CN114965986A - Kit for detecting soluble growth stimulation expression gene 2 protein (ST2) in blood - Google Patents
Kit for detecting soluble growth stimulation expression gene 2 protein (ST2) in blood Download PDFInfo
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Abstract
The invention discloses a kit for detecting soluble growth stimulation expression gene 2 protein (ST2) in blood, which can quickly and accurately detect the concentration of the soluble growth stimulation expression gene 2 protein in the blood by synchronous application of a latex immune scattering rate method and a latex enhanced immune transmission turbidimetric method, and has the characteristics of accurate result, high specificity and the like. Can be used for detecting the clinical ST2 protein.
Description
Technical Field
The invention relates to biotechnology, in particular to a kit for detecting soluble growth stimulation expression gene 2 protein (ST2) in a sample and a using method thereof.
Background
The growth-stimulating expression gene 2 protein (ST2) is a member of the IL-1 receptor family, and mainly comprises two isomers, namely a transmembrane type (ST2L) and a soluble type ST 2. Soluble ST2 is secreted by heart disease or injury, and if its concentration in blood is low, IL-33 will bind, and can achieve cardioprotective effect to maintain cardiac function. On the other hand, when the concentration in blood is high, ST2 competitively binds to IL-33, and the heart defense signal transmission system of IL-33 fails to operate, thereby inducing inflammatory reaction of myocardial cells, natural death of cells, fibrosis, myocardial function degradation, etc., and accelerating heart failure. The ST2 test, unlike current heart failure markers (BNP, NT-proBNP), provides prognostic information. The method is not affected by age, body mass index, renal dysfunction and the like, can be used for continuous detection, and provides independent prognosis information for heart failure patients.
Currently, enzyme immunoassay (ELI) is adopted for detecting soluble growth stimulation expression gene 2 protein (ST2) in the market, and the method comprises the steps of coupling an antibody on an enzyme label plate, adsorbing an antigen in a sample, adding a second antibody for color development and measuring the concentration of ST2 in the sample, and according to the difference of conjugates of the second antibody, enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay (CLI) can be divided. The method has the problems of long detection time, high cost and the like, and has high clinical use difficulty and low accessibility.
Disclosure of Invention
In view of the problems involved in the prior art for detecting soluble growth-stimulating expression gene 2 protein (ST2), it is an object of an aspect of the present invention to provide a kit for detecting soluble growth-stimulating expression gene 2 protein (ST 2).
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the kit for detecting soluble growth-stimulating expressed gene 2 protein (ST2) in blood comprises a first reagent (also referred to as reagent R1), a second reagent (also referred to as reagent R2), a calibrator, wherein the first reagent comprises 3-50g/L of electrolyte, 1-50g/L of promoter, 0.5-20g/L of preservative, 0.5-40g/L of surfactant, and 1-100mmol/L of buffer solution with pH of 6-9; the second reagent comprises ST2 antibody or antigen binding fragment thereof, 0.05% -5% of polystyrene microspheres with carboxyl groups in w/v and 1-200mmol/L buffer solution with pH of 5.5-9.5, and the calibrator comprises antigen with known concentration.
Preferably, the electrolyte is selected from sodium ions or potassium ions,
preferably, the promoter is selected from polyethylene glycol 4000-20000, more preferably polyethylene glycol 6000.
Preferably, the preservative is selected from one or more of sodium azide, sodium benzoate, sodium nitrite, thimerosal, ProClin100, ProClin200, ProClin250, ProClin300 and ProClin 350.
More preferably, the preservative is selected from ProClin 300.
Preferably, the surfactant is selected from one or more of Tween20, Tween40, Span40, Span80 and TritonX-100.
The surfactant is selected from Tween 20.
Preferably, the buffer is selected from PB buffer, EMS buffer or Tris buffer.
Preferably, the preparation method of the second reagent comprises the following steps:
(1) adding 10mg of polystyrene microspheres with carboxyl into 1mL of 1-100mmol/L EMS buffer solution with pH6.5,
(2) adding 10-30 μ L of 15-25 mg/mL EDC, activating for 20-30 min, centrifuging, suspending the precipitate in 1mL of 1-100mmol/L EMS buffer solution with pH of 6.5,
(3) adding 0.2-0.8 mg of ST2 antibody, mixing uniformly at 18-28 deg.C for 40-80 min, centrifuging, removing supernatant, adding 1mL of 1-100mmol/L EMS buffer solution with pH6.5 into the precipitate, performing ultrasonic treatment for 1-3 min,
(4) adding 0.1-0.3 mL of 10% BSA, mixing uniformly at 18-28 ℃ for 40-80 min, performing ultrasonic treatment for 1-3 min,
(5) adding 1-200mmol/L PB buffer solution with pH of 5.5-9.5 to 5mL, sealing, and storing at 2-8 deg.C.
Preferably, the calibrator in the method for preparing the second reagent comprises: ST2 antigen at a concentration of 0.0ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, and buffers and protectants.
Preferably, the buffer in the preparation method of the second reagent is selected from PB buffer, EMS buffer or Tris buffer.
Preferably, the second reagent is prepared by a method in which the protective agent is selected from bovine serum albumin and trehalose.
Preferably, the kit further comprises a quality control product.
Preferably, the quality control product comprises: antigen at concentrations of 33ng/mL and 90ng/mL and buffers and protective agents,
preferably, the buffer in the quality control product is selected from PB buffer, EMS buffer or Tris buffer,
preferably, the protective agent in the quality control product is selected from bovine serum albumin and trehalose.
Preferably, the ST2 antibody in the kit for detecting soluble growth-stimulating expressed gene 2 protein (ST2) in blood of the present invention is a monoclonal antibody.
Preferably, the kit for detecting soluble growth-stimulating expressed gene 2 protein in blood (ST2) comprises:
a first reagent, comprising: 2.2-9.5ng/mL PB buffer solution, pH 6-9, 3-5g/L NaCl, 0.5-40mL/L Tween20, 1-50g/L PEG6000, 0.5-20mL/L ProClin 300;
a second reagent, comprising: 0.05-5% by w/v of 60-300nm polystyrene microspheres with carboxyl, 1-200mMol/L PB buffer solution pH 505-9.5, 0.1-8g/L surfactant and 1-50g/L BSA;
a calibrator, comprising: 1-100mMol/L PB buffer solution, pH 6-8, 10-100g/L trehalose, 1-100g/L BSA, 0.5-20g/L Proclin-300;
optionally, a quality control comprising: 1-100mMol/L PB buffer solution, pH 6-8, 10-100g/L trehalose, 1-100g/L BSA, 0.5-20g/L Proclin-300.
In another aspect of the present invention, there is provided a method for detecting soluble growth-stimulating expressed gene 2 protein (ST2) using the above kit, comprising the steps of:
(1) respectively adding the sample and the calibrator into the first reagent, uniformly mixing, and incubating at the constant temperature of 37 ℃ for 3-8 minutes;
(2) adding the second reagent into the mixture of the sample, the calibrator and the first reagent respectively,
(3) measuring absorbance of the mixed solution immediately after mixing as A1 and absorbance of the mixed solution 2-7 minutes after mixing as A2, respectively, and calculating an absorbance difference Delta A of the concentration of the calibrator as calibrator A2-calibrator A1;
(4) obtaining a calibration curve according to the concentration of the calibrator and the absorbance difference delta A corresponding to each concentration,
(5) measuring absorbance of the sample by the same method as the calibrator to obtain the absorbance difference of the sample, obtaining the corresponding concentration of the sample on the calibration curve according to the absorbance of the sample,
optionally, the method further comprises the step of obtaining the absorbance difference of the quality control product according to the same steps as the sample, and performing quality control.
Preferably, the parameters measured in step (3) are: the temperature is 37 ℃, the wavelength is 400-800nm, the sample is 0.01-mul, the calibrator is 2-20 mul, the quality control is 2-20 mul, the reagent R1 is 50-600 mul, the reagent R2 is 10-200 mul,
optionally, the quality control product is 2-20 μ l.
The invention also provides a preparation method of the polystyrene microsphere combined with the ST2 antibody, which comprises the following steps:
(1) 10mg of polystyrene microspheres with carboxyl groups are added into 1mL of 1-100mmol/LEMS buffer solution with pH6.5,
(2) adding 10-30 μ L of 15-25 mg/mL EDC, activating for 20-30 min, centrifuging, suspending the precipitate in 1mL of 1-100mmol/L EMS buffer solution with pH of 6.5,
(3) adding 0.2-0.8 mg of ST2 antibody, mixing uniformly at 18-28 deg.C for 40-80 min, centrifuging, removing supernatant, adding 1mL of 1-100mmol/L EMS buffer solution with pH6.5 into the precipitate, performing ultrasonic treatment for 1-3 min,
(4) adding 0.1-0.3 mL of 10% BSA, mixing uniformly at 18-28 ℃ for 40-80 min, performing ultrasonic treatment for 1-3 min,
(5) adding 1-200mmol/L PB buffer solution with pH of 5.5-9.5 to 5mL, sealing, and storing at 2-8 deg.C.
The kit disclosed by the invention has the advantages that the polystyrene microspheres coupled with ST2 antibody and provided with carboxyl groups and carboxyl groups are subjected to specific aggregation reaction with ST2 in a sample, and the concentration of ST2 in the sample is represented by the change of transmitted light and scattered light before and after the incident light with a specific wavelength and light intensity is subjected to reaction of a reagent-sample mixed solution. The method adopts the latex immunoturbidimetry to measure the ST2 content in the sample, and particularly adopts the synchronous application of the latex immunoscattering velocity method and the latex enhanced immunotransmission turbidimetry, thereby enlarging the use range, quickening the measuring time and improving the sensitivity.
Drawings
FIG. 1 is calibration data (curve) obtained using the standard of example 3.
FIG. 2 is a graph showing the correlation between the sample assay in example 4 using the enzyme immunoassay product on the market and the kit of the present invention.
FIG. 3 shows the sensitivity of the kit of the invention.
Detailed Description
In view of the disadvantages of the prior art, the inventors of the present invention have conducted extensive studies and found that the concentration of ST2 antibody in a sample can be accurately and rapidly measured by coupling high affinity ST2 antibody to carboxyl group-containing polystyrene microspheres, allowing ST2 antibody to form an immunoreaction with ST2 in the sample, mediating the aggregation of coupled carboxyl group-containing polystyrene microspheres to form a turbidity change, and quantitatively measuring the concentration of ST2 in the sample by measuring the turbidity change. The present invention has been completed based on this finding.
The first reagent (reagent R1) of the kit has the functions of pretreating a sample, diluting the sample and ensuring the accuracy, the qualification and the specificity of a detection result.
The second reagent (reagent R2) of the kit has the function of adsorbing a substance to be detected to form aggregates, so that the absorbance is changed, the change of the absorbance is correlated with the concentration of the substance to be detected, and a calibration curve is drawn by the change of the absorbance and the concentration of a calibrator.
The standard substance and the quality control substance solution of the kit can be independently packaged.
In the description of the present invention, the particle size of the polystyrene microsphere with carboxyl groups is 60-300nm, and more preferably 200-250 nm.
In the context of the present invention, the ST2 antibody is preferably a monoclonal antibody, e.g., an american rapin monoclonal antibody.
When the kit of the present invention is used, the method for detecting the change in turbidity can be carried out by using instruments and methods commonly used in the art (e.g., MACO240/MACAO 600/Hitachi 7180/7060/7100/7600, Beckmann AU480/AU580 Meyer BS2000, Toshiba 120/2000/equi-wavelength cover 340-.
The process of collecting and processing the sample to be tested when using the kit of the invention is also conventional in the art: the collected blood sample is naturally coagulated, and then supernatant (serum) is taken as a test sample. The sample can be stored at 2-8 deg.C for 24h and at-20 deg.C for 1 month. This makes the handling of the kit of the invention during use easily accessible to the operator.
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention will be further described in detail through the embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. Percentages are by weight unless otherwise indicated.
Example 1
Selection of the ratio of the amount of antibody to the amount of microspheres
(1) Taking 10 centrifugal tubes, respectively adding 10mg of polystyrene microsphere Japanese JSR220nm microspheres with carboxyl, and adding 25mMol/LMES buffer solution with pH of 6.5 to 1 mL;
(2) adding 20 μ L of 20mg/mLEDC for activation for 25min, centrifuging, and suspending the precipitate in 1mL of 25mMol/L MES buffer solution with pH of 6.5;
(3) adding 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.25mg, 0.3mg, 0.4mg, 0.5mg, 0.7mg and 1.0mg of antibody respectively, and coupling for 2 h;
(4) centrifuging, taking the precipitate, suspending the precipitate in 1mL of pH6.5MES buffer solution, adding 1mL of PB buffer solution, adding 0.2mL of 10% BSA, and blocking for 1 h;
(5) fixing the volume to 3/5/8mL by using a PB buffer solution to obtain a reagent R2 with different concentrations;
(6) reagent R1 was formulated as follows:
(6.1) taking a clean beaker, labeling the product batch number, name, production amount, date and operator on a label, putting a stirrer, adding 800mL of purified water, and stirring;
(6.2) weighing 0.56g of sodium dihydrogen phosphate dihydrate according to the process formula, adding the sodium dihydrogen phosphate dihydrate into a beaker, stirring the mixture by using a magnetic stirrer until the sodium dihydrogen phosphate dihydrate is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(6.3) weighing 4.65g of disodium hydrogen phosphate dodecahydrate according to the process formula, adding the disodium hydrogen phosphate dodecahydrate into a beaker, stirring the mixture by using a magnetic stirrer until the disodium hydrogen phosphate is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(6.4) weighing 29.25g of sodium chloride according to the process formula, adding the sodium chloride into a beaker, stirring the sodium chloride by using a magnetic stirrer until the sodium chloride is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(6.5) weighing 600010.0 g of polyethylene glycol according to the process formula, adding the polyethylene glycol into a beaker, stirring the mixture by using a magnetic stirrer until the polyethylene glycol is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist; (6.6) weighing 202 mL of Tween according to the process formula, adding into a beaker, stirring by using a magnetic stirrer until the Tween is completely dissolved, and observing the solution by naked eyes without obvious particles;
(6.7) weighing Proclin-3001 mL according to the process formula, adding into a beaker, stirring with a magnetic stirrer until the Proclin-3001 mL is completely dissolved, and observing the solution with naked eyes without obvious particles;
adding purified water to 1000mL, mixing with magnetic stirrer for about 10min, measuring pH to 7.0-7.4, and adjusting pH to 7.0-7.4 with hydrochloric acid/sodium hydroxide when pH exceeds the range; 10mL of the diluted solution is taken and checked under light, the solution is colorless, no turbid, no precipitate and clear liquid, and the measured pH value is 7.0-7.4.
(7) Preparing a calibration and quality control product according to the following steps
(7.1) taking a clean beaker, labeling the label with the batch number, name, production amount, date and operator, putting a stirrer, adding 800mL of purified water, and stirring;
(7.2) weighing 0.56g of sodium dihydrogen phosphate dihydrate according to the process formula, adding into a beaker, stirring by using a magnetic stirrer until the sodium dihydrogen phosphate dihydrate is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(7.3) weighing 4.65g of disodium hydrogen phosphate dodecahydrate according to the process formula, adding the disodium hydrogen phosphate dodecahydrate into a beaker, stirring the mixture by using a magnetic stirrer until the disodium hydrogen phosphate is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(7.4) weighing 9g of sodium chloride according to the process formula, adding the sodium chloride into a beaker, stirring the sodium chloride by using a magnetic stirrer until the sodium chloride is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(7.5) weighing 5g of BSA according to the process formula, adding the BSA into a beaker, stirring the BSA by using a magnetic stirrer until the BSA is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(7.6) weighing 50g of trehalose according to the process formula, adding the trehalose into a beaker, stirring the trehalose by using a magnetic stirrer until the trehalose is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
(7.7) weighing Proclin-3001 mL according to the process formula, adding into a beaker, stirring with a magnetic stirrer until the Proclin-3001 mL is completely dissolved, and observing the solution with naked eyes without obvious particles;
(7.8) adding the rest purified water into a beaker, and uniformly mixing for about 10min by using a magnetic stirrer to obtain 1000mL of protein diluent;
(7.9) determining the concentration of ST2 antigen (American Reidesmoplants), with the determined antigen concentration of 2130 ng/mL; according to the antigen concentration measurement, 1mL of antigen solution is taken, and 9.65mL of protein diluent is added to obtain a calibrator S6 with the concentration of 200 ng/mL;
(7.10) diluting S6 with a protein diluent according to a 2-fold gradient to obtain calibrators with the concentrations of 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL and 0 ng/mL;
(7.12) according to the antigen concentration, taking 0.1mL of antigen solution, and adding 2.27mL of protein diluent to obtain a quality control level 2 with the concentration of 90 ng/mL;
(7.13) according to the antigen concentration, 0.05mL of the antigen solution was taken and 3.2mL of the protein diluent was added to obtain a quality control level 1 of 33 ng/mL.
(8) And (3) scaling the 30 reagents prepared in the step (5) by the calibrator diluted in the step (7), and measuring the quality control substances with the identification concentrations of 33ng/ml and 90ng/ml (specifically, a calibration curve is made by the calibrator concentration and corresponding delta A, and the ST-2 concentration in the quality control substances is read from the calibration curve by the delta A of the quality control substances).
The deviation from the marker concentration is calculated as the difference between the measured value and the marker concentration. The formula is as follows:
relative deviation of ═ C Measurement of -C Identification )/C Identification *100％
In the formula: c Measurement of ..
C Identification ..
The ratio of the amount of the ST2 antibody to the polystyrene microsphere having carboxyl groups was obtained by measuring the relative deviation of the quality control products, and is shown in Table 1 below.
TABLE 1
As can be seen from table 1: the dosage of the fixed microspheres is 10mg, the antibody is 0.5mg, and the optimal effect is achieved when the final volume is 5mL, namely the optimal effect is achieved when the concentration of the carboxyl microsphere-antibody compound in the reagent R2 is 0.21% (W/V).
Example 2:
optimization of reaction system parameters
(1) Taking 9 centrifugal tubes, respectively adding 10mg of polystyrene microspheres with carboxyl groups and the particle sizes of 200 nm, 260nm and 300nm into each centrifugal tube, adding polystyrene microspheres with the same particle size into each three centrifugal tubes, respectively adding MES buffer solution with the pH5.5, 6.5 and 7.5 into the centrifugal tubes containing the polystyrene microspheres with the same particle size to 1mL, and respectively adding 3 centrifugal tubes under each pH condition, wherein the centrifugal tubes are respectively marked as A1, A2, A3, B1, B2, B3, C1, C2 and C3 (microsphere activation pH selection);
(2) respectively adding 10 mu L of 20mg/mLEDC into centrifuge tubes of A1, B1 and C1, respectively adding 20 mu L of 20mg/mLEDC into centrifuge tubes of A2, B2 and C2, respectively adding 30 mu L of 20mg/mLEDC into centrifuge tubes of A3, B3 and C3, respectively activating for 25min, centrifuging, taking precipitates, and respectively suspending the precipitates in 1mL of MES buffer solution (the dosage of EDC) with the pH of 5.5, 6.5 and 7.5;
(3) adding 0.1mg of antibody, and coupling with 1H;
(4) centrifuging, taking the precipitate, suspending in corresponding 1mL of MES buffer solution with pH of 5.5-7.5, adding 0.2mL of 10% BSA (adding 0.2mL of 0.1 casein in parallel in group of 260 nm), and blocking for 1 h;
(5) the volume of PB buffer solution is determined to be 5mL, and a reagent R2 is prepared;
(6) reagent R1 was prepared according to the method of example 1 (6.1-6.7);
(7) preparing a calibrator and a quality control product according to the method (7.1-7.13) of example 1;
(8) the deviation from the labeled concentration was obtained in accordance with the method of step (8) of example 1, and the optimum was obtained from the relative deviation of the measured quality control materialParameters of reaction system. The results are shown in table 2 below.
TABLE 2
As can be seen from table 2: this was best achieved when microspheres with a particle size of 260nm were used, the activation environment was pH6.5, EDC was used in20 μ L20 mg/mLEDCm, and finally blocking was performed with 0.2mL 10% BSA.
Example 3:
kits prepared according to the invention with the parameters optimized above
3.1 preparation of reagent R1:
3.1.1 taking a clean beaker, labeling the product batch number, name, production amount, date and operator on a label, putting a stirrer, adding 800mL of purified water, and stirring;
3.1.2 weighing 0.43g of sodium dihydrogen phosphate according to the process formula, adding into a beaker, stirring by using a magnetic stirrer until the sodium dihydrogen phosphate is completely dissolved, and observing the solution by naked eyes without obvious particles;
3.1.3 weighing 1.84g of disodium hydrogen phosphate according to the process formula, adding into a beaker, stirring by using a magnetic stirrer until the disodium hydrogen phosphate is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
3.1.4 weighing 9g of sodium chloride according to the process formula, adding the sodium chloride into a beaker, stirring the sodium chloride by using a magnetic stirrer until the sodium chloride is completely dissolved, and observing the solution by naked eyes to ensure that no obvious particles exist;
3.1.5 adding 600010.0 g of polyethylene glycol into a beaker according to the process formula, stirring by a magnetic stirrer until the polyethylene glycol is completely dissolved, and observing the solution by naked eyes without obvious particles;
3.1.7 weighing 202 mL of Tween according to the process formula, adding into a beaker, stirring with a magnetic stirrer until the Tween is completely dissolved, and observing the solution with naked eyes without obvious particles;
3.1.8 taking Proclin-3001 mL according to the technical formula, adding into a beaker, stirring with a magnetic stirrer until the Proclin-3001 mL is completely dissolved, and observing the solution with naked eyes without obvious particles;
3.1.9 measuring pH, adding the rest purified water into a beaker, and mixing with a magnetic stirrer for about 10 min; the prepared product is sealed and then is labeled and put in a refrigeration house (2 ℃ -8 ℃).
3.2 preparation of reagent R2:
3.2.1 dissolving 10mg of polystyrene microspheres with carboxyl groups and the particle size of 260nm in25 MES buffer solution with the concentration of pH6.5 to ensure that the total volume is 1mL, then adding 20 mul of 20mg/mL EDC, and uniformly mixing for 25min at 18-28 ℃;
3.2.2 centrifugation of the carboxyl-bearing polystyrene microspheres activated with EDC at 15000g x 15min, aspiration of the supernatant, addition of 1ml MES pH6.5, sonication for 2 min;
3.2.3 adding 0.5mgST2 antibody, mixing well for 60min at 18-28 deg.C;
3.2.415000 g 15min, sucking supernatant, adding 1ml of MES (pH 6.5, 25 mM), and performing ultrasonic treatment for 2 min;
3.2.5 adding 0.2ml 10% BSA, mixing well at 18-28 deg.C for 60 min;
3.2.6 sonication for 2min, PB buffer was added to 5ml.
3.2.7 sticking the label, sealing and storing at 2-8 ℃.
3.4 preparation of calibrator:
3.4.1 preparation of protein Diluent
3.4.1.1 labeling a clean beaker, placing a stirrer with batch number, name, production amount, date and operator, adding 800mL of purified water, and stirring;
3.4.1.2 adding 0.43g sodium dihydrogen phosphate into a beaker according to the formula, stirring with a magnetic stirrer until the sodium dihydrogen phosphate is completely dissolved, and observing the solution with naked eyes without obvious particles;
3.4.1.3 adding 1.84g disodium hydrogen phosphate into a beaker according to the formula, stirring with a magnetic stirrer until the disodium hydrogen phosphate is completely dissolved, and observing the solution with naked eyes to ensure that no obvious particles exist;
3.4.1.4 adding 9g sodium chloride into a beaker according to the formula, stirring with a magnetic stirrer until the sodium chloride is completely dissolved, and observing the solution with naked eyes without obvious particles;
3.4.1.5 according to the technical formula, 50g of trehalose is added into a beaker, stirred by a magnetic stirrer until the trehalose is completely dissolved, and the solution is observed by naked eyes without obvious particles;
3.4.1.6 weighing 9g of BSA according to the process formula, adding into a beaker, stirring with a magnetic stirrer until the BSA is completely dissolved, and observing the solution with naked eyes without obvious particles;
3.4.1.7 weighing Proclin-3001 mL according to the process formula, adding into a beaker, stirring with a magnetic stirrer until the Proclin-3001 mL is completely dissolved, and observing the solution with naked eyes without obvious particles;
3.4.1.8 adding the rest purified water to 1000mL in a beaker, and mixing uniformly for about 10min by a magnetic stirrer;
3.4.1.9 the self-checking product should be colorless clear liquid, the measured pH should be 7.32, the prepared product is sealed and labeled and put in a cold storage (2-8 ℃) for standby, and the product character will not change obviously within 180 days.
3.4.2 preparation of quality control product for calibration
The amount of antigen and antigen diluent was calculated according to the following formula, measured accurately and added, after stirring for 20 minutes in a magnetic stirrer.
V B ＝V p -V 0 ....................(2)
In the formula: v 0 : the volume of the raw materials to be added;
C 0 : the effective concentration of the raw material;
C i : preparing a target concentration value;
V p : preparing batches;
V B : adding the volume of the calibrator and the volume of the quality control dilution buffer I/II;
the concentration of ST2 antigen (American Reid) was measured, and the antigen concentration was measured to be 2130 ng/mL;
according to the antigen concentration measurement, 1mL of antigen solution is taken, and 9.65mL of protein diluent is added to obtain a calibrator S6 with the concentration of 200 ng/mL;
diluting S6 with protein diluent according to 2 times gradient to obtain calibrator with concentration of 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL and 0 ng/mL;
3.5 preparation of quality control Material
3.5.1 preparation of protein Diluent
The same as 3.4.1 (3.4.1.1-3.4.1.9).
3.5.2 preparation of quality control products
Similar to the method of the calibrator. According to the antigen concentration measurement, 0.1mL of antigen solution is taken, and 2.27mL of protein diluent is added to obtain a quality control level 2 with the concentration of 90 ng/mL;
according to the antigen concentration, 0.05mL of antigen solution was taken and 3.2mL of protein diluent was added to obtain a quality control level 1 of 33 ng/mL.
The kit prepared in this example includes: individually packaged reagent R1 (preparation 3.1), reagent R2 (preparation 3.2), individually packaged calibrator solution (preparation 3.4) and individually packaged quality control solution (preparation 3.5).
The method of use of the kit prepared in this example is as follows:
1. the reagent R1 and the reagent R2 are not required to be prepared and can be directly used after being opened.
2. Test conditions
The method comprises the following steps: two-point end point method or fixed time method
Wavelength: wavelength minor 570 nm: is free of
Reaction temperature: reaction time at 37 ℃: 7min
The standard curve simulates the equation: 6-point calibration, and a nonlinear calculation mode such as logit and Spline is adopted.
The automatic biochemical analyzer has its own program parameter input method, and the basic parameters need to be combined with the program parameter input method of the automatic biochemical analyzer, so that the reagent can be automatically determined by the matched instrument after the computer parameters are input.
The procedure is shown in table 3 below:
TABLE 3
3. And (3) calibration procedure:
direct vial opening was 6-point calibrated with 6-point liquid calibrators of different concentrations (i.e., calibration curves were plotted with calibrant concentration versus the corresponding Δ a, as shown in fig. 1).
4. Quality control procedure:
the quality control product verification test program matched with the kit is used.
Once the quality control result is not within the allowable range, correction is required.
5. And (4) calculating a result:
the concentration of ST-2 in the sample is read from the calibration curve by the Δ a of the sample.
Example 4: 40 samples were tested using the product prepared in example 3 above and compared to a commercial enzyme immune product.
Control reagent information: soluble growth stimulation expression gene 2 protein determination kit (enzyme-linked immunosorbent assay)
The manufacturer: american Reinforcement organism
The product specification is as follows: and 96 parts of human body.
And (4) calibrating concentration: 200ng/ml
The test time is 60min
100ml samples were prepared according to the above examples (3.1-3.5);
the comparison with the above control reagent is shown in the following tables 4 and 5:
TABLE 4.10-50 ng/mL ST2 Range calculated Absolute deviation
| Serial number | Control | The invention | Absolute deviation | Serial number | Control | The | Absolute deviation | |
| 1 | 34.6 | 35.3 | 0.7 | 13 | 45.4 | 48.6 | 3.2 | |
| 2 | 10 | 11.0 | 1.0 | 14 | 15.9 | 17.3 | 1.4 | |
| 3 | 15 | 15.8 | 0.8 | 15 | 47 | 43.2 | -3.8 | |
| 4 | 24.5 | 25.7 | 1.2 | 16 | 38.1 | 34.7 | -3.4 | |
| 5 | 25 | 22.8 | -2.3 | 17 | 38.1 | 38.5 | 0.4 | |
| 6 | 26.4 | 28.2 | 1.8 | 18 | 20.7 | 20.3 | -0.4 | |
| 7 | 35.7 | 35.3 | -0.4 | 19 | 47.3 | 48.2 | 0.9 | |
| 8 | 40.2 | 44.2 | 4.0 | 20 | 17.2 | 17.2 | 0.0 | |
| 9 | 26.4 | 27.7 | 1.3 | 21 | 12.9 | 12.3 | -0.6 | |
| 10 | 32.2 | 31.9 | -0.3 | 22 | 28.9 | 27.2 | -1.7 | |
| 11 | 41.5 | 44.0 | 2.5 | 23 | 20.4 | 21.2 | 0.8 | |
| 12 | 11.3 | 10.4 | -0.9 | 24 | 15.5 | 16.1 | 0.6 |
TABLE 5.51-200 ng/mL ST2 relative deviations calculated
| Serial number | Control | The invention | Absolute deviation | Serial number | Control | The | Absolute deviation | |
| 25 | 168.0 | 151.2 | -10.0％ | 33 | 189.8 | 189.6 | -0.1％ | |
| 26 | 127.0 | 137.8 | 8.5％ | 34 | 78.3 | 85.0 | 8.5％ | |
| 27 | 58.3 | 61.8 | 5.9％ | 35 | 95.3 | 94.8 | -0.5％ | |
| 28 | 101.8 | 95.8 | -5.9％ | 36 | 51.4 | 56.1 | 9.2％ | |
| 29 | 62.9 | 59.4 | -5.5％ | 37 | 108.3 | 116.3 | 7.4％ | |
| 30 | 196.2 | 193.0 | -1.6％ | 38 | 72.8 | 70.6 | -3.0％ | |
| 31 | 93.4 | 95.6 | 2.4％ | 39 | 170.4 | 165.8 | -2.7％ | |
| 32 | 161.6 | 154.3 | -4.5％ | 40 | 162.2 | 155.1 | -4.4％ |
As can be seen from the above tables 4 and 5, the detection results of the product and the control product have no obvious difference, the maximum relative deviation is 10%, and the maximum absolute deviation is 4.0 ng/ml. The novel fitting was performed with the measurement result of the enzyme-free product on the market as X axis and the measurement result of the kit of the present invention as Y axis (see fig. 1), and the a value was 0.9708, which was close to 1.00, and the correlation coefficient r was 0.9968. When the kit is used for detecting the product ST2, the result can be obtained in less than 10min, and the time is far shorter than the time for detecting ST2 by using the existing product in the market.
Therefore, when the kit is used for detecting a product ST2, the detection speed is high, the detection result is accurate and the repeatability is good, and the full-automatic operation can be realized.
The following performance analyses: assay sensitivity, linear range, accuracy, reproducibility were tested using the formulated product of example 3. Accuracy the control reagent was the soluble growth expressing gene 2 protein assay kit (enzyme linked immunosorbent assay) of american rapin.
Sensitivity analysis
Taking a sample with the concentration of 35 +/-10 ng/mL, adding the reagent R2, obtaining the absorbance change value which is the analysis sensitivity according to the absorbance A1 at about 1min and the absorbance A2 at about 4min after the reagent R2 is added and obtaining the absorbance change value which is the analysis sensitivity according to (A2-A1)/c 35
Note: c is the sample assay concentration.
As can be seen from FIG. 3, the absorbance change at a concentration of 35ng/mL was 0.01 or more.
Linear range analysis
Diluting a high-value sample close to the linear range and a low-value sample close to the linear range into 5 different concentrations, measuring each concentration for 3 times, fitting the mean value of the 3 measurements and the theoretical concentration, obtaining an estimated value according to a fitting function, and calculating the estimated value and the mean value of the 3 measurements. The fitting function dependent dilution R is required to be greater than or equal to 0.99. The results are shown in Table 6.
TABLE 6
As can be seen from the above Table 6, the correlation coefficient r is more than or equal to 0.990 in the linear range of [5, 200] ng/ml; linear deviation: when [5,50] ng/mL, the absolute deviation of linearity is not more than +/-5 ng/mL; at [50,200] ng/mL, the relative deviation of linearity is not more than. + -. 10%.
Accuracy analysis
40 samples were simultaneously assayed using the reagent prepared in example 3 and the kit for assaying soluble growth-expressing gene 2 protein of American Redy organism (ELISA), and the accuracy of the product prepared in example 3 was examined using American Redy organism as a reference standard. The correlation coefficient should be more than or equal to 0.975, within the range of 5-50ng/ml, the absolute deviation should not exceed +/-5 ng/ml, and the relative deviation should not exceed +/-15% within the range of 50-200 ng/ml. The results are shown in Table 7.
TABLE 7
As can be seen from table 7 above, reagent accuracy: 5-50ng/mL, and the absolute deviation is not more than 5 ngf/mL. The relative deviation is not more than +/-15% within the range of 50-200ng/mL, and the absolute deviation is not more than 5ng/mL within the range of 5-50 ng/mL.
Precision analysis
The measurement was repeated 10 times using the quality control prepared in example 3, and the CV was calculated for 10 measurements. The results are shown in Table 8.
TABLE 8
| |
37.5 | 86.3 |
| Measured |
29.8 | 95.2 |
| Measured value 3 | 30.7 | 100 |
| Measured value 4 | 32.6 | 89.2 |
| Measured |
35.6 | 97 |
| Measured value 6 | 29.7 | 95.2 |
| Measured value 7 | 34.9 | 87.3 |
| Measured value 8 | 30.5 | 81.2 |
| Measured value 9 | 35 | 84.5 |
| Measured |
29.1 | 95.4 |
| Mean value | 32.54 | 91.13 |
| SD | 2.991543637 | 6.225226457 |
| CV | 9.2％ | 6.8％ |
As can be seen from the above table, the repeatability CV is less than or equal to 10%.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A kit for detecting soluble growth-stimulating expressed gene 2 protein (ST2) in blood, comprising a first reagent, a second reagent, a calibrator,
wherein the first reagent comprises 3-50g/L of electrolyte, 1-50g/L of promoter, 0.5-20g/L of preservative, 0.5-40g/L of surfactant and 1-100mmol/L of buffer solution with pH of 6-9;
the second reagent comprises: ST2 antibody or antigen binding fragment thereof, 0.05-5% of polystyrene microsphere with carboxyl based on w/v, and 1-200mmol/L buffer solution with pH of 5.5-9.5,
the calibrator contains a known concentration of antigen.
2. The kit according to claim 1,
the electrolyte is selected from sodium ions or potassium ions,
the promoter is selected from polyethylene glycol 4000-20000, more preferably polyethylene glycol 6000,
the preservative is selected from one or more of sodium azide, sodium benzoate, sodium nitrite, thimerosal, ProClin100, ProClin200, ProClin250, ProClin300, ProClin350, more preferably ProClin300,
the surfactant is selected from one or more of Tween20, Tween40, Span40, Span80 and TritonX-100, more preferably Tween20,
the buffer is selected from PB buffer, EMS buffer or Tris buffer.
3. The kit of claim 1, wherein the second reagent is prepared by a method comprising the steps of:
(1) adding 10mg of polystyrene microspheres with carboxyl into 1mL of 1-100mmol/L EMS buffer solution with pH6.5,
(2) adding 10-30 μ L of 15-25 mg/mL EDC, activating for 20-30 min, centrifuging, suspending the precipitate in 1mL of 1-100mmol/L EMS buffer solution with pH of 6.5,
(3) adding 0.2-0.8 mg of ST2 antibody, mixing uniformly at 18-28 deg.C for 40-80 min, centrifuging, removing supernatant, adding 1mL of 1-100mmol/L EMS buffer solution with pH6.5 into the precipitate, performing ultrasonic treatment for 1-3 min,
(4) adding 0.1-0.3 mL of 10% BSA, mixing uniformly at 18-28 ℃ for 40-80 min, performing ultrasonic treatment for 1-3 min,
(5) adding 1-200mmol/L PB buffer solution with pH of 5.5-9.5 to 5mL, sealing, and storing at 2-8 deg.C.
4. The kit of claim 1, wherein the calibrator comprises: ST2 antigen at a concentration of 0.0ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, and buffers and protectants,
preferably, the buffer is selected from PB buffer, EMS buffer or Tris buffer,
preferably, the protective agent is selected from bovine serum albumin, trehalose.
5. The kit of claim 1, further comprising a quality control.
6. The kit of claim 5, wherein the quality control product comprises: antigen at concentrations of 33ng/mL and 90ng/mL and buffers and protective agents,
preferably, the buffer is selected from PB buffer, EMS buffer or Tris buffer,
preferably, the protective agent is selected from bovine serum albumin, trehalose.
7. The kit according to claim 1, wherein the ST2 antibody is a monoclonal antibody.
8. A kit for detecting soluble growth-stimulating expressed gene 2 protein (ST2) in blood, the kit comprising:
a reagent R1 comprising: 1-100mMol/L PB buffer solution, pH 6-9, 3-50g/L NaCl, 0.5-4mL/L Tween20, 1-50g/L PEG6000, 0.5-20mL/L ProClin 300;
a reagent R2 comprising: 0.05-0.5% w/v of 60-300nm polystyrene microspheres with carboxyl, 0.1mol/L PB buffer solution pH5.5-9.5, 0.1-10g/L surfactant and 1-50g/L BSA;
a calibrator, comprising: 1-100mMol/L PB buffer solution, pH 6-8, 10-100g/L trehalose, 1-100g/L BSA, 0.5-20g/L Proclin-300;
optionally, a quality control comprising: 1-100mMol/L PB buffer solution, pH 6-8, 10-100g/L trehalose, 1-100g/L BSA, 0.5-20g/L Proclin-300.
9. Method for the detection of soluble growth-stimulating expressed gene 2 protein (ST2) using a kit according to any one of claims 1 to 8, characterized in that it comprises the following steps:
(1) respectively adding the sample and the calibrator into the first reagent, uniformly mixing, and incubating at the constant temperature of 37 ℃ for 3-8 minutes;
(2) adding the second reagent into the mixture of the sample, the calibrator and the first reagent respectively,
(3) measuring absorbance of the mixed solution immediately after mixing as A1 and absorbance of the mixed solution 2-7 minutes after mixing as A2, respectively, and calculating an absorbance difference Delta A of the concentration of the calibrator as calibrator A2-calibrator A1;
(4) obtaining a calibration curve according to the concentration of the calibrator and the absorbance difference delta A corresponding to each concentration,
(5) measuring absorbance of the sample by the same method as the calibrator to obtain the absorbance difference of the sample, obtaining the corresponding concentration of the sample on the calibration curve according to the absorbance of the sample,
optionally, the method further comprises the step of obtaining the absorbance difference of the quality control product according to the same steps as the sample, and performing quality control.
10. The method of claim 9, wherein the parameters measured in step (3) are: temperature 37 ℃, wavelength 400-: 50-600. mu.l, reagent R2:10-200 μ l, and optionally, 2-20 μ l.
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