcin magnetic microsphere electrochemiluminescence kit, comprising: N-MID Osteocalcin reagent Ra, N-MID Osteocalcin reagent Rb, streptavidin superparamagnetic microspheres, a calibration product, tripropylamine cleaning solution, dibutylethanolamine cleaning solution and pipeline cleaning solution.
The N-MID Osteocalcin magnetic microsphere electrochemiluminescence kit comprises an N-MID Osteocalcin reagent Ra, a biotin-labeled anti-N-MID Osteocalcin monoclonal antibody, wherein the number of biotin molecular markers on the surface of each antibody molecule is 2-5, the buffer solution is 20 mM-200 mM phosphate buffer solution, the pH is 7.4-7.8 or 20 mM-200 mM tris (hydroxymethyl) aminomethane buffer solution, and the pH is 7.4-7.8. The N-MID Osteocalcin reagent Rb is an anti-N-MID Osteocalcin monoclonal antibody containing a terpyridyl ruthenium marker, the number of ruthenium molecular markers on the surface of each antibody molecule is 2-10, the buffer solution is 20 mM-200 mM phosphate buffer solution, the pH value is 7.4-7.8 or 20 mM-200 mM tris (hydroxymethyl) aminomethane buffer solution, and the pH value is 7.4-7.8.
The N-MID Osteocalcin magnetic microsphere electrochemiluminescence kit comprises a streptavidin superparamagnetic microsphere, a magnetic microsphere and a magnetic particle coating buffer solution, wherein the surface of the streptavidin superparamagnetic microsphere is coated with the streptavidin superparamagnetic microsphere, the particle size of the magnetic microsphere is 1.5-5.0 micrometers, the magnetic particle coating buffer solution is 20 mM-200 mM phosphate buffer solution, the pH value is 7.4-7.8 or 20 mM-200 mM tris buffer solution, and the pH value is 7.4-7.8.
The N-MID Osteocalcin magnetic microsphere electrochemiluminescence kit comprises a kit body, a kit body and a kit body, wherein the kit body is used for carrying out electrochemiluminescence on the N-MID Osteocalcin magnetic microsphere electrochemiluminescence kit body.
The detection method of the N-MID Osteocalcin magnetic microsphere electrochemiluminescence kit comprises the following steps of (1) taking tripropylamine with the concentration of 180mmol/L as a cleaning solution, wherein the tripropylamine with the concentration of 300mmol/L contains a phosphate buffer solution; or 90mmol/L dibutylethanolamine containing phosphate buffer solution with concentration of 300 mmol/L.
The invention provides a detection method of an N-MID Osteocalcin magnetic microsphere electrochemiluminescence kit, which comprises the following steps:
1) adding the sample into a reaction tube, sequentially adding an N-MID Osteocalcin reagent Ra and an N-MID Osteocalcin reagent Rb, incubating at 37 ℃ for 9min, finally adding streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 9 min;
2) sucking the reaction tube after the incubation reaction into an electrochemical flow cell through a liquid absorption steel needle, and adsorbing the reaction tube by a magnet of the flow cell;
3) and (3) sucking a cleaning solution (tripropylamine or DBAE) by a liquid suction steel needle, cleaning the ruthenium-labeled antibody which is not bound to the superparamagnetic microspheres and the sample, electrifying the flow cell, and emitting light by the terpyridyl ruthenium under the condition that the tripropylamine or DBAE exists.
4) Recording the luminous value by the photomultiplier, establishing a standard curve, and calculating the concentration of the N-MID Osteocalcin in the sample according to the established standard curve.
The streptavidin and the biotin have high-specificity binding capacity, and the streptavidin and the biotin-labeled high-purity antibody are specifically bound through non-covalent bonds, so that the streptavidin-labeled high-purity antibody has the effect of cascade amplification, and the reaction is highly specific. Therefore, the sensitivity is improved, non-specific interference is not increased, and the binding property is not affected by the high dilution of the reaction reagent, so that the non-specific action of the reaction reagent can be reduced to the maximum extent in practical application.
The invention combines the high specificity of antibody-antigen reaction with the high sensitivity of ruthenium terpyridyl luminescence, utilizes the photons generated by ruthenium terpyridyl under tripropylamine or DBAE to detect the product concentration, and has the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.
The raw materials and reagents used in the magnetic microsphere electrochemiluminescence immunoassay kit for detecting N-MID Osteocalcin (N-MID osteopalcin) provided by the invention are all available in the market.
All components of the test kit of the present invention can be commercially obtained from biological or chemical reagents companies. The device used was a full-automatic chemiluminescence immunoassay analyzer (model UD90DT) manufactured by tek technologies ltd, yokyo.
The invention is further illustrated by the following examples:
example 1: preparation of biotin-labeled N-MID Osteocalcin (N-MID osteoalcin) antibody and reagent Ra
The N-MID Osteocalcin (N-MID Osteocalcin) antibody for labeling biotin is purchased from Beijing edge Tianxin Yechow science and technology Co., Ltd, and has the product number of YT-N-MID Osteocalcin-002 and the clone number of 6F 4.
2.0mg of an antibody for labeling biotin N-MID Osteocalcin (N-MID Osteocalcin) was taken, the buffer was changed to a phosphate buffer (pH 7.8) using a desalting column PD10, the mixture was concentrated using an ultrafiltration tube and then adjusted to a concentration of 2.0mg/mL, 80 μ g of biotin (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled biotin was removed using a desalting column PD 10. An N-MID Osteocalcin (N-MID Osteocalcin) antibody labeled with biotin was diluted to 1mg/L with a phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as an N-MID Osteocalcin reagent Ra. The number of biotin molecular markers on the surface of each antibody molecule is 2-3.
Example 2: preparation of ruthenium-labeled N-MID Osteocalcin (N-MID osteoalcin) antibody and reagent Rb
The N-MID Osteocalcin (N-MID Osteocalcin) antibody for labeling biotin is purchased from Beijing edge Tianxin Yechow science and technology Co., Ltd, with the product number of YT-N-MID Osteocalcin-003 and the clone number of 1E 2.
2.0mg of an N-MID Osteocalcin (N-MID Osteocalcin) antibody for labeling ruthenium terpyridyl was taken, the buffer was changed to a phosphate buffer (pH 7.8) using a desalting column PD10, the mixture was concentrated using an ultrafiltration tube and adjusted to a concentration of 2.0mg/mL, 80. mu.g of succinamide ruthenium terpyridyl (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled ruthenium was removed using a desalting column PD 10. Ruthenium-labeled N-MID Osteocalcin (N-MID Osteocalcin) antibody was diluted to 1mg/L with 1% bovine serum albumin-containing phosphate buffer (pH 7.4) as N-MID Osteocalcin reagent Rb. The number of the ruthenium molecular markers on the surface of each antibody molecule is 5-6.
Example 3: preparation of the calibration articles
The antigen for preparing the calibration sample is purchased from Ji-shi limited of Tianxin, Beijing, with the product number YT-N-MID Osteocalcin-001. For recombinant expression of the protein.
The antigen was diluted to 3.5ng/mL and 160ng/mL at the indicated concentrations using 1% bovine serum albumin in phosphate buffer (pH 7.4). Used as a calibrator for establishing a standard curve.
Example 4: preparation of tripropylamine cleaning solution and dibutylethanolamine cleaning solution
300mmol/L phosphate buffer solution is prepared, tripropylamine is added to 180mmol/L, and the mixture is mixed and dissolved. As a tripropylamine cleaning solution.
Preparing 300mmol/L phosphate buffer solution, adding dibutyl ethanolamine to 90mmol/L, and mixing and dissolving. As a cleaning solution of dibutylethanolamine.
Example 5:
the determination of N-MID Osteocalcin (N-MID Osteocalcin) adopts a sandwich method, and the detection method comprises the following steps:
1) adding 20 μ l of sample into a reaction tube, sequentially adding 80 μ l of N-MID Osteocalcin reagent Ra prepared in example 1 and 75 μ l of N-MID Osteocalcin reagent Rb prepared in example 2, incubating at 37 deg.C for 9min, adding 30 μ l of streptavidin superparamagnetic microsphere (particle size of 3.0 μm), and incubating at 37 deg.C for 9 min;
2) the reaction mixed liquid pipe after the incubation reaction is sucked into the measuring tank through a liquid suction steel needle, and the superparamagnetic microspheres are adsorbed through a magnet;
3) a liquid-absorbing steel needle absorbs the cleaning solution (dibutylethanolamine), and a mark Ru (bpy) which is not combined on the superparamagnetic microspheres3 2+After the antibody and the sample were washed, the flow cell was charged, and Ru (bpy) was performed in the presence of dibutylethanolamine3 2+And (4) emitting light.
4) The photomultiplier tube records the luminous value, and the concentration of N-MID Osteocalcin in the sample is calculated according to a standard curve established after the calibration is carried out by using the luminous value of the calibration sample.
Example 6:
the determination of N-MID Osteocalcin (N-MID Osteocalcin) adopts a sandwich method, and the detection method comprises the following steps:
1) adding 20 μ l of sample into a reaction tube, sequentially adding 80 μ l of N-MID Osteocalcin reagent Ra prepared in example 1 and 75 μ l of N-MID Osteocalcin reagent Rb prepared in example 2, incubating at 37 deg.C for 9min, adding 30 μ l of streptavidin superparamagnetic microsphere (particle size of 3.0 μm), and incubating at 37 deg.C for 9 min;
2) the reaction mixed liquid pipe after the incubation reaction is sucked into the measuring tank through a liquid suction steel needle, and the superparamagnetic microspheres are adsorbed through a magnet;
3) a liquid-absorbing steel needle absorbs the cleaning solution (dibutylethanolamine), and a mark Ru (bpy) which is not combined on the superparamagnetic microspheres3 2+After the antibody and the sample were washed, the flow cell was charged, and Ru (bpy) was performed in the presence of dibutylethanolamine3 2+And (4) emitting light.
4) The photomultiplier tube records the luminous value, and the concentration of N-MID Osteocalcin in the sample is calculated according to a standard curve established after the calibration is carried out by using the luminous value of the calibration sample.
Example 7: margin test
(1) The reaction method in example 5 was used, and the RLU value (relative luminescence value) of 20 measurements was obtained using the zero-concentration diluent as the sample, and the average (M) and Standard Deviation (SD) thereof were calculated to obtain M +2SD, and at the same time, samples of adjacent concentrations were repeatedly tested 2 times, and two-point regression fitting was performed according to the concentration-RLU between the zero-concentration diluent and the adjacent low-concentration samples to obtain a linear equation, and the RLU value of M +2SD was substituted into the above equation to obtain the corresponding concentration value, which was the margin.
TABLE 1
Figure BDA0003022871830000091
(2) The reaction method in example 6 was used, and a zero-concentration diluent was used as a sample to obtain RLU values (relative luminescence values) of 20 measurements, and the average (M) and Standard Deviation (SD) thereof were calculated to obtain M +2SD, and samples of adjacent concentrations were repeatedly tested 2 times, and two-point regression fitting was performed according to the concentration-RLU between the zero-concentration diluent and the adjacent low-concentration samples to obtain a linear equation, and the RLU values of M +2SD were substituted into the above equation to obtain the corresponding concentration value, which was the blank limit.
TABLE 2
Figure BDA0003022871830000101
Example 8: verification of linear range
(1) Using the reaction method of example 5, high-value samples near the upper limit of the linear range (300ng/ml) were diluted to at least 5 concentrations in a proportion in which the low-value samples had to be near 0.5 ng/ml. And (3) repeatedly detecting the samples with each concentration for 2 times, calculating the average value of the samples to obtain the measured concentration, performing straight line 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 a straight line fit of the diluted concentrations to the measured concentrations are shown in figure 1.
TABLE 3
Figure BDA0003022871830000102
Figure BDA0003022871830000111
(2) The reaction method of example 6, wherein the high value sample near the upper limit of the linear range (300ng/ml) is diluted to at least 5 concentrations in a certain proportion, and the low value sample is near 0.5 g/ml. And operating according to the kit specification, repeatedly detecting the sample with each concentration for 2 times, calculating the average value of the sample 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 the r is not less than 0.99. The results of a straight line fit of the diluted concentrations to the measured concentrations are shown in figure 2.
TABLE 4
Item Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
Concentration of dilution 2 76.98 156.4 220.4 289.6
Measured concentration 1 1.95 77.51 158.2 218.9 293.6
Measured concentration 2 1.93 72.15 155.9 227.9 286.9
Measured concentration 1.940 74.830 157.050 223.4 290.25
Figure BDA0003022871830000112
Summary of the comparative kit conditions:
TABLE 5
Examples 7 to 8 Comparative example 1 Comparative example 2
Methodology of Electrochemiluminescence sandwich method Enzymatic chemiluminescence method Enzyme linked immunosorbent assay
Sensitivity of the probe 0.5ng/ml 2ng/ml 2ng/ml
Linear range 2.0-300ng/ml 2-200ng/ml 5-200ng/ml
Time of detection 18 minutes 20 minutes 120 minutes
Antibody treatment 60 minutes Greater than 10 hours Greater than 10 hours
The electrochemical luminescence immunoassay technology has the advantages of high sensitivity, rapidness, accuracy, good repeatability, safety, no toxicity, no pollution and the like. Luminol, isoluminol and its derivatives are the first type of chemiluminescent species used, but their application to chemiluminescent immunoassays requires the use of catalysts and enhancers, which leads to an increase in background luminescence, thereby limiting the sensitivity of this technology and its application and development. Simple acridinium ester luminescent systemWithout catalyst, in H2O2The acridinium ester can emit light in the solution without a catalytic process or an enhancer, so background light emission is reduced, sensitivity is improved, interference effect is small, but the acridinium ester is easy to hydrolyze, and the light emission of the acridinium ester is released rapidly. The peak value of luminescence is 0.4s, so in-situ sample injection is needed, and the requirement on equipment is high. The ruthenium terpyridyl is easy to be connected with protein, has small molecular weight, has small influence on the conformation of the connected antibody, and has good stability because the marker is metal ions and controllable luminescence because the luminescence is required under the condition of electrification. Therefore, the application of the electrochemical method in the detection of the N-MID Osteocalcin can improve the sensitivity of the product, shorten the process marking time, improve the linear range and shorten the test time, and provides a basis for clinically dealing with the treatment of the brain trauma in time.
The property of the electrochemiluminescence marker ruthenium pyridine is very stable, and the luminous efficiency of the electrochemiluminescence marker ruthenium pyridine is not influenced by factors such as temperature, pH and ionic strength. The signal value of the electrochemiluminescence reagent is reduced within 3 percent compared with that of a fresh reagent. The bottle opening period is three months, and the bottle can be stabilized at 2-8 ℃ for more than 15 months.
TABLE 6
Light-emitting system Horseradish enzyme-luminol Alkaline phosphatase Electrochemiluminescence
Time stamping Greater than 24 hours Greater than 24 hours 60 minutes
Test time 60 minutes 30 minutes 18 minutes
Expiration date of reagent 12 months old 12 months old More than 15 months
The electrochemical labeling reaction is rapid, and the whole reaction only needs half an hour. The marking efficiency reaches 70%. The proportion of the marks can be controlled by the feed ratio, and the productivity is improved by over 50 percent. Ruthenium has small molecular weight (800D), small steric hindrance and good antibody activity. An absorption peak at 455nm, the feed ratio can be controlled to control the batch-to-batch difference.
The results show that the reaction mode of the sandwich method adopted by the invention utilizes the principle of magnetic microsphere electrochemistry to quantitatively detect the content of N-MID Osteocalcin (N-MID osteopalcin) in human serum or plasma samples, thereby ensuring the detection sensitivity. And is suitable for use in fully automatic equipment. The detection speed and the detection flux are increased, the detection efficiency is improved, and errors caused by manual operation are avoided.
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to those examples; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (10)

1. The composition for detecting N-MID Osteocalcin (N-MID Osteocalcin) is characterized by comprising an N-MID Osteocalcin reagent Ra, an N-MID Osteocalcin reagent Rb and streptavidin superparamagnetic microspheres;
the N-MID Osteocalcin reagent Ra comprises an anti-N-MID Osteocalcin monoclonal antibody containing a biotin label;
the N-MID Osteocalcin reagent Rb comprises an anti-N-MID Osteocalcin monoclonal antibody containing a terpyridyl ruthenium marker;
the streptavidin superparamagnetic microspheres comprise superparamagnetic microspheres with streptavidin coated on the surfaces.
2. The composition of claim 1, wherein the superparamagnetic microspheres have a particle size of 1.5 to 5.0 μm.
3. The composition of claim 1, wherein the amount of biotin molecular markers on the surface of each antibody molecule in the N-MID Osteocalcin reagent Ra is 2 to 5; in the N-MID Osteocalcin reagent Rb, the number of ruthenium molecular markers on the surface of each antibody molecule is 2-10.
4. A composition according to any one of claims 1 to 3, wherein the N-MID Osteocalcin reagent Ra is prepared by: mixing an anti-N-MID Osteocalcin monoclonal antibody with biotin in the presence of a buffer solution to prepare an N-MID Osteocalcin reagent Ra; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.
5. A composition according to any one of claims 1 to 3, wherein the N-MID Osteocalcin reagent Rb is prepared by: mixing an anti-N-MID Osteocalcin monoclonal antibody with terpyridyl ruthenium in the presence of a buffer solution to prepare an N-MID Osteocalcin reagent Rb; the buffer solution comprises a phosphate buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM or a tris (hydroxymethyl) aminomethane buffer solution with the pH value of 7.4-7.8 and the pH value of 20 mM-200 mM.
6. A composition according to any one of claims 1 to 3, further comprising a standard and/or a cleaning solution; the cleaning solution comprises tripropylamine with the concentration of 150-200 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L; or dibutylethanolamine with the concentration of 80-100 mmol/L and phosphate buffer solution with the concentration of 200-400 mmol/L.
7. The composition of any one of claims 1 to 3, wherein the volume ratio of the N-MID Osteocalcin reagent Ra, the N-MID Osteocalcin reagent Rb to the streptavidin superparamagnetic microspheres is (50-80): (50-80): (20-40).
8. Use of a composition according to any one of claims 1 to 7 in the preparation of a magnetic microsphere electrochemiluminescence immunoassay kit for N-MID Osteocalcin (N-MID osteoalcin).
9. A magnetic microsphere electrochemiluminescence immunoassay kit for N-MID Osteocalcin (N-MID osteoalcin), comprising the composition according to any one of claims 1 to 7 and a reagent acceptable for detection.
10. A magnetic microsphere electrochemiluminescence immunoassay method of N-MID Osteocalcin (N-MID osteoalcin), based on the composition according to any one of claims 1 to 7 or the kit according to claim 9, comprising the steps of:
step 1: taking a sample, sequentially adding an N-MID Osteocalcin reagent Ra and an N-MID Osteocalcin reagent Rb, incubating at 37 ℃ for 8-12 min, finally adding streptavidin superparamagnetic microspheres, and incubating at 37 ℃ for 8-12 min to obtain a reaction solution; wherein the volume ratio of the sample, the N-MID Osteocalcin reagent Ra, the N-MID Osteocalcin reagent Rb to the streptavidin superparamagnetic microspheres is 15: (50-80): (50-80): (20-40);
step 2: adsorbing the reaction solution by using a magnet;
and step 3: taking a cleaning solution, cleaning the ruthenium-labeled antibody and the sample which are not bonded to the superparamagnetic microspheres, electrifying, and enabling the terpyridyl ruthenium to emit light under the condition of the presence of the cleaning solution;
and 4, step 4: and recording the luminous value, establishing a standard curve, and obtaining the concentration of the N-MID Osteocalcin in the sample according to the established standard curve.
CN202110407515.3A 2021-04-15 2021-04-15 Composition for N-MID osteocalcin detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method Pending CN113125752A (en)

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JPH0658932A (en) * 1992-08-12 1994-03-04 Yamasa Shoyu Co Ltd Measuring method of active osteocalcine
JPH0755804A (en) * 1993-08-06 1995-03-03 Yamasa Shoyu Co Ltd Measuring method for active osteocalcine
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CN106353506A (en) * 2016-08-12 2017-01-25 江苏泽成生物技术有限公司 Kit for detecting osteocalcin content and testing method thereof
CN107831314A (en) * 2017-10-31 2018-03-23 太原瑞盛生物科技有限公司 A kind of N middle-ends BGP chemiluminescence detection kit and preparation method thereof
CN109061175A (en) * 2018-06-29 2018-12-21 江南大学 A kind of kit and application method detecting osteocalcin
CN112649616A (en) * 2020-12-30 2021-04-13 北京联众泰克科技有限公司 Composition for detecting cardiac troponin I, application thereof, magnetic microsphere electrochemiluminescence immunoassay kit and detection method

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US5506111A (en) * 1989-02-10 1996-04-09 Teijin Limited Method of immunological assaying of human osteocalcin, reagent and kit therefor, antibody to human osteocalcin, hybridoma producing said antibody, and method of producing it
JPH0658932A (en) * 1992-08-12 1994-03-04 Yamasa Shoyu Co Ltd Measuring method of active osteocalcine
JPH0755804A (en) * 1993-08-06 1995-03-03 Yamasa Shoyu Co Ltd Measuring method for active osteocalcine
CN106353506A (en) * 2016-08-12 2017-01-25 江苏泽成生物技术有限公司 Kit for detecting osteocalcin content and testing method thereof
CN107831314A (en) * 2017-10-31 2018-03-23 太原瑞盛生物科技有限公司 A kind of N middle-ends BGP chemiluminescence detection kit and preparation method thereof
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