CN112014555B - Enzyme-labeled antibody buffer solution, reagent R1 and creatine kinase isozyme determination kit - Google Patents

Enzyme-labeled antibody buffer solution, reagent R1 and creatine kinase isozyme determination kit Download PDF

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CN112014555B
CN112014555B CN202010893094.5A CN202010893094A CN112014555B CN 112014555 B CN112014555 B CN 112014555B CN 202010893094 A CN202010893094 A CN 202010893094A CN 112014555 B CN112014555 B CN 112014555B
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antibody
reagent
solution
alkaline phosphatase
creatine kinase
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CN112014555A (en
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刘磊
来祥兵
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Wuhan Life Origin Biotech Joint Stock Co ltd
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Abstract

The invention provides an enzyme-labeled antibody buffer solution, a reagent R1 and a creatine kinase isoenzyme determination kit, wherein the formulation 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 pH value of 6.4-6.6; wherein the surfactant is one or two of Tetronic701 and polyoxyethylene oil ether. The reagent R1 is a working solution obtained by diluting the anti-CK-MB antibody-alkaline phosphatase marker by using 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

Enzyme-labeled antibody buffer solution, reagent R1 and creatine kinase isozyme determination kit
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 isozyme determination kit.
Background
Acute myocardial infarction (acute myocardial infarction, AMI) is myocardial necrosis caused by acute and persistent ischemia and hypoxia of the coronary arteries, and can be complicated with arrhythmia, shock or heart failure, often endangering life. The disease is most common in europe and america, and about 150 tens of thousands of myocardial infarction occur annually in the united states. In recent years, china has a remarkable rising trend, and at least 50 ten thousand new people are found each year, and at least 200 ten thousand new people are found each year.
Creatine Kinase (CK), also known as Creatine phosphokinase. CK is a dimer composed of two subunits, M and B, with three isozymes, CKMM, CKMB, CKBB, in different intramuscular isozymes in different proportions. CK-MB is mainly distributed in myocardial cells, rises 3-4 hours after myocardial injury, reaches the highest point in 24 hours, and can be recovered to be normal within 3 days. CK-MB is one of the most widely used myocardial injury markers nowadays, and has higher specificity for judging myocardial necrosis, and because the detection window period is relatively short, the CK-MB measurement is also suitable for diagnosing recurrent myocardial infarction, if the rising of CK-MB is detected, the CK-MB is predicted to be myocardial necrosis, and is an important prognosis index in ACS patient assessment, especially NSTE-ACS patients, the concentration of CK-MB is positively correlated with the death rate of 30 days after admission, the death rate of CK-MB is 1.8% when the concentration of CK-MB is lower than the upper limit of the reference range by 1-2 times, the death rate of CK-MB is 3.3% when the concentration of CK-MB is higher than the upper limit of the reference range by 10 times, and the concentration of CK-MB is 8.3% when the concentration of CK-MB is higher than the upper limit of the reference range, so that the CK-MB has important or even irreplaceable effects in the diagnosis and treatment of myocardial infarction.
In vitro diagnostic reagents, the stability and anti-interference capability of the reagent are essential, as mentioned in CN103278623A, the reagent can still keep activity unchanged after being placed for 7 days at 37 ℃, and has better anti-interference capability for the specific cross reaction of CK-MM and CK-BB, and the reagent performance is superior. However, in the current clinical setting, the anti-interference capability required by the kit should be stronger, for example, both Rheumatoid Factor (RF) and human anti-mouse antibodies (HAMAs) existing in the patient body can affect the detection result, and the single 37 ℃ condition stability cannot be suitable for daily clinical diagnosis.
As can be seen, the current kits require higher specificity and long-term effective activity under the conditions of storage of reagents of clinical analytical value.
Therefore, how to prepare an enzyme-labeled antibody buffer solution with good stability and capability of resisting non-specific adsorption, a reagent R1 and a creatine kinase isoenzyme assay kit 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 assay kit, which have good stability: can be stable for 12 months at 2-8 ℃ and has good biological activity; in terms of anti-interference, the nonionic surfactant has a better resistance effect on nonspecific adsorption.
The invention is realized by the following technical scheme:
the invention aims at providing 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 pH value of 6.4-6.6;
wherein the surfactant is one or two of Tetronic701 and polyoxyethylene oil ether.
Further, the mass ratio of Tetronic701 to polyoxyethylene oleyl ether is (0.8-1.2): (0.8-1.2).
Further, the buffer is selected from the group consisting of: tris, HEPES, MES, MOPSO buffer; the preservative is selected from the group consisting of: sodium azide, proClin-300.
Further, the protective agent is one of bovine serum albumin, PEG-1000 and glycine; the inorganic salt is MgCl 2 Or/and ZnCl 2 。
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 measuring creatine kinase isozyme, 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.
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 CK-MB antibody in PBS solution to obtain CK-MB antibody solution with concentration of 0.5-2 mg/ml;
adding alkaline phosphatase into the CK-MB antibody solution, uniformly mixing, adding glutaraldehyde, and dialyzing to remove unreacted glutaraldehyde after the reaction is completed to obtain 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, wherein the final concentration of glutaraldehyde is 0.1-0.4%.
It is still another object of the present invention to provide a creatine kinase isozyme assay kit comprising: reagent M and said reagent R1.
Further, the reagent M is a working solution obtained by dissolving magnetic particles coated with the anti-CK-MB antibody in a magnetic particle buffer solution; the preparation method of the magnetic particles coated with the anti-CK-MB antibody comprises the following steps:
cleaning and resuspending the magnetic bead particles to obtain a magnetic particle solution;
activating the magnetic particle solution by using an activating solution to obtain an activated magnetic particle solution;
and coating the CK-MB antibody with the activated magnetic particle solution to obtain the magnetic particles coated with the anti-CK-MB antibody.
The invention aims at providing a preparation method of a creatine kinase isoenzyme assay kit, which is prepared by adopting the formula.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
according to the enzyme-labeled antibody buffer solution, the reagent R1 and the creatine kinase isoenzyme determination kit, the formula of the reagent R1 is improved by improving the enzyme-labeled antibody buffer solution in the chemiluminescent kit, so that the stability and the capacity of resisting non-specific interference are improved simultaneously; in the enzyme-labeled antibody buffer, the applicant found through experiments that: the polymers of 1, 2-ethylenediazo tetrapropanol with ethylene oxide and methyl propylene oxide (Tetronic 701) and polyoxyethylene (20) oleyl ether as two nonionic surfactants have special interactions with proteases, and the interaction forces can improve the stability and the non-specific adsorption resistance of enzymes under proper conditions (namely the formulation composition and the pH condition). The creatine kinase isoenzyme assay kit prepared finally has good stability: can be stable for 12 months at 2-8 ℃ and has good biological activity; in terms of anti-interference, the nonionic surfactant has a better resistance effect on nonspecific adsorption.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing a correlation analysis between the measured value of the kit of example 1 of the present invention and the measured concentration value of the Roche kit.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning 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. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The technical scheme provided by the embodiment of the invention aims to solve the technical problems, and the overall thought is as follows:
according to one exemplary embodiment of the present invention, there is provided an enzyme-labeled antibody buffer, wherein the formulation of the enzyme-labeled antibody buffer 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 pH value of 6.4-6.6;
wherein the surfactant is one or two of Tetronic701 and polyoxyethylene oil ether.
According to the invention, the enzyme-labeled antibody buffer solution in the chemiluminescent kit is improved, so that the formula of the reagent R1 is improved, and the stability and the capacity of resisting non-specific interference are improved at the same time; in the enzyme-labeled antibody buffer, the applicant found through experiments that: the polymers of 1, 2-ethylenediazo tetrapropanol with ethylene oxide and methyl propylene oxide (Tetronic 701) and polyoxyethylene (20) oleyl ether as two nonionic surfactants have special interactions with proteases, and the interaction forces can improve the stability and the non-specific adsorption resistance of enzymes under proper conditions (namely the formulation composition and the pH condition). The creatine kinase isoenzyme assay kit prepared finally has good stability: can be stable for 12 months at 2-8 ℃ and has good biological activity; in terms of anti-interference, the nonionic surfactant has a better resistance effect on nonspecific adsorption.
Nonionic surfactants, zwitterionic surfactants, and the like are neutral surfactants that bind proteins in solution with relatively weak forces, such as hydrophobic forces, hydrogen bonding, etc., and such interactions typically do not cause strong changes in protein conformation. Furthermore, the interaction of surfactants with proteins is not only type dependent, but also concentration dependent. The same surfactant interacts differently with proteins at different concentrations. The stability of the protease can be improved by selecting a suitable surfactant and an appropriate concentration. Tetronic701 has an HLB value of 1 to 7 and is completely miscible with water, and is a nonionic surfactant without physical and chemical hazards; polyoxyethylene (20) oil ether is a hydrophilic nonionic surfactant, has an HLB value of 15, and is soluble in water.
In the present embodiment of the present invention,
the mass ratio of Tetronic701 to polyoxyethylene oleyl ether is (0.8-1.2): (0.8-1.2).
The buffer is selected from the group consisting of: tris, HEPES, MES, MOPSO buffer; the preservative is selected from the group consisting of: sodium azide, proClin-300.
The protective agent is one of bovine serum albumin, PEG-1000 and glycine; the inorganic salt is MgCl 2 Or/and ZnCl 2 。
The stabilizer is one of 4-aminoantipyrine; the blocking agent is mouse IgG. In general, HAMA present in a patient or in an experimental pharmaceutical composition of animal origin at the time of detection can interfere with many immunological detection methods, leading to a false negative (positive) detection result. The mouse IgG can effectively shield interference of HAMA on a sample test, so that the accuracy of the sample test is improved.
In a preferred embodiment, the mass ratio of Tetronic701 to polyoxyethylene oleyl ether is (0.8 to 1.2): (0.8-1.2). The applicant found that the mass ratio of the two is (0.8-1.2): (0.8-1.2) the effect is better than that of adding only 1 of them. A too high or too low mass ratio is detrimental to an improvement in stability.
According to another exemplary embodiment of the present invention, there is provided a reagent R1 for measuring creatine kinase isozymes, 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.
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 components:
s11, dissolving the CK-MB antibody in PBS solution to obtain 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, and dialyzing to remove unreacted glutaraldehyde after the reaction is completed to obtain 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, wherein the final concentration of 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 alkaline phosphatase is excessively used, 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 test precision of the high-value sample is affected; when the mass ratio is reduced and the antibodies are too high, unlabeled free antibodies compete with alkaline phosphatase-anti-CK-MB antibody complexes for antigenic sites, reducing low-end sample sensitivity.
The final concentration of glutaraldehyde is 0.1-0.4%. A glutaraldehyde concentration that is too high may increase the nonspecific reaction, while a concentration that is too low may affect the sensitivity.
The CK-MB antibody (creatine kinase isozyme antibody) used in this example was derived from a mouse, sheep or rabbit. The creatine kinase isozyme antibody is a monoclonal antibody.
According to another exemplary embodiment of the present invention, there is provided a creatine kinase isozyme assay kit comprising: reagent M and said reagent R1.
As a preferred embodiment, the reagent M is a working solution obtained by dissolving magnetic particles coated with an anti-CK-MB antibody in a magnetic particle buffer; the preparation method of the magnetic particles coated with the anti-CK-MB antibody comprises the following steps:
s21, cleaning and resuspending the magnetic bead particles to obtain a magnetic particle solution;
step S22, activating the magnetic particle solution by using an activating solution to obtain an activated magnetic particle solution;
and S23, coating the CK-MB antibody on the activated magnetic particle solution to obtain the magnetic particles coated with the anti-CK-MB antibody.
Wherein, in the step S21, obtaining the magnetic particle solution includes: and (3) taking magnetic bead particles, cleaning 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, an MES buffer solution and a boric acid buffer solution.
In the step S22, EDC is adopted as the activating solution, and the mass ratio of the EDC to the magnetic particles is 1 (90-110), preferably 1:100; when the mass ratio is increased and the EDC usage amount is excessive, the activation rate of the magnetic particles is increased, and the concentration of the activated magnetic particles is increased, so that the magnetic particles are easy to agglomerate and sink; when the mass ratio is reduced and the magnetic particles are too large, the activation rate of the magnetic particles is reduced, resulting in a reduction 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 antibody amount is excessive, the antibody which is 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 reduced and the magnetic particles are too many, nonspecific adsorption easily occurs among the magnetic particles, and then the background value of the test system is increased.
Coating the activated magnetic particle solution with CK-MB antibody, which specifically comprises the following steps: adding the CK-MB antibody into the activated magnetic particle solution, uniformly mixing, adding a sealing solution for sealing, uniformly mixing, performing magnetic separation, removing supernatant to obtain magnetic particles coated with the anti-CK-MB antibody, and then adding a magnetic particle buffer solution for uniformly mixing to obtain the magnetic particle reagent M coated with the anti-CK-MB antibody.
The invention provides a creatine kinase isoenzyme assay kit, which comprises the following detection principles:
the creatine kinase isoenzyme assay kit (magnetic particle chemiluminescence immunoassay) uses a double-antibody sandwich method. In the measurement, a reagent M (magnetic particle working solution coated with an anti-CK-MB antibody) and a reagent R1 (an alkaline phosphatase-labeled anti-CK-MB antibody) were mixed with a sample. The 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 label. After washing to remove free enzyme-labeled antibodies, chemiluminescent substrate is added to the immune complex. The luminescence signal generated by the enzyme reaction is detected by the full-automatic chemiluminescence immunoassay analyzer, the detected luminescence intensity is related to the concentration of CK-MB in the sample, and the full-automatic chemiluminescence immunoassay analyzer can calculate the concentration value of CK-MB in the sample.
A creatine kinase isoenzyme assay kit of the present application will be described in detail with reference to examples, comparative examples and experimental data.
The present embodiment provides a CK-MB quantitative assay kit comprising a calibrator, a quality control, an anti-CK-MB antibody-alkaline phosphatase marker reagent R1, and a magnetic particle 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 is as follows:
the CK-MB antigen is dissolved by using a calibrator buffer, and a CK-MB calibrator and a quality control product are prepared, wherein the calibrator contains the 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 concentration of CK-MB in the calibrator is 0, 5, 10, 50, 100, 200pmol/L, respectively, and the concentration of CK-MB in the quality control is 5pmol/L, 100pmol/L, respectively; 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-hydroxy propane sulfonic acid (HEPPSO) buffer TM 300, the pH value is 6.0-8.0, and the solution is stirred and dissolved and then is treated by a filter membrane with the thickness of 0.22 mu m;
2. anti-CK-MB antibody-alkaline phosphatase marker reagent R1
The CK-MB antibody-alkaline phosphatase label was dissolved in a buffer of the enzyme-labeled antibody.
Wherein, the CK-MB antibody-alkaline phosphatase marker is prepared by dissolving 10mg alkaline phosphatase in 1ml PBS (0.01 mol/L pH 7.0) containing 1mg of anti-CK-MB antibody, adding 4ml of 1% glutaraldehyde solution after complete dissolution to make the final concentration of glutaraldehyde be 0.2%, dialyzing to remove excessive glutaraldehyde, and storing at 4 ℃.
Wherein, the formula of the enzyme-labeled antibody buffer solution is as follows:
weighing 12.114g Tris, 5g NaCl, 1g PC-300, 0.1%Tetronice 701, 0.019g MgCl 2 、0.02706gZnCl 2 10g BSA, 10g Glycine, 50g PEG-1000, 10g mouse IgG were dissolved in 800mL ultra pure water, pH was adjusted to 6.5, and the volume was set to 1L.
3. Magnetic particle reagent M coated with anti-CK-MB antibody
Dissolving the magnetic particles coated with the anti-CK-MB antibody by using a buffer solution of the magnetic particles coated with the antibody; the preparation method of the magnetic particles coated with the anti-CK-MB antibody comprises the following steps:
(1) cleaning: taking a proper amount of magnetic bead particles, cleaning the magnetic bead particles for 3 times by using 0.1mol/L MES, and re-suspending the magnetic bead particles by using 0.1mol/L MES to ensure that the concentration of the magnetic bead particles is 2mg/ml;
(2) activating: taking the EDC solution prepared at present, adding the EDC solution into the resuspended magnetic particles according to the mass ratio of EDC to the magnetic particles of 1:100, and uniformly mixing at room temperature for 20min after vortex mixing;
(3) coating: adding the CK-MB antibody to be coated into the activated magnetic particles according to the coating proportion of the CK-MB antibody to the magnetic particles in a mass ratio of 1:500, and uniformly mixing by vortex at room temperature for 12 hours;
(4) closing: adding 5% BSA solution into the magnetic particles, and horizontally and uniformly mixing for 2 hours at room temperature;
(5) and (3) preserving: removing supernatant by magnetic separation, and adding into magnetic bead preservation solution for uniform mixing.
Example 2
In this example, "0.1%Tetronice 701" was replaced with "0.2% polyoxyethylene (20) oil ether" in the R1 reagent, and the other components and processes were the same as in example 1.
Example 3
In this example, "0.1%Tetronice 701" was replaced with "0.1% by mass of the total mass fraction" in the R1 reagent, and the mass ratio was 1: tetronice 701 and polyoxyethylene (20) oil ether "of 1, the other processes and components are the same as in example 1.
Example 4
In this example, "0.1%Tetronice 701" was replaced with "0.1% by mass total of R1 reagent, and the mass ratio was 0.8: tetronice 701 and polyoxyethylene (20) oil ether ", 1.2, other processes and components are the same as in example 1.
Example 5
In this example, "0.1%Tetronice 701" was replaced with "0.1% by mass of the total mass fraction of the R1 reagent, and the mass ratio was 1.2: tetronice 701 and polyoxyethylene (20) oil ether "0.8, the other processes and components are the same as in example 1.
Example 6
In this example, "0.1%Tetronice 701" was replaced with "0.1% by mass total of R1 reagent, and the mass ratio was 0.5: tetronice 701 and polyoxyethylene (20) oil ether ", 1.2, other processes and components are the same as in example 1.
Example 7
In this example, "0.1%Tetronice 701" was replaced with "0.1% by mass of the total mass fraction of the R1 reagent, and the mass ratio was 1.2: tetronice 701 and polyoxyethylene (20) oil ether ", 0.5, the other processes and components are the same as in example 1.
Comparative example 1
The enzyme-labeled antibody buffer in the R1 reagent in this comparative example is: 12.114g Tris, 5g NaCl, 1g PC-300, 1g T-20, 0.019g MgCl2, 0.02706g ZnCl2, 10g BSA, 10g Glycine, 50g PEG-1000 and 10g mouse IgG are weighed and dissolved in 800mL ultra pure water, the pH is adjusted to 6.5, and the volume is fixed to 1L.
Comparative example 2
T-20 was removed on the basis of comparative example 1, and other components and processes were the same as those of comparative example 1.
Comparative example 3
The R1 reagent was prepared according to the method of CN 103278623A. The M reagent is the magnetic bead working solution described in CN 103278623A.
Test example 1
1. The serum sample with a certain concentration is measured according to the operation of the instruction book of the kit, and the concentration of the sample is recorded. Mixing the serum sample with anti-interference substances with different concentrations according to the proportion of 9:1, respectively measuring and recording the concentration of the mixed sample; the interference degree was less than 10% when the interference concentration was evaluated to be 1.0g/L bilirubin, the interference concentration was evaluated to be 30g/L triglyceride, and the interference concentration was evaluated to be 10.0g/L hemoglobin.
TABLE 1 evaluation of anti-interference ability
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 nonspecifically adsorbing an anti-protein, so that example 1 and example 2 have a good anti-interference effect. And in the embodiment 3-5, the mass ratio of the Tetronic701 to the polyoxyethylene oleyl ether is (0.8-1.2): (0.8-1.2) is added simultaneously, the anti-jamming capability is stronger than that of the embodiment 1-2, and the mass ratio of the embodiment 3 is 1:1 is added at the same time, and the anti-interference capability is strongest. However, the mass ratio in examples 6 to 7 was not in the range of (0.8 to 1.2): in the range of (0.8-1.2), the stability is poor.
2. Stability evaluation
And (3) testing the same serum quality control product after the prepared kits are placed for different times at 37 ℃ and 2-8 ℃, and checking the ratio of the tested value to the tested value of the 0 th month after 3, 6, 9 and 12 months by taking the tested value of the 0 th month as a reference, wherein the ratio of the tested value to the tested value of the same serum quality control product is in a 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, examples 1,2 and 3 were excellent in stability at 2 to 8℃and were stable for 12 months at 2 to 8 ℃.
3. Surfactant concentration screening
Six kits containing 0.1%, 1%,2% Tetronic701 and 0.1%, 1%,2% polyoxyethylene (20) oleyl ether were prepared according to the preparation methods of the kits 2 and 3, respectively, and the effect of the apparent concentration on the stability of the reagent R1 was analyzed. And checking the ratio of the test value after 3, 6, 9 and 12 months to the test value of 0 month by taking the test value of 0 month as a reference, wherein the ratio of the test value to the test 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 is clear from the data in Table 3, the concentrations of the two surfactants were optimized, and 0.1% to 1% tetronic701 was preferable, and the stability was good. The stability of 0.1 to 1 percent of polyoxyethylene (20) oil ether is also better.
4. Linear detection
High value samples near the upper end of the linear range are diluted to at least 5 concentrations in proportion, with low value concentration samples necessarily near the lower end of the linear range. According to the operation description of the kit, the sample with each concentration is subjected to three repeated tests, an average value is calculated, the average value and the dilution ratio of the result are subjected to linear fitting according to a least square method, and a linear correlation coefficient r is calculated, 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 greater 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 detection linear correlation coefficient r of the kit is more than or equal to 0.9900, and the linear detection is qualified;
5. correlation with Roche and measured value bias
According to the preparation method of the kit 2, 100 samples are tested by taking the Roche kit as a comparison example, the measured value deviation of the measured value of the kit 2 and the Roche kit is checked respectively, the measured value relative deviation is more than or equal to 4.5ng/mL and less than 15%, the measured value is less than 4.5ng/mL, and the absolute deviation is less than or equal to 0.5; the linear correlation of the measured values should be > 0.975. The results are shown in Table 5:
TABLE 5 negative-positive compliance with Roche reagent comparison
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From the data in Table 5, it can be seen that: the qualification rate of the measured value deviation of the comparison between the kit 1 and the Roche kit is 96%, 4 cases of larger deviation values occur in 100 cases of sample tests, and the overall linear correlation between the kit 1 and the Roche kit is 0.9923.
In summary, in order to obtain the creatine kinase isoenzyme detection reagent R1 with good performance, the invention optimizes the diluent of the R1 reagent to solve the problem of the long-term stability and anti-interference performance of the reagent at 2-8 ℃ which are relatively lacking in the current reagent development. Under the action of Tetronic701 and polyoxyethylene (20) oil ether, the stability of the reagent R1 is obviously improved, the reagent R1 can be stabilized for 7 days at 37 ℃ relative to CN103278623A, and after the suitable concentration of the surfactant is optimized, the reagent R1 can be stabilized for 12 months at 2-8 ℃ and has good biological activity when 0.5% Tetronic701 is used as an additive. In terms of anti-interference, the nonionic surfactant has a better resistance effect on nonspecific adsorption.
Finally, it is also 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. It is therefore intended that the following claims be interpreted as including the 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 modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (6)

1. An alkaline phosphatase-labeled CK-MB antibody buffer, wherein the alkaline phosphatase-labeled CK-MB antibody buffer is formulated 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 pH value of 6.4-6.6;
wherein the surfactant is Tetronic701 and polyoxyethylene (20) oleyl ether;
the mass ratio of Tetronic701 to polyoxyethylene (20) oil ether is (0.8-1.2): (0.8-1.2);
the buffer solution is Tris buffer solution; the preservative is ProClin-300;
the protective agent is bovine serum albumin; the inorganic salt is MgCl 2 And ZnCl 2 ；
The stabilizer is 4-aminoantipyrine; the blocking agent is mouse IgG and the electrolyte is NaCl.
2. A reagent R1 for measuring creatine kinase isozymes, wherein the reagent R1 is a working solution obtained by diluting an anti-CK-MB antibody-alkaline phosphatase marker with the alkaline phosphatase-labeled CK-MB antibody buffer according to claim 1.
3. The reagent R1 for measuring creatine kinase isozymes according to claim 2, wherein the anti-CK-MB antibody-alkaline phosphatase marker is obtained by reacting alkaline phosphatase, CK-MB antibody and glutaraldehyde, and specifically comprises:
dissolving CK-MB antibody in PBS solution to obtain CK-MB antibody solution with concentration of 0.5-2 mg/ml;
adding alkaline phosphatase into the CK-MB antibody solution, uniformly mixing, adding glutaraldehyde, and dialyzing to remove unreacted glutaraldehyde after the reaction is completed to obtain 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, wherein the final concentration of glutaraldehyde is 0.1-0.4%.
4. A creatine kinase isozyme assay kit, comprising: reagent M and reagent R1 for measuring creatine kinase isozyme according to any one of claims 2 to 3.
5. The creatine kinase isoenzyme assay kit according to claim 4 wherein the reagent M
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 magnetic particles coated with the anti-CK-MB antibody comprises the following steps:
cleaning and resuspending the magnetic bead particles to obtain a magnetic particle solution;
activating the magnetic particle solution by using an activating solution to obtain an activated magnetic particle solution;
and coating the CK-MB antibody with the activated magnetic particle solution to obtain the magnetic particles coated with the anti-CK-MB antibody.
6. A method for preparing a creatine kinase isozyme assay kit, which is characterized by adopting the formula of any one of claims 4-5.
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