CN112834756A - Composition for detecting interleukin 6, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method - Google Patents
Composition for detecting interleukin 6, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method Download PDFInfo
- Publication number
- CN112834756A CN112834756A CN202011626346.4A CN202011626346A CN112834756A CN 112834756 A CN112834756 A CN 112834756A CN 202011626346 A CN202011626346 A CN 202011626346A CN 112834756 A CN112834756 A CN 112834756A
- Authority
- CN
- China
- Prior art keywords
- reagent
- composition
- value
- buffer solution
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
-
- 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
-
- 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
-
- 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
-
- 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
- G01N33/577—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
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/5412—IL-6
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to the field of electrochemical detection, in particular to a composition for detecting interleukin 6(IL-6), 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 interleukin 6(IL-6), 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
IL-6 is a pleiotropic cytokine with a wide range of functions. Originally known as beta 2-interferon, plasmacytoma growth factor and stem cell stimulating factor. Later called human B cell stimulating factor 2(BSF 2). A large number of researches show that the protein has physiological activity on B cells and T cells, hematopoietic stem cells, liver cells and brain cells. IL-6 is expressed as a single gene and has a molecular weight of about 22-27 kDa.
IL-6 is rapidly produced during acute inflammatory reactions in the presence of internal and external trauma, surgery, stress, infection, brain death, tumor production and other conditions. IL-6 concentrations in surgical patients can be over-predictive of the occurrence of surgical complications.
Continuous monitoring of IL-6 levels in the serum or plasma of Intensive Care Unit (ICU) patients is useful in assessing the severity of Systemic Inflammatory Response Syndrome (SIRS), the prognosis of sepsis and septic shock. IL-6 also serves as an early warning indicator of sepsis. IL-6 and in chronic inflammatory responses (such as rheumatoid arthritis) play an important role.
To date, the methods used to detect IL-6 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 interleukin 6(IL-6), 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 interleukin 6(IL-6), which comprises an IL-6 reagent Ra, an IL-6 reagent Rb and streptavidin superparamagnetic microspheres;
the IL-6 reagent Ra comprises an anti-IL-6 monoclonal antibody containing a biotin label;
the IL-6 reagent Rb comprises an anti-IL-6 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 IL-6 reagent Ra has 2-5 molecular markers per antibody molecule; in the IL-6 reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.
In some embodiments of the invention, the IL-6 reagent Ra is prepared by: mixing an anti-IL-6 monoclonal antibody with biotin in the presence of a buffer solution to prepare an IL-6 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 invention, the IL-6 reagent Ra is prepared by: after 2.0mg of an antibody for labeling biotin interleukin 6(IL-6) was taken, the buffer was changed to a phosphate buffer (pH 7.8) using desalting column PD10, the buffer was concentrated using an ultrafiltration tube and 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 desalting column PD 10. An interleukin 6(IL-6) antibody labeled biotin was diluted to 1mg/L with a phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as an IL-6 reagent Ra.
In some embodiments of the invention, the IL-6 agent Rb is prepared by a method comprising: mixing an anti-IL-6 monoclonal antibody with terpyridyl ruthenium in the presence of a buffer solution to prepare the IL-6 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 IL-6 agent Rb is prepared by a method comprising: taking 2.0mg of interleukin 6(IL-6) antibody for labeling ruthenium terpyridyl, changing the buffer to phosphate buffer (pH 7.8) using desalting column PD10, concentrating the mixture using an ultrafiltration tube, adjusting the concentration to 2.0mg/mL, adding 30 to 80 μ g of succinamide ruthenium terpyridyl (dissolved in DMF), mixing the mixture for 30 minutes, and removing the unlabeled ruthenium using desalting column PD 10. As the IL-6 reagent Rb, a ruthenium-labeled interleukin 6(IL-6) antibody was diluted to 1mg/L with a phosphate buffer solution (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 dibutyl ethanolamine with the concentration of 90mmol/L and phosphate buffer with the concentration of 300 mmol/L.
In some embodiments of the invention, the volume ratio of the IL-6 reagent Ra, the IL-6 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 for interleukin 6 (IL-6).
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay kit for interleukin 6(IL-6), which comprises the composition and an acceptable detection reagent.
The invention also provides a magnetic microsphere electrochemiluminescence immunoassay method for interleukin 6(IL-6), which is based on the composition or the kit and comprises the following steps:
step 1: taking a sample, sequentially adding an IL-6 reagent Ra and an IL-6 reagent Rb, incubating for 8-12 min at 37 ℃, finally adding streptavidin superparamagnetic microspheres, and incubating for 8-12 min at 37 ℃ to obtain a reaction solution; wherein the volume ratio of the sample, the IL-6 reagent Ra, the IL-6 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 luminescence value, establishing a standard curve, and obtaining the concentration of the IL-6 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 30 μ l of sample into a reaction tube, sequentially adding 75 μ l of IL-6 reagent Ra and 65 μ l of IL-6 reagent Rb, incubating at 37 deg.C for 9min, adding 30 μ l of 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: the photomultiplier tube records the light emission value, establishes a standard curve, and calculates the concentration of IL-6 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 interleukin 6(IL-6), 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: an IL-6 magnetic microsphere electrochemiluminescence kit comprising: IL-6 reagent Ra, IL-6 reagent Rb, streptavidin superparamagnetic microspheres, a calibrator, tripropylamine cleaning solution and dibutylethanolamine cleaning solution.
The IL-6 magnetic microsphere electrochemiluminescence kit comprises an IL-6 reagent Ra, a buffer solution, a pH value of the buffer solution is 7.4-7.8 or 20 mM-200 mM tris buffer solution, wherein the reagent Ra is an anti-IL-6 monoclonal antibody containing biotin labels, the molecular weight of each biotin on the surface of each antibody molecule is 2-5, and the pH value of the buffer solution is 7.4-7.8. The IL-6 reagent Rb is an anti-IL-6 monoclonal antibody containing a terpyridyl ruthenium mark, the mark amount of ruthenium molecules on the surface of each antibody molecule is 2-10, and the buffer solution is 20 mM-200 mM phosphate buffer solution with the pH value of 7.4-7.8 or 20 mM-200 mM tris buffer solution with the pH value of 7.4-7.8.
The IL-6 magnetic microsphere electrochemiluminescence kit comprises a magnetic microsphere coated with streptavidin, a magnetic particle coating buffer solution, a phosphate buffer solution and a trihydroxymethyl aminomethane buffer solution, wherein the streptavidin superparamagnetic microsphere is coated with streptavidin, the particle size of the magnetic microsphere is 1.5-5.0 micrometers, the magnetic particle coating buffer solution is 20 mM-200 mM, the pH value is 7.4-7.8 or 20 mM-200 mM, and the pH value is 7.4-7.8.
The IL-6 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 kit body, and the kit body is used for carrying out electrochemiluminescence on the kit body.
The detection method of the IL-6 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 IL-6 magnetic microsphere electrochemiluminescence kit, which comprises the following steps:
1) adding 30 μ l of sample into a reaction tube, sequentially adding 75 μ l of IL-6 reagent Ra and 65 μ l of IL-6 reagent Rb, incubating at 37 deg.C for 9min, adding 30 μ l of streptavidin superparamagnetic microsphere, and incubating at 37 deg.C 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) The photomultiplier tube records the light emission value, establishes a standard curve, and calculates the concentration of IL-6 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 interleukin 6(IL-6) provided by the invention is commercially available in raw materials and reagents.
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 interleukin 6(IL-6) antibody and reagent Ra
The interleukin 6(IL-6) antibody for labeling biotin was purchased from Tianxin Jakoku technologies, Inc., Beijing, having a product number of YT-IL-6-002 and a clone number of 3E 4.
After 2.0mg of an antibody for labeling biotin interleukin 6(IL-6) was taken, the buffer was changed to a phosphate buffer (pH 7.8) using desalting column PD10, the buffer was concentrated using an ultrafiltration tube and 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 desalting column PD 10. An interleukin 6(IL-6) antibody labeled biotin was diluted to 1mg/L with a phosphate buffer solution (pH 7.4) containing 1% bovine serum albumin as an IL-6 reagent Ra. The number of biotin molecular markers on the surface of each antibody molecule is 2-3.
Example 2: preparation of ruthenium-labeled interleukin 6(IL-6) antibody and reagent Rb
The interleukin 6(IL-6) antibody for labeling biotin was purchased from Tianxin Jakoku technologies, Inc., Beijing, having a product number of YT-IL-6-003 and a clone number of 7H 2.
After 2.0mg of an interleukin 6(IL-6) 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 μ g of ruthenium succinamide terpyridyl (dissolved in DMF) was added thereto, the mixture was mixed and reacted for 30 minutes, and the unlabeled ruthenium was removed using a desalting column PD 10. As the IL-6 reagent Rb, a ruthenium-labeled interleukin 6(IL-6) antibody was diluted to 1mg/L with a phosphate buffer solution (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 Beijing edge Tian Xin, and has a product number of YT-IL-6-001. For recombinant expression of the protein.
The antigen was diluted to 18.0pg/mL and 700.0pg/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 interleukin 6(IL-6) is measured by a sandwich method, and the detection method comprises the following steps:
1) adding 30 μ l of sample into a reaction tube, sequentially adding 75 μ l of IL-6 reagent Ra prepared in example 1 and 65 μ l of IL-6 reagent Rb prepared in example 2, incubating at 37 deg.C for 9min, adding 30 μ l of streptavidin magnetic microsphere (particle size of 3.0 μm), and incubating at 37 deg.C 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 IL-6 in the sample is calculated from a standard curve created after calibration using the luminescence value of the calibration sample.
Example 6:
the interleukin 6(IL-6) is measured by a sandwich method, and the detection method comprises the following steps:
1) adding 30 μ l of sample into a reaction tube, sequentially adding 75 μ l of IL-6 reagent Ra prepared in example 1 and 65 μ l of IL-6 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) 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 IL-6 in the sample is calculated from a standard curve created after calibration using the luminescence 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
(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, the high value sample near the upper limit of the linear range (5000pg/ml) was diluted to at least 5 concentrations in a proportion in which the low value concentration sample was close to 5 pg/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
(2) The reaction method of example 6, wherein the high value sample near the upper limit of the linear range (5000pg/ml) is diluted to at least 5 concentrations in a proportion, wherein the low value concentration sample should be near 5 pg/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
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 | 1.5pg/ml | 2pg/ml | 3pg/ml |
| Linear range | 5-5000pg/mL | 2.0-1500pg/ml | 3.0-1000pg/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. The acridinium ester luminescent system is simple, does not need a catalyst and is placed 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 IL-6 can improve the sensitivity of the product, shorten the process labeling 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 steps 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 interleukin 6(IL-6) in a human serum or plasma sample, 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 interleukin 6(IL-6) is characterized by comprising an IL-6 reagent Ra, an IL-6 reagent Rb and streptavidin superparamagnetic microspheres;
the IL-6 reagent Ra comprises an anti-IL-6 monoclonal antibody containing a biotin label;
the IL-6 reagent Rb comprises an anti-IL-6 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 in the IL-6 agent Ra is 2-5; in the IL-6 reagent Rb, the labeling quantity of ruthenium molecules on the surface of each antibody molecule is 2-10.
4. The composition of any one of claims 1 to 3, wherein the IL-6 agent Ra is prepared by: mixing an anti-IL-6 monoclonal antibody with biotin in the presence of a buffer solution to prepare an IL-6 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. The composition according to any one of claims 1 to 3, wherein the IL-6 agent Rb is prepared by: mixing an anti-IL-6 monoclonal antibody with terpyridyl ruthenium in the presence of a buffer solution to prepare the IL-6 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 IL-6 reagent Ra, the IL-6 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 interleukin 6 (IL-6).
9. A magnetic microsphere electrochemiluminescence immunoassay kit for interleukin 6(IL-6), comprising the composition of any one of claims 1 to 7 and a reagent acceptable for detection.
10. A magnetic microsphere electrochemiluminescence immunoassay method for interleukin 6(IL-6), based on the composition of any one of claims 1 to 7 or the kit of claim 9, comprising the steps of:
step 1: taking a sample, sequentially adding an IL-6 reagent Ra and an IL-6 reagent Rb, incubating for 8-12 min at 37 ℃, finally adding streptavidin superparamagnetic microspheres, and incubating for 8-12 min at 37 ℃ to obtain a reaction solution; wherein the volume ratio of the sample, the IL-6 reagent Ra, the IL-6 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 luminescence value, establishing a standard curve, and obtaining the concentration of the IL-6 in the sample according to the established standard curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011626346.4A CN112834756A (en) | 2020-12-30 | 2020-12-30 | Composition for detecting interleukin 6, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011626346.4A CN112834756A (en) | 2020-12-30 | 2020-12-30 | Composition for detecting interleukin 6, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112834756A true CN112834756A (en) | 2021-05-25 |
Family
ID=75924587
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011626346.4A Pending CN112834756A (en) | 2020-12-30 | 2020-12-30 | Composition for detecting interleukin 6, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112834756A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118091145A (en) * | 2024-04-28 | 2024-05-28 | 江西赛基生物技术有限公司 | Acridinium ester conjugate concentrated solution, working solution and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030108973A1 (en) * | 1999-11-03 | 2003-06-12 | Science And Technology Corp. | Immunoassay and reagents and kits for performing the same |
| CN102721804A (en) * | 2012-05-29 | 2012-10-10 | 西安金磁纳米生物技术有限公司 | Electrical chemiluminescence immunoassay based on gold magnetic particles |
| CN107402308A (en) * | 2017-06-21 | 2017-11-28 | 北京科跃中楷生物技术有限公司 | Immue quantitative detection reagent boxes of IL 6 and preparation method thereof |
| CN108519487A (en) * | 2018-06-15 | 2018-09-11 | 郑州安图生物工程股份有限公司 | A kind of quantitative detecting method and detection kit of interleukin-6 |
| CN109541227A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection pepsinogen Cgene kit and preparation method thereof |
| CN109541202A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection anti-HBs kit and preparation method thereof |
| CN111007250A (en) * | 2019-12-10 | 2020-04-14 | 江苏三联生物工程有限公司 | Electrochemiluminescence kit for detecting TAT (thrombin-antithrombin complex) and preparation method |
| CN111044719A (en) * | 2019-12-10 | 2020-04-21 | 江苏三联生物工程有限公司 | Electrochemiluminescence kit for detecting titin antibody and preparation method thereof |
| CN111983242A (en) * | 2019-05-23 | 2020-11-24 | 郑州迪安图医学检验所有限公司 | Electrochemical luminescence immunoassay method and kit |
-
2020
- 2020-12-30 CN CN202011626346.4A patent/CN112834756A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030108973A1 (en) * | 1999-11-03 | 2003-06-12 | Science And Technology Corp. | Immunoassay and reagents and kits for performing the same |
| CN102721804A (en) * | 2012-05-29 | 2012-10-10 | 西安金磁纳米生物技术有限公司 | Electrical chemiluminescence immunoassay based on gold magnetic particles |
| CN107402308A (en) * | 2017-06-21 | 2017-11-28 | 北京科跃中楷生物技术有限公司 | Immue quantitative detection reagent boxes of IL 6 and preparation method thereof |
| CN108519487A (en) * | 2018-06-15 | 2018-09-11 | 郑州安图生物工程股份有限公司 | A kind of quantitative detecting method and detection kit of interleukin-6 |
| CN109541227A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection pepsinogen Cgene kit and preparation method thereof |
| CN109541202A (en) * | 2018-11-09 | 2019-03-29 | 广州源起健康科技有限公司 | A kind of detection anti-HBs kit and preparation method thereof |
| CN111983242A (en) * | 2019-05-23 | 2020-11-24 | 郑州迪安图医学检验所有限公司 | Electrochemical luminescence immunoassay method and kit |
| CN111007250A (en) * | 2019-12-10 | 2020-04-14 | 江苏三联生物工程有限公司 | Electrochemiluminescence kit for detecting TAT (thrombin-antithrombin complex) and preparation method |
| CN111044719A (en) * | 2019-12-10 | 2020-04-21 | 江苏三联生物工程有限公司 | Electrochemiluminescence kit for detecting titin antibody and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王敏: "《医学免疫学实验指导》", 31 January 2018, 北京邮电大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118091145A (en) * | 2024-04-28 | 2024-05-28 | 江西赛基生物技术有限公司 | Acridinium ester conjugate concentrated solution, working solution and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112649616A (en) | Composition for detecting cardiac troponin I, application thereof, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN109975557B (en) | IL-6/PCT combined detection time resolution detection kit and method | |
| EP2972351B1 (en) | Improved assay methods | |
| CN103674935B (en) | A kind of method of measuring gibberellin based on hybridization chain reaction signal amplification technique | |
| CN107817354A (en) | A kind of chemiluminescence detection kit of interleukin 6 and preparation method thereof | |
| WO2021258618A1 (en) | Biological sample testing method and test kit | |
| CN111751527A (en) | Novel coronavirus IgG and IgM antibody detection kit and preparation method and application thereof | |
| CN107841527A (en) | It is a kind of to utilize aptamer and the fluorescence detection method of magnetic material detection fibrin ferment | |
| CN113092786B (en) | Buffer solution and application thereof in central nervous system specific protein detection kit | |
| CN113125760A (en) | Composition for detecting N-terminal brain natriuretic peptide precursor, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN112834756A (en) | Composition for detecting interleukin 6, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN117434274A (en) | Single interleukin 6 magnetic particle chemiluminescence kit and determination method thereof | |
| CN113125761A (en) | Composition for procalcitonin detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN112798781A (en) | Composition for detecting human epididymis protein 4, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN114509569A (en) | Kit for detecting interleukin 6, detection method and application | |
| CN111650369A (en) | Helicobacter pylori magnetic particle chemiluminescence detection kit and preparation method thereof | |
| US20210096075A1 (en) | Kit for quickly detecting cadmium content in sample | |
| CN109085343B (en) | Kit for determining anti-Jo-1 antibody and detection method | |
| CN111398598A (en) | Chemiluminescence detection kit for insulin-like growth factor binding protein-3 and preparation method thereof | |
| CN112816703A (en) | Composition for anti-mullerian hormone detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN112816716A (en) | Composition for C peptide detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN113075406A (en) | Composition for pregnancy-associated plasma protein A detection, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN112946301A (en) | Composition for detecting special beta-collagen sequence, application thereof, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| CN111487409A (en) | Chemiluminescence detection kit for S100B protein and use method thereof | |
| CN113125752A (en) | Composition for N-MID osteocalcin detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |