CN107918022B - cTnI detection kit and use method thereof - Google Patents

cTnI detection kit and use method thereof Download PDF

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CN107918022B
CN107918022B CN201711146344.3A CN201711146344A CN107918022B CN 107918022 B CN107918022 B CN 107918022B CN 201711146344 A CN201711146344 A CN 201711146344A CN 107918022 B CN107918022 B CN 107918022B
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ctni
antibody
alkaline phosphatase
magnetic bead
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CN107918022A (en
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王保君
汤双双
欧卫军
徐艳
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NANTONG EGENS BIOTECHNOLOGY CO Ltd
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NANTONG EGENS BIOTECHNOLOGY CO Ltd
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Abstract

The invention discloses a cTnI detection kit, which comprises a calibrator, a cleaning solution, a substrate solution, a pretreatment solution, an enzyme conjugate working solution and a magnetic bead conjugate working solution; the pretreatment solution contains imidazole, the enzyme conjugate working solution contains an enzyme-labeled cTnI antibody, and the magnetic bead conjugate working solution contains magnetic beads labeled with the cTnI antibody. The cTnI detection kit can realize accurate determination of cTnI in a whole blood sample, simplifies detection steps and improves detection efficiency. The kit has the advantages of low detection limit of 0.02ng/ml, linear range of 0.02-50ng/ml, high detection sensitivity, wide linear range and accurate detection result. The invention also discloses a using method of the cTnI detection kit, which has simple using steps, effectively shortens the detection time of the cTnI and is beneficial to realizing the rapid and sensitive detection of the cTnI.

Description

cTnI detection kit and use method thereof
Technical Field
The invention belongs to the technical field of immunochemistry detection, and particularly relates to a cTnI detection kit and a using method thereof.
Background
Cardiovascular diseases are major fatal diseases which harm the health and life of people in China, according to the statistics of Ministry of health, two hundred and thirty-five people die of cardiovascular and cerebrovascular diseases in every hundred thousand in cities in China, account for the first death caused by various diseases, and are increased by 2% every year. Acute Myocardial Infarction (AMI) is the leading cause of death in cardiovascular patients. AMI is a clinical paroxysmal disease, and the paroxysm is often accompanied by chest pain, nausea, fever, arrhythmia, a series of symptoms such as serum marker rise and the like, and has important significance for saving dying cardiac muscle, improving prognosis, reducing acute fatality rate and the like if the acute paroxysmal disease can be diagnosed as soon as possible and intervened in treatment.
Troponin (Tn) is a contraction regulation protein of cardiac and skeletal muscles, and cTn is cardiac troponin, which is composed of three subunits, cTnI, cTnT and cTnC. cTnI, cTnC and cTnT play important regulatory roles in muscle relaxation and contraction, but cTnC has no myocardial specificity and is not generally used for myocardial injury detection. Under normal conditions, both cTnI and cTnT can not penetrate cell membranes to enter blood, so that the cTnI and cTnT in the blood of healthy people are extremely low; such as myocardial cell damage, cTnI and cTnT enter the human intercellular matrix and blood. However, since the content of cTnT in blood is also greatly increased in diseases such as renal failure, pneumonia, and sepsis, the specificity of detecting AMI using cTnT is low. The cTnI rises 3-5 hours after the onset of AMI, reaches a peak 15-24 hours, lasts for a long time, and can be reduced to normal after 5-10 days. At present, the cTnI is used for diagnosing myocardial infarction and myocardial cell injury and is a new 'gold standard' for diagnosing AMI.
The NT-proBNP can be determined by electrophoresis, ion-exchange column chromatography, ELISA, radioimmunoassay, etc. The conventional electrophoresis method and the ELISA method are more, but the electrophoresis method cannot be applied to a full-automatic biochemical analyzer, and the used instrument is expensive, so that the electrophoresis method cannot be popularized; the ELISA method has complex operation process and overlong detection time, and is not beneficial to realizing clinical rapid detection of cTnI.
Chinese patent document CN104330575A discloses a troponin I detection reagent, which utilizes the characteristics of homogeneous colloidal gold particles to label specific cTnI antibodies on the surface of the colloidal gold particles, when there is cTnI in the detection system or detection environment, the antibodies on the surface of the colloidal gold particles will capture the corresponding antigens and form antigen-antibody complexes, further causing the aggregation or accumulation of local colloidal gold particles, so that the transmission spectrum of the homogeneous colloidal gold reagent shifts from red to blue, and the shift mainly reflects the decrease of absorbance at 540nm and the increase of absorbance at 660nm, thereby achieving the purpose of quantitatively detecting the cTnI antigens in human body. The cTnI detection reagent has the detection speed and sensitivity of cTnI to a certain extent, but the detection reagent can only be used for detecting the content of cTnI in serum or plasma, and cannot realize accurate and rapid determination of the content of cTnI in whole blood. In practical clinical application, the collected whole blood sample needs to be subjected to centrifugal separation, so that the detection step of cTnI is complicated, and the detection cost is increased; on the other hand, the colloidal gold detection method has the phenomena of poor detection repeatability, unfavorable quality control, easy occurrence of misreading and misjudgment and the like.
Disclosure of Invention
Therefore, the technical problem to be solved by the present invention is to overcome the defect that the detection of a whole blood sample cannot be realized by the cTnI detection in the prior art.
Therefore, the invention provides a cTnI detection kit, which comprises: the kit comprises a calibrator, a cleaning solution, a substrate solution, a pretreatment solution, an enzyme conjugate working solution and a magnetic bead conjugate working solution; the pretreatment solution contains imidazole, the enzyme conjugate working solution contains an enzyme-labeled cTnI antibody, and the magnetic bead conjugate working solution contains magnetic beads labeled with the cTnI antibody.
In the cTnI detection kit, the enzyme-labeled cTnI antibody is an alkaline phosphatase-labeled cTnI antibody.
In the above cTnI detection kit, the alkaline phosphatase-labeled cTnI antibody is prepared by the following method:
A. activating cTnI antibodies
Adding a cTnI antibody of 2mg/ml into a TCEP solution of 0.5M until the final concentration of TCEP is 1.25mM, uniformly mixing, and standing at room temperature for reaction;
desalting the reacted antibody solution and replacing the antibody solution into 100mM triethanolamine buffer solution with pH 7.3;
B. activating alkaline phosphatase
③ adding 17.5mg/mL of Sulfo-SMCC solution into the alkaline phosphatase solution, uniformly mixing, and standing at room temperature; the sulfol-SMCC solution is prepared by adding a sulfol-SMCC reagent into dimethylformamide, wherein the molar ratio of the sulfol-SMCC reagent to the alkaline phosphatase is (10-15): 1;
fourthly, continuously adding 1M glycine solution, uniformly mixing, and standing at room temperature for reaction; wherein the molar ratio of the addition amount of the glycine to the Sulfo-SMCC reagent is (10-20): 1;
fifthly, desalting the alkaline phosphatase solution after reaction, and replacing the alkaline phosphatase solution into triethanolamine buffer solution with the pH value of 7.3 and the concentration of 100 mM;
C. uniformly mixing the cTnI antibody solution activated in the step A and the alkaline phosphatase solution activated in the step B, and standing at the temperature of 2-8 ℃ for reaction for 18-24 hours; wherein the molar ratio of the cTnI antibody to the alkaline phosphatase is 1: 2;
D. and D, adding 12.5mg/ml of N-ethylmaleimide solution into the solution reacted in the step C, uniformly mixing, standing at room temperature for reaction, and purifying the reacted solution to obtain the alkaline phosphatase labeled cTnI antibody.
In the cTnI detection kit, the cleaning solution is Tris buffer solution containing SBS, Tween-20 and Proclin-300, the substrate solution is enzymatic chemiluminescence substrate solution, and the calibrator is cTnI HEPES buffer solution prepared by calibrator diluent.
The cTnI detection kit further comprises: and the quality control product is a cTnI HEPES buffer solution prepared by using a calibrator diluent.
In the cTnI detection kit, the calibrator diluent comprises: 20-50 mM BES, 150-300 mM NaCI, 1% horse serum, 0.5-5 mM Proclin-300 and 1% mannitol, wherein the pH of the calibrator diluent is 6.5-7.5.
According to the cTnI detection kit, the concentration of imidazole is 0.8-1.5 mM, the concentration of the enzyme-labeled cTnI antibody is 0.6-1.8 mu g/ml, and the concentration of the magnetic bead-labeled cTnI antibody is 0.2-0.5 mg/ml.
The above cTnI detection kit, the pretreatment solution further comprises: 25-100 mM Tris,150mM NaCl, 1-5% sucrose, 1-5% glycerol, 0.1% BSA, 0.05-0.2% Tween-20 and 0.05-0.2% Proclin-300, wherein the pH of the pretreatment solution is 7.0-7.5;
the enzyme conjugate working solution further comprises: 20-50 mM Tris, 150-300 mM NaCl, 1% BSA, 5% sucrose, 5% glycerol, 0.1% Tween-20, 0.02-5 mM ZnCl20.02-5 mM MgCl2And 0.05-0.2% of Proclin300, wherein the pH of the working solution of the enzyme conjugate is 7.0-9.0;
the working solution of the magnetic bead conjugate further comprises: 20-50 mM Tris, 150-300 mM NaCl, 1% BSA, 1% sucrose, 1% gelatin, 0.1% Tween-20, 1% PVP360 and 0.05-0.2% Proclin300, wherein the pH of the working solution of the magnetic bead conjugate is 7.0-9.0.
In the cTnI detection kit, the magnetic beads have the particle size of 3 microns.
In the cTnI detection kit, the magnetic beads labeled with the cTnI antibody are prepared by the following method:
E. adding a 100mM MES buffer solution with the pH value of 5.0 into the cTnI antibody until the concentration of the antibody solution is 1-5 mg/ml;
F. activated magnetic bead
1) Placing the magnetic bead solution of 100mg/ml on a magnetic frame for standing, and magnetically separating and removing supernate; wherein the mass ratio of the magnetic bead solution to the cTnI antibody is 100: 1;
2) dissolving the magnetic beads obtained in the step 1) by using a MES buffer solution with the pH of 5.0 and the concentration of 100mM, and magnetically separating and discarding a supernatant;
3) dissolving the magnetic beads obtained in the step 2) in a 100mM MES buffer solution, continuously adding a 10mg/ml NHS solution with the weight of 0.23 time that of the magnetic beads and a 10mg/ml EDC solution with the weight of 0.1 time that of the magnetic beads, and carrying out oscillation reaction at room temperature; wherein the NHS solution is prepared by dissolving NHS reagent in MES buffer solution with pH of 5.0 and 100mM, and the EDC solution is prepared by dissolving EDC reagent in MES buffer solution with pH of 5.0 and 100 mM;
4) magnetically separating the magnetic bead solution reacted in the step 3), discarding the supernatant, and dissolving in 100mM MES buffer solution;
G. mixing the cTnI antibody solution obtained in the step E with the magnetic bead solution activated in the step F, carrying out crosslinking reaction for 16-24 hours at room temperature, carrying out magnetic separation, discarding supernatant, continuously adding the magnetic bead sealing solution CE210, uniformly mixing, and carrying out reaction for 16-24 hours at room temperature;
H. and G, magnetically separating the magnetic bead solution obtained in the step G, then discarding the supernatant, and then cleaning the magnetic beads to obtain magnetic beads marked by the cTnI antibody.
The use method of the cTnI detection kit comprises the following steps:
(1) adding a pretreatment solution into a sample to be detected, uniformly mixing, adding a magnetic bead working solution and an enzyme conjugate working solution, uniformly mixing, and reacting at the temperature of 37-42 ℃; the volume ratio of the pretreatment solution to the sample to be detected is not more than 4, and the volume ratio of the pretreatment solution to the magnetic bead working solution to the enzyme conjugate working solution is 1: 5: 5;
(2) carrying out magnetic separation on the reaction liquid obtained in the step (1), and collecting magnetic beads;
(3) and (3) cleaning the magnetic beads, adding an enzymatic chemiluminescence substrate solution, uniformly mixing, and detecting a luminescence value.
Compared with the prior art, the invention has the following advantages:
1. the cTnI detection kit provided by the invention comprises a calibrator, a cleaning solution, a substrate solution, a pretreatment solution, an enzyme conjugate working solution and a magnetic bead conjugate working solution; the pretreatment solution contains imidazole, the enzyme conjugate working solution contains an enzyme-labeled cTnI antibody, and the magnetic bead conjugate working solution contains magnetic beads labeled with the cTnI antibody.
When the cTnI detection kit is used for detecting a sample to be detected, an enzyme-labeled cTnI antibody and a magnetic bead labeled by the cTnI antibody are respectively combined with different epitopes of a cTnI antigen in the sample to be detected through the cTnI antibody to form a sandwich structure. Directly precipitate in an external magnetic field and can be separated without centrifugation. The supernatant is decanted, the precipitated complex is washed, and then the enzymatic chemiluminescent substrate is added. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, photons are emitted when the excited state intermediate returns to a ground state to form a luminescence reaction, namely, a luminometer can be used for detecting the luminescence intensity of the reaction, the luminescence intensity is in direct proportion to the concentration of cTnI in a sample to be detected, and the accurate determination of the content of cTnI in the sample to be detected can be realized by detecting the luminescence intensity. The cTnI detection kit provided by the invention combines a chemiluminescence immunoassay method with a magnetic particle separation technology, and has the advantages of high detection sensitivity, strong specificity and accurate detection result.
According to the cTnI detection kit provided by the invention, the pretreatment liquid contains imidazole, 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 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.
The kit provided by the invention is used for detecting chemiluminescence intensity under different concentrations of cTnI, and a standard curve for detecting the kit can be drawn. When the subsequent kit is used for detecting the cTnI antigen in the sample to be detected, the cTnI content in the sample can be calculated according to the standard curve, so that accurate quantitative detection is realized.
2. The concentrations, components and contents of all components of the pretreatment solution, the enzyme conjugate working solution and the magnetic bead conjugate working solution provided by the invention can provide a stable working environment for an enzyme-labeled cTnI antibody and a magnetic bead labeled by the cTnI antibody, can realize specific identification and detection on cTnI when the concentration of the cTnI antigen in a sample is 0.02ng/ml, has a detection range of 0.02-50ng/ml, and is favorable for realizing high sensitivity and wide-range detection on cTnI; meanwhile, the stability of the detection of the kit is improved, the repeatability of the detection result of the kit is high, and the misreading misjudgment which possibly occurs in AMI diagnosis is reduced. In addition, the solution system ensures high catalytic activity of the enzyme, so that the enzymatic reaction substrate can emit light for a long time under the action of the enzymatic reaction substrate. The high detection performance of the cTnI detection kit is realized.
3. The invention provides a preparation method of a cTnI antibody marked by alkaline phosphatase, which comprises the steps of activating alkaline phosphatase by utilizing Sulfo-SMCC, reducing the cTnI antibody by TCEP, enabling the cTnI antibody to have a sulfhydryl group capable of being combined with the activated alkaline phosphatase, and realizing the coupling of the antibody and enzyme through the combination of the sulfhydryl group and an amino group. The invention realizes the coupling of alkaline phosphatase and cTnI antibody by using a reduction method, has high coupling efficiency, and can improve the selectivity of coupling reaction and the uniformity of coupling products; and a triethanolamine buffer solution is used in the reaction body fluid, the triethanolamine has tertiary amino and does not contain primary amino, interference is not generated in the coupling process, and a stable coupling environment is provided for coupling of alkaline phosphatase and the NT-proBNP antibody. The alkaline phosphatase-labeled cTnI antibody obtained by the preparation process has high enzyme catalysis performance and high binding efficiency with the cTnI antigen, so that the enzyme conjugate working solution containing the alkaline phosphatase-labeled cTnI antibody has the advantages of high sensitivity, strong specificity, wide detection range and the like when being used for cTnI antigen detection.
In the preparation process, a glycine solution is added 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 conjugate of the cTnI antibody and the enzyme 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.
4. The invention provides a preparation method of magnetic beads marked by a cTnI antibody, wherein NHS and EDC are adopted to activate the magnetic beads in the preparation process, so that the coupling efficiency of the cTnI antibody and the magnetic beads is improved; the MES buffer solution is used for dissolving the magnetic beads, so that the dispersion stability of the magnetic beads is ensured, the influence on detection signals due to the condensation of the magnetic beads is effectively prevented, and the accuracy of a detection result of the kit is ensured; when the kit is used for whole blood detection, the components in blood are complex, and the preparation method provided by the invention has the advantages that the cTnI antibody is coupled with the magnetic beads and then the magnetic bead sealing liquid is added, so that the sites which are not combined with the antibody on the magnetic beads are effectively sealed, the non-specific combination which is possibly generated in the later detection of a whole blood sample is avoided, and the accuracy of the detection result is further ensured. The magnetic bead sealing liquid CE210 is used for effectively sealing the magnetic beads, ensuring the activity of the cTnI antibody and improving the detection performance of the kit; the using amount, temperature and pH condition of each reaction substance in the coupling reaction are limited, and the coupling reaction condition is optimized.
5. The cTnI 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 cTnI is realized, and the accuracy and the detection efficiency are greatly improved.
6. The invention provides a using method of a cTnI 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 with reference to the accompanying drawings, and it should be understood 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.
The following examples are provided to illustrate embodiments of the present invention, and unless otherwise indicated, the experimental methods disclosed herein are all conventional in the art, and the percentages used herein are by weight. The cTnI antibodies in the following examples were purchased from Hytest corporation; 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
This example provides a method for preparing an alkaline phosphatase-labeled cTnI antibody, comprising the steps of:
1. the storage concentration of the cTnI-labeled antibody (i.e., cTnI antibody) was 2mg/mL, and 0.5M of a TECP solution was added to the cTnI-labeled antibody to a final concentration of 1.25mM of the TECP-labeled antibody, which was immediately mixed, followed by allowing the mixture to stand at 25 ℃ for 60 minutes.
2. The cTnI labeled antibody solution obtained in step 1 was immediately desalted and replaced with 100mM triethanolamine solution having pH7.3, and the concentration of the antibody solution was measured by UV spectrophotometry.
3. An appropriate amount of Sulfo-SMCC reagent is weighed and prepared into a solution with the concentration of 17.5mg/mL by using dimethylformamide DMF.
4. Adding the sulfoc-SMCC solution prepared in the step 2 into an alkaline phosphatase solution (AP solution with the concentration of 20mg/ml), wherein the molar ratio of the alkaline phosphatase to the sulfoc-SMCC is 1: immediately, mix well, and let stand to react at 25 ℃ for 15 minutes.
5. And (3) adding a 1M glycine solution into the alkaline phosphatase solution obtained in the step (4), immediately mixing the alkaline phosphatase solution and the sulfoc-SMCC reagent at a molar ratio of 10:1, and standing the mixture at 25 ℃ for reaction for 10 minutes.
6. Immediately desalting the alkaline phosphatase solution obtained in the step 5, replacing the alkaline phosphatase solution with triethanolamine solution with the pH value of 7.3 and the concentration of the alkaline phosphatase solution being 100mM, and measuring the concentration of the alkaline phosphatase solution by using an ultraviolet spectrophotometry;
7. mixing the activated antibody solution of the step 2 with the activated alkaline phosphatase solution of the step 6, wherein the molar ratio of the activated cTnI labeled antibody to the activated alkaline phosphatase is 1: 2, fully and uniformly mixing, and standing and reacting for 24 hours at the temperature of 2 ℃.
8. A suitable amount of N-ethylmaleimide (NEM) reagent was weighed and prepared into a 12.5mg/mL NEM solution using 100mM triethanolamine solution at pH 7.3.
9. And (3) adding one percent volume of the NEM solution prepared in the step (8) into the solution after the crosslinking reaction in the step (7), fully mixing the solution, and standing the mixture at 25 ℃ for reaction for 30 minutes to block redundant sulfydryl.
10. The solution of the crosslinked product after the termination reaction of step 9 is purified by dialysis, desalting, ultrafiltration concentration or the like.
11. And (3) determining the concentration of the purified alkaline phosphatase-labeled cTnI antibody concentrated solution, adding glycerol with the same volume, fully and uniformly mixing, and storing at-20 ℃ for later use.
Example 2
This example provides a method for preparing an alkaline phosphatase-labeled cTnI antibody, comprising the steps of:
1. the storage concentration of the cTnI-labeled antibody (i.e., cTnI antibody) was 2mg/mL, and 0.5M of a TECP solution was added to the cTnI-labeled antibody to a final concentration of 1.25mM of the TECP-labeled antibody, which was immediately mixed, followed by allowing the mixture to stand at 25 ℃ for 60 minutes.
2. The cTnI labeled antibody solution obtained in step 1 was immediately desalted and replaced with 100mM triethanolamine solution having pH7.3, and the concentration of the antibody solution was measured by UV spectrophotometry.
3. An appropriate amount of Sulfo-SMCC reagent is weighed and prepared into a solution with the concentration of 17.5mg/mL by using dimethylformamide DMF.
4. Adding the sulfoc-SMCC solution prepared in the step 2 into an alkaline phosphatase solution (AP solution with the concentration of 20mg/ml), wherein the molar ratio of the alkaline phosphatase to the sulfoc-SMCC is 1: 15, immediately mixing, standing and reacting for 15 minutes at 25 ℃.
5. And (3) adding a 1M glycine solution into the alkaline phosphatase solution obtained in the step (4), immediately mixing the alkaline phosphatase solution and the sulfoc-SMCC reagent at a molar ratio of 10:1, and standing the mixture at 25 ℃ for reaction for 10 minutes.
6. Immediately desalting the alkaline phosphatase solution obtained in the step 5, replacing the alkaline phosphatase solution with triethanolamine solution with the pH value of 7.3 and the concentration of the alkaline phosphatase solution being 100mM, and measuring the concentration of the alkaline phosphatase solution by using an ultraviolet spectrophotometry;
7. mixing the activated antibody solution of the step 2 with the activated alkaline phosphatase solution of the step 6, wherein the molar ratio of the activated cTnI labeled antibody to the activated alkaline phosphatase is 1: 2, fully mixing the mixture, and standing the mixture at the temperature of 8 ℃ for 18 hours.
8. A suitable amount of N-ethylmaleimide (NEM) reagent was weighed and prepared into a 12.5mg/mL NEM solution using 100mM triethanolamine solution at pH 7.3.
9. And (3) adding one percent volume of the NEM solution prepared in the step (8) into the solution after the crosslinking reaction in the step (7), fully mixing the solution, and standing the mixture at 25 ℃ for reaction for 30 minutes to block redundant sulfydryl.
10. The solution of the crosslinked product after the termination reaction of step 9 is purified by dialysis, desalting, ultrafiltration concentration or the like.
11. And (3) determining the concentration of the purified alkaline phosphatase-labeled cTnI antibody concentrated solution, adding glycerol with the same volume, fully and uniformly mixing, and storing at-20 ℃ for later use.
Example 3
The embodiment provides a preparation method of magnetic beads labeled by cTnI antibodies, which comprises the following steps:
1. the cTnI coated antibody (namely cTnI antibody) is preserved in 100mM MES buffer solution with the pH value of 5.0, and the concentration of the antibody is 1 mg/mL;
2. taking a carboxyl magnetic bead solution with the weight of 100 times of that of the antibody, wherein the concentration of the carboxyl magnetic bead solution is 100mg/ml, the particle size of the carboxyl magnetic bead is 3 mu m, and magnetically separating and discarding the supernatant;
3. washing: the magnetic beads obtained in the above step were redissolved in 100mM MES buffer at pH5.0, and the supernatant was removed by magnetic separation.
4. Repeat step 3 once.
5. And (4) adding an appropriate amount of 100mM MES buffer to dissolve the magnetic beads obtained in the step (4).
6. Weighing an appropriate amount of NHS (N-hydroxysuccinimide) reagent, and dissolving into a solution with the concentration of 10mg/mL by using a MES buffer solution with the pH value of 5.0 and the concentration of 100 mM;
7. weighing an appropriate amount of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) reagent, and dissolving the EDC reagent into a solution with the concentration of 10mg/mL by using a MES buffer solution with the pH of 5.0 and the concentration of 100 mM;
8. the NHS solution of step 6 and the EDC solution of step 7 were added to the magnetic bead solution obtained in step 5 at 0.23 times the weight of the magnetic beads, and reacted for 30 minutes at 25 ℃ in a homogenizer.
9. And magnetically separating the magnetic bead solution after the reaction is finished, and discarding the supernatant.
10. The magnetic beads obtained in step 9 were dissolved by adding an appropriate amount of 100mM MES buffer.
11. And (3) adding the cTnI coated antibody obtained in the step (1) into the magnetic beads obtained in the step (10), wherein the mass ratio of the cTnI coated antibody to the carboxyl magnetic bead solution is 1:100, and carrying out crosslinking reaction on the mixture at room temperature for 16 hours on a mixing machine.
12. After the crosslinking, the mixture was magnetically separated, and the supernatant was collected to determine the crosslinking rate. Repeat the above steps 3 times.
13. And (3) adding a magnetic bead sealing solution into the magnetic beads obtained in the step (12), and reacting for 24 hours at room temperature on a mixing machine.
14. The magnetic bead solution after the reaction was magnetically separated, and the supernatant was discarded.
15. The beads were washed 3 times with bead wash.
16. And (4) resuspending the washed magnetic beads in a magnetic bead preservation solution, and preserving at 2 ℃ for later use.
Example 4
The embodiment provides a preparation method of magnetic beads labeled by cTnI antibodies, which comprises the following steps:
1. the cTnI coated antibody (namely cTnI antibody) is preserved in 100mM MES buffer solution with the pH value of 5.0, and the concentration of the antibody is 1 mg/mL;
2. taking a carboxyl magnetic bead solution with the weight of 100 times of that of the antibody, wherein the concentration of the carboxyl magnetic bead solution is 100mg/ml, the particle size of the carboxyl magnetic bead is 3 mu m, and magnetically separating and discarding the supernatant;
3. washing: the magnetic beads obtained in the above step were redissolved in 100mM MES buffer at pH5.0, and the supernatant was removed by magnetic separation.
4. Repeat step 3 once.
5. And (4) adding an appropriate amount of 100mM MES buffer to dissolve the magnetic beads obtained in the step (4).
6. Weighing an appropriate amount of NHS reagent, and dissolving into a solution with the concentration of 10mg/mL by using a MES buffer solution with the pH of 5.0 and the concentration of 100 mM;
7. weighing an appropriate amount of EDC reagent, and dissolving the EDC reagent into a solution with the concentration of 10mg/mL by using a MES buffer solution with the pH of 5.0 and the concentration of 100 mM;
8. the NHS solution of step 6 and the EDC solution of step 7 were added to the magnetic bead solution obtained in step 5 at 0.23 times the weight of the magnetic beads, and reacted for 30 minutes at 25 ℃ in a homogenizer.
9. And magnetically separating the magnetic bead solution after the reaction is finished, and discarding the supernatant.
10. The magnetic beads obtained in step 9 were dissolved by adding an appropriate amount of 100mM MES buffer.
11. And (3) adding the cTnI coated antibody obtained in the step (1) into the magnetic beads obtained in the step (10), wherein the mass ratio of the cTnI coated antibody to the carboxyl magnetic bead solution is 1:100, and carrying out crosslinking reaction on the mixture at room temperature for 24 hours on a mixing machine.
12. After the crosslinking, the mixture was magnetically separated, and the supernatant was collected to determine the crosslinking rate. Repeat the above steps 3 times.
13. And (3) adding a magnetic bead sealing solution into the magnetic beads obtained in the step (12), and reacting for 16 hours at room temperature on a mixing machine.
14. The magnetic bead solution after the reaction was magnetically separated, and the supernatant was discarded.
15. The beads were washed 3 times with bead wash.
16. And (4) resuspending the washed magnetic beads in a magnetic bead preservation solution, and preserving at 8 ℃ for later use.
Example 5
The invention provides a cTnI detection kit, which comprises: pretreatment solution, enzyme conjugate working solution, magnetic bead conjugate working solution, cleaning solution, substrate solution, calibrator and quality control product. The components of each reagent in the cTnI detection kit are as follows:
1. calibrator and quality control product
And (3) determining the concentration of the cTnI antigen through tracing, and diluting with a calibrator diluent to prepare a cTnI calibrator and a cTnI quality control product. For example, the concentrations of the calibrant are: 0.05, 0.5, 1, 5, 25, 50ng/ml and 5ng/ml of quality control material.
The specific components of the calibrator diluent are as follows: BES (N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid) at 20mM, NaCl at 300mM, horse serum at 1%, Proclin-300 at 0.5mM, and mannitol at 1%, and the pH of the calibrator dilution was 6.5.
2. Pretreatment liquid
The pretreatment solution contains 1.5mM of imidazole; in addition, the method also comprises the following steps: 25mM Tris,150mM NaCl, 1% sucrose, 5% glycerol, 0.1% BSA, 0.05% Tween-20 and 0.2% Proclin-300, and the pH of the pretreatment solution was 7.0.
3. Enzyme conjugate working solution
The enzyme conjugate working solution contained 0.6. mu.g/ml of the alkaline phosphatase-labeled cTnI antibody prepared by the preparation method shown in example 1.
The enzyme conjugate working solution also comprises: 50mM Tris,150mM NaCl, 1% BSA, 5% sucrose, 5% glycerol, 0.1% Tween-20, 0.2mM ZnCl25mM MgCl2And 0.05% Proclin300, the pH of the enzyme conjugate working solution was 7.0.
4. Working solution of magnetic bead conjugate
The working solution of the magnetic bead conjugate contained 0.5mg/ml of a magnetic bead-labeled cTnI antibody prepared by the preparation method shown in example 3.
The working solution of the magnetic bead conjugate further comprises: 20mM Tris,300mM NaCl, 1% BSA, 1% sucrose, 1% gelatin, 0.1% Tween-20, 1% PVP360 and 0.05% Proclin300, pH of the magnetic bead conjugate working solution was 7.0.
5. Substrate solution
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.
6. Cleaning liquid
Adding 1211.4mg of Tris, 9g of NaCl, 3g of SBS, 201 g of Tween, 3001g of Proclin and 900mL of water into a container in sequence, mixing uniformly 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, mixing uniformly for 30 minutes, and filtering at 0.22 mu m to obtain the cleaning solution.
Example 6
The invention provides a cTnI detection kit, which comprises: pretreatment solution, enzyme conjugate working solution, magnetic bead conjugate working solution, cleaning solution, substrate solution, calibrator and quality control product. The components of each reagent in the cTnI detection kit are as follows:
1. calibrator and quality control product
And (3) determining the concentration of the cTnI antigen through tracing, and diluting with a calibrator diluent to prepare a cTnI calibrator and a cTnI quality control product. For example, the concentrations of the calibrant are: 0.05, 0.5, 1, 5, 25, 50ng/ml and 5ng/ml of quality control material.
The specific components of the calibrator diluent are as follows: BES 50mM, NaCl 150mM, 1% horse serum, Proclin-300 mM 5mM and mannitol 1%, the pH of the calibrator dilution being 7.5.
2. Pretreatment liquid
The pretreatment solution contains 0.8mM of imidazole; in addition, the method also comprises the following steps: 100mM Tris,150mM NaCl, 5% sucrose, 1% glycerol, 0.1% BSA, 0.2% Tween-20 and 0.05% Proclin-300, and the pH of the pretreatment solution was 7.5.
3. Enzyme conjugate working solution
The enzyme conjugate working solution contained 1.8. mu.g/ml of the alkaline phosphatase-labeled cTnI antibody prepared by the preparation method shown in example 2.
The enzyme conjugate working solution also comprises: 20mM Tris,300mM NaCl, 1% BSA, 5% sucrose, 5% glycerol, 0.1% Tween-20, 5mM ZnCl20.02mM MgCl2And 0.2% Proclin300, pH of the enzyme conjugate working solution was 9.0.
4. Working solution of magnetic bead conjugate
The working solution of the magnetic bead conjugate contained 0.2mg/ml of a magnetic bead-labeled cTnI antibody prepared by the preparation method shown in example 4.
The working solution of the magnetic bead conjugate further comprises: 50mM Tris,150mM NaCl, 1% BSA, 1% sucrose, 1% gelatin, 0.1% Tween-20, 1% PVP360 and 0.2% Proclin300, pH of the working solution of the magnetic bead conjugate was 9.0.
5. Substrate solution
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.
6. Cleaning liquid
Adding 1211.4mg of Tris, 9g of NaCl, 3g of SBS, 201 g of Tween, 3001g of Proclin and 900mL of water into a container in sequence, mixing uniformly 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, mixing uniformly for 30 minutes, and filtering at 0.22 mu m to obtain the cleaning solution.
Example 7
This example provides the procedure for determining cTnI antigen in whole blood using the cTnI detection kit provided in example 5, taking finger tip whole blood as the sample to be tested:
1. respectively adding 10 mul of cTnI calibrator, cTnI quality control material and sample to be tested into the three reaction tubes;
2. adding 40 mul of pretreatment solution into each reaction tube and mixing uniformly;
3. adding 50 mul of enzyme conjugate working solution and 50 mul of magnetic bead working solution into each test tube, and uniformly mixing; reacting at 42 ℃ for 5 min;
4. carrying out magnetic separation on the solution reacted in the step 3, and collecting magnetic beads; adding 300 mul of cleaning solution into each reaction tube to clean the magnetic beads, repeating for 3-5 times, and removing the cleaning solution;
5. add 100 μ l substrate solution into each reaction tube, mix well and detect the luminescence value.
6. Drawing a marking curve by using the concentration and the luminous value of the standard substance;
7. and substituting the luminous value of the sample to be detected into the standard curve to obtain the content of the cTnI in the sample to be detected.
The cTnI detection method provided in this example is also applicable to cTnI detection of anticoagulated venous whole blood, serum, or plasma.
Example 8
This example provides the procedure for determining cTnI antigen in whole blood using the cTnI detection kit provided in example 6, taking finger tip whole blood as the sample to be tested:
1. respectively adding 10 mul of cTnI calibrator, cTnI quality control material and sample to be tested into the three reaction tubes;
2. adding 25 mul of pretreatment solution into each reaction tube and mixing uniformly;
3. adding 50 mul of enzyme conjugate working solution and 50 mul of magnetic bead working solution into each test tube, and uniformly mixing; reacting at 37 ℃ for 10 min;
4. carrying out magnetic separation on the solution reacted in the step 3, and collecting magnetic beads; adding 300 mul of cleaning solution into each reaction tube to clean the magnetic beads, repeating for 3-5 times, and removing the cleaning solution;
5. add 100 μ l substrate solution into each reaction tube, mix well and detect the luminescence value.
6. Drawing a marking curve by using the concentration and the luminous value of the standard substance;
7. and substituting the luminous value of the sample to be detected into the standard curve to obtain the content of the cTnI in the sample to be detected.
The cTnI detection method provided in this example is also applicable to cTnI detection of anticoagulated venous whole blood, serum, or plasma.
Experimental example 1 Linear verification
Taking a standard substance or a quality control substance with traceability, measuring the concentration of the standard substance or the quality control substance to the maximum extent, diluting a sample by using normal saline, measuring for 3 times at each point, taking an average value, making a regression straight line between the result and the expected concentration, and calculating to obtain a regression coefficient r larger than 0.99, which shows that the dilution linearity of the kit provided by the invention is good.
Experimental example 2 precision verification
Taking each part of high-value quality control product and low-value quality control product of retrospective serum, performing 10 times of detection on each part of quality control product, calculating average value and standard value of 10 times of detection results, and calculating CV according to variation coefficient CV (standard deviation/average value) × 100%, wherein CV is obtained by calculation1(25ng/mL)＝4％，CV2(5ng/mL) ═ 2%. Therefore, the kit provided by the invention has higher precision.
Experimental example 3 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 4 verification of sensitivity
Diluting the quality control product with traceability to the vicinity of the lower limit (0.02ng/mL) of the detection range, measuring 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 5 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 0.02-50ng/ml, and the detection range of the kit disclosed by the invention is 0.02-50 ng/ml.
Experimental example 6 correlation verification
200 serum samples were taken, and the correlation between the detection results of the kit of the present invention and the cTnI detection kit from 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 R20.9774, as can be seen from fig. 1, the test results of the sample of the present invention are not significantly different from the test results of the products of the well-known company 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 (9)

1. A cTnI detection kit, comprising: the kit comprises a calibrator, a cleaning solution, a substrate solution, a pretreatment solution, an enzyme conjugate working solution and a magnetic bead conjugate working solution; the pretreatment solution contains imidazole, the enzyme conjugate working solution contains an enzyme-labeled cTnI antibody, the magnetic bead conjugate working solution contains magnetic beads labeled with the cTnI antibody, and the concentration of the imidazole is 0.8-1.5 mM; the pretreatment liquid further comprises: 25-100 mM Tris,150mM NaCl, 1-5% sucrose, 1-5% glycerol, 0.1% BSA, 0.05-0.2% Tween-20 and 0.05-0.2% Proclin-300, wherein the pH of the pretreatment solution is 7.0-7.5; the enzyme conjugate working solution further comprises:20-50 mM Tris, 150-300 mM NaCl, 1% BSA, 5% sucrose, 5% glycerol, 0.1% Tween-20, 0.02-5 mM ZnCl20.02-5 mM MgCl2And 0.05-0.2% of Proclin300, wherein the pH of the working solution of the enzyme conjugate is 7.0-9.0;
the working solution of the magnetic bead conjugate further comprises: 20-50 mM Tris, 150-300 mM NaCl, 1% BSA, 1% sucrose, 1% gelatin, 0.1% Tween-20, 1% PVP360 and 0.05-0.2% Proclin300, wherein the pH of the working solution of the magnetic bead conjugate is 7.0-9.0.
2. The cTnI detection kit of claim 1, wherein the enzyme-labeled cTnI antibody is an alkaline phosphatase-labeled cTnI antibody.
3. The cTnI detection kit of claim 2, wherein the alkaline phosphatase-labeled cTnI antibody is prepared by:
A. activating cTnI antibodies
1, adding a cTnI antibody of 2mg/ml into a TCEP solution of 0.5M until the final concentration of TCEP is 1.25mM, uniformly mixing, and standing at room temperature for reaction;
2 desalting the reacted antibody solution, and replacing the antibody solution into 100mM triethanolamine buffer solution with pH 7.3;
B. activating alkaline phosphatase
3 adding 17.5mg/mL Sulfo-SMCC solution into the alkaline phosphatase solution, uniformly mixing, and standing at room temperature; the sulfol-SMCC solution is prepared by adding a sulfol-SMCC reagent into dimethylformamide, wherein the molar ratio of the sulfol-SMCC reagent to the alkaline phosphatase is (10-15): 1;
4, continuously adding 1M glycine solution, uniformly mixing, and standing at room temperature for reaction; wherein the molar ratio of the addition amount of the glycine to the Sulfo-SMCC reagent is (10-20): 1;
5 desalting the alkaline phosphatase solution after the reaction, and replacing the alkaline phosphatase solution into 100mM triethanolamine buffer solution with pH 7.3;
C. uniformly mixing the cTnI antibody solution activated in the step A and the alkaline phosphatase solution activated in the step B, and standing at the temperature of 2-8 ℃ for reaction for 18-24 hours; wherein the molar ratio of the cTnI antibody to the alkaline phosphatase is 1: 2;
D. and D, adding 12.5mg/ml of N-ethylmaleimide solution into the solution reacted in the step C, uniformly mixing, standing at room temperature for reaction, and purifying the reacted solution to obtain the alkaline phosphatase labeled cTnI antibody.
4. The cTnI detection kit of any one of claims 1-3, wherein the wash solution is Tris buffer containing SBS, Tween-20 and Proclin-300, the substrate solution is an enzymatic chemiluminescent substrate solution, and the calibrator is cTnI HEPES buffer in calibrator diluent.
5. The cTnI detection kit according to any one of claims 1 to 3, further comprising: and the quality control product is a cTnI HEPES buffer solution prepared by using a calibrator diluent.
6. The cTnI detection kit of claim 4, wherein the calibrator diluent comprises: 20-50 mM BES, 150-300 mM NaCI, 1% horse serum, 0.5-5 mM Proclin-300 and 1% mannitol, wherein the pH of the calibrator diluent is 6.5-7.5.
7. The cTnI detection kit according to any one of claims 1 to 3, wherein the concentration of the enzyme-labeled cTnI antibody is 0.6 to 1.8 μ g/ml, and the concentration of the cTnI antibody-labeled magnetic beads is 0.2 to 0.5 mg/ml.
8. The cTnI assay kit of any of claims 1-3, wherein the magnetic beads have a particle size of 3 μm.
9. The cTnI detection kit of claim 1, wherein the cTnI antibody-labeled magnetic beads are prepared by:
E. adding a 100mM MES buffer solution with the pH value of 5.0 into the cTnI antibody until the concentration of the antibody solution is 1-5 mg/ml;
F. activated magnetic bead
1) Placing the magnetic bead solution of 100mg/ml on a magnetic frame for standing, and magnetically separating and removing supernate; wherein the mass ratio of the magnetic bead solution to the cTnI antibody is 100: 1;
2) dissolving the magnetic beads obtained in the step 1) by using a MES buffer solution with the pH of 5.0 and the concentration of 100mM, and magnetically separating and discarding a supernatant;
3) dissolving the magnetic beads obtained in the step 2) in a 100mM MES buffer solution, continuously adding a 10mg/ml NHS solution with the weight of 0.23 time that of the magnetic beads and a 10mg/ml EDC solution with the weight of 0.1 time that of the magnetic beads, and carrying out oscillation reaction at room temperature; wherein the NHS solution is prepared by dissolving NHS reagent in MES buffer solution with pH of 5.0 and 100mM, and the EDC solution is prepared by dissolving EDC reagent in MES buffer solution with pH of 5.0 and 100 mM;
4) magnetically separating the magnetic bead solution reacted in the step 3), discarding the supernatant, and dissolving in 100mM MES buffer solution;
G. mixing the cTnI antibody solution obtained in the step E with the magnetic bead solution activated in the step F, carrying out crosslinking reaction for 16-24 hours at room temperature, carrying out magnetic separation, discarding supernatant, continuously adding the magnetic bead sealing solution CE210, uniformly mixing, and carrying out reaction for 16-24 hours at room temperature;
H. and G, magnetically separating the magnetic bead solution obtained in the step G, then discarding the supernatant, and then cleaning the magnetic beads to obtain magnetic beads marked by the cTnI antibody.
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