CN112269025A - Interleukin-6 chemiluminescence assay kit and preparation method thereof - Google Patents
Interleukin-6 chemiluminescence assay kit and preparation method thereof Download PDFInfo
- Publication number
- CN112269025A CN112269025A CN202011270410.XA CN202011270410A CN112269025A CN 112269025 A CN112269025 A CN 112269025A CN 202011270410 A CN202011270410 A CN 202011270410A CN 112269025 A CN112269025 A CN 112269025A
- Authority
- CN
- China
- Prior art keywords
- interleukin
- antibody
- solution
- enzyme
- kit
- 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
- G01N33/543—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
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of medical diagnosis, and discloses an interleukin-6 chemiluminescence assay kit and a preparation method thereof. The kit comprises an interleukin-6 antibody A coupled superparamagnetic particle working solution, an enzyme-labeled interleukin-6 antibody B working solution and a chemiluminescent substrate solution. Wherein the enzyme-labeled interleukin-6 antibody B is obtained by coupling enzyme activated by Traut's Reagent and interleukin-6 antibody B activated by Sulfo-SMCC. The invention takes Traut's Reagent and Sulfo-SMCC as a bridge to mark the alkaline phosphatase and the interleukin-6 antibody, compared with the conventional enzyme-labeled antibody obtained by EDC-NHS enzyme labeling, the proposal of the invention can obviously improve the sensitivity of the interleukin-6 detection kit and can be applied to the chemiluminescence immunoassay detection kit.
Description
Technical Field
The invention relates to the technical field of medical diagnosis, in particular to an interleukin-6 chemiluminescence assay kit and a preparation method thereof.
Background
Interleukin-6, abbreviated as interleukin 6(IL-6), also known as interferon- β 2, hepatocyte stimulating factor (BSF2), hybridoma growth factor, etc. (HGF), is a multifunctional cytokine, which is produced mainly by a variety of cells such as fibroblasts, macrophages, T lymphocytes, B lymphocytes, epithelial cells, etc., and regulates immune response, hematopoiesis, acute phase reaction at the time of tissue injury, inflammatory reaction, etc.
The content of interleukin-6 in the serum or plasma of an intensive care patient is continuously monitored, so that the severity of Systemic Inflammatory Response Syndrome (SIRS), sepsis and the prognosis of septic shock can be effectively evaluated. The time from the onset of septic shock to the start of effective antibiotic therapy is positively correlated with the death risk value, and interleukin-6 can be used as an early warning index of sepsis and also plays an important role in chronic inflammatory reactions.
At present, reagents for detecting IL-6 clinically mainly adopt an immunochromatography method and a chemiluminescence method, compared with the immunochromatography method, the chemiluminescence method has the advantages of high sensitivity, wide linear range and the like, and at present, imported reagents (including Roche, Siemens and Beckmann) adopt the chemiluminescence method, but the cost is high, so that the development of domestic chemiluminescence reagents with high sensitivity, short detection time and excellent performance is necessary.
Disclosure of Invention
In view of the above, the present invention aims to provide a chemical luminescence assay kit for interleukin-6, which has high sensitivity, short detection time and excellent performance;
another object of the present invention is to provide a method for preparing the above kit.
In order to achieve the above purpose, the invention provides the following technical scheme:
an interleukin-6 chemiluminescence assay kit comprises an interleukin-6 antibody A coupled superparamagnetic particle working solution, an enzyme-labeled interleukin-6 antibody B working solution and a chemiluminescence substrate solution; wherein the enzyme-labeled interleukin-6 antibody B is obtained by coupling enzyme activated by Traut's Reagent (2-iminothiolane hydrochloride) and interleukin-6 antibody B activated by Sulfo-SMCC.
The specific reaction process of the enzyme-labeled interleukin-6 antibody B is as follows:
preferably, a corresponding monoclonal antibody can be used for both the interleukin-6 antibody A and the interleukin-6 antibody B;
preferably, the enzyme is alkaline phosphatase; the chemiluminescent substrate solution is APS-5 (4-chlorobenzenethiol) (10-methyl-9, 10-acridan methylene) disodium phosphate, the substrate is a glow type substrate, the sensitivity is high, the platform period can be quickly reached, the reaction time is shortened, and the sensitivity is improved;
preferably, the superparamagnetic particles coupled with the interleukin-6 antibody A are obtained by coupling superparamagnetic particles with tosyl groups on the surfaces with the interleukin-6 antibody A.
Preferably, the interleukin-6 antibody A coupled superparamagnetic particle working solution comprises interleukin-6 antibody A coupled superparamagnetic particles, MES, BSA and Triton X-100. In a specific embodiment of the invention, the working solution of the interleukin-6 antibody A-coupled superparamagnetic particles comprises 0.2mg/mL interleukin-6 antibody A-coupled superparamagnetic particles, 0.1M MES, 1% BSA, and 0.1% Triton X-100.
Preferably, the enzyme-labeled interleukin-6 antibody B working solution comprises enzyme-labeled interleukin-6 antibody B, MES, BSA, Triton X-100 and ZnCl2And MgCl2. In the specific embodiment of the invention, the working solution of the enzyme-labeled interleukin-6 antibody B comprises 2ug/mL enzyme-labeled interleukin-6 antibody B, 0.1M MES, 1% BSA, 0.1% Triton X-100 and 0.1mMZnCl2And 5mM MgCl2。
Further, the kit of the invention also comprises a cleaning solution which is Tris containing Tween 20; in a specific embodiment of the invention, the wash solution is a 0.1M Tris and 0.2% Tween 20 solution.
Meanwhile, the invention also provides a preparation method of the kit, which comprises the steps of coupling the superparamagnetic particles with the interleukin-6 antibody A, then sealing the magnetic bead sealing liquid, and diluting with the buffer solution to obtain the interleukin-6 antibody A coupled superparamagnetic particle working solution; wherein, the surface active group of the superparamagnetic particle is tosyl;
activating enzyme with Traut's Reagent, coupling with interleukin-6 antibody B activated with Sulfo-SMCC, adding enzyme blocking liquid for blocking, and diluting with buffer solution to obtain enzyme-labeled interleukin-6 antibody B working solution;
the kit consists of a super paramagnetic particle working solution coupled with an interleukin-6 antibody A, an enzyme-labeled interleukin-6 antibody B working solution and a chemiluminescent substrate solution.
In the preparation process, the diameter of the superparamagnetic particles is 1-3 mu m, and the concentration of the magnetic particles is 0.1-1.0 mg/mL; the magnetic bead blocking solution is BSA aqueous solution, more specifically 10% BSA aqueous solution.
When the superparamagnetic particles are coupled with the interleukin-6 antibody A, 10mg of superparamagnetic particles are required to be added with a magnetic bead coating solution 1 for treatment, magnetic separation is carried out, supernatant is discarded, 900 mu L (A mu L) of the magnetic bead coating solution 1 is added, after vortex mixing, 200 mu g (B mu L) of the IL-6 monoclonal antibody A is added, and then (A + B)/2 mu L of the magnetic bead coating solution 2 is added for reaction; sealing the reaction solution by using a magnetic bead sealing solution, cleaning the reaction solution by using a magnetic bead cleaning solution, and adding a buffer solution to dilute the reaction solution into a superparamagnetic particle working solution coupled with the interleukin-6 antibody A;
wherein the magnetic bead coating solution 1 is boric acid buffer solution, specifically 0.1M boric acid buffer solution, and has pH of 9.5; the magnetic bead coating solution 2 is a boric acid buffer solution containing ammonium sulfate, specifically 3M ammonium sulfate/0.1M boric acid buffer solution, and has a pH of 9.5; the magnetic bead cleaning solution is Tris-HCl containing sodium chloride and Tween 20, specifically 25mM Tris-HCl, 0.15M NaCl, 0.05% Tween 20, and pH 7.2.
When preparing the enzyme-labeled interleukin-6 antibody B working solution, the molar ratio of the enzyme activated by Traut's Reagent to the interleukin-6 antibody B activated by Sulfo-SMCC is 1: 3-5: 1, reacting the two at room temperature; in a specific embodiment, the molar ratio of the enzyme activated by Traut's Reagent to the interleukin-6 antibody B activated by Sulfo-SMCC is 2:1, and the reaction is carried out for 60min at 22 ℃; adding enzyme-labeled confining liquid, sealing for 10min, ultrafiltering with an ultrafiltration tube to remove small molecular substances, and adding enzyme-labeled buffer solution to dilute into enzyme-labeled working solution of interleukin-6 antibody B coupled alkaline phosphatase;
preferably, the enzyme blocking solution is preferably an N-ethylmaleimide solution and an ethanolamine solution.
Preferably, the buffer comprises MES, BSA and Triton X-100, or MES, BSA, Triton X-100, ZnCl2And MgCl2The former is preferably used for the working solution preparation of the interleukin-6 antibody A coupled superparamagnetic particles, and the latter is preferably used for the working solution preparation of the enzyme-labeled antibody. In a specific embodiment of the invention, the buffer is 0.1M MES, 1% BSA, 0.1% Triton X-100, or 0.1M MES, 1% BSA, 0.1% Triton X-100, 0.1mM ZnCl2、5mM MgCl2。
In an EDC-NHS enzyme labeling method and an enzyme labeling method sensitivity comparison experiment, an enzyme-labeled interleukin-6 antibody B obtained by the enzyme labeling method and an enzyme-labeled interleukin-6 antibody B obtained by a conventional EDC-NHS enzyme labeling method adopt the same other kit components to form a kit, and the minimum detection limits of the two kits are 0.6pg/mL and 4.8pg/mL respectively, which shows that the enzyme-labeled antibody prepared by the enzyme labeling method can obviously improve the detection sensitivity of the kit.
According to the technical scheme, Traut's Reagent and Sulfo-SMCC are used as bridges to label the alkaline phosphatase and the interleukin-6 antibody, and compared with the enzyme-labeled antibody obtained by conventional EDC-NHS enzyme labeling, the technical scheme provided by the invention can obviously improve the sensitivity of the interleukin-6 detection kit and can be applied to a chemiluminescence immunoassay detection kit.
Drawings
FIG. 1 is a graph showing the linear relationship between the theoretical concentration and the measured concentration after sample dilution;
FIG. 2 is a graph showing a comparison of the measurement value of the kit of the present invention and the measurement value of Elecsys Roche.
Detailed Description
The embodiment of the invention discloses an interleukin-6 chemiluminescence assay kit and a preparation method thereof, and a person skilled in the art can realize the assay by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the reagents and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations or appropriate modifications and combinations of the reagents and methods of making the reagents and methods described herein can be used in the practice or application of the techniques of the invention without departing from the spirit and scope of the invention.
The detection principle of the invention is as follows: when the sample contains interleukin-6, the magnetic bead coated antibody A and the enzyme-labeled antibody B have immunoreaction with the interleukin-6 to form a magnetic bead coated antibody A-interleukin-6 antigen-enzyme-labeled antibody B sandwich immune complex; removing unreacted substances after magnetic separation and cleaning, adding chemiluminescent substrate solution, allowing alkaline phosphatase on the sandwich immune complex to act on a luminescent substrate to emit light, detecting a luminescent signal, and calculating the concentration of interleukin-6 in the sample.
The specific detection method comprises the following steps:
firstly, taking an antibody A coupled superparamagnetic particle working solution and an enzyme labeled antibody B working solution to perform a specific reaction with a sample to be detected to obtain a double-antibody sandwich immune complex;
secondly, adding chemiluminescent substrate liquid after cleaning with cleaning liquid, carrying out enzymatic reaction with the immune complex sandwiched by the double antibodies, and measuring luminescent signals to obtain the concentration of interleukin-6;
the reaction temperature of the first step is 37 ℃, and the reaction time is 10 min; the reaction temperature in the second step was 37 ℃ and the reaction time was 1 min.
The interleukin-6 chemiluminescence assay kit and the preparation method thereof provided by the invention are further described below.
Example 1: preparation of the kit of the invention
1. Preparation of various buffers
Magnetic bead coating liquid 1: 0.1M boric acid buffer, pH 9.5;
magnetic bead coating liquid 2: 3M ammonium sulfate/0.1M boric acid buffer, pH 9.5;
cleaning liquid for magnetic beads: 25mM Tris-HCl, 0.15M NaCl, 0.05% Tween 20, pH 7.2;
magnetic bead sealing liquid: 10% BSA/H2O；
2. Preparation of antibody-coupled superparamagnetic microparticles
Transferring 10mg of superparamagnetic particles (the diameter is 1-3 mu m) with surface active groups being p-toluenesulfonyl groups, placing the superparamagnetic particles on a magnetic separator for magnetic separation to remove supernatant, adding 1mL of magnetic bead coating solution 1, performing vortex mixing, performing magnetic separation to remove the supernatant, adding 900 mu L (A mu L) of magnetic bead coating solution 1, performing vortex mixing, adding 200 mu g (B mu L) of IL-6 monoclonal antibody A, and then adding (A + B)/2 mu L of magnetic bead coating solution 2; vortex, uniformly mixing, placing on an oscillator, reacting at 37 ℃ for 18h, adding 10 mu L of magnetic bead sealing liquid, placing on the oscillator, reacting at 37 ℃ for more than 6h, after the reaction is finished, placing on a magnetic separator, carrying out magnetic separation to remove supernatant, adding 500 mu L of magnetic bead cleaning liquid, uniformly mixing, carrying out magnetic separation to remove supernatant, repeatedly cleaning for 3 times by using the magnetic separation cleaning liquid, removing supernatant, adding a buffer solution suitable for subsequent processes, uniformly mixing, and storing at 2-8 ℃ until use.
3. Preparation of enzyme-labeled antibody
Alkaline phosphatase was activated with Traut's Reagent and desalted and purified with a desalting column, IL-6 monoclonal antibody B was activated with Sulfo-SMCC and desalted and purified with a desalting column, and then the activated antibody and the activated alkaline phosphatase were mixed at a molar ratio of 1: 2, uniformly mixing by vortex, standing at 22 ℃ for reaction for 60min, adding an N-ethylmaleimide solution and an ethanolamine solution, sealing for 10min, performing ultrafiltration by using an ultrafiltration tube to remove small molecular substances, finally adding a buffer solution suitable for subsequent procedures, uniformly mixing, and storing at 2-8 ℃ until use.
4. Preparation of the kit
Diluting the prepared antibody coupled superparamagnetic particles to 0.1mg/mL by using 0.1M MES, 1% Bovine Serum Albumin (BSA) and 0.1% Triton X-100 solution to obtain an interleukin-6 monoclonal antibody A coupled superparamagnetic particle working solution;
using 0.1M MES, 1% Bovine Serum Albumin (BSA), 0.1% Triton X-100, 0.1mM ZnCl2、5mM MgCl2Diluting the prepared enzyme-labeled antibody to 2ug/mL by using the solution to obtain an interleukin-6 monoclonal antibody B-coupled alkaline phosphatase enzyme-labeled antibody working solution;
preparing a cleaning solution by using 0.1M Tris and 0.2% Tween 20 solution;
200 mul of chemical luminescence substrate solution APS-5 and 1600 mul of cleaning solution are taken to be combined with 50 mul of the prepared interleukin-6 monoclonal antibody A coupled superparamagnetic particle working solution and 50 mul of the interleukin-6 monoclonal antibody B coupled alkaline phosphatase enzyme-labeled antibody working solution, and the interleukin-6 chemical luminescence quantitative detection kit can be obtained.
Example 2: performance evaluation of the kit prepared according to the invention
1. Linear evaluation
Clinical samples with a fixed IL-6 concentration of 5066pg/mL from the Roche Elecsys system were diluted with 4% BSA solution according to the following Table 1, and the results are shown in Table 1, and the linear relationship between the measured concentration and the theoretical concentration is shown in FIG. 1;
TABLE 1 Linear evaluation test data
As can be seen from Table 1 and FIG. 1, the linear range of the kit of the present invention is wide, and is in the range of 1.5 pg/mL-5066 pg/mL, R2The value reaches 0.9986.
2. Methodological comparison evaluation
40 clinical samples are taken and respectively detected by using the Roche Elecsys reagent and the reagent prepared in the embodiment 1 of the invention, the correlation of the detection results of the two kits is compared, the detection data are shown in the table 2, and the comparison graph of the methodology is shown in the figure 2.
Table 2: method for comparing IL-6 concentration values measured by two kits
As can be seen from Table 2 and FIG. 2, the kit of the present invention is expected to have better correlation and superior performance when the imported kit is used as a control standard.
Example 3: sensitivity comparison of EDC-NHS enzyme labeling method and enzyme labeling method
Activating ALP with EDC and Sulfo-NHS at 25 deg.C in dark for 1h, ultrafiltering, centrifuging, adding interleukin 6 antibody B, and reacting at 25 deg.C for 3 h; and (3) performing ultrafiltration and centrifugation after sealing, adding the enzyme-labeled buffer solution to obtain an alkaline phosphatase enzyme-labeled antibody working solution coupled with the interleukin-6 monoclonal antibody B, combining the alkaline phosphatase enzyme-labeled antibody working solution with other reagent components prepared in the example 1 to form a kit (kit 2), and simultaneously testing the lowest detection limit with the kit (kit 1) prepared in the example 1, wherein the detection data are shown in a table 3.
TABLE 3 IL-6 minimum detection limits determined by two kits
As can be seen from Table 3, the detection result of the lowest detection limit of the kit 1 is obviously lower than that of the kit 2, and the kit 1 has higher sensitivity, which indicates that the enzyme-labeled antibody prepared by the enzyme labeling method can obviously improve the detection sensitivity of the kit.
The foregoing is only for the purpose of understanding the method of the present invention and the core concept thereof, and it will be understood by those skilled in the art that various changes and modifications may be made without departing from the principle of the invention, and the invention also falls within the scope of the appended claims.
Claims (10)
1. An interleukin-6 chemiluminescence assay kit is characterized by comprising an interleukin-6 antibody A coupled superparamagnetic particle working solution, an enzyme-labeled interleukin-6 antibody B working solution and a chemiluminescence substrate solution; wherein the enzyme-labeled interleukin-6 antibody B is obtained by coupling enzyme activated by Traut's Reagent and interleukin-6 antibody B activated by Sulfo-SMCC.
2. The kit of claim 1, wherein the enzyme is alkaline phosphatase.
3. The kit according to claim 1, wherein the chemiluminescent substrate solution is APS-5.
4. The kit of claim 1, wherein the working solution of the interleukin-6 antibody A-conjugated superparamagnetic particles comprises interleukin-6 antibody A-conjugated superparamagnetic particles, MES, BSA and Triton X-100.
5. The kit of claim 1, wherein the enzyme-labeled interleukin-6 antibody B working solution comprises enzyme-labeled interleukin-6 antibody B, MES, BSA, Triton X-100, ZnCl2And MgCl2。
6. The kit of any one of claims 1 to 5, further comprising a wash solution.
7. The preparation method of the kit according to claim 1, wherein the superparamagnetic particles are coupled with interleukin-6 antibody A, then the magnetic bead blocking solution is closed, and the magnetic bead blocking solution is diluted by buffer solution to obtain the superparamagnetic particle working solution coupled with interleukin-6 antibody A;
activating enzyme with Traut's Reagent, coupling with interleukin-6 antibody activated by Sulfo-SMCC, adding enzyme blocking liquid for blocking, and diluting with buffer solution to obtain enzyme-labeled interleukin-6 antibody B working solution;
the kit consists of a super paramagnetic particle working solution coupled with an interleukin-6 antibody A, an enzyme-labeled interleukin-6 antibody B working solution and a chemiluminescent substrate solution.
8. The method of claim 7, wherein the blocking solution for magnetic beads is an aqueous BSA solution.
9. The method according to claim 7, wherein the enzyme blocking solution is an N-ethylmaleimide solution and an ethanolamine solution.
10. The method of claim 7, wherein the buffer comprises MES, BSA or Triton X-100.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011270410.XA CN112269025A (en) | 2020-11-13 | 2020-11-13 | Interleukin-6 chemiluminescence assay kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011270410.XA CN112269025A (en) | 2020-11-13 | 2020-11-13 | Interleukin-6 chemiluminescence assay kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112269025A true CN112269025A (en) | 2021-01-26 |
Family
ID=74340086
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011270410.XA Pending CN112269025A (en) | 2020-11-13 | 2020-11-13 | Interleukin-6 chemiluminescence assay kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112269025A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114113626A (en) * | 2021-10-25 | 2022-03-01 | 江苏纳迪芯生命科技研究院有限公司 | Cytokine detection kit based on magnetic particle luminescence method |
| CN114137203A (en) * | 2021-10-21 | 2022-03-04 | 广州万孚生物技术股份有限公司 | Method for preparing antigen and alkaline phosphatase conjugate |
| CN114910641A (en) * | 2022-05-09 | 2022-08-16 | 山东中鸿特检生物科技有限公司 | Detection kit for interleukin, detection method and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102901812A (en) * | 2012-11-13 | 2013-01-30 | 江阴泽成生物技术有限公司 | Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb) |
| CN103344633A (en) * | 2013-07-17 | 2013-10-09 | 江阴泽成生物技术有限公司 | Chemiluminiscence substrate liquid of alkaline phosphatase |
| CN104330551A (en) * | 2014-11-17 | 2015-02-04 | 南方医科大学南方医院 | Interleukin 6 quantitative determination kit and preparation method thereof |
| CN105392895A (en) * | 2013-03-13 | 2016-03-09 | 中尺度技术有限责任公司 | Improved assay methods |
| CN107942047A (en) * | 2017-11-17 | 2018-04-20 | 南通伊仕生物技术股份有限公司 | Interleukin-18 detection kit |
| CN108535494A (en) * | 2018-06-29 | 2018-09-14 | 湖南师范大学 | A kind of IL-6ELISA detection kits and its application method |
| CN109444419A (en) * | 2018-10-26 | 2019-03-08 | 成都普利泰生物科技有限公司 | A kind of chemical luminescence immune analysis reagent box detecting dog thyrotropic hormone |
| CN109596835A (en) * | 2018-11-23 | 2019-04-09 | 深圳天辰医疗科技有限公司 | A kind of detection method of detection kit and preparation method thereof and troponin T |
| CN111521777A (en) * | 2020-04-30 | 2020-08-11 | 四川携光生物技术有限公司 | Treatment method for reducing non-specific adsorption in autoimmune antibody detection and kit thereof |
-
2020
- 2020-11-13 CN CN202011270410.XA patent/CN112269025A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102901812A (en) * | 2012-11-13 | 2013-01-30 | 江阴泽成生物技术有限公司 | Magnetic particle chemiluminescence immunoassay kit and assay method for human thyroglobulin antibodies (TGAb) |
| CN105392895A (en) * | 2013-03-13 | 2016-03-09 | 中尺度技术有限责任公司 | Improved assay methods |
| CN103344633A (en) * | 2013-07-17 | 2013-10-09 | 江阴泽成生物技术有限公司 | Chemiluminiscence substrate liquid of alkaline phosphatase |
| CN104330551A (en) * | 2014-11-17 | 2015-02-04 | 南方医科大学南方医院 | Interleukin 6 quantitative determination kit and preparation method thereof |
| CN107942047A (en) * | 2017-11-17 | 2018-04-20 | 南通伊仕生物技术股份有限公司 | Interleukin-18 detection kit |
| CN108535494A (en) * | 2018-06-29 | 2018-09-14 | 湖南师范大学 | A kind of IL-6ELISA detection kits and its application method |
| CN109444419A (en) * | 2018-10-26 | 2019-03-08 | 成都普利泰生物科技有限公司 | A kind of chemical luminescence immune analysis reagent box detecting dog thyrotropic hormone |
| CN109596835A (en) * | 2018-11-23 | 2019-04-09 | 深圳天辰医疗科技有限公司 | A kind of detection method of detection kit and preparation method thereof and troponin T |
| CN111521777A (en) * | 2020-04-30 | 2020-08-11 | 四川携光生物技术有限公司 | Treatment method for reducing non-specific adsorption in autoimmune antibody detection and kit thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114137203A (en) * | 2021-10-21 | 2022-03-04 | 广州万孚生物技术股份有限公司 | Method for preparing antigen and alkaline phosphatase conjugate |
| CN114113626A (en) * | 2021-10-25 | 2022-03-01 | 江苏纳迪芯生命科技研究院有限公司 | Cytokine detection kit based on magnetic particle luminescence method |
| CN114910641A (en) * | 2022-05-09 | 2022-08-16 | 山东中鸿特检生物科技有限公司 | Detection kit for interleukin, detection method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112269025A (en) | Interleukin-6 chemiluminescence assay kit and preparation method thereof | |
| US6027904A (en) | Platelet count assay using thrombospondin or β-thromboglobulin | |
| CN108318680B (en) | Detection method and detection kit for anti-drug antibody | |
| CN112014575B (en) | CYFRA21-1 determination kit and preparation method thereof | |
| CN111983217A (en) | Sample treatment fluid and application thereof | |
| CN111175491A (en) | sBCMA magnetic particle chemiluminescence immunoassay kit and preparation method and application thereof | |
| CN202916286U (en) | Latex enhanced turbidimetric immunoassay kit for quantitatively detecting procalcitonin (PCT) | |
| CN109324181B (en) | Sealing agent composition for immunochromatography, application and preparation method of immunochromatography kit | |
| EP4130741A1 (en) | Method for immunoassay of amyloid beta in blood, and kit for same | |
| CN113640511A (en) | Magnetic particle electrochemiluminescence kit | |
| CN109073641B (en) | Immunological assay using sulfated polysaccharides | |
| CN106366199B (en) | Troponin I monoclonal antibody magnetic particle, preparation method thereof and detection kit | |
| CN116183931A (en) | Galectin-3 magnetic particle chemiluminescence immune quantitative detection kit | |
| CN111487409A (en) | Chemiluminescence detection kit for S100B protein and use method thereof | |
| CN112710841B (en) | ST2 protein detection kit and detection method | |
| CN115166229A (en) | Direct chemiluminescence method kit for determining content of neurofilament light chain protein and preparation method thereof | |
| CN113777326A (en) | Kit for high-specificity detection of heparin binding protein and application thereof | |
| JP2004012434A (en) | Method for measuring pepsinogen and measuring kit | |
| CN113125761A (en) | Composition for procalcitonin detection, application of composition, magnetic microsphere electrochemiluminescence immunoassay kit and detection method | |
| JPH02257063A (en) | Reagent and method for immunoassay | |
| CN111381046A (en) | Calprotectin chemiluminescence immunoassay kit and preparation method thereof | |
| JPH11344494A (en) | Immunological agglutination reaction reagent and method for suppressing prozone phenomenon by using the same | |
| EP1412471B1 (en) | Apparatus and method for determining the onset and presence of sepsis conditions | |
| CN113030470B (en) | Creatine kinase isoenzyme determination kit | |
| CN113252909B (en) | Preparation method of immunofluorescence detection kit based on quantum dots |
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 |