阅读说明：本技术 检测肌钙蛋白及复合物的双标记试剂盒及制备与检测方法 (Double-labeling kit for detecting troponin and compound, and preparation and detection methods thereof ) 是由 黄飚 任志奇 静亚茹 于 2019-09-09 设计创作，主要内容包括：本发明涉及检测肌钙蛋白及复合物的双标记试剂盒及制备与检测方法,属于免疫检测分析技术和纳米生物技术领域,在所述试剂盒中,包括集成在试剂条上的反应缓冲液、清洗液、增强液、包被cTnI单克隆抗体的磁微粒溶液、铕标记的cTnC单克隆抗体溶液和钐标记的cTnI单克隆抗体溶液,另附2-6瓶不同浓度天然cTnI抗原(含cTnC)的冻干校准品。上述试剂盒可以提供一种接近均相的双标记反应体系,保证同时检测完整片段cTnI和cTnI-C复合物的含量,缩短检测时间,提高了效率,为临床使用提供便利。(The invention relates to a double-labeled kit for detecting troponin and a compound, and a preparation method and a detection method thereof, belonging to the fields of immunoassay analysis technology and nano-biotechnology, wherein the kit comprises a reaction buffer solution, a cleaning solution, an enhancement solution, a magnetic particle solution coating a cTnI monoclonal antibody, a europium-labeled cTnC monoclonal antibody solution and a samarium-labeled cTnI monoclonal antibody solution which are integrated on a reagent strip, and 2-6 bottles of freeze-dried calibrators of natural cTnI antigens (containing cTnC) with different concentrations are additionally attached. The kit can provide a near-homogeneous double-label reaction system, ensures that the contents of complete fragments cTnI and cTnI-C are detected simultaneously, shortens the detection time, improves the efficiency and provides convenience for clinical use.)

1. The double-labeling kit for detecting troponin and compounds is characterized by comprising a plurality of reagent strips and a kit, wherein the reagent strips are arranged in the kit and are stacked up and down, the number of the reagent strips is five, a plurality of preformed holes, a plurality of cleaning holes, an enhancement liquid hole, a detection hole, an europium/samarium marker hole, a reaction buffer hole and a magnetic particle solution hole are formed in each reagent strip, the reagent strips comprise a reaction buffer solution, a cleaning solution, an enhancement liquid, a magnetic particle solution coated with a cTnI monoclonal antibody, a europium-labeled cTnC monoclonal antibody solution and a samarium-labeled cTnI monoclonal antibody solution which are integrated on the reagent strips, and 2-6 bottles of freeze-dried calibrators of natural cTnI (containing cTnC) with different concentrations are attached.

2. The dual-labeled kit for detecting troponin and complexes according to claim 1, wherein the europium-labeled cTnC monoclonal antibody is dissolved in Eu³⁺-N₂- [ P-Isocyanato-benzyl group]The-diethylenetriamine tetracetic acid sodium marked cTnC monoclonal antibody is prepared, and the cTnC monoclonal antibody and the Eu are³⁺-N₂- [ P-Isocyanato-benzyl group]The mass ratio of sodium diethylenetriamine tetracetate is 5: 1;

the samarium-labeled cTnI monoclonal antibody solution is Sm³⁺-N₂- [ P-Isocyanato-benzyl group]The-diethylenetriamine tetracetic acid sodium mark cTnI monoclonal antibody is prepared, and the cTnI monoclonal antibody and the Sm are³⁺-N₂- [ P-Isocyanato-benzyl group]The mass ratio of sodium diethylenetriamine tetracetate is 5: 1;

the magnetic particle solution coated with the cTnI monoclonal antibody is prepared by connecting, washing, sealing and washing activated magnetic particles with the diameter of 100-5000nm and the cTnI monoclonal antibody.

3. The kit for detecting troponin and its complexes as claimed in claim 2, wherein the washing solution is a buffer solution containing 0.48g/L Tris, 12.49g/L NaCl, 1.11g/L Tween-20, 0.04g/L Triton X-100, pH7.8 adjusted with hydrochloric acid;

the enhancement solution is a mixed solution containing glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, β -naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand;

the reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, contains 8mmol/L NaCl, 0.1% BSA, 50 mu mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN_3；

The calibrator solution of the cTnI-C complex is prepared by2g/L BSA and 1g/L NaN₃50mmol/L of reaction buffer (pH7.8Tris-HCl), the natural cTnI antigen (containing cTnC) is prepared into calibrator solutions with different concentrations, and then the calibrator solutions are freeze-dried and stored.

4. A double-labeled kit for the detection of troponin and complexes according to any one of claims 1 to 3, wherein each of said kits has the formulation: reaction buffer solution, cleaning solution, enhancing solution, magnetic particle solution coated with a cTnI monoclonal antibody, europium-labeled cTnC monoclonal antibody solution and samarium-labeled cTnI monoclonal antibody solution are integrated on the reagent strips, 5 reagent strips can be placed on each reagent rack, one box contains 25-100 reagent strips, and 2-6 bottles of freeze-dried calibrators with different concentrations of cTnI are attached.

5. A method for preparing the double-labeled kit for detecting troponin and complexes according to any one of claims 1 to 4, characterized by comprising any one or more of the following preparation methods:

preparation of the natural cTnI-C complex calibrator solution: taking a natural cTnI-C complex antigen solution with the concentration of 1mg/mL, using 2g/L BSA and 1g/L NaN₃Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, lyophilizing, and storing at 2-8 deg.C;

the preparation of the magnetic particle solution coated with the cTnI monoclonal antibody comprises the following steps: activating ferroferric oxide microspheres with the diameter of 100-5000nm by using glutaraldehyde with the mass concentration of 5-10%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L PBS buffer solution with the pH value of 7.2, and suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 μ L of the magnetic particle suspension, adding 1mL of 0.05mol/L PBS buffer solution with pH of 7.2 and 50 μ g of cTnI monoclonal antibody, mixing and incubating at 25 deg.C for 2 hr, performing magnetic separation, retaining magnetic particles, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, blocking with 1mL of 0.0lmol/L PBS buffer solution with pH of 7.2 containing 5% BSA by mass at 25 deg.C for 30 min, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, and washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 timesBSA at a mass concentration of 0.5% and NaN at a mass concentration of 0.1%₃0.05mol/L Tris-HCl buffer solution with pH value of 7.2 is used for resuspension, subpackaging and storing at 2-8 ℃;

the preparation of the samarium-labeled cTnI monoclonal antibody comprises the steps of taking 1mL of the cTnI antibody, converting the buffer condition by a PD-10 column, and eluting 50mmol/L Na containing 0.155mol/L NaCl and having the pH value of 8.5₂CO₃-NaHCO₃Buffer solution, collecting protein peak to obtain cTnI monoclonal antibody solution concentrated to 2g/L, adding 500 mu L of the cTnI monoclonal antibody solution into 0.2mg of the benzyldiethylenetriamine tetraacetic acid samarium sodium isothiocyanate, magnetically stirring and reacting for 20 hours at 25 ℃, then transferring the reaction solution to a Sepharose CL-6B column which is balanced by 80mmol/L Tris buffer solution with pH7.8 in advance for chromatography, collecting protein peak, diluting, subpackaging, vacuum freeze-drying, and storing at 2-8 ℃;

the preparation of the europium-labeled cTnC monoclonal antibody solution: taking 1mL of cTnC antibody, converting the buffer condition by a PD-10 column, and eluting with 50mmol/L Na containing 0.155mol/L NaCl and having a pH value of 8.5₂CO₃-NaHCO₃Buffer solution, collecting protein peak to obtain cTnC monoclonal antibody solution concentrated to 2g/L, adding 0.2mg of benzyl diethylene triamine tetraacetic acid europium sodium isothiocyanate into 500 mu L of the cTnC monoclonal antibody solution, reacting for 20 hours under magnetic stirring at 25 ℃, transferring the reaction solution to Sepharose CL-6B column balanced by 80mmol/L Tris buffer solution with pH7.8 in advance for chromatography, collecting protein peak, diluting, subpackaging, vacuum freeze drying, and storing at 2-8 ℃;

preparing the enhancement solution by uniformly mixing 3.6mL of glacial acetic acid, 0.5g of sodium acetate, 0.05g of β -naphthoyl trifluoroacetone, 0.03g of trioctylphosphine oxide and 1mL of Triton X-100, adding deionized water to a constant volume of 1000mL, subpackaging, and storing at 2-8 ℃;

the reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, contains 8mmol/L NaCl, 0.1% BSA, 50 mu mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN₃；

The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and 0.04g/L of Triton X-100, and the pH value is adjusted to be 7.8 by hydrochloric acid.

6. A method for the simultaneous detection of cTnI and cTnI-C using the double-labeled kit for the detection of troponins and complexes according to any one of claims 1 to 4, characterized in that it comprises the following steps, which can be carried out by fully automatic equipment:

(1) respectively taking a calibrator solution or a sample to be tested of the natural cTnI antigen (containing cTnC), mixing the calibrator solution or the sample to be tested with the magnetic particle solution coated with the cTnI monoclonal antibody, adding the samarium-labeled cTnI monoclonal antibody solution and the europium-labeled cTnC monoclonal antibody solution diluted by the reaction buffer solution into the mixed magnetic particle solution, incubating, then cleaning by the cleaning solution, performing magnetic separation, then sucking and removing the supernatant, repeating the cleaning step, and then adding the enhancement solution;

(2) respectively measuring the fluorescence values of europium and samarium in the solution added with the enhancement solution by using a time-resolved fluoroimmunoassay analyzer to respectively obtain the fluorescence values of a calibrator solution of the cTnI-C complex and a sample to be detected, then respectively drawing a standard curve according to the concentration of the cTnI-C antigen in the calibrator solution, the concentration of the cTnI antigen (containing cTnC) and the fluorescence values corresponding to the concentrations, and then substituting the corresponding fluorescence values of the sample to be detected into the corresponding cTnI-C standard curve or cTnI standard curve to calculate the contents of the cTnI-C complex and the cTnI of the complete segment in the sample to be detected.

Technical Field

The invention belongs to the fields of immunoassay and nano-biotechnology, and particularly relates to a double-labeled kit for detecting a troponin and a compound, and a preparation method and a detection method thereof.

Background

Cardiac troponin (cTn) is a regulatory protein of myocardial muscle contraction. cTn is composed of subunits of three different genes: cardiac troponin I (cTn I) and troponin C (TnC), cardiac troponin T (cTnT).

cTn exists in myocardial cells in the form of a cTnI-C-T complex and free cTnI, which can be further broken down into a cTnI-C complex and free cTnT after release into the blood circulation upon myocardial injury, and which is the predominant form in blood. Due to their minimal content in normal serum, they are markedly elevated in Acute Myocardial Infarction (AMI). When myocardial cells are destroyed by ischemia, hypoxia and the like, free cTnI and cTnT can be firstly and rapidly released into blood from the cells, and then bound cTnI and cTnT bound in structural proteins of cardiac muscle are gradually decomposed and slowly released into circulating blood, and in AMI, the cTnI and cTnT appear earlier in the circulating blood and can last for a long time. Although troponin has a short half-life (cTnT 2 hours, free cTnI 2 h-5 d), its degradation process from myofibrils lasts for a long time and can be maintained in the blood for a long time. Therefore, cTnI is more in the early stage of onset, and cTnI-C is dominant as the onset time continues.

Troponin is mainly measured by an immunological method using a double antibody sandwich. When the cTnI of the blood of a patient is quantitatively detected, the influence is different according to different antigen epitopes of antibodies used by detection reagents. Monomeric cTnI molecules in serum are very susceptible to degradation, while the middle region of cTnI molecules is more stable in troponin complexes due to the protection of TnC. Therefore, an antibody recognizing an epitope of the middle stable region of the cTnI molecule should be preferably used. In cardiomyocytes, cTnI exists as a ternary complex of I-T-C. In the blood of patients, cTnI exists in the form of an I-C complex rather than an I-T-C ternary complex. The ability of the antibody used for the cTnI detection to recognize the cTnI-C complex is therefore crucial for the detection.

The most stable region of the cTnI molecule is the 30-110 amino acid region. Since this region is protected by endogenous cTnC in necrotic tissue as well as in blood. Due to the lack of protection by cTnC, at least partial truncation of the N-and C-termini of cTnI occurs, especially after 20 hours of onset of clinical characterization. cTnI exists in blood mainly as a binary complex of cTnI-C. Free cTnI is present only in trace amounts. The cTnC is not affected by phosphorylation, is not easy to proteolysis, and is not sensitive to autoantibodies and heparin in the sample. These characteristics all help to improve the analysis sensitivity, precision and repeatability of the detection system. Thus, cTnI can be detected using an antibody pair that recognizes the cTnI-C complex. Therefore, the simultaneous detection of the complete fragments cTnI and cTnI-C complex has certain guiding significance on the AMI attack time.

Therefore, the invention combines the double-label time-resolved fluoroimmunoassay technology with the magnetic particles, provides a double-label time-resolved fluoroimmunoassay kit based on the cTnI and the cTnI-C compound, a preparation method and a detection method thereof, can simultaneously detect the levels of the complete fragments cTnI and the cTnI-C compound in a sample, has higher detection sensitivity and specificity, and achieves better performance parameters.

Disclosure of Invention

The reaction principle is shown in the figure, an antibody aiming at the cTnI region protected by endogenous cTnC is used as a solid phase antibody to capture all cTnI, an antibody mark Eu aiming at the cTnC is used for detecting a cTnI-C complex, an antibody mark Sm aiming at the tail end of the cTnI is used for detecting the cTnI of a complete fragment, and the tail end of the cTnI is easy to be hydrolyzed after entering blood, so that the complete cTnI is reduced, and the cTnI-C complex exists stably, so that the complete cTnI and the cTnI-C complex are detected, and the ratio of the complete cTnI and the cTnI-C complex is calculated, so that the judgment of the duration time of the AMI is facilitated.

The reaction principle is shown in the figure, an antibody aiming at the cTnI region protected by endogenous cTnC is used as a solid phase antibody to capture all cTnI, an antibody mark Eu aiming at the cTnC is used for detecting a cTnI-C complex, an antibody mark Sm aiming at the tail end of the cTnI is used for detecting the cTnI of a complete fragment, and the tail end of the cTnI is easy to be hydrolyzed after entering blood, so that the complete cTnI is reduced, and the cTnI-C complex exists stably, so that the complete cTnI and the cTnI-C complex are detected, and the ratio of the complete cTnI and the cTnI-C complex is calculated, so that the judgment of the duration time of the AMI is facilitated.

Therefore, the invention provides a dual-labeling time-resolved fluorescence immunoassay kit based on cTnI and a cTnI-C compound, which comprises a reaction buffer solution, a cleaning solution, an enhancement solution, a magnetic particle solution coated with a cTnI monoclonal antibody, a europium-labeled cTnC monoclonal antibody solution and a samarium-labeled cTnI monoclonal antibody solution which are integrated on a reagent strip, and 2-6 bottles of freeze-dried calibrators of cTnI with different concentrations are attached.

The kit for double-labeling time-resolved fluorescence immunoassay based on the cTnI and cTnI-C compound comprises a europium-labeled cTnC monoclonal antibody solution and Eu³⁺-N₂- [ P-Isocyanato-benzyl group]The monoclonal antibody of-DTTA sodium tag cTnC is prepared, and the cTnC monoclonal antibody and the Eu are³⁺-N₂- [ P-Isocyanato-benzyl group]The mass ratio of sodium diethylenetriamine tetracetate is 5: 1;

the samarium-labeled cTnI monoclonal antibody solution is Sm³⁺-N₂- [ P-Isocyanato-benzyl group]The monoclonal antibody of-DTPA sodium marker cTnI is prepared, and the cTnI monoclonal antibody and the Sm are³⁺-N₂- [ P-Isocyanato-benzyl group]The mass ratio of sodium diethylenetriamine tetracetate is 5: 1;

the magnetic particle solution coated with the cTnI monoclonal antibody is prepared by connecting, washing, sealing and washing activated magnetic particles with the diameter of 100-5000 nanometers with the cTnI monoclonal antibody;

the double-labeling time-resolved fluorescence immunoassay kit based on the cTnI and the cTnI-C complex is characterized in that the cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and 0.04g/L of Triton X-100, and the pH value is adjusted to 7.8 by hydrochloric acid;

the enhancement solution is a mixed solution containing glacial acetic acid with the volume concentration of 3.6 per thousand, sodium acetate with the volume concentration of 0.5g/L, β -naphthoyl trifluoroacetone with the volume concentration of 0.05g/L, trioctylphosphine oxide with the volume concentration of 0.03g/L and Triton X-100 with the volume concentration of 1 per thousand;

the reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, contains 8mmol/L NaCl, 0.1% BSA, 50 mu mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN₃；

The calibrator solution of the cTnI-C complex is prepared by using 2g/L BSA and 1g/L NaN₃50mmol/L reaction buffer, pH7.8 Tris-HCl. The natural cTnI antigen (containing cTnC) is prepared into calibrator solutions with different concentrations and then is freeze-dried and stored.

The cTnI and cTnI-C complex-based double-labeling time-resolved fluorescence immunoassay kit comprises:

reaction buffer solution, cleaning solution, enhancement solution, magnetic particle solution coated with cTnI monoclonal antibody, europium-labeled cTnC monoclonal antibody solution and samarium-labeled cTnI monoclonal antibody solution which are integrated on the reagent strip, and 2-6 bottles of freeze-dried calibrators with different concentrations of cTnI are attached.

The invention provides a method for preparing the dual-labeling time-resolved fluoroimmunoassay kit based on cTnI and cTnI-C complexes, which comprises any one or more of the following preparation methods:

preparation of the natural cTnI-C complex calibrator solution: taking a natural cTnI-C complex antigen solution with the concentration of 1mg/mL, using 2g/L BSA and 1g/L NaN₃Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, lyophilizing, and storing at 2-8 deg.C;

the preparation of the magnetic particle solution coated with the cTnI monoclonal antibody comprises the following steps: activating ferroferric oxide microspheres with the diameter of 100-5000nm by using glutaraldehyde with the mass concentration of 5-10%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L PBS buffer solution with the pH value of 7.2, and then suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 μ L of the magnetic particle suspension, adding 1mL of 0.05mol/L PBS buffer solution with pH of 7.2 and 50 μ g of cTnI monoclonal antibody, mixing and incubating at 25 deg.C for 2 hr, performing magnetic separation, retaining magnetic particles, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, blocking with 1mL of 0.0lmol/L PBS buffer solution with pH of 7.2 containing 5% BSA by mass at 25 deg.C for 30 min, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, washing with 0.5% BSA by mass and 0.1% NaN by mass₃0.05mol/L Tris-HCl buffer solution with pH value of 7.2 is used for resuspension, subpackaging and is placed at 2-8 ℃ for storage;

the preparation of the samarium-labeled cTnI monoclonal antibody comprises the steps of taking 1mL of the cTnI antibody, converting the cTnI antibody into buffer conditions through a PD-10 column, and eluting 50mmol/L Na containing 0.155mol/L NaCl and having the pH value of 8.5₂CO₃-NaHCO₃Buffer solution, collecting protein peak, obtaining cTnI monoclonal antibody solution concentrated to 2g/L, adding 500 mu L of the cTnI monoclonal antibody solution into 0.2mg of the samarium sodium benzyldiethylenetriamine tetraacetate, magnetically stirring and reacting for 20 hours at 25 ℃, then transferring the reaction solution to a Sepharose CL-6B column balanced by 80mmol/L of Tris buffer solution with pH7.8 in advance for chromatography, collecting protein peak, diluting, subpackaging, vacuum freeze-drying, and storing at 2-8 ℃;

the preparation of the europium-labeled cTnC monoclonal antibody solution: taking 1mL of cTnC antibody, converting the buffer condition by a PD-10 column, and eluting with 50mmol/L Na containing 0.155mol/L NaCl and having a pH value of 8.5₂CO₃-NaHCO₃Buffer solution, collecting protein peak to obtain cTnC monoclonal antibody solution concentrated to 2g/L, taking 500 microliter of the cTnC monoclonal antibody solution, adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate, magnetically stirring at 25 ℃ for reaction for 20 hours, transferring the reaction solution to a Sepharose CL-6B column which is balanced by 80mmol/L Tris buffer solution with pH7.8 in advance for chromatography, collecting protein peak, diluting, subpackaging, vacuum freeze drying, and storing at 2-8 ℃;

the enhancing solution is prepared by mixing 3.6mL of glacial acetic acid, 0.5g of sodium acetate, 0.05g of β -naphthoyl trifluoropropionone, 0.03g of trioctylphosphine oxide and 1mL of Triton X-100, adding deionized water to a constant volume of 1000mL, packaging, and storing at 2-8 deg.C;

the reaction buffer solution is 50mmol/L Tris-HCl with pH7.8, contains 8mmol/L NaCl, 0.1% BSA, 50 mu mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN₃；

The cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and 0.04g/L of Triton X-100, and the pH value is adjusted to be 7.8 by hydrochloric acid;

the method for simultaneously detecting the cTnI and the cTnI-C by the double-labeling time-resolved fluoroimmunoassay kit based on the cTnI and the cTnI-C is characterized by comprising the following steps,

(1) respectively taking 100ul of the calibrator solution of the cTnI-C complex and 100ul of a sample to be detected, mixing the calibrator solution and the sample to be detected with 50ul of the magnetic particle solution coated with the cTnI monoclonal antibody, adding 100ul of samarium-labeled cTnI monoclonal antibody solution and europium-labeled cTnC monoclonal antibody solution diluted by the reaction buffer solution into the mixed magnetic particle solution, incubating for 10 minutes, then washing by using the washing solution, performing magnetic separation, repeating the washing step for 4 times, and then adding the enhancement solution;

(2) respectively measuring the fluorescence values of europium and samarium in the solution added with the enhancement solution by using a time-resolved fluoroimmunoassay analyzer to respectively obtain the fluorescence values of a calibrator solution of the cTnI-C complex and a sample to be detected, then respectively drawing standard curves according to the concentration of the cTnI-C antigen in the calibrator solution, the concentration of the cTnI antigen (containing cTnC) and the fluorescence values corresponding to the cTnI antigen and the concentration of the cTnI antigen (containing cTnC) and the fluorescence values corresponding to the cTnI antigen, and then substituting the corresponding fluorescence values of the sample to be detected into the corresponding cTnI-C standard curve or cTnI standard curve to calculate the contents of the cTnI-C complex and the complete segment cTnI in the sample to be detected.

Compared with the prior art, the technical scheme of the invention has the following advantages:

(1) the invention provides a double-labeling time-resolved fluorescence immunoassay kit based on cTnI and a cTnI-C compound, which comprises a reaction buffer solution, a cleaning solution, an enhancing solution, a magnetic particle solution coating a cTnI monoclonal antibody, a europium-labeled cTnC monoclonal antibody solution and a samarium-labeled cTnI monoclonal antibody solution which are integrated on a reagent strip, is fully-automatic in operation and simple and easy to implement. The content of the complete fragment cTnI and the cTnI-C compound in the serum can be simultaneously detected within 15min, the detection result is accurate and reliable, the method has outstanding advantages particularly for improving the detection accuracy of the cTnI/cTnI-C ratio parameter, has the advantages of high sensitivity, long storage time, no radioactive pollution, wide standard curve range and the like of the TRFIA technology, can also greatly shorten the reaction time and improve the detection sensitivity by the enrichment function of immune magnetic particles and the characteristic that the magnetic particles are fully diffused in liquid to expand the combined surface area, simultaneously the magnetic particles and the antibodies are directionally connected through chemical groups, greatly reduces the consumption of paired antibodies and obviously improves the detection precision, realizes automation simultaneously, overcomes the defect that the traditional microplate type TRFIA technology can detect the samples only when the samples are accumulated to a certain amount, and realizes the instant detection of the samples, the efficiency is high;

the kit is a high-efficiency double-marking time-resolved fluorescence technology taking nano magnetic particles as carriers: the magnetic particles are connected with free amino groups of the cTnI monoclonal antibody to form immune magnetic particles coated with the cTnI monoclonal antibody, the immune magnetic particles can capture a large amount of antigen-antibody complexes marked with Eu or Sm in a short time by means of the overlarge specific surface area of the immune magnetic particles, and after the Eu and Sm are dissociated from the complexes by using enhancement solution, chelating with a chelating agent in the enhancing liquid to form a colloidal molecular group, wherein the molecular group can emit strong fluorescence with different emission wavelengths (615nm and 643nm) and lifetimes (820 mu s and 880 mu s) under the excitation of ultraviolet light, the signal is enhanced by million times, and the two signals are easily distinguished, the content of the cTnI and the cTnI-C compound in serum can be detected simultaneously, three results of the cTnI and the cTnI-C compound and the cTnI/cTnI-C compound can be obtained simultaneously by one-time measurement, and the ratio is more accurate due to consistent conditions.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings.

FIG. 1 is a graph of the cTnI-C standard curve;

fig. 2 is a graph of the cTnI standard.

FIG. 3 is a schematic view of the structure of the reagent strip.

Fig. 4 is a schematic structural diagram of a kit integrating 5-person reagent strips.

The corresponding reference numbers for the component names in the figures are as follows:

1. a reagent strip; 2. a kit; 3. reserving a hole; 4. cleaning the holes; 5. a reinforcement liquid hole; 6. a detection hole; 7. europium/samarium marker pores; 8. a reaction buffer well; 9. magnetic particle solution pores.

Detailed Description

The invention is described in further detail below with reference to the following figures and examples:

as an example of the double labeling kit for detecting troponin and its complex and the method for preparing and detecting the same according to the present invention, the reaction principle is shown in FIGS. 1-2, an antibody against the cTnI region protected by endogenous cTnC as a solid phase antibody captures all cTnI, an antibody against cTnC as Eu for detecting cTnI-C complex, an antibody against the end of cTnI as Sm for detecting the complete fragment of cTnI, since the end of cTnI is easily hydrolyzed after entering blood, the complete cTnI is decreased, and the cTnI-C complex is stable, thus detecting the complete cTnI and cTnI-C complexes, and calculating the ratio thereof, will help to judge the duration of onset of AMI.

In this example, a dual-labeled time-resolved fluoroimmunoassay kit based on cTnI and cTnI-C complexes, as shown in fig. 3-4, comprises a plurality of reagent strips 1 and a reagent box 2, wherein the reagent strips 1 are arranged inside the reagent box 2, and are stacked up and down, the number of the reagent strips 1 is five, a plurality of preformed holes 3, a plurality of cleaning holes 4, an enhancement liquid hole 5, a detection hole 6, an europium/samarium marker hole 7, a reaction buffer hole 8 and a magnetic particle solution hole 9 are arranged on the reagent strips 1, the reagent strip 1 comprises reaction buffer solution, cleaning solution, enhancing solution, magnetic particle solution coated with cTnI monoclonal antibody, europium-labeled cTnC monoclonal antibody solution and samarium-labeled cTnI monoclonal antibody solution which are integrated on the reagent strip, and 2-6 bottles of freeze-dried calibrators of natural cTnI (containing cTnC) with different concentrations are attached.

In this embodiment, in the dual-labeled time-resolved fluorescence immunoassay kit based on cTnI and cTnI-C complex, Eu is used as the europium-labeled cTnC monoclonal antibody solution³⁺-N₂- [ P-Isocyanato-benzyl group]The-diethylenetriamine tetracetic acid sodium marked cTnC monoclonal antibody is prepared, and the cTnC monoclonal antibody and the Eu are³⁺-N₂- [ P-Isocyanato-benzyl group]The mass ratio of sodium diethylenetriamine tetracetate is 5: 1;

in this embodiment, the samarium-labeled cTnI monoclonal antibody solution is Sm³⁺-N₂- [ P-Isocyanato-benzyl group]The monoclonal antibody is prepared from a-diethylenetriamine tetra-sodium acetate marked cTnI monoclonal antibodyAntibody against Sm as described³⁺-N₂- [ P-Isocyanato-benzyl group]The mass ratio of sodium diethylenetriamine tetracetate is 5: 1;

in this embodiment, the magnetic particle solution coated with the cTnI monoclonal antibody is prepared by connecting, washing, sealing and washing the activated magnetic particles with a diameter of 100-;

in this embodiment, the double-labeling time-resolved fluoroimmunoassay kit based on the cTnI and cTnI-C complex comprises a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20, and 0.04g/L of Triton X-100, and the pH value is adjusted to 7.8 by hydrochloric acid;

in the embodiment, the enhancing solution is a mixed solution containing 3.6 per thousand of glacial acetic acid by volume concentration, 0.5g/L of sodium acetate, 0.05g/L of β -naphthoyl trifluoroacetone, 0.03g/L of trioctylphosphine oxide and 1 per thousand of Triton X-100 by volume concentration;

in this example, the reaction buffer is 50mmol/L Tris-HCl pH7.8 containing 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN₃；

In this example, the calibrator solution of the cTnI-C complex was prepared by using 2g/L BSA and 1g/L NaN₃50mmol/L reaction buffer, pH7.8 Tris-HCl. The natural cTnI antigen (containing cTnC) is prepared into calibrator solutions with different concentrations and then is freeze-dried and stored.

In this embodiment, the dual-labeled time-resolved fluoroimmunoassay kit based on cTnI and cTnI-C complexes comprises: reaction buffer solution, cleaning solution, enhancement solution, magnetic particle solution coated with cTnI monoclonal antibody, europium-labeled cTnC monoclonal antibody solution and samarium-labeled cTnI monoclonal antibody solution which are integrated on the reagent strip, and 2-6 bottles of freeze-dried calibrators with different concentrations of cTnI are attached.

In this embodiment, the method for preparing the dual-labeled time-resolved fluorescence immunoassay kit based on the cTnI and the cTnI-C complex includes any one or more of the following preparation methods:

in this example, the natural cPreparation of TnI-C Complex calibrator solution: taking a natural cTnI-C complex antigen solution with the concentration of 1mg/mL, using 2g/L BSA and 1g/L NaN₃Preparing 50mmol/L Tris-HCl reaction buffer solution with pH of 7.8 into calibrator solutions with different concentrations, subpackaging, lyophilizing, and storing at 2-8 deg.C;

in this example, the preparation of the magnetic particle solution coated with the cTnI monoclonal antibody: activating ferroferric oxide microspheres with the diameter of 100-5000nm by using glutaraldehyde with the mass concentration of 5-10%, uniformly mixing for 4 hours at room temperature, washing for 1-3 times by using 0.05mol/L PBS buffer solution with the pH value of 7.2, and then suspending by using the buffer solution to prepare magnetic particle suspension with the concentration of 100 mg/mL; taking 100 μ L of the magnetic particle suspension, adding 1mL of 0.05mol/L PBS buffer solution with pH of 7.2 and 50 μ g of cTnI monoclonal antibody, mixing and incubating at 25 deg.C for 2 hr, performing magnetic separation, retaining magnetic particles, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, blocking with 1mL of 0.0lmol/L PBS buffer solution with 5% BSA at 25 deg.C for 30 min, washing with 0.05mol/L PBS buffer solution with pH of 7.2 for 1-3 times, washing with 0.5% BSA at mass concentration and 0.1% NaN at mass concentration₃0.05mol/L Tris-HCl buffer solution with pH value of 7.2 is used for resuspension, subpackaging and storing at 2-8 ℃;

in this example, the samarium-labeled cTnI monoclonal antibody was prepared by subjecting 1mL of the cTnI antibody to PD-10 column conversion under buffer conditions, and eluting with 50mmol/L Na containing 0.155mol/L NaCl and having a pH of 8.5₂CO₃-NaHCO₃Buffer solution, collecting protein peak to obtain cTnI monoclonal antibody solution concentrated to 2g/L, adding 500 mu L of the cTnI monoclonal antibody solution into 0.2mg of samarium sodium benzyldiethylenetriamine tetraacetate, magnetically stirring and reacting for 20 hours at 25 ℃, then transferring reaction liquid to a Sepharose CL-6B column which is balanced by Tris buffer solution with pH of 7.8 and concentration of 80mmol/L for chromatography, collecting protein peak, diluting, subpackaging, vacuum freeze-drying, and storing at 2-8 ℃;

in this example, the europium-labeled cTnC monoclonal antibody solution was prepared: taking 1mL of cTnC antibodyPassing through PD-10 column to convert buffer condition, eluting with 50mmol/L Na containing 0.155mol/L NaCl and having pH of 8.5₂CO₃-NaHCO₃Buffer solution, collecting protein peak to obtain cTnC monoclonal antibody solution concentrated to 2g/L, taking 500 microliter of the cTnC monoclonal antibody solution, adding 0.2mg of benzyldiethylenetriamine tetraacetic acid europium sodium isothiocyanate, magnetically stirring at 25 ℃ for reaction for 20 hours, transferring the reaction solution to a Sepharose CL-6B column which is balanced by Tris buffer solution with pH7.8 and concentration of 80mmol/L for chromatography, collecting protein peak, diluting, subpackaging, vacuum freeze drying, and storing at 2-8 ℃;

in this example, the enhancing solution was prepared by mixing 3.6mL of glacial acetic acid, 0.5g of sodium acetate, 0.05g of β -naphthoyl trifluoroacetone, 0.03g of trioctylphosphine oxide and 1mL of Triton X-100, adding deionized water to a constant volume of 1000mL, packaging, and storing at 2-8 deg.C;

in this example, the reaction buffer is 50mmol/L Tris-HCl pH7.8 containing 8mmol/L NaCl, 0.1% BSA, 50. mu. mol/L DTPA, 0.1ml/L Tween-80 and 0.1% NaN₃；

In the embodiment, the cleaning solution is a buffer solution containing 0.48g/L of Tris, 12.49g/L of NaCl, 1.11g/L of Tween-20 and 0.04g/L of Triton X-100, and the pH value is adjusted to 7.8 by hydrochloric acid;

in this embodiment, the method for simultaneously detecting cTnI and cTnI-C based on the cTnI and cTnI-C double-labeled time-resolved fluoroimmunoassay kit is characterized by comprising the following steps,

(1) respectively taking 100ul of the calibrator solution of the cTnI-C complex and 100ul of a sample to be detected, mixing the calibrator solution and the sample to be detected with 50ul of the magnetic particle solution coated with the cTnI monoclonal antibody, adding 100ul of samarium-labeled cTnI monoclonal antibody solution and europium-labeled cTnC monoclonal antibody solution diluted by the reaction buffer solution into the mixed magnetic particle solution, incubating for 10 minutes, then washing by using the washing solution, performing magnetic separation, repeating the washing step for 4 times, and then adding the enhancement solution;

(2) respectively measuring the fluorescence values of europium and samarium in the solution added with the enhancement solution by using a time-resolved fluoroimmunoassay analyzer to respectively obtain the fluorescence values of a calibrator solution of the cTnI-C complex and a sample to be detected, then respectively drawing standard curves according to the concentration of the cTnI-C antigen in the calibrator solution, the concentration of the cTnI antigen (containing cTnC) and the fluorescence values corresponding to the cTnI antigen and the concentration of the cTnI antigen (containing cTnC) and the fluorescence values corresponding to the cTnI antigen, and then substituting the corresponding fluorescence values of the sample to be detected into the corresponding cTnI-C standard curve or cTnI standard curve to calculate the contents of the cTnI-C complex and the complete segment cTnI in the sample to be detected.

The basic principles and the main features of the invention and the advantages of the invention have been shown and described above, it being obvious to a person skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Moreover, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art will be able to make the description as a whole, and the embodiments may be appropriately combined to form other embodiments as will be apparent to those skilled in the art.