CN112946301A - Composition for detecting special beta-collagen sequence, application thereof, magnetic microsphere electrochemiluminescence immunoassay kit and detection method - Google Patents
Composition for detecting special beta-collagen sequence, application thereof, magnetic microsphere electrochemiluminescence immunoassay kit and detection method Download PDFInfo
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Abstract
The invention relates to the field of electrochemical detection, in particular to a composition for detecting a beta-collagen special sequence (beta-Crosslaps), 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
Technical Field
The invention relates to the field of electrochemical detection, in particular to a composition for detecting a beta-collagen special sequence (beta-Crosslaps), 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
The content of type I collagen in the organic components of the bone matrix is over 90% 1. There is catabolic and anabolic regulation of this basic substance in bone. The mature type I collagen is degraded during normal bone metabolism and the small fragments formed are excreted through the kidneys after entering the bone bloodstream. When physiological or pathological bone resorption is enhanced (such as in the elderly or osteoporosis), the degradation of type I collagen is increased, and the content of collagen fragments in blood is correspondingly increased. Osteoclast activity can be measured by detecting these markers of bone resorption. A particularly relevant type I collagen fragment is the β -isomerized C-terminal peptide (β -CTx). These isomerized telopeptides are highly specific for type I collagen degradation products that are predominantly present in bone.
To date, the methods used to detect β -Crosslaps in human serum are mainly: 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 a beta-collagen special sequence (beta-Crosslaps), 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 a beta-collagen special sequence (beta-Crosslaps), which comprises a beta-Crosslaps reagent Ra, a beta-Crosslaps reagent Rb and a streptavidin superparamagnetic microsphere;
the beta-Crosslaps reagent Ra comprises an anti-beta-Crosslaps monoclonal antibody containing a biotin label;
the beta-Crosslaps reagent Rb comprises an anti-beta-Crosslaps monoclonal antibody marked by terpyridyl ruthenium;
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 biotin molecular markers on the surface of each antibody molecule in the beta-Crosslaps reagent Ra is 2-5; in the beta-Crosslaps reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.
In some embodiments of the present invention, the preparation method of the β -Crosslaps reagent Ra is: mixing an anti-beta-Crosslaps monoclonal antibody with biotin in the presence of a buffer solution to prepare a beta-Crosslaps 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 preparation method of the β -Crosslaps reagent Ra is: 2.0mg of an antibody for labeling a biotin β -collagen specific sequence (. beta. -Crosslaps) was taken, the buffer was changed to a phosphate buffer (pH 7.8) using desalting column PD10, the mixture was concentrated using an ultrafiltration tube and adjusted to a concentration of 2.0mg/mL, 80. mu.g of biotin (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled biotin was removed using desalting column PD 10. Beta-collagen specific sequence (beta-Crosslaps) antibody labeled biotin was diluted to 1mg/L with phosphate buffer (pH 7.4) containing 1% bovine serum albumin as beta-Crosslaps reagent Ra.
In some embodiments of the invention, the beta-Crosslaps reagent Rb is prepared by: mixing an anti-beta-Crosslaps monoclonal antibody with terpyridyl ruthenium in the presence of a buffer solution to prepare a beta-Crosslaps 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 beta-Crosslaps reagent Rb is prepared by: 2.0mg of a β -collagen specific sequence (β -Crosslaps) 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, 80 μ 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. As the β -Crosslaps reagent Rb, a ruthenium-labeled β -collagen specific sequence (β -Crosslaps) antibody was diluted to 1mg/L with a phosphate buffer (pH 7.4) containing 1% bovine serum albumin.
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 beta-Crosslaps reagent Ra, the beta-Crosslaps reagent Rb to the streptavidin superparamagnetic microspheres is (50-80): (50-80): (20-40).
On the basis of the research, the invention also provides application of the composition in preparing a magnetic microsphere electrochemiluminescence immunoassay kit of a beta-collagen special sequence (beta-Crosslaps).
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay kit of the beta-collagen special sequence (beta-Crosslaps), which comprises the composition and a reagent acceptable for detection.
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay method of beta-collagen special sequences (beta-Crosslaps), which is based on the composition or the kit and comprises the following steps:
step 1: taking a sample, sequentially adding a beta-Crosslaps reagent Ra and a beta-Crosslaps 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 beta-Crosslaps reagent Ra, the beta-Crosslaps 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 beta-Crosslaps 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 15 μ l of sample into a reaction tube, sequentially adding 70 μ l of beta-Crosslaps reagent Ra and 70 μ l of beta-Crosslaps reagent Rb, incubating at 37 ℃ for 9min, finally adding 40 μ l of streptavidin superparamagnetic microspheres, and incubating at 37 ℃ 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 beta-Crosslaps 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 a beta-collagen special sequence (beta-Crosslaps), which can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. 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: a beta-Crosslaps magnetic microsphere electrochemiluminescence kit comprises: beta-Crosslaps reagent Ra, beta-Crosslaps reagent Rb, streptavidin superparamagnetic microspheres, a calibration product, tripropylamine cleaning solution, dibutylethanolamine cleaning solution and pipeline cleaning solution.
The beta-Crosslaps magnetic microsphere electrochemiluminescence kit comprises a beta-Crosslaps reagent Ra, a biotin-labeled anti-beta-Crosslaps monoclonal antibody, wherein the biotin molecular labeling amount of each antibody molecule surface is 2-5, 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 beta-Crosslap reagent Rb is an anti-beta-Crosslap monoclonal antibody containing a terpyridyl ruthenium mark, the marking amount of ruthenium molecules 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 buffer solution, and the pH value is 7.4-7.8.
The beta-Crosslaps 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 beta-Crosslaps magnetic microsphere electrochemiluminescence kit comprises a biotin-labeled beta-Crosslaps antibody, a monoclonal antibody, a fluorescent probe and a fluorescent probe.
The detection method of the beta-Crosslaps magnetic microsphere electrochemiluminescence kit comprises the following steps of (1) taking tripropylamine with the concentration of 180mmol/L and phosphate buffer with the concentration of 300mmol/L as a cleaning solution; or 90mmol/L dibutylethanolamine containing phosphate buffer solution with concentration of 300 mmol/L.
The invention provides a detection method of a beta-Crosslaps magnetic microsphere electrochemiluminescence kit, which comprises the following steps:
1) adding 15 ul of sample into a reaction tube, sequentially adding 70 ul of beta-Crosslaps reagent Ra and 70 ul of beta-Crosslaps reagent Rb, incubating at 37 ℃ for 9min, finally adding 40ul of 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 beta-Crosslaps 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 magnetic microsphere electrochemiluminescence immunoassay kit for detecting the special beta-collagen sequence (beta-Crosslaps) provided by the invention has the advantages that raw materials and reagents used in the kit can be purchased from 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 beta-collagen special sequence (beta-Crosslaps) antibody and reagent Ra
The beta-collagen specific sequence (beta-Crosslaps) antibody for labeling biotin is purchased from Beijing Yuan Tianxin Jane science and technology Co., Ltd, and has the product number of YT-beta-Crosslaps-002 and the clone number of 5A 7.
2.0mg of an antibody for labeling a biotin β -collagen specific sequence (. beta. -Crosslaps) was taken, the buffer was changed to a phosphate buffer (pH 7.8) using desalting column PD10, the mixture was concentrated using an ultrafiltration tube and adjusted to a concentration of 2.0mg/mL, 80. mu.g of biotin (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and unlabeled biotin was removed using desalting column PD 10. Beta-collagen specific sequence (beta-Crosslaps) antibody labeled biotin was diluted to 1mg/L with phosphate buffer (pH 7.4) containing 1% bovine serum albumin as beta-Crosslaps reagent Ra. The number of biotin molecular markers on the surface of each antibody molecule is 2-3.
Example 2: preparation of ruthenium-labeled beta-collagen specific sequence (beta-Crosslaps) antibody and reagent Rb
The beta-collagen specific sequence (beta-Crosslaps) antibody for labeling biotin is purchased from Jimaki technologies, Inc. of Beijing Yuan Tian, and has the product number of YT-beta-Crosslaps-003 and the clone number of 4F 8.
2.0mg of a β -collagen specific sequence (β -Crosslaps) 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, 80 μ 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. As the β -Crosslaps reagent Rb, a ruthenium-labeled β -collagen specific sequence (β -Crosslaps) antibody was diluted to 1mg/L with a phosphate buffer (pH 7.4) containing 1% bovine serum albumin. 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 Tech Co Ltd of Tian Xin of Beijing, and has the product number of YT-beta-Crosslaps-001. For recombinant expression of the protein.
The antigen was diluted to 0.2ng/mL and 4.0ng/mL at the indicated concentrations using a phosphate buffer containing 1% bovine serum albumin (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 the beta-collagen special sequence (beta-Crosslaps) adopts a sandwich method, and the detection method comprises the following steps:
1) adding 15 μ l of sample into a reaction tube, sequentially adding 80 μ l of the beta-Crosslaps reagent Ra prepared in example 1 and 80 μ l of the beta-Crosslaps reagent Rb prepared in example 2, incubating at 37 ℃ for 9min, finally adding 40 μ l of streptavidin magnetic microspheres (with the particle size of 3.0 μm), 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) 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 in the presence of the tripropylamine.
4) The photomultiplier tube records the luminescence value, and the concentration of beta-Crosslaps in the sample is calculated according to a standard curve established after the calibration is carried out by using the luminescence value of the calibration object.
Example 6:
the determination of the beta-collagen special sequence (beta-Crosslaps) adopts a sandwich method, and the detection method comprises the following steps:
1) adding 15 μ l of sample into a reaction tube, sequentially adding 80 μ l of the beta-Crosslaps reagent Ra prepared in example 1 and 80 μ l of the beta-Crosslaps reagent Rb prepared in example 2, incubating at 37 ℃ for 9min, finally adding 40 μ l of streptavidin superparamagnetic microspheres (with the particle size of 3.0 μm), and incubating at 37 ℃ for 9 min;
2) adding a streptavidin-coated superparamagnetic microsphere for incubation, and allowing the formed immune complex to be bound to the superparamagnetic microsphere through the interaction between biotin and streptavidin;
3) after incubation, absorbing the reaction mixture into a measuring cell, adsorbing the superparamagnetic microspheres onto an electrode through a magnet, absorbing the cleaning solution (dibutylethanolamine) by a liquid absorbing steel needle, and absorbing a mark Ru (bpy) which is not combined with 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 luminescence value, and the concentration of beta-Crosslaps in the sample is calculated according to a standard curve established after the calibration is carried out by using the luminescence value of the calibration object.
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
(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
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 (6ng/ml) were diluted to at least 5 concentrations in a proportion in which the low-value samples were close to 0.05 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
| Item | Sample 1 | |
Sample 3 | |
Sample 5 |
| Concentration of dilution | 0.05 | 0.9 | 3 | 5.1 | 6 |
| Measured concentration 1 | 0.051 | 0.917 | 3.222 | 5.344 | 5.837 |
| |
0.055 | 0.971 | 3.029 | 5.24 | 5.95 |
| Measured concentration | 0.053 | 0.944 | 3.126 | 5.292 | 5.8935 |
(2) The reaction method of example 6, wherein the high value sample near the upper limit of the linear range (6ng/ml) is diluted to at least 5 concentrations in a certain proportion, and the low value sample is near 0.05 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 2.
TABLE 4
| Item | Sample 1 | |
Sample 3 | |
Sample 5 |
| Concentration of dilution | 0.05 | 0.9 | 3 | 5.1 | 6 |
| Measured concentration 1 | 0.053 | 0.939 | 3.175 | 5.172 | 5.855 |
| |
0.059 | 0.984 | 3.085 | 5.192 | 5.877 |
| Measured concentration | 0.056 | 0.962 | 3.130 | 5.182 | 5.866 |
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 | 0.01ng/ml | 0.05ng/ml | 0.1ng/mL |
| Linear range | 0.05-6.0ng/mL | 0.05-5.0ng/mL | 0.1-5.0ng/mL |
| Time of detection | 18 minutes | 190 minutes | 270 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. The acridinium ester luminescent system is simple, does not need a catalyst, and is placedIn 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 electrochemical method is applied to the detection of the beta-Crosslaps, the sensitivity of the product can be improved, the process labeling time can be shortened, the linear range can be improved, the test time can be shortened, and a basis is provided for clinically and timely dealing with the treatment of brain trauma.
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 steps show that the reaction mode of the sandwich method is adopted, the magnetic microsphere electrochemical principle is utilized, the content of the beta-collagen special sequence (beta-Crosslaps) in the human serum or plasma sample is quantitatively detected, and the detection sensitivity is ensured. 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 the beta-collagen special sequence (beta-Crosslaps) is characterized by comprising a beta-Crosslaps reagent Ra, a beta-Crosslaps reagent Rb and a streptavidin superparamagnetic microsphere;
the beta-Crosslaps reagent Ra comprises an anti-beta-Crosslaps monoclonal antibody containing a biotin label;
the beta-Crosslaps reagent Rb comprises an anti-beta-Crosslaps monoclonal antibody marked by terpyridyl ruthenium;
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 labels per antibody molecule surface is 2-5 in the β -Crosslaps reagent Ra; in the beta-Crosslaps reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.
4. A composition according to any one of claims 1 to 3, wherein the β -Crosslaps reagent Ra is prepared by: mixing an anti-beta-Crosslaps monoclonal antibody with biotin in the presence of a buffer solution to prepare a beta-Crosslaps 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 β -Crosslaps reagent Rb is prepared by: mixing an anti-beta-Crosslaps monoclonal antibody with terpyridyl ruthenium in the presence of a buffer solution to prepare a beta-Crosslaps 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 beta-Crosslaps reagent Ra, the beta-Crosslaps 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 specific sequences of beta-collagen (β -Crosslaps).
9. A magnetic microsphere electrochemiluminescence immunoassay kit for specific beta-collagen sequences (β -Crosslaps), comprising a composition according to any one of claims 1 to 7 and a reagent acceptable for detection.
10. A method for the electrochemiluminescence immunoassay of magnetic microspheres of specific sequence β -collagen (β -Crosslaps), based on a composition according to any one of claims 1 to 7 or a kit according to claim 9, comprising the steps of:
step 1: taking a sample, sequentially adding a beta-Crosslaps reagent Ra and a beta-Crosslaps 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 beta-Crosslaps reagent Ra, the beta-Crosslaps reagent Rb to the streptavidin superparamagnetic microspheres is 15: (50-80): (50-80): (20 to 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 beta-Crosslaps in the sample according to the established standard curve.
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