CN112014555A - Enzyme-labeled antibody buffer solution, reagent R1 and creatine kinase isoenzyme determination kit - Google Patents
Enzyme-labeled antibody buffer solution, reagent R1 and creatine kinase isoenzyme determination kit Download PDFInfo
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Abstract
The invention provides an enzyme-labeled antibody buffer solution, a reagent R1 and a creatine kinase isozyme determination kit, wherein the enzyme-labeled antibody buffer solution comprises the following components in parts by weight: 50 mM-150 mM buffer solution, 5 g/L-10 g/L electrolyte, 0.1% -1% preservative, 0.1% -2% surfactant, 2 mM-20 mM inorganic salt, 0.3 g/L-1 g/L stabilizer, 0.1% -3% blocking agent, 0.1% -1% protective agent, and the pH value is 6.4-6.6; wherein the surfactant is one or two of Tetronic701 and polyoxyethylene oleyl ether. The reagent R1 is a working solution obtained by diluting an anti-CK-MB antibody-alkaline phosphatase marker by adopting the enzyme-labeled antibody buffer solution. The invention can be stable for 12 months at 2-8 ℃ and has good biological activity; has better resistance effect on nonspecific adsorption.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and relates to an enzyme-labeled antibody buffer solution, a reagent R1 and a creatine kinase isoenzyme determination kit.
Background
Acute Myocardial Infarction (AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of coronary artery, and can be accompanied by arrhythmia, shock or heart failure, which is often life-threatening. The disease is most common in europe and the united states, and about 150 million people suffer myocardial infarction in the united states each year. China has a remarkable rising trend in recent years, newly issues at least 50 ten thousand every year, and finds out at least 200 ten thousand.
Creatine Kinase (CK), also known as Creatine phosphokinase. CK is a dimer consisting of two subunits, M and B, and has three isozymes, namely CKMM, CKMB and CKBB, and the proportions of the isozymes in different muscles are different. CK-MB is mainly distributed in myocardial cells, and rises 3-4 hours after myocardial injury, reaches the highest point in 24 hours, and can recover within 3 days. CK-MB is one of the most extensive myocardial injury markers at present, CK-MB also has higher specificity to judge myocardial necrosis, because the detection window period is relatively short, CK-MB determination is also suitable for diagnosing recurrent myocardial infarction, if CK-MB increase is detected, myocardial necrosis is indicated, the CK-MB is an important prognostic index in ACS patient evaluation, especially NSTE-ACS patients, CK-MB concentration and 30-day mortality are in positive correlation after admission, 30-day mortality of CK-MB concentration lower than the upper limit of the reference range is 1.8%, 3.3% of CK-MB concentration 1-2 times of the upper limit of the reference range, 8.3% of CK-MB concentration higher than the upper limit of the reference range is 10 times of the upper limit of the reference range, and CK-MB plays a very important role in myocardial diseases, especially in the diagnosis and treatment process of myocardial infarction, even irreplaceable role.
In vitro diagnostic reagents, the stability and the anti-interference capability of the reagent are essential, for example, in CN103278623A, the reagent can be kept unchanged in activity after being placed at 37 ℃ for 7 days, and the anti-interference capability of the reagent on the specific cross reaction of CK-MM and CK-BB is better, and the performance of the reagent is superior. However, in the current clinical background, the anti-interference capability required by the kit should be stronger, for example, Rheumatoid Factors (RF) and human anti-mouse antibodies (HAMAs) existing in a patient body can influence a detection result, and the stability of a single condition at 37 ℃ cannot be suitable for daily clinical diagnosis.
It follows that there is a need for current kits that are more specific and have long-term effective activity under clinically valuable reagent storage conditions.
Therefore, how to prepare an enzyme-labeled antibody buffer solution, a reagent R1 and a creatine kinase isoenzyme assay kit which have good stability and can resist nonspecific adsorption becomes a technical problem to be solved urgently.
Disclosure of Invention
In order to solve the technical problems, the invention provides an enzyme-labeled antibody buffer solution, a reagent R1 and a creatine kinase isoenzyme determination kit, which has good stability: can be stable for 12 months at the temperature of 2-8 ℃ and has good biological activity; in the aspect of anti-interference, the non-ionic surfactant has a better resistance effect on non-specific adsorption.
The invention is realized by the following technical scheme:
one of the purposes of the invention is to provide an enzyme-labeled antibody buffer solution, which comprises the following components in percentage by weight:
50 mM-150 mM buffer solution, 5 g/L-10 g/L electrolyte, 0.1% -1% preservative, 0.1% -2% surfactant, 2 mM-20 mM inorganic salt, 0.3 g/L-1 g/L stabilizer, 0.1% -3% blocking agent, 0.1% -1% protective agent, and the pH value is 6.4-6.6;
wherein the surfactant is one or two of Tetronic701 and polyoxyethylene oleyl ether.
Further, the mass ratio of the Tetronic701 to the polyoxyethylene oleyl ether is (0.8-1.2): (0.8 to 1.2).
Further, the buffer is selected from: one of Tris, HEPES, MES and MOPSO buffer solution; the preservative is selected from: sodium azide and ProClin-300.
Further, the protective agent is one of bovine serum albumin, PEG-1000 and glycine; the inorganic salt is MgCl2Or/and ZnCl2。
Further, the stabilizer is one of 4-aminoantipyrine; the blocking agent is mouse IgG.
The second object of the present invention is to provide a reagent R1 for creatine kinase isoenzyme assay, wherein the reagent R1 is a working solution obtained by diluting an anti-CK-MB antibody-alkaline phosphatase marker with the enzyme-labeled antibody buffer solution.
Further, the anti-CK-MB antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a CK-MB antibody and glutaraldehyde, and specifically comprises the following steps:
dissolving a CK-MB antibody into a PBS (phosphate buffer solution) to obtain a CK-MB antibody solution with the concentration of 0.5-2 mg/ml;
adding alkaline phosphatase into the CK-MB antibody solution, uniformly mixing, adding glutaraldehyde, reacting completely, dialyzing to remove unreacted glutaraldehyde, and obtaining an anti-CK-MB antibody-alkaline phosphatase marker; the mass ratio of the alkaline phosphatase to the anti-CK-MB antibody is (8-12):1, the final concentration of the glutaraldehyde is 0.1-0.4%.
The invention also aims to provide a creatine kinase isoenzyme determination kit, which comprises: reagent M and said reagent R1.
Further, the reagent M is a working solution obtained by dissolving the magnetic particles coated with the anti-CK-MB antibody in a magnetic particle buffer solution; the preparation method of the anti-CK-MB antibody-coated magnetic particle comprises the following steps:
washing and re-suspending the magnetic bead particles to obtain a magnetic particle solution;
activating the magnetic particle solution by using an activation solution to obtain an activated magnetic particle solution;
and coating the CK-MB antibody by the activated magnetic particle solution to obtain the anti-CK-MB antibody-coated magnetic particles.
The fourth purpose of the invention is to provide a preparation method of the creatine kinase isoenzyme determination kit, which is prepared by adopting the formula.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
according to the enzyme-labeled antibody buffer solution, the reagent R1 and the creatine kinase isoenzyme determination kit provided by the invention, the formula of the reagent R1 is improved by improving the enzyme-labeled antibody buffer solution in the chemiluminescence kit, so that the stability and the non-specific interference resistance are improved simultaneously; in the enzyme-labeled antibody buffer solution, the applicant finds through experiments that: the polymer of 1, 2-ethylenedioxy tetrapropanol and ethylene oxide and methyl propylene oxide (Tetronic 701) and polyoxyethylene (20) oleyl ether are used as two nonionic surfactants, and have special interaction with protease, and the interaction force can improve the stability and the nonspecific adsorption resistance of the enzyme under proper conditions (namely the formula composition and the pH condition of the invention). The creatine kinase isoenzyme determination kit prepared finally has good stability: can be stable for 12 months at the temperature of 2-8 ℃ and has good biological activity; in the aspect of anti-interference, the non-ionic surfactant has a better resistance effect on non-specific adsorption.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a graph showing correlation between the values measured by the kit of example 1 of the present invention and the concentration values measured by the Roche kit.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
according to one exemplary embodiment of the present invention, an enzyme-labeled antibody buffer is provided, and the enzyme-labeled antibody buffer has a formula of:
50 mM-150 mM buffer solution, 5 g/L-10 g/L electrolyte, 0.1% -1% preservative, 0.1% -2% surfactant, 2 mM-20 mM inorganic salt, 0.3 g/L-1 g/L stabilizer, 0.1% -3% blocking agent, 0.1% -1% protective agent, and the pH value is 6.4-6.6;
wherein the surfactant is one or two of Tetronic701 and polyoxyethylene oleyl ether.
According to the invention, by improving the enzyme-labeled antibody buffer solution in the chemiluminescence kit, the formula of the reagent R1 is further improved, so that the stability and the non-specific interference resistance are simultaneously improved; in the enzyme-labeled antibody buffer solution, the applicant finds through experiments that: the polymer of 1, 2-ethylenedioxy tetrapropanol and ethylene oxide and methyl propylene oxide (Tetronic 701) and polyoxyethylene (20) oleyl ether are used as two nonionic surfactants, and have special interaction with protease, and the interaction force can improve the stability and the nonspecific adsorption resistance of the enzyme under proper conditions (namely the formula composition and the pH condition of the invention). The creatine kinase isoenzyme determination kit prepared finally has good stability: can be stable for 12 months at the temperature of 2-8 ℃ and has good biological activity; in the aspect of anti-interference, the non-ionic surfactant has a better resistance effect on non-specific adsorption.
Nonionic surfactants, and zwitterionic surfactants, bind proteins in solution with relatively weak forces, such as hydrophobic forces, hydrogen bonding, and the like, and such interactions generally do not cause a strong change in protein conformation. Furthermore, the interaction of the surfactant with the protein is not only related to the type, but also to the concentration. The same surfactant will interact with the protein differently at different concentrations. The stability of the protease can be improved by selecting a proper surfactant and a proper concentration. The HLB value of the Tetronic701 is 1-7, the Tetronic701 and water are completely mutually soluble, and the Tetronic701 is a nonionic surfactant without physical and chemical dangers; polyoxyethylene (20) oleyl ether is a hydrophilic nonionic surfactant with an HLB value of 15 and is soluble in water.
In the present embodiment, the first and second electrodes are,
the mass ratio of the Tetronic701 to the polyoxyethylene oleyl ether is (0.8-1.2): (0.8 to 1.2).
The buffer is selected from: one of Tris, HEPES, MES and MOPSO buffer solution; the preservative is selected from: sodium azide and ProClin-300.
The protective agent is one of bovine serum albumin, PEG-1000 and glycine; the inorganic salt is MgCl2Or/and ZnCl2。
The stabilizer is one of 4-aminoantipyrine; the blocking agent is mouse IgG. In general, HAMA present in a patient or in a pharmaceutical composition for animal-derived assays interferes with many immunological assays, resulting in false negative (positive) results. The mouse IgG can effectively shield the interference of HAMA on sample test, thereby improving the accuracy of sample test.
In a preferred embodiment, the mass ratio of Tetronic701 to polyoxyethylene oleyl ether is (0.8 to 1.2): (0.8 to 1.2). The applicant finds that the two are simultaneously mixed in a mass ratio of (0.8-1.2): (0.8-1.2), the effect is better than that of only 1 of the above-mentioned materials. The quality ratio is too high or too low, which is not favorable for improving the stability.
According to another exemplary embodiment of the present invention, there is provided a reagent R1 for creatine kinase isozyme assay, wherein the reagent R1 is a working solution obtained by diluting an anti-CK-MB antibody-alkaline phosphatase label with the enzyme-labeled antibody buffer solution.
The anti-CK-MB antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a CK-MB antibody and glutaraldehyde, and specifically comprises the following steps:
step S11, dissolving the CK-MB antibody into a PBS solution to obtain a CK-MB antibody solution with the concentration of 0.5-2 mg/ml;
step S12, adding alkaline phosphatase into the CK-MB antibody solution, uniformly mixing, adding glutaraldehyde, dialyzing after complete reaction to remove unreacted glutaraldehyde, and obtaining an anti-CK-MB antibody-alkaline phosphatase marker; the mass ratio of the alkaline phosphatase to the anti-CK-MB antibody is (8-12):1, the final concentration of the glutaraldehyde is 0.1-0.4%.
The mass ratio of the alkaline phosphatase to the anti-CK-MB antibody is (8-12):1, when the mass ratio is increased and the dosage of the alkaline phosphatase is excessive, the background value of the system is increased, and the photon value of the high-value sample exceeds the upper limit of the measured value of the instrument, so that the testing precision of the high-value sample is influenced; when the mass ratio is reduced and the antibodies are too high, unlabeled free antibody competes with alkaline phosphatase-anti-CK-MB antibody complex for antigenic sites, reducing the sensitivity of the low-end sample.
The final concentration of glutaraldehyde is 0.1-0.4%. Too high a concentration of glutaraldehyde may increase non-specific reactions, while too low a concentration may affect sensitivity.
The CK-MB antibody (creatine kinase isoenzyme antibody) used in this example was derived from mouse, sheep or rabbit. The creatine kinase isoenzyme antibody is a monoclonal antibody.
According to another exemplary embodiment of the present invention, there is provided a creatine kinase isoenzyme assay kit, including: reagent M and said reagent R1.
In a preferred embodiment, the reagent M is a working solution obtained by dissolving magnetic particles coated with anti-CK-MB antibodies in a magnetic particle buffer solution; the preparation method of the anti-CK-MB antibody-coated magnetic particle comprises the following steps:
step S21, washing and resuspending the magnetic bead particles to obtain a magnetic particle solution;
step S22, activating the magnetic particle solution by using an activation solution to obtain an activated magnetic particle solution;
and step S23, coating the CK-MB antibody with the activated magnetic particle solution to obtain the anti-CK-MB antibody-coated magnetic particles.
Wherein, in the step S21, obtaining the magnetic particle solution includes: and taking magnetic bead particles, washing the magnetic bead particles for multiple times by using a buffer solution, and then re-suspending the magnetic bead particles by using the buffer solution to obtain a magnetic particle solution with the concentration of 1-4mg/ml, wherein the buffer solution is one or more of a phosphate buffer solution, a HEPES buffer solution, a carbonate buffer solution, a MES buffer solution and a boric acid buffer solution.
In the step S22, EDC is used as the activating solution, and the mass ratio of EDC to magnetic particles is 1 (90-110), preferably 1: 100; when the mass ratio is increased and the use amount of EDC is excessive, the activation rate of the magnetic particles is increased, and the concentration of the activated magnetic particles is increased to easily cause the coagulation of the magnetic particles; when the mass ratio is decreased and the number of magnetic particles is too large, the activation rate of the magnetic particles is decreased, resulting in a decrease in the antibody coating rate.
In the step S23, the mass ratio of the CK-MB antibody to the magnetic particles is 1 (480-520), preferably 1: 500. When the mass ratio is increased and the amount of the antibody is excessive, the antibody not coated on the magnetic particles forms free antibody, so that the long-term stability of the M reagent is easily reduced; when the mass ratio is decreased and the number of magnetic particles is too large, nonspecific adsorption between the magnetic particles is likely to occur, which in turn increases the background value of the test system.
The method for coating the CK-MB antibody by the activated magnetic particle solution specifically comprises the following steps: and adding the CK-MB antibody into the activated magnetic particle solution, uniformly mixing, adding a sealing liquid for sealing, uniformly mixing, performing magnetic separation to remove supernatant liquid to obtain magnetic particles coated with the anti-CK-MB antibody, adding a magnetic particle buffer solution, and uniformly mixing to obtain the magnetic particle reagent M coated with the anti-CK-MB antibody.
The invention provides a detection principle of a creatine kinase isoenzyme determination kit, which comprises the following steps:
the creatine kinase isoenzyme determination kit (magnetic particle chemiluminescence immunoassay) adopts a double-antibody sandwich method. For the measurement, a reagent M (magnetic particle working solution coated with anti-CK-MB antibody) and a reagent R1 (alkaline phosphatase-labeled anti-CK-MB antibody) were mixed with the sample. CK-MB in the sample is combined with the anti-CK-MB antibody to form a magnetic particle immune complex of the anti-CK-MB antibody-CK-MB-anti-CK-MB antibody enzyme marker. After washing to remove free enzyme-labeled antibody, a chemiluminescent substrate is added to the immunocomplex. The luminous signal generated by enzyme reaction is detected by a full-automatic chemiluminescence immunoassay analyzer, the detected luminous intensity is related to the concentration of CK-MB in the sample, and the concentration value of CK-MB in the sample can be calculated by the full-automatic chemiluminescence immunoassay analyzer.
The creatine kinase isoenzyme assay kit of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
This example provides a kit for quantitative measurement of CK-MB, which comprises a calibrator, a quality control, an anti-CK-MB antibody-alkaline phosphatase marker reagent R1, and a magnetic microparticle reagent M coated with an anti-CK-MB antibody.
1. The preparation method of the CK-MB calibrator and the CK-MB quality control product comprises the following steps:
and dissolving the CK-MB antigen by using a calibrator buffer solution to prepare a CK-MB calibrator and a quality control product, wherein the calibrator comprises CK-MB with known concentration, and the concentration of the CK-MB is in the range of 0-200 pmol/L. In a specific embodiment, the concentrations of CK-MB in the calibrator and the quality control are respectively 0, 5, 10, 50, 100 and 200pmol/L and 5pmol/L and 100 pmol/L; wherein the calibrator buffer is prepared by adding 0.5-3% sorbitol, 0.5-3% trehalose, 0.5-1% bovine serum albumin, 150-300mmol/L sodium chloride and 0.03-0.1% ProClin into 4- (2-hydroxyethyl) piperazine-1-2-hydroxypropanesulfonic acid (HEPPSO) buffer TM300, the pH value is 6.0-8.0, and the product is prepared by stirring and dissolving and then treating with a 0.22 mu m filter membrane;
2. anti-CK-MB antibody-alkaline phosphatase marker reagent R1
The CK-MB antibody-alkaline phosphatase marker was dissolved in a buffer solution labeled with an enzyme.
The CK-MB antibody-alkaline phosphatase marker was prepared by dissolving 10mg of alkaline phosphatase in 1ml of PBS (0.01mol/L pH7.0) containing 1mg of anti-CK-MB antibody, adding 4ml of 1% glutaraldehyde solution to a final concentration of 0.2% after complete dissolution, dialyzing to remove excess glutaraldehyde, and storing at 4 ℃.
The enzyme-labeled antibody buffer solution comprises the following components in percentage by weight:
weighing 12.114g Tris, 5g NaCl, 1g PC-300, 0.1% tetronic701, 0.019g MgCl2、0.02706gZnCl210g BSA, 10g Glycine, 50g PEG-1000, 10g mouse IgG dissolved in 800mL ultrapure water, pH adjusted to 6.5, constant volume to 1L.
3. Magnetic particle reagent M coated with anti-CK-MB antibody
Dissolving the anti-CK-MB antibody-coated magnetic particles in a buffer solution of the antibody-coated magnetic particles; the preparation method of the anti-CK-MB antibody-coated magnetic particle comprises the following steps:
cleaning: taking a proper amount of magnetic bead particles, washing the magnetic bead particles for 3 times by using 0.1mol/L MES, and then re-suspending the magnetic bead particles by using 0.1mol/L MES to ensure that the concentration of the magnetic bead particles is 2 mg/ml;
activation: taking the prepared EDC solution, adding the EDC solution into the resuspended magnetic particles according to the mass ratio of EDC to the magnetic particles of 1:100, and horizontally mixing for 20min at room temperature after uniformly mixing in a vortex manner;
coating: according to the coating proportion of the CK-MB antibody to the magnetic particles in a mass ratio of 1:500, adding the CK-MB antibody to be coated into the activated magnetic particles, uniformly mixing in a vortex mode, and horizontally uniformly mixing for 12 hours at room temperature;
sealing: adding 5% BSA solution into the magnetic particles, and horizontally and uniformly mixing for 2h at room temperature;
preservation: removing supernatant by magnetic separation, adding into magnetic bead preservation solution, and mixing.
Example 2
In this example R1 reagent "0.1% tetronic 701" was replaced by "0.2% polyoxyethylene (20) oleyl ether", the other components and procedures were the same as in example 1.
Example 3
In the R1 reagent in this example, "0.1% tetronic 701" was replaced with "0.1% total mass fraction, 1:1 tetronic701 and polyoxyethylene (20) oleyl ether ", the other processes and components were the same as in example 1.
Example 4
In the reagent R1 in this example, "0.1% tetronic 701" was replaced with "0.1% total mass fraction, 0.8: 1.2 tetronic701 and polyoxyethylene (20) oleyl ether ", the other processes and components being the same as in example 1.
Example 5
In the reagent R1 in this example, "0.1% tetronic 701" was replaced with "total mass fraction of 0.1%, mass ratio of 1.2: 0.8 tetronic701 and polyoxyethylene (20) oleyl ether ", the other processes and components were the same as in example 1.
Example 6
In the reagent R1 in this example, "0.1% tetronic 701" was replaced with "0.1% total mass fraction, 0.5 mass ratio: 1.2 tetronic701 and polyoxyethylene (20) oleyl ether ", the other processes and components being the same as in example 1.
Example 7
In the reagent R1 in this example, "0.1% tetronic 701" was replaced with "total mass fraction of 0.1%, mass ratio of 1.2: 0.5 tetronic701 and polyoxyethylene (20) oleyl ether ", the other processes and components were the same as in example 1.
Comparative example 1
The enzyme-labeled antibody buffer in the R1 reagent in this comparative example was: 12.114g of Tris, 5g of NaCl, 1g of PC-300, 1g T-20, 0.019g of MgCl2, 0.02706g of ZnCl2, 10g of BSA, 10g of Glycine, 50g of PEG-1000 and 10g of mouse IgG are weighed and dissolved in 800mL of ultrapure water, the pH value is adjusted to 6.5, and the volume is adjusted to 1L.
Comparative example 2
On the basis of comparative example 1, T-20 was removed and the other components and processes were the same as in comparative example 1.
Comparative example 3
Reagent R1 was prepared according to the method of CN 103278623A. The M reagent is a magnetic bead working solution described in CN 103278623A.
Test example 1
1. And (4) according to the operation of the kit specification, measuring a serum sample with a certain concentration, and recording the concentration of the sample. Then mixing the serum sample with anti-interference substances with different concentrations according to a ratio of 9:1, respectively measuring and recording the concentrations of the mixed samples; evaluation of interference degree of less than 10% when the concentration of the interfering substance is 1.0g/L bilirubin, the concentration of the interfering substance is 30g/L triglyceride, and the concentration of the interfering substance is 10.0g/L hemoglobin.
TABLE 1 evaluation of interference rejection
As can be seen from the data in Table 1, the kits with better anti-interference performance are example 1, example 2 and comparative example 3; among them, Tetronic701 and polyoxyethylene (20) oleyl ether have a good effect of inhibiting nonspecific adsorption of proteins, and thus examples 1 and 2 have a good anti-interference effect. In examples 3 to 5, the mass ratio of Tetronic701 to polyoxyethylene oleyl ether is (0.8-1.2): (0.8-1.2) and the anti-interference capability is stronger than that of the embodiment 1-2, and the mass ratio of the embodiment 3 is 1:1, the anti-interference capability is strongest. However, in examples 6 to 7, the mass ratio was not (0.8 to 1.2): (0.8-1.2), poor stability.
2. Evaluation of stability
The prepared kits are placed at 37 ℃ and 2-8 ℃ for different time and then the same serum quality control product is tested, the test value of the 0 th month is taken as a reference, the ratio of the test value after the 3 rd, 6 th, 9 th and 12 th months to the measured value of the 0 th month is checked, and the ratio of the measured value of the same serum quality control product is within the range of 85-115%. The results are shown in Table 2:
TABLE 2 stability evaluation data (2-8 ℃ C.)
As is clear from the data in Table 2, the stability of examples 1,2 and 3 was good at 2 to 8 ℃ and the storage stability was stable at 2 to 8 ℃ for 12 months.
3. Surfactant concentration screening
Six kits, including 0.1%, 1%, 2% Tetronic701 and 0.1%, 1%, 2% polyoxyethylene (20) oleyl ether, were prepared according to the methods for preparing kits 2 and 3, respectively, and the influence of the apparent concentration on the stability of reagent R1 was analyzed. By taking the test value of the 0 th month as a reference, the ratio of the test value after the 3 rd, 6 th, 9 th and 12 th months to the measured value of the 0 th month is checked, and the ratio of the measured value to the measured value of the same serum sample is in the range of 85-115%. The results are shown in Table 3:
TABLE 3 surfactant concentration screening
As can be seen from the data in table 3, the concentrations of the two surfactants were optimized, and 0.1% to 1% Tetronic701 was preferable, which is good in stability. The stability of 0.1% -1% polyoxyethylene (20) oleyl ether is also good.
4. Linear detection
The high value samples near the upper end of the linear range are diluted to at least 5 concentrations in a certain proportion, wherein the low value concentration samples must be near the lower end of the linear range. According to the kit operation instruction, carrying out three times of repeated tests on samples with each concentration, calculating an average value, carrying out straight line fitting on the result average value and the dilution ratio according to a least square method, and calculating a linear correlation coefficient r, wherein the result is in accordance with the linear range of 0.3 ng/mL-300.0 ng/mL, and the linear correlation coefficient r is more than or equal to 0.9900. The results are shown in Table 4:
TABLE 4 Linear data
From the data in table 4, it can be seen that: the kit provided by the invention has the advantages that the linear correlation coefficient r is greater than or equal to 0.9900 when the kit is used for detecting, and the linear detection is qualified;
5. correlation with Roche and measured value bias
Preparing a kit according to the preparation method of the kit 2, testing 100 samples by taking a Roche kit as a comparative example, and respectively checking the measured value deviation of the kit 2 and the Roche kit, wherein the relative deviation of the measured value of the kit 2 is more than or equal to 4.5ng/mL is less than 15%, the measured value of the kit is less than 4.5ng/mL, and the absolute deviation of the kit is less than or equal to 0.5; the linear correlation should be measured to be > 0.975. The results are shown in Table 5:
TABLE 5 Yin Yang coincidence rates compared to Roche reagents
From the data in table 5, it can be seen that: the deviation qualification rate of the measured values of the kit 1 and the Roche kit in comparison is 96%, 4 samples with larger deviation appear in 100 samples, and the overall linear correlation of the kit 1 and the Roche kit is 0.9923.
In conclusion, in order to obtain the creatine kinase isoenzyme detection reagent R1 with good performance and solve the problem of long-term stability and anti-interference performance of the reagent at 2-8 ℃ which is relatively deficient in the current reagent development, the diluent of the R1 reagent is optimized. Under the action of Tetronic701 and polyoxyethylene (20) oleyl ether, the stability of the reagent R1 is obviously improved, the reagent R1 can be stable for 7 days at 37 ℃ compared with CN103278623A, and after the applicable concentration of the surfactant is optimized, the reagent R1 can be stable for 12 months at 2-8 ℃ and has good bioactivity when 0.5% of Tetronic701 is used as an additive. In the aspect of anti-interference, the non-ionic surfactant has a better resistance effect on non-specific adsorption.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. An enzyme-labeled antibody buffer solution is characterized in that the formula of the enzyme-labeled antibody buffer solution is as follows:
50 mM-150 mM buffer solution, 5 g/L-10 g/L electrolyte, 0.1% -1% preservative, 0.1% -2% surfactant, 2 mM-20 mM inorganic salt, 0.3 g/L-1 g/L stabilizer, 0.1% -3% blocking agent, 0.1% -1% protective agent, and the pH value is 6.4-6.6;
wherein the surfactant is one or two of Tetronic701 and polyoxyethylene oleyl ether.
2. The enzyme-labeled antibody buffer solution according to claim 1, wherein the mass ratio of Tetronic701 to polyoxyethylene oleyl ether is (0.8-1.2): (0.8 to 1.2).
3. An enzyme-labeled antibody buffer according to claim 1, wherein the buffer is selected from the group consisting of: one of Tris, HEPES, MES and MOPSO buffer solution; the preservative is selected from: sodium azide and ProClin-300.
4. The enzyme-labeled antibody buffer solution of claim 1, wherein the protective agent is one of bovine serum albumin, PEG-1000, and glycine; the inorganic salt is MgCl2Or/and ZnCl2。
5. The enzyme-labeled antibody buffer solution of claim 1, wherein the stabilizer is one of 4-aminoantipyrine; the blocking agent is mouse IgG.
6. A reagent R1 for creatine kinase isozyme assay, characterized in that the reagent R1 is a working solution obtained by diluting an anti-CK-MB antibody-alkaline phosphatase marker with the enzyme-labeled antibody buffer solution according to any one of claims 1 to 6.
7. The kit for measuring the creatine kinase isoenzyme according to claim 6, wherein the anti-CK-MB antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, a CK-MB antibody and glutaraldehyde, and specifically comprises:
dissolving a CK-MB antibody into a PBS (phosphate buffer solution) to obtain a CK-MB antibody solution with the concentration of 0.5-2 mg/ml;
adding alkaline phosphatase into the CK-MB antibody solution, uniformly mixing, adding glutaraldehyde, reacting completely, dialyzing to remove unreacted glutaraldehyde, and obtaining an anti-CK-MB antibody-alkaline phosphatase marker; the mass ratio of the alkaline phosphatase to the anti-CK-MB antibody is (8-12):1, the final concentration of the glutaraldehyde is 0.1-0.4%.
8. A creatine kinase isoenzyme assay kit, comprising: reagent M and reagent R1 according to any one of claims 6 to 7.
9. The creatine kinase isoenzyme assay kit according to claim 8, wherein the reagent M is a working solution obtained by dissolving magnetic microparticles coated with an anti-CK-MB antibody in a magnetic microparticle buffer; the preparation method of the anti-CK-MB antibody-coated magnetic particle comprises the following steps:
washing and re-suspending the magnetic bead particles to obtain a magnetic particle solution;
activating the magnetic particle solution by using an activation solution to obtain an activated magnetic particle solution;
and coating the CK-MB antibody by the activated magnetic particle solution to obtain the anti-CK-MB antibody-coated magnetic particles.
10. A method for preparing a creatine kinase isoenzyme assay kit, characterized in that it is prepared by using the formulation according to any one of claims 8 to 9.
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