CN107907691B - Myoglobin detection kit and use method thereof - Google Patents
Myoglobin detection kit and use method thereof Download PDFInfo
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Abstract
The invention belongs to the field of in-vitro diagnostic kits, and particularly relates to a myoglobin detection kit and a using method thereof, wherein the myoglobin detection kit comprises: the reagent R1 contains an imidazole component, so that blood cells in whole blood can be rapidly eliminated, and the possibility that the blood cells swallow magnetic beads is effectively avoided; the cleaning solution contains sodium dodecyl sulfate, so that the cleaning effect is enhanced, and the nonspecific combination of a chemiluminescent substrate is avoided. The kit can directly use finger tip whole blood or anticoagulated vein whole blood as a sample to be detected, and can directly detect without pre-treating a whole blood sample, thereby greatly improving the detection speed, simplifying the operation steps, enlarging the application range of the kit and being convenient for large-scale popularization and application.
Description
Technical Field
The invention belongs to the field of in-vitro diagnostic kits, and particularly relates to a myoglobin detection kit and a use method thereof.
Background
Myoglobin (MB) is a skeletal muscle and myocardial specific oxygen binder protein consisting of a single polypeptide chain of 153 amino acid residues and a blood red prosthetic group with a molecular weight of 17.8kD, and has the function of transporting and storing oxygen in muscle cells. Normal human myocardium and skeletal muscle contain a large amount of MB and little blood, but when myocardial or skeletal muscle is injured, MB is released from muscle tissue into circulating blood, and is filtered by glomeruli and appears in urine. Therefore, MB measurement in blood and urine can be used for diagnosis of certain myopathies and heart diseases, such as Acute Myocardial Infarction (AMI), acute muscle injury, acute and chronic renal failure, severe congestive heart failure, long-term shock, muscular dystrophy, muscular atrophy and polymyositis.
AMI is a disease seriously endangering human life, the concentration of MB in the blood serum of a patient is rapidly increased within 2-4 hours after AMI occurs, the peak is reached within 8-10 hours, the blood serum can be continued for 12 hours, and the AMI has high sensitivity. Since the increase of MB is caused by skeletal muscle diseases, renal function impairment, inflammation, systemic lupus erythematosus and dermatitis, the specificity of the diagnosis index as AMI is low, but the diagnosis index can be generally used as a check index to assist the early diagnosis of AMI due to the high sensitivity of the diagnosis index. Since MB has a short half-life in blood and returns to a normal level within 24 hours after the increase, MB can be used as an index for observing the presence or absence of re-infarction and the presence or absence of extension of infarction.
The existing MB detection methods mainly comprise a radioactive immunoassay, an enzyme-linked immunosorbent assay, a chemiluminescence assay, an enzyme-linked fluorescence assay and an immunoturbidimetry. Such as: chinese patent document CN102565419B discloses a myoglobin assay kit, which comprises reagent R1 and reagent R2, wherein: r1 is a buffer solution, and R2 is a buffer solution in which the polystyrene latex particles sensitized with the MB antibody are placed. The detection principle used by the kit is that after latex particles coated with the MB antibody and MB in a sample have immunoreaction, aggregate particles are formed, and the content of the MB in the sample can be measured by measuring the turbidity generated by the formation of the aggregate under a certain wavelength. However, the techniques disclosed in the above documents coat latex microparticles with MB antibodies, which are polyclonal antibodies, and the specificity of the polyclonal antibodies is poor, and even if polyclonal antibodies prepared using the same antigen are used, there are differences between different batches. Therefore, the specificity of the obtained kit is not ideal, false positive phenomena are easy to occur, and the requirement of clinical diagnosis cannot be met. In addition, the kit adopts a plasma or serum sample for detection, needs to take blood from veins and has large blood collection amount, the sample needs to be centrifuged before use, trace finger whole blood cannot be directly adopted as a detection sample, and the detection speed is low, so the kit cannot well meet the requirements of quick diagnosis in emergency and outpatient service of hospitals.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is that the existing myoglobin detection kit has low specificity, adopts blood plasma or blood serum as a sample, and has the defects of complex operation and narrow detection range, thereby providing the myoglobin detection kit which has high specificity and can directly use whole blood as the sample; further, the invention also provides a using method of the detection kit.
In order to solve the above technical problems, the present invention provides a myoglobin assay kit, comprising:
(1) a calibrator;
(2) reagent R1;
(3) enzyme conjugate working solution R2;
(4) a magnetic bead conjugate working solution M;
(5) cleaning fluid;
(6) a chemiluminescent substrate;
the reagent R1 contains an imidazole component, and the cleaning solution contains a sodium lauryl sulfate component.
Preferably, the reagent R1 comprises: 25-100 mmol/L of trihydroxymethyl aminomethane, 150mmol/L of NaCl, 1-5% of sucrose, 1-5% of glycerol, 0.1% of bovine serum albumin, 0.4-2 mmol/L of imidazole, 0.05-0.2% of Tween 20 and 0.05-0.2% of Proclin300, and the pH value is 7.0-7.5.
Preferably, the calibrator is prepared by adding myoglobin to a calibrator diluent, and the calibrator diluent comprises: 20-50 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 150-300 mmol/L NaCl, 1% bovine serum albumin, 0.5-5 mmol/L Proclin300, and pH 6.5-7.5.
Preferably, the concentration of the enzyme conjugate working solution R2 is 0.5-2 μ g/mL, and the enzyme conjugate working solution is prepared by adding an enzyme conjugate solution into an enzyme conjugate diluent, wherein the diluent comprises: 20-50 mmol/L2- (N-morpholine) ethanesulfonic acid, 150-300 mmol/L NaCl, 1% bovine serum albumin, 5% sucrose, 5% glycerol, 0.1% Tween 20, 0.05-5 mmol/L LZnCl2、0.05～5mmol/LMgCl20.05-0.2% Proclin300, and pH 6.0-6.5.
Preferably, the concentration of the working solution M of the magnetic bead conjugate is 0.1-0.5 mg/mL, and the working solution M is prepared by adding a magnetic bead conjugate solution into a magnetic bead diluent, wherein the diluent comprises: 20-50 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 150-300 mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone, 0.5-5% Proclin300, and the pH is 7.0-8.0.
Preferably, the main component of the enzyme conjugate is an alkaline phosphatase-labeled myoglobin-labeled antibody.
Preferably, the main component of the magnetic bead conjugate is carboxyl magnetic beads coupled with the myoglobin coated antibody, and the particle size of the carboxyl magnetic beads is 1.3-4 mu m.
Preferably, the alkaline phosphatase-labeled myoglobin-labeled antibody is prepared by contacting activated myoglobin-labeled antibody with activated alkaline phosphatase in a molar ratio of 1: (1-2), and reacting in a triethanolamine buffer solution with the pH value of 7.3 at the temperature of 2-8 ℃ for 18-24 hours to obtain the alcohol-based catalyst.
Preferably, the activated myoglobin marker antibody is prepared by mixing a solution of Traut's with a solution of myoglobin marker antibody in a molar ratio of 1: (15-30), and reacting in triethanolamine buffer solution with the pH value of 8.5 for 12-18 min at room temperature to obtain the product; the activated alkaline phosphatase is prepared by mixing a sodium salt solution of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester with an alkaline phosphatase solution in a molar ratio of 1: (10-15), and reacting in an N, N-dimethylformamide solution at room temperature for 12-18 min to obtain the compound.
More preferably, the concentration of the Traut's solution is 1.3-1.5 mg/mL; the concentration of the myoglobin marker antibody solution is 2-5 mg/mL; the concentration of the 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt solution is 16-18 mg/mL; the concentration of the alkaline phosphatase solution is 16-20 mg/mL.
Preferably, the preparation of the alkaline phosphatase-labeled myoglobin-labeled antibody further comprises: and adding a glycine solution into the activated myoglobin marker antibody, wherein the amount of the glycine is 10-20 times of that of the Traut's reagent substance, and reacting at room temperature for 8-12 min after fully and uniformly mixing.
Preferably, the preparation of the alkaline phosphatase-labeled myoglobin-labeled antibody further comprises: adding a glycine solution into the activated alkaline phosphatase, wherein the amount of glycine is 10-20 times of that of the sodium salt of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester, fully mixing uniformly, and reacting at room temperature for 8-12 min.
Preferably, the preparation of the alkaline phosphatase-labeled myoglobin-labeled antibody further comprises: and adding an N-ethyl maleimide solution into the myoglobin labeled antibody marked by the alkaline phosphatase, wherein the amount of the N-ethyl maleimide is 5-20 times of that of the myoglobin labeled antibody marked by the alkaline phosphatase, and reacting at room temperature for 25-35 min after fully and uniformly mixing.
Preferably, the preparation of the alkaline phosphatase-labeled myoglobin-labeled antibody further comprises: adding glycerol into the purified myoglobin labeled antibody labeled by alkaline phosphatase and storing at the temperature of-25 to-15 ℃.
Preferably, the myoglobin coated antibody coupled carboxyl magnetic bead is obtained by mixing a myoglobin coated antibody solution and a carboxyl magnetic bead solution according to the mass ratio of 1 (80-120), and performing crosslinking reaction in a 2- (N-morpholine) ethanesulfonic acid buffer solution with the pH value of 5.0 for 16-24 h at room temperature.
More preferably, the concentration of the myoglobin coating antibody solution is 1-5 mg/mL; the concentration of the carboxyl magnetic bead solution is 100 mg/mL.
Preferably, the preparation of the myoglobin coated antibody coupled carboxyl magnetic beads further comprises: before the cross-linking reaction, performing activation treatment on carboxyl magnetic beads, wherein the activation treatment comprises the following steps:
(1) placing the carboxyl magnetic beads on a magnetic frame and standing for 2min, magnetically separating and removing supernatant, washing twice with 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution with the pH value of 5.0, adding into 2- (N-morpholine) ethanesulfonic acid buffer solution, and performing ultrasonic treatment until the carboxyl magnetic beads are completely dissolved;
(2) respectively adding an N-N-hydroxysuccinimide solution and a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution into the system in the step (1), wherein the mass of the N-N-hydroxysuccinimide solution is 0.20-0.25 times of that of the carboxyl magnetic beads, and the mass of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution is 0.08-0.12 times of that of the carboxyl magnetic beads, uniformly mixing, and stirring and reacting at room temperature for 25-35 min.
More preferably, the concentration of the N-N-hydroxysuccinimide solution is 10 mg/mL; the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution is 10 mg/mL.
Preferably, the preparation of the myoglobin coated antibody coupled carboxyl magnetic beads further comprises: and adding a magnetic bead sealing solution into the carboxyl magnetic beads coupled with the myoglobin coated antibody, and reacting for 16-24 h at room temperature.
More preferably, the magnetic bead blocking solution is CE 210.
Preferably, the kit further comprises a myoglobin quality control.
The invention also provides a using method of the myoglobin detection kit, which comprises the following steps:
(1) adding 25 microliter of calibrator, 25 microliter of quality control material and 25 microliter of sample to be tested into the three test tubes respectively;
(2) adding 0-40 mu L of reagent R1, 50 mu L of enzyme conjugate working solution R2 and 50 mu L of magnetic bead conjugate working solution M into each test tube respectively, fully mixing, and reacting at 40-45 ℃ for 5-10 min;
(3) magnetic separation, pouring out supernatant, adding 300 mu L of cleaning solution into each test tube, mixing uniformly, performing magnetic separation, and pouring out supernatant;
(4) each test tube was added with 100. mu.L of chemiluminescent substrate, mixed well and the intensity of luminescence was measured with a chemiluminescence apparatus.
The technical scheme of the invention has the following advantages:
(1) the myoglobin detection kit provided by the invention contains a reagent R1, and the reagent R1 contains an imidazole component, so that blood cells in whole blood can be quickly eliminated, and the possibility that the blood cells swallow magnetic beads is effectively avoided. The kit can directly use finger tip whole blood or anticoagulated vein whole blood as a sample to be detected, and can directly detect without pre-treating a whole blood sample, so that the detection speed is greatly improved, the operation steps are simplified, and the application range of the kit is expanded; the method can be operated in a full-automatic and one-key mode, the detection result can be obtained within about 15 minutes, the requirements of quick diagnosis in emergency and outpatient service of hospitals can be well met, and the method is convenient to popularize and apply in a large range.
(2) According to the myoglobin detection kit provided by the invention, the cleaning solution contains the sodium dodecyl sulfate component, so that the cleaning effect is improved, and the nonspecific combination of a chemiluminescent substrate is effectively avoided.
(3) The myoglobin detection kit provided by the invention combines a chemiluminescence technology with immunomagnetic particles, provides a reaction system close to homogeneous phase, and adopts a one-step reaction mode, so that the detection sensitivity and precision are greatly improved; the carboxyl magnetic beads are used as solid phase carriers, the uniformity of the size and the shape of the solid phase carriers enables target substances to be rapidly and effectively combined on the magnetic beads, and the spherical structure of the solid phase carriers can eliminate non-specific binding substances related to particles with irregular shapes, so that the specificity of kit products is improved.
(4) The myoglobin detection kit provided by the invention comprises an enzyme conjugate, wherein the main component of the enzyme conjugate is an alkaline phosphatase-labeled myoglobin labeled antibody, a glycine solution is added in the preparation process to terminate the activation reaction, and an N-ethylmaleimide solution seals redundant sulfydryl to terminate the labeling reaction, so that the occurrence of non-specific reaction is effectively avoided, and the stability of the alkaline phosphatase-labeled myoglobin labeled antibody is ensured; the conditions of the labeling reaction are optimized by limiting the amount of each reactant used in the labeling reaction, the temperature, the pH condition, and the like.
(5) According to the myoglobin detection kit provided by the invention, the enzyme conjugate is stored in glycerol, so that the activity of the enzyme is effectively ensured.
(6) The myoglobin detection kit provided by the invention comprises an enzyme conjugate magnetic bead conjugate, wherein the enzyme conjugate magnetic bead conjugate is carboxyl magnetic beads coupled with a myoglobin coated antibody, and N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are adopted to activate the carboxyl magnetic beads in the preparation process, so that the coupling efficiency of the myoglobin coated antibody and the carboxyl magnetic beads is improved; the dissolution of the carboxyl magnetic beads is accelerated by adopting an ultrasonic mode during the activation treatment of the carboxyl magnetic beads, so that the carboxyl magnetic beads are more uniformly dispersed in a system, the influence on detection signals caused by the coagulation of the magnetic beads is effectively prevented, and the accuracy of the detection result of the kit is ensured; after the coupling reaction is finished, the magnetic bead sealing liquid is added, so that the probability of cross reaction is effectively reduced, the specificity of the kit is improved, and the accuracy of the detection result of the kit is further ensured; the using amount, temperature and pH condition of each reaction substance in the coupling reaction are limited, and the coupling reaction condition is optimized.
(7) The myoglobin detection kit provided by the invention has the advantages that all components in the detection kit are optimal formulas under the reaction system, and the detection performance of the detection kit is powerfully guaranteed.
(8) The myoglobin detection kit provided by the invention can be used for simultaneously detecting a plurality of samples on a full-automatic chemiluminescence apparatus, so that high-flux rapid detection of myoglobin is realized, and the accuracy and the detection efficiency are greatly improved.
(9) The invention provides a using method of a myoglobin detection kit, which adopts a one-step reaction mode, has a uniform reaction system and greatly improves the reaction rate.
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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the correlation between the kit of the present invention and Beckmann's kit for detecting clinical serum.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
In the following examples, myoglobin-labeled antibodies were purchased from medix; myoglobin-coated antibody was purchased from medix; traut's Reagent was purchased from Thermo; alkaline phosphatase was purchased from Roche; the carboxyl magnetic beads were purchased from JSR corporation of japan; CE210 was purchased from JSR corporation, japan.
EXAMPLE 1 preparation of enzyme conjugates
1. Weighing 3mg of Traut's reagent, and preparing a solution with the concentration of 1.376mg/mL by using 100mmol/L triethanolamine buffer (pH 8.5 +/-0.05); 0.5mg of the myoglobin marker antibody was weighed, a solution having a concentration of 2mg/mL was prepared using 100mmol/L triethanolamine buffer (pH 8.5. + -. 0.05), and Traut's solution having a concentration of 1.376mg/mL was added thereto at a molar ratio of the myoglobin marker antibody to the Traut's reagent of 1:15, and the mixture was immediately mixed and reacted at room temperature for 15 minutes. A1 mmol/L glycine solution was added in an amount of 10 times that of the sodium salt of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester, and the mixture was immediately mixed and reacted at room temperature for 10 minutes. Desalted and replaced into 100mmol/L triethanolamine cross-linking buffer (pH 7.3 ± 0.05).
2. Weighing 3mg of 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfo-group succinimide ester sodium salt, and preparing a solution with the concentration of 17.5mg/mL by using dimethylformamide; to a 16mg/mL alkaline phosphatase solution was added 17.5mg/mL 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide sodium salt solution in a molar ratio of 1:10 of alkaline phosphatase to 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide sodium salt, and the mixture was immediately mixed and reacted at room temperature for 15 minutes. A1 mmol/L glycine solution was added in an amount of 10 times that of Traut's reagent substance, and the mixture was immediately mixed and reacted at room temperature for 8 minutes. Desalted and replaced into 100mmol/L triethanolamine cross-linking buffer (pH 7.3 ± 0.05).
3. Mixing the activated myoglobin marker antibody solution obtained in the step 1 with the activated alkaline phosphatase solution obtained in the step 2, wherein the molar ratio of the activated myoglobin marker antibody to the activated alkaline phosphatase is 1:1, fully and uniformly mixing, and reacting for 18 hours at the temperature of 2-8 ℃.
4. 3mg of N-ethylmaleimide was weighed out and prepared into a solution of 12.5mg/mL using 100mmol/L triethanolamine buffer (pH 7.3. + -. 0.05).
5. Adding the N-ethylmaleimide solution obtained in the step 4 into the system obtained in the step 3, wherein the molar ratio of the alkaline phosphatase-labeled myoglobin-labeled antibody to the N-ethylmaleimide is 1:10, mix well and react for 30 minutes at room temperature to block excess thiol groups. After the reaction is finished, the final myoglobin labeled antibody conjugate marked by alkaline phosphatase is obtained by purification by a molecular sieve chromatography method. And measuring the concentration of the purified enzyme conjugate concentrated solution, adding glycerol with the same volume, fully and uniformly mixing, and storing at-20 ℃ for later use.
EXAMPLE 2 preparation of enzyme conjugates
1. Weighing 3mg of Traut's reagent, and preparing a solution with the concentration of 1.3mg/mL by using 100mmol/L triethanolamine buffer (pH 8.5 +/-0.05); 0.5mg of the myoglobin marker antibody was weighed, a 5mg/mL solution was prepared using 100mmol/L triethanolamine buffer (pH 8.5. + -. 0.05), and 1.3mg/mL Traut's solution was added thereto at a molar ratio of the myoglobin marker antibody to the Traut's reagent of 1:10, and the mixture was immediately mixed and reacted at room temperature for 12 minutes. A1 mmol/L glycine solution in an amount 20 times the amount of the sodium salt of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester was added, mixed immediately, and reacted at room temperature for 12 minutes. Desalted and replaced into 100mmol/L triethanolamine cross-linking buffer (pH 7.3 ± 0.05).
2. Weighing 3mg of 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt, and preparing a solution with the concentration of 16mg/mL by using dimethylformamide; to a 20mg/mL alkaline phosphatase solution was added 16mg/mL sodium sulfosuccinimate 4- (N-maleimidomethyl) cyclohexane-1-carboxylate in a molar ratio of 1:15, and the mixture was immediately mixed and reacted at room temperature for 15 minutes. A1 mmol/L glycine solution was added in an amount of 20 times the amount of Traut's reagent substance, and the mixture was immediately mixed and reacted at room temperature for 12 minutes. Desalted and replaced into 100mmol/L triethanolamine cross-linking buffer (pH 7.3 ± 0.05).
3. Mixing the activated myoglobin marker antibody solution obtained in the step 1 with the activated alkaline phosphatase solution obtained in the step 2, wherein the molar ratio of the activated myoglobin marker antibody to the activated alkaline phosphatase is 1: and 2, fully and uniformly mixing, and reacting for 24 hours at the temperature of 2-8 ℃.
4. 3mg of N-ethylmaleimide was weighed out and prepared into a solution of 12.5mg/mL using 100mmol/L triethanolamine buffer (pH 7.3. + -. 0.05).
5. Adding the N-ethylmaleimide solution obtained in the step 4 into the system obtained in the step 3, wherein the molar ratio of the alkaline phosphatase-labeled myoglobin-labeled antibody to the N-ethylmaleimide is 1: 20, mix well and react at room temperature for 35 minutes to block excess thiol groups. After the reaction is finished, the final myoglobin labeled antibody conjugate marked by alkaline phosphatase is obtained by purification by an ultrafiltration concentration method. And measuring the concentration of the purified enzyme conjugate concentrated solution, adding glycerol with the same volume, fully and uniformly mixing, and storing at-20 ℃ for later use.
EXAMPLE 3 preparation of enzyme conjugates
1. Weighing 3mg of Traut's reagent, and preparing a solution with the concentration of 1.5mg/mL by using 100mmol/L triethanolamine buffer (pH 8.5 +/-0.05); 0.5mg of myoglobin marker antibody was weighed, a solution having a concentration of 2.5mg/mL was prepared using 100mmol/L triethanolamine buffer (pH 8.5. + -. 0.05), and Traut's solution having a concentration of 1.5mg/mL was added thereto at a molar ratio of myoglobin marker antibody to Traut's reagent of 1:15, and the mixture was immediately mixed and reacted at room temperature for 18 minutes. A1 mmol/L glycine solution was added in an amount of 15 times the amount of the sodium salt of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester, and the mixture was immediately mixed and reacted at room temperature for 10 minutes. Desalted and replaced into 100mmol/L triethanolamine cross-linking buffer (pH 7.3 ± 0.05).
2. Weighing 3mg of 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfo-group succinimide ester sodium salt, and preparing a solution with the concentration of 18mg/mL by using dimethylformamide; to an alkaline phosphatase solution (18 mg/mL), a solution of sodium sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (molar ratio of alkaline phosphatase to sodium sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (molar ratio of 1: 12) was added, and the mixture was immediately mixed and reacted at room temperature for 12 minutes. A1 mmol/L glycine solution was added in an amount 15 times the amount of Traut's reagent substance, and the mixture was immediately mixed and reacted at room temperature for 10 minutes. Desalted and replaced into 100mmol/L triethanolamine cross-linking buffer (pH 7.3 ± 0.05).
3. Mixing the activated myoglobin marker antibody solution obtained in the step 1 with the activated alkaline phosphatase solution obtained in the step 2, wherein the molar ratio of the activated myoglobin marker antibody to the activated alkaline phosphatase is 1: 1.5, fully and uniformly mixing, and reacting for 20 hours at the temperature of 2-8 ℃.
4. 3mg of N-ethylmaleimide was weighed out and prepared into a solution of 12.5mg/mL using 100mmol/L triethanolamine buffer (pH 7.3. + -. 0.05).
5. Adding the N-ethylmaleimide solution obtained in the step 4 into the system obtained in the step 3, wherein the molar ratio of the alkaline phosphatase-labeled myoglobin-labeled antibody to the N-ethylmaleimide is 1: 5, mixing well, and reacting at room temperature for 25 minutes to block the excess thiol groups. After the reaction is finished, the final myoglobin labeled antibody conjugate marked by alkaline phosphatase is obtained by purification by an ultrafiltration concentration method. And measuring the concentration of the purified enzyme conjugate concentrated solution, adding glycerol with the same volume, fully and uniformly mixing, and storing at-20 ℃ for later use.
Example 4 preparation of magnetic bead conjugates
1. Weighing 3mg of N-N-hydroxysuccinimide, and preparing a solution with the concentration of 10mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (the pH value is 5.0 +/-0.05); weighing 3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and preparing a solution with the concentration of 10mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05);
2. placing carboxyl magnetic beads with the particle size of 3 mu m on a magnetic frame and standing for 2min, magnetically separating and discarding supernate, washing twice by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05), adding into 2- (N-morpholine) ethanesulfonic acid buffer solution, and carrying out ultrasonic treatment until the carboxyl magnetic beads are completely dissolved; adding 10mg/mL of N-hydroxysuccinimide solution and 10mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution, wherein the mass of the N-hydroxysuccinimide solution and the mass of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution are 0.23 time and 0.1 time respectively of the mass of the carboxyl magnetic beads, uniformly mixing, and stirring at room temperature for reaction for 30 minutes. After the reaction is finished, magnetic beads are precipitated by a magnetic plate to remove supernatant, the reaction is repeated again, and the activated carboxyl magnetic beads are dissolved in 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05) for later use;
3. weighing 0.5mg of myoglobin-coated antibody, and preparing a solution with the concentration of 2mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (the pH value is 5.0 +/-0.05);
4. and (3) mixing the myoglobin coated antibody solution in the step (3) with the carboxyl magnetic bead solution in the step (2) according to the mass ratio of 1:100, and carrying out crosslinking reaction on the mixture for 20 hours at room temperature on a blending instrument. After the reaction was completed, the magnetic beads were precipitated with a magnetic plate to remove the supernatant, and the procedure was repeated 2 times. Adding the magnetic bead blocking solution CE210, and reacting for 20 hours at room temperature on a mixing instrument. After the reaction is finished, the magnetic beads are precipitated by a magnetic plate to remove supernatant, the magnetic beads are washed for 3 times by a magnetic bead washing solution, and the obtained magnetic beads are resuspended in a magnetic bead preservation solution (pH is 6.0 +/-0.05, 30mmol/L2- (N-morpholine) ethanesulfonic acid, 200mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone and 3% Proclin300) and are preserved at the temperature of 2-8 ℃ for later use.
Example 5 preparation of magnetic bead conjugates
1. Weighing 3mg of N-N-hydroxysuccinimide, and preparing a solution with the concentration of 10mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (the pH value is 5.0 +/-0.05); weighing 3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and preparing a solution with the concentration of 10mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05);
2. placing carboxyl magnetic beads with the particle size of 1.3 mu m on a magnetic frame to stand for 2min, magnetically separating and discarding supernate, washing twice by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05), adding the washed solution into the 2- (N-morpholine) ethanesulfonic acid buffer solution, and carrying out ultrasonic treatment until the carboxyl magnetic beads are completely dissolved; respectively adding 10mg/mL of N-hydroxysuccinimide solution and 10mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution, wherein the mass of the N-hydroxysuccinimide solution and the mass of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution are 0.20 time and 0.08 time respectively of that of the carboxyl magnetic beads, uniformly mixing, and stirring for reaction for 25 minutes at room temperature. After the reaction is finished, magnetic beads are precipitated by a magnetic plate to remove supernatant, the reaction is repeated again, and the activated carboxyl magnetic beads are dissolved in 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05) for later use;
3. weighing 0.5mg of myoglobin-coated antibody, and preparing a solution with the concentration of 5mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (the pH value is 5.0 +/-0.05);
4. and (3) mixing the myoglobin coated antibody solution in the step (3) with the carboxyl magnetic bead solution in the step (2) according to the mass ratio of 1:80, and carrying out crosslinking reaction for 16 hours at room temperature on a blending instrument. After the reaction was completed, the magnetic beads were precipitated with a magnetic plate to remove the supernatant, and the procedure was repeated 2 times. Adding the magnetic bead blocking solution CE210, and reacting for 16 hours at room temperature on a mixing instrument. After the reaction is finished, the magnetic beads are precipitated by a magnetic plate to remove supernatant, the magnetic beads are washed for 3 times by a magnetic bead washing solution, and the obtained magnetic beads are resuspended in a magnetic bead preservation solution (pH is 6.0 +/-0.05, 30mmol/L2- (N-morpholine) ethanesulfonic acid, 200mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone and 3% Proclin300) and are preserved at the temperature of 2-8 ℃ for later use.
Example 6 preparation of magnetic bead conjugates
1. Weighing 3mg of N-N-hydroxysuccinimide, and preparing a solution with the concentration of 10mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (the pH value is 5.0 +/-0.05); weighing 3mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and preparing a solution with the concentration of 10mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05);
2. placing carboxyl magnetic beads with the particle size of 4 mu m on a magnetic frame and standing for 2min, magnetically separating and discarding supernate, washing twice by using 2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05) with the concentration of 100mmol/L, adding the washed solution into 2- (N-morpholine) ethanesulfonic acid buffer solution, and carrying out ultrasonic treatment until the carboxyl magnetic beads are completely dissolved; respectively adding 10mg/mL of N-hydroxysuccinimide solution and 10mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution, wherein the mass of the N-hydroxysuccinimide solution and the mass of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution are 0.25 time and 0.12 time respectively of that of the carboxyl magnetic beads, uniformly mixing, and stirring at room temperature for reaction for 35 minutes. After the reaction is finished, magnetic beads are precipitated by a magnetic plate to remove supernatant, the reaction is repeated again, and the activated carboxyl magnetic beads are dissolved in 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (pH is 5.0 +/-0.05) for later use;
3. weighing 0.5mg of myoglobin-coated antibody, and preparing a solution with the concentration of 5mg/mL by using 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution (the pH value is 5.0 +/-0.05);
4. and (3) mixing the myoglobin coated antibody solution in the step (3) with the carboxyl magnetic bead solution in the step (2) according to the mass ratio of 1:120, and carrying out crosslinking reaction on the mixture for 24 hours at room temperature on a blending instrument. After the reaction was completed, the magnetic beads were precipitated with a magnetic plate to remove the supernatant, and the procedure was repeated 2 times. Adding the magnetic bead blocking solution CE210, and reacting for 24 hours at room temperature on a mixing instrument. After the reaction is finished, the magnetic beads are precipitated by a magnetic plate to remove supernatant, the magnetic beads are washed for 3 times by a magnetic bead washing solution, and the obtained magnetic beads are resuspended in a magnetic bead preservation solution (pH is 6.0 +/-0.05, 30mmol/L2- (N-morpholine) ethanesulfonic acid, 200mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone and 3% Proclin300) and are preserved at the temperature of 2-8 ℃ for later use.
Example 7 Myoglobin assay kit of the present invention
Myoglobin assay kit comprising: myoglobin calibrator, reagent R1, enzyme conjugate working solution R2, magnetic bead conjugate working solution M, cleaning solution and chemiluminescent substrate.
Wherein: the myoglobin calibrator is prepared by diluting a calibration product with traceability to 1500ng/mL by a calibrator diluent, wherein the calibrator diluent comprises the following components: 30 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin, 3mmol/L Proclin300, and pH 7.0 +/-0.05;
wherein: the reagent R1 comprises the following specific components: 60mmol/L of tris (hydroxymethyl) aminomethane, 150mmol/L NaCl, 3% sucrose, 3% glycerol, 0.1% bovine serum albumin, 1.2mmol/L of imidazole, 0.15% Tween 20 and 0.15% Proclin300, and the pH is 7.3 +/-0.05;
wherein: the concentration of the enzyme conjugate working solution R2 was 1.3. mu.g/mL, which was prepared by adding an enzyme conjugate diluent to an enzyme conjugate solution. The specific components of the enzyme conjugate diluent are as follows: 35 mmol/L2- (N-morphine) ethanesulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin, 5% sucrose, 5% glycerol, 0.1% Tween 20, 2.5mmol/LZnCl2、2.5mmol/LMgCl20.15% Proclin300, pH 6.3 + -0.05;
wherein: the concentration of the working solution M of the magnetic bead conjugate is 0.25mg/mL, and the working solution M is prepared by adding a magnetic bead conjugate solution into a magnetic bead conjugate diluent. The magnetic bead conjugate diluent comprises the following specific components: 35 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone, 3% Proclin300, and the pH is 7.5 +/-0.05.
Wherein: the preparation process of the cleaning solution comprises the following steps: sequentially adding 1211.4mg of tris (hydroxymethyl) aminomethane, 9g of NaCl, 201 g of Tween, 2.5g of sodium dodecyl sulfate and 900mL of water into a container, uniformly mixing until all components are dissolved, adjusting the pH of the solution to 8.0 +/-0.5 by using a sodium hydroxide solution, adding water to a constant volume of 1000mL, uniformly mixing for 30 minutes, and filtering with a particle size of 0.22 μm to obtain the cleaning solution.
Wherein: the preparation process of the chemiluminescence substrate comprises the following steps:
accurately weighing 0.5g of 3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxygen cyclohexane disodium salt (AMPPD), 18g of trihydroxymethyl aminomethane, 100g of NaCl, 0.02g of hexadecyl trimethyl ammonium chloride and water to a constant volume of 1000mL, and adjusting the pH value of a chemiluminescent substrate solution to 9.4 +/-0.05; the chemiluminescent substrate is an ethylene dioxides substrate for alkaline phosphatase, needs to be stored in a dark place at the temperature of 2-8 ℃, and is slowly and uniformly mixed when used.
Example 8 Myoglobin assay kit of the present invention
Myoglobin assay kit comprising: myoglobin calibrator, reagent R1, enzyme conjugate working solution R2, magnetic bead conjugate working solution M, cleaning solution and chemiluminescent substrate.
Wherein: the myoglobin calibrator is prepared by diluting a calibrator with traceability to 1ng/mL by a calibrator diluent, wherein the calibrator diluent comprises the following components: 20 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 150mmol/L NaCl, 1% bovine serum albumin, 0.5mmol/L Proclin300, and pH 6.5 +/-0.05;
wherein: the reagent R1 comprises the following specific components: 25mmol/L of tris (hydroxymethyl) aminomethane, 150mmol/L NaCl, 1% sucrose, 1% glycerol, 0.1% bovine serum albumin, 0.4mmol/L imidazole, 0.05% Tween 20 and 0.05% Proclin300, and the pH is 7.3 +/-0.05;
wherein: the concentration of the enzyme conjugate working solution R2 was 0.5. mu.g/mL, which was prepared by adding an enzyme conjugate diluent to an enzyme conjugate solution. The specific components of the enzyme conjugate diluent are as follows: 20 mmol/L2- (N-morphine) ethanesulfonic acid, 150mmol/L NaCl, 1% bovine serum albumin, 5% sucrose, 5% glycerol, 0.1% Tween 20, 0.05mmol/LZnCl2、0.05mmol/LMgCl20.05% Proclin300, pH 6.0 + -0.05;
wherein: the concentration of the working solution M of the magnetic bead conjugate is 0.1mg/mL, and the working solution M is prepared by adding a magnetic bead conjugate solution into a magnetic bead conjugate diluent. The magnetic bead conjugate diluent comprises the following specific components: 20 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 150mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone, 0.5% Proclin300, and pH of 7.0 +/-0.05.
In this example, the composition and preparation process of the cleaning solution and the chemiluminescent substrate were the same as those in example 7.
Example 9 Myoglobin assay kit of the present invention
Myoglobin assay kit comprising: myoglobin calibrator, reagent R1, enzyme conjugate working solution R2, magnetic bead conjugate working solution M, cleaning solution and chemiluminescent substrate.
Wherein: the myoglobin calibrator is prepared by diluting a calibrator with traceability to 3000ng/mL by a calibrator diluent, wherein the calibrator diluent comprises the following components: 100 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 150mmol/L NaCl, 1% bovine serum albumin, 5mmol/L Proclin300, and pH 7.5 +/-0.05;
wherein: the reagent R1 comprises the following specific components: 100mmol/L of tris (hydroxymethyl) aminomethane, 150mmol/L NaCl, 5% sucrose, 5% glycerol, 0.1% bovine serum albumin, 2mmol/L of imidazole, 0.2% Tween 20 and 0.2% Proclin300, and the pH is 7.5 +/-0.05;
wherein: the concentration of the enzyme conjugate working solution R2 was 2. mu.g/mL, which was prepared by adding an enzyme conjugate diluent to an enzyme conjugate solution. The specific components of the enzyme conjugate diluent are as follows: 50mmol/L2- (N-morphine) ethanesulfonic acid, 300mmol/L NaCl, 1% bovine serum albumin, 5% sucrose, 5% glycerol, 0.1% Tween 20, 5mmol/LZnCl2、5mmol/LMgCl20.2% Proclin300, pH 6.5 + -0.05;
wherein: the concentration of the working solution M of the magnetic bead conjugate is 0.5mg/mL, and the working solution M is prepared by adding a magnetic bead conjugate solution into a magnetic bead conjugate diluent. The magnetic bead conjugate diluent comprises the following specific components: 50 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 300mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone, 5% Proclin300, and the pH is 8.0 +/-0.05.
In this example, the composition and preparation process of the cleaning solution and the chemiluminescent substrate were the same as those in example 7.
Example 10 Myoglobin assay kit of the present invention
Myoglobin assay kit comprising: myoglobin calibrator, quality control material, reagent R1, enzyme conjugate working solution R2, magnetic bead conjugate working solution M, cleaning solution and chemiluminescent substrate.
Wherein: the myoglobin calibrator or quality control product is prepared by diluting a traceable calibrator or quality control product to 3000ng/mL by a calibrator diluent, wherein the calibrator diluent comprises the following components: 30 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin, 3mmol/L Proclin300, and pH of 7.0 +/-0.05;
wherein: the reagent R1 comprises the following specific components: 60mmol/L of tris (hydroxymethyl) aminomethane, 150mmol/L NaCl, 3% sucrose, 3% glycerol, 0.1% bovine serum albumin, 1.2mmol/L of imidazole, 0.15% Tween 20 and 0.15% Proclin300, and the pH is 7.3 +/-0.05;
wherein: the concentration of the enzyme conjugate working solution R2 was 1.3. mu.g/mL, which was prepared by adding an enzyme conjugate diluent to an enzyme conjugate solution. The specific components of the enzyme conjugate diluent are as follows: 35 mmol/L2- (N-morphine) ethanesulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin, 5% sucrose, 5% glycerol, 0.1% Tween 20, 2.5mmol/LZnCl2、2.5mmol/LMgCl20.15% Proclin300, pH 6.35 + -0.05;
wherein: the concentration of the working solution M of the magnetic bead conjugate is 0.25mg/mL, and the working solution M is prepared by adding a magnetic bead conjugate solution into a magnetic bead conjugate diluent. The magnetic bead conjugate diluent comprises the following specific components: 35 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 250mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone, 3% Proclin300, and the pH is 7.5 +/-0.05.
In this example, the composition and preparation process of the cleaning solution and the chemiluminescent substrate were the same as those in example 7.
Example 11 procedure for detecting Myoglobin in finger peripheral Whole blood with kit of the present invention
1. Adding 25 μ L of sample, calibrator or quality control material into the test tube;
2. adding 20 μ L of reagent R1 into the test tube, and gently shaking the test tube back and forth;
3. add 50. mu.L of enzyme conjugate working solution R2 to the tube;
4. adding 50 mu L of magnetic bead conjugate working solution M into a test tube;
5. the test tube is prevented from being mixed on a vortex mixer for 30 seconds and then is placed at 42 ℃ for reaction for 8 minutes;
6. placing the test tube on a magnetic frame, standing for 2 minutes, pouring out supernatant, patting to dry, adding 300 mu L of cleaning solution, and repeating the step for 3 times;
7. the reaction tube is transferred to a luminescence detector for detection, and 100. mu.L of chemiluminescent substrate is added to the luminescence detector and the luminescence value of each test sample is read.
8. Making a dose-response curve by using the concentration and the luminous value of the standard substance or the quality control substance;
9. and substituting the luminous value of the sample to be detected into the curve to obtain the content of the myoglobin in the sample to be detected.
The myoglobin detection method provided in the embodiment is also suitable for myoglobin detection of anticoagulated venous whole blood, serum or plasma.
Experimental example 1
The kit of the invention shows a luminescence phenomenon when 25 mul of anticoagulated venous whole blood is used as a sample, the detection procedure according to the embodiment 11 of the invention can obtain the content of myoglobin in the whole blood, and the detection by the technology disclosed in the Chinese patent document CN107255723A does not detect the concentration of myoglobin in the whole blood.
Experimental example 2 Linear verification
Taking a traceable standard substance or a quality control substance with high concentration to the utmost extent, diluting a sample by using normal saline, measuring for 3 times at each point, taking an average value, taking a regression line between the result and the expected concentration, and calculating to obtain a regression coefficient r which is 0.9989 and more than 0.99, which shows that the dilution linearity of the kit provided by the invention is good.
Experimental example 3 verification of precision
And taking one part of the high-value quality control product and one part of the low-value quality control product of the traceable serum, carrying out detection on each part of the quality control product for 10 times, and calculating the average value and the standard value of 10 detection results. According toThe coefficient of variation CV is (standard deviation/average) × 100%, calculated as: CV of1(1000ng/mL)＝3％，CV2(111ngg/mL) 5%. Therefore, the kit provided by the invention has higher precision.
Experimental example 4 verification of accuracy
And taking one part of the high-value quality control product and one part of the low-value quality control product of the traceable serum, respectively detecting for 5 times by using the kit, calculating an average value, and then contrasting with a target value of the quality control product. The result shows that the detection value of the kit is close to the target value, which indicates that the kit provided by the invention has higher accuracy.
Experimental example 5 verification of sensitivity
Diluting the quality control product with traceability to the vicinity of the lower limit (1ng/mL) of the detection range, measuring repeatedly for 3 times, calculating the average value, and comparing with the target value of the quality control product. The result shows that the detection value of the kit is close to the target value, which indicates that the kit provided by the invention has higher sensitivity.
Experimental example 6 verification of detection Range
And (3) taking the traceable standard substance, diluting by different times, and respectively detecting whether the luminescence phenomenon exists, wherein the result shows that the luminescence phenomenon exists in the range of the concentration of 1-300 ng/mL, and the detection range of the kit is 1-300 ng/mL.
Experimental example 7 correlation verification
200 serum samples were taken, and the correlation between the detection results of the kit of the present invention and the myoglobin detection kit of beckmann corporation was compared, and the results are shown in fig. 1. Wherein: the abscissa is the detection result of the Beckmann's kit, the ordinate is the detection result of the kit of the invention, and the correlation coefficient R20.9709, the regression line equation is: y 1.0261x + 10.8821. As can be seen from FIG. 1, the results of the sample detection of the present invention are not significantly different from the results of the detection of products of the known companies in the industry.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. It is not necessary or necessary to exhaustively enumerate all embodiments herein, and obvious variations or modifications can be made without departing from the scope of the invention.
Claims (15)
1. A myoglobin assay kit, comprising:
(1) a calibrator;
(2) reagent R1;
(3) enzyme conjugate working solution R2;
(4) a magnetic bead conjugate working solution M;
(5) cleaning fluid;
(6) a chemiluminescent substrate;
the reagent R1 comprises: 25-100 mmol/L of trihydroxymethyl aminomethane, 150mmol/L of NaCl, 1-5% of sucrose, 1-5% of glycerol, 0.1% of bovine serum albumin, 0.4-2 mmol/L of imidazole, 0.05-0.2% of Tween 20 and 0.05-0.2% of Proclin300, wherein the pH value is 7.0-7.5, the cleaning solution contains a sodium dodecyl sulfate component, and the kit directly uses finger tip whole blood or anticoagulation vein whole blood as a sample to be detected;
the calibrator is prepared by adding myoglobin into a calibrator diluent, wherein the calibrator diluent comprises: 20-50 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 150-300 mmol/L NaCl, 1% bovine serum albumin, 0.5-5 mmol/L proclin300, and the pH value is 6.5-7.5;
the concentration of the enzyme conjugate working solution R2 is 0.5-2 mug/mL, and the enzyme conjugate working solution is prepared by adding an enzyme conjugate solution into an enzyme conjugate diluent, wherein the diluent comprises: 20-50 mmol/L2- (N-morpholine) ethanesulfonic acid, 150-300 mmol/L NaCl, 1% bovine serum albumin, 5% sucrose, 5% glycerol, 0.1% Tween 20, 0.05-5 mmol/LZnCl2、0.05~5mmol/L MgCl20.05 to 0.2 percent of Proclin300, and the pH value is 6.0 to 6.5;
the concentration of the working solution M of the magnetic bead conjugate is 0.1-0.5 mg/mL, and the working solution M is prepared by adding a magnetic bead conjugate solution into a magnetic bead diluent, wherein the diluent comprises: 20-50 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 150-300 mmol/L NaCl, 1% bovine serum albumin, 1% sucrose, 1% gelatin, 0.1% Tween 20, 1% polyvinylpyrrolidone, 0.5-5% Proclin300, and the pH value is 7.0-8.0;
the main component of the enzyme conjugate is an alkaline phosphatase-labeled myoglobin-labeled antibody;
the main component of the magnetic bead conjugate is a carboxyl magnetic bead coupled with a myoglobin coated antibody, and the particle size of the carboxyl magnetic bead is 1.3-4 mu m.
2. The myoglobin assay kit of claim 1, wherein said alkaline phosphatase-labeled myoglobin labeled antibody is prepared by contacting activated myoglobin-labeled antibody with activated alkaline phosphatase in a molar ratio of 1: (1-2), and reacting in a triethanolamine buffer solution with the pH value of 7.3 at the temperature of 2-8 ℃ for 18-24 hours to obtain the alcohol-based catalyst.
3. The myoglobin assay kit of claim 2, wherein said activated myoglobin labeled antibody is prepared by contacting a solution of Traut's with a solution of myoglobin labeled antibody at a molar ratio of 1: (15-30), and reacting in triethanolamine buffer solution with the pH value of 8.5 for 12-18 min at room temperature to obtain the product; the activated alkaline phosphatase is prepared by mixing a sodium salt solution of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester with an alkaline phosphatase solution in a molar ratio of 1: (10-15), and reacting in an N, N-dimethylformamide solution at room temperature for 12-18 min to obtain the compound.
4. The myoglobin detection kit according to claim 3, wherein the concentration of said Traut's solution is 1.3-1.5 mg/mL; the concentration of the myoglobin marker antibody solution is 2-5 mg/mL; the concentration of the 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt solution is 16-18 mg/mL; the concentration of the alkaline phosphatase solution is 16-20 mg/mL.
5. The myoglobin detection kit of claim 4, further comprising: and adding a glycine solution into the activated myoglobin marker antibody, wherein the amount of the glycine is 10-20 times of that of the Traut's reagent substance, and reacting at room temperature for 8-12 min after fully and uniformly mixing.
6. The myoglobin detection kit of claim 5, further comprising: adding a glycine solution into the activated alkaline phosphatase, wherein the amount of glycine is 10-20 times of that of the sodium salt of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester, fully mixing uniformly, and reacting at room temperature for 8-12 min.
7. The myoglobin detection kit of claim 6, further comprising: and adding an N-ethyl maleimide solution into the myoglobin labeled antibody marked by the alkaline phosphatase, wherein the amount of the N-ethyl maleimide is 5-20 times of that of the myoglobin labeled antibody marked by the alkaline phosphatase, and reacting at room temperature for 25-35 min after fully and uniformly mixing.
8. The myoglobin detection kit of claim 7, further comprising: adding glycerol into the purified myoglobin marked antibody marked by the alkaline phosphatase, and storing at the temperature of minus 25 to minus 15 ℃.
9. The myoglobin detection kit according to claim 1, wherein the carboxyl magnetic beads coupled with the myoglobin-coated antibody are obtained by mixing a myoglobin-coated antibody solution and a carboxyl magnetic bead solution according to a mass ratio of 1 (80-120), and performing crosslinking reaction in a 2- (N-morpholine) ethanesulfonic acid buffer solution with the pH of 5.0 at room temperature for 16-24 hours.
10. The myoglobin assay kit of claim 9, wherein said myoglobin coated antibody solution has a concentration of 1-5 mg/mL; the concentration of the carboxyl magnetic bead solution is 100 mg/mL.
11. The myoglobin detection kit of claim 10, further comprising: before the cross-linking reaction, performing activation treatment on carboxyl magnetic beads, wherein the activation treatment comprises the following steps:
(1) placing the carboxyl magnetic beads on a magnetic frame and standing for 2min, magnetically separating and discarding supernatant, washing twice with 100mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution with the pH value of 5.0, adding into the 2- (N-morpholine) ethanesulfonic acid buffer solution, and performing ultrasonic treatment until the carboxyl magnetic beads are completely dissolved;
(2) respectively adding an N-N-hydroxysuccinimide solution and a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution into the system in the step (1), wherein the mass of the N-N-hydroxysuccinimide solution is 0.20-0.25 times of that of the carboxyl magnetic beads, and the mass of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution is 0.08-0.12 times of that of the carboxyl magnetic beads, uniformly mixing, and stirring and reacting at room temperature for 25-35 min.
12. The myoglobin assay kit of claim 11, wherein said N-hydroxysuccinimide solution is present at a concentration of 10 mg/mL; the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution is 10 mg/mL.
13. The myoglobin detection kit of claim 12, further comprising: and adding a magnetic bead sealing solution into the carboxyl magnetic beads coupled with the myoglobin coated antibody, and reacting for 16-24 h at room temperature.
14. The myoglobin detection kit of claim 13, wherein said bead blocking solution is CE 210.
15. The myoglobin detection kit of any one of claims 1-14 wherein said kit further comprises a myoglobin quality control agent.
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| CN108931652A (en) * | 2018-06-20 | 2018-12-04 | 泰州泽成生物技术有限公司 | A kind of kit with Magnetism particulate immuno chemistry luminescence method detection myoglobin content |
| CN109254153B (en) * | 2018-08-27 | 2021-10-12 | 广东菲鹏生物有限公司 | Antioxidant of myoglobin and application thereof |
| CN112051404B (en) * | 2020-09-10 | 2023-08-08 | 武汉生之源生物科技股份有限公司 | Myoglobin detection kit and application thereof |
| CN112129951A (en) * | 2020-09-16 | 2020-12-25 | 武汉生之源生物科技股份有限公司 | Enzyme-labeled diluent for SAA chemiluminescence enzyme immunoassay and application thereof |
| CN112505334B (en) * | 2020-11-23 | 2021-12-28 | 厦门宝太生物科技股份有限公司 | NT-proBNP homogeneous phase chemiluminescence detection kit |
| CN113030458A (en) * | 2021-02-26 | 2021-06-25 | 武汉菲恩生物科技有限公司 | Sample diluent for ELISA detection kit and preparation method thereof |
| CN113311174B (en) * | 2021-05-14 | 2022-05-13 | 宁波瑞源生物科技有限公司 | Myoglobin antibody-enzyme marker and preparation and application thereof |
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| CN1945331B (en) * | 2006-10-20 | 2011-06-08 | 邹明强 | Method for preparing and using reagent for simultaneously detecting multiple small molecular compounds |
| CN102435752B (en) * | 2011-08-31 | 2014-04-02 | 刘起中 | Quantitative determination kit for human myoglobin and detection method thereof |
| 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) |
| CN103278651B (en) * | 2013-06-14 | 2015-07-15 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit |
| CN105203748A (en) * | 2015-09-28 | 2015-12-30 | 成都博奥新景医学科技有限公司 | Quantitative detection kit for full-measuring-range C reaction protein |
| CN105403693B (en) * | 2015-10-27 | 2018-07-10 | 北京九强生物技术股份有限公司 | A kind of preparation method of magnetic microparticle chemiluminescence reagent |
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