CN112858678A

CN112858678A - Creatine kinase isoenzyme detection kit, preparation method and application

Info

Publication number: CN112858678A
Application number: CN201911101424.6A
Authority: CN
Inventors: 张瑞; 刘一凡; 石晓强; 徐建新; 李福刚
Original assignee: Shanghai Aopu Biomedical Co ltd
Current assignee: Shanghai Aopu Biomedical Co ltd
Priority date: 2019-11-12
Filing date: 2019-11-12
Publication date: 2021-05-28
Anticipated expiration: 2039-11-12
Also published as: CN112858678B

Abstract

The invention relates to a creatine kinase isoenzyme detection kit, a preparation method and application. Specifically, the creatine kinase isoenzyme detection kit of the invention contains a sample diluent, wherein the diluent comprises: a buffer solution with pH value of 7.2-8.5, an ion chelating agent and a protective agent. The creatine kinase isoenzyme calibration product has the advantages of long redissolution stability, long protection period, and stable storage and transportation. Meanwhile, the detection kit provided by the invention has better sensitivity and specificity, reduces the non-specific binding of the antibody to the creatine kinase isoenzyme while saving the cost, improves the detection sensitivity and the detection stability, reduces the non-specific binding, can quickly detect the low-concentration creatine kinase isoenzyme without adding other catalysts, and can be used for early diagnosis of myocardial infarction.

Description

Creatine kinase isoenzyme detection kit, preparation method and application

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a creatine kinase isoenzyme detection kit, a preparation method and application.

Background

In 10 months 2007, Acute Myocardial Infarction (AMI) was defined as myocardial cell death due to myocardial ischemia by the unified definition of european heart disease society (ESC), american heart disease society (ACC), american heart society (AHA), and world heart association (WHF) global myocardial infarction, which is unchanged in the new edition. AMI is a serious disease of coronary atherosclerotic heart disease, the disease is extremely dangerous, the death rate is high, and the optimal treatment time is within 120 minutes after the disease, so that the clinical rapid detection is very key to diagnosing and controlling the AMI disease. Modern medical research shows that the early, rapid and correct diagnosis of AMI in clinic can be improved by jointly detecting a myocardial early Marker (MYO), a definitive marker (cTnI) and an injury marker (CK-MB). The detection of creatine kinase-MB (CK-MB) can be used for the diagnosis and prognosis of Acute Myocardial Infarction (AMI), the diagnosis and prognosis of myocarditis, the diagnosis of muscle diseases, the diagnosis of brain diseases such as cerebral infarction, meningitis, encephalitis and the like.

Creatine Kinase (CK) is known as Creatine Phosphokinase (CPK). CK has a molecular weight of 81000 and consists of two subunits. It is an important kinase that is usually present in the cytoplasm and mitochondria of tissues such as heart, muscle and brain of animals, has a direct relation with intracellular energy movement, muscle contraction and ATP regeneration, and reversibly catalyzes the transphosphoryl reaction between creatine and ATP. CK catalyzes the phosphorylation of creatine in the presence of ATP to produce ATP and phosphocreatine. This enzyme-catalyzed reaction is reversible. In 1979, the content of creatine kinase in serum of prostate cancer patients was first found to be significantly increased, and the same finding was found in other types of cancer patients. With the progress of research, people have started to search for new antitumor drugs by screening creatine kinase inhibitors in recent years. Due to the important physiological function of creatine kinase, it has attracted much attention and intensive research.

The N-terminus of each subunit of creatine kinase isozyme (CK-MB) dimer has a small domain linked to a large domain at the C-terminus by a linker sequence. In the whole dimer, 98% of tyrosine residues are wrapped inside the spatial structure, and only 2% of tyrosine residues are exposed on the surface of the structure; in addition, each subunit surface contains 2 reactive histidine residues and 2 reactive thiol groups. The complex spatial structure thereof brings great difficulty to the application of in vitro recombination technology. Therefore, the recombination difficulty is high, and the CK-MB with high quality on the market at present is all blood source type. But the sources are limited and the price is high, so that the research and development of CK-MB diagnostic products are seriously hindered. Although there are many reports about CK-MB at home and abroad, most of the research belongs to academic research, and the structure and the function of the protease are mainly concerned; and there is very little research really on the application of its products. In 2001, Yoshiko Sunahara and the like simultaneously express MB, MM and BB isoenzymes, respectively purify the three isoenzymes, mix the purified isoenzymes according to the proportion of 50:40:10, and complete the later folding of MB by applying the protein dynamics principle to obtain the protein with higher CK activity. However, the study was completed as it is, and no study was made on the stability, specificity, sensitivity and application of the protein as a biological raw material. In addition, in the research, the expression and mixing ratio of MB are complex, the process stability is difficult to control, and the MB cannot be used for the production of products. Huang Y et al (2006) chose to express its isozyme MM in vitro, but the spatial structure of MM or BB isozymes is very different from MB, and is far from the natural structure of MB, and it is impossible to replace CK-MB as a calibration quality control product for clinical detection.

The creatine kinase isoenzyme is a biological macromolecular protein and exists in a dimer form, so that the creatine kinase isoenzyme is extremely unstable, and severe storage, transportation, use conditions and the like of the creatine kinase isoenzyme bring about inconvenience for the use of the protein. The calibrator of most of the company kits on the market is preserved in the form of freeze-dried product, and low-temperature freeze-drying is the most effective and most common method for dry preservation of biological products such as protein, microorganism and the like at present. At present, the common recombinant protein freeze-drying protective agent comprises saccharides and polymers, polyhydric alcohols, surfactant-free, water-soluble amino acid, certain salts, amines and the like, but the stability is not good enough. At present, there are many immunoassay methods for creatine kinase isozyme, including Radioisotope Immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), colloidal gold Immunochromatography (ICA), and chemiluminescence method (CLIA) which has been widely used in recent years.

The detection principle of Radioisotopic Immunoassay (RIA) is to perform a competitive binding reaction between an isotopically labeled antigen and an unlabeled antigen to be detected and an insufficient amount of a specific antibody, and then measure the radioactivity of the reaction to obtain the amount of the antigen to be detected. This method is an ultra-micro analysis technique that has been increasingly widely used since the 80 s.

The enzyme-linked immunosorbent assay (ELISA) detection principle, taking the double-antibody sandwich method as an example, is to coat a specific antibody on a solid phase carrier, add a sample to be detected, add another enzyme-labeled antibody to form a double-antibody sandwich compound, and then enable the instrument to quantitatively detect immunoreaction through the color development of a luminescent substrate.

The colloidal gold immunochromatography method as a POCT (point of care testing) test method has the advantages of small sample loading amount, simplicity, convenience and rapidness, and is suitable for bedside test. The principle is as follows, still taking the double antibody sandwich method as an example, when a sample containing an antigen is dropped into an absorption hole, the antigen is bound with an antibody originally labeled with colloidal gold to form an antigen-antibody complex, and then another antigen immobilized on a sample pad captures the complex to form a double antibody sandwich complex, and a color band is generated, so that the double antibody sandwich complex is detected.

Chemiluminescence is a product obtained by closely combining an immune reaction system and chemiluminescence technology, which is an immunoassay technique that has been developed in recent years. The chemiluminescence technology is a detection means which utilizes a luminescent substance, such as acridinium ester and the like, to form an excited intermediate after being oxidized by an oxidant and catalyzed by a catalyst, and then utilizes a corresponding measuring instrument to measure the yield of light quantum generated when the intermediate returns to the ground state. The immunoreaction system is to mark the luminous labeling matter on antigen or antibody and then to form antigen-antibody complex for detection. The technology has the advantages of strong specificity, high sensitivity, good precision, wide linear range, high flux and easy realization of automation.

The direct chemiluminescence immunoassay refers to directly labeling an antibody (antigen) with acridinium ester, forming a solid phase coated antibody-antigen to be detected-acridinium ester labeled antibody compound after immunoreaction with the corresponding antigen (antibody) in a sample to be detected, and reacting with an oxidant (H)₂O₂) Under the action of NaOH, the acridinium ester can be decomposed and emit light without a catalyst. The strong direct luminescence is finished within one second, the fast flash luminescence is realized, the luminous intensity is in direct proportion to the amount of the antigen to be detected, and the content of the antigen to be detected can be calculated from a standard curve.

At present, although chemiluminescence method kits for detecting creatine kinase isozyme exist in the market, many of the kits have the defects of insufficient sensitivity, low specificity and the like. Meanwhile, the calibrator has poor stability, is unstable in a liquid environment, is easy to hydrolyze, and is not favorable for long-term storage.

For example, CN102435738A, CN107389941A, CN109490549A, and CN109613242A all disclose a kit for detecting creatine kinase isozyme by a chemiluminescence method. However, none of the calibrators of the creatine kinase isoenzyme kit disclosed in the above documents has sufficiently high stability.

Radioisotopic Immunoassay (RIA) has the problems of complex operation, long time consumption, poor result repeatability, poor sensitivity and specificity and the like, has the risk of radioactive contamination, is not suitable for large-scale detection, and is not used basically at present.

Enzyme-linked immunosorbent assay (ELISA) has the defects of complex operation, low sensitivity, narrow linear range, long detection period and the like. When early AMI occurs, the content of creatine kinase isoenzyme in serum is often very low, and the method cannot quickly and accurately detect early AMI due to low sensitivity and long detection period, so that the use of ELISA in myocardial detection is further limited.

The colloidal gold immunochromatography method as a POCT (point of care testing) test method has the advantages of small sample loading amount, simplicity, convenience and rapidness, and is suitable for bedside test. However, the sensitivity of the method is poor, so that the application of the method in clinical detection of creatine kinase isoenzyme is further limited.

Enzymatic chemiluminescence is a horseradish peroxidase system and an alkaline phosphatase system. The method has the defects of long excitation luminescence time, low test speed, easy influence of enzyme on environmental temperature and the like, and the stability of the reagent is not as good as that of direct chemiluminescence.

The electrochemical luminescence method is that the terpyridyl ruthenium marked antigen-antibody compound generates visible light after oxidation-reduction reaction under the action of tripropylamine. The excitation process is complex and long, and each light-emitting speed is about 25 seconds.

The relatively short storage time of liquid CK-MB calibrators limits the useful life of CK-MB kits.

The redissolution stability of the lyophilized CK-MB calibrator is not good enough, which affects the performance.

Therefore, it is desirable to provide a stable CK-MB calibrator diluent which can be dried at low temperature and quickly absorb water to restore the CK-MB to a fresh state before drying. Meanwhile, the defects of the prior art need to be overcome so as to solve the problems of short storage time and inconvenient transportation of the creatine kinase isoenzyme calibration product; the freeze-dried product has poor re-solubility.

Disclosure of Invention

Problems to be solved by the invention

The invention aims to provide a creatine kinase isoenzyme sample diluent and a creatine kinase isoenzyme detection kit containing the diluent.

Another object of the present invention is to provide a method for preparing the creatine kinase isoenzyme detection kit.

Another object of the present invention is to provide a use of the diluted solution of the acid kinase isoenzyme sample and a creatine kinase isoenzyme detection kit containing the diluted solution.

Means for solving the problems

Specifically, the present invention describes the following means.

【1】 The creatine kinase isoenzyme sample diluent comprises: a buffer solution with pH value of 7.2-8.5, an ion chelating agent and a protective agent, wherein,

the protective agent comprises one or more selected from the group consisting of bovine serum albumin, Casein, a complex enzyme protective agent AFP and a complex enzyme protective agent HPD.

【2】 The diluent according to [ 1 ], wherein the buffer is MOPSO buffer; preferably, the MOPSO buffer has a pH value of 7.2.

【3】 The diluent according to any one of [ 1 ] to [ 2 ], wherein the protective agent is selected from bovine serum albumin, a complex enzyme protective Agent (AFP) and a complex enzyme protective agent (HPD), and the ion chelating agent is EDTA; wherein the content of the first and second substances,

the content of the bovine serum albumin is 1% -10%, preferably 2-5%, and more preferably 3%;

the content of the compound enzyme protective agent AFP is 3-10%, preferably 5%;

the content of the compound enzyme protective agent HPD is 3-10%, preferably 5%;

the concentration of EDTA is 5-15mM, preferably 10 mM.

【4】 The diluent according to any one of [ 1 ] to [ 3 ], wherein the sample is a calibrator; preferably, the calibrator is a lyophilized calibrator.

【5】 A creatine kinase isoenzyme detection kit, wherein the kit comprises: a first antibody coated on the magnetic microsphere, a second antibody marked with a tracer marker and the creatine kinase isoenzyme sample diluent as any one of [ 1 ] to [ 4 ]; wherein the content of the first and second substances,

the first antibody and the second antibody recognize different specific binding sites for creatine kinase isozyme;

optionally, the test kit further comprises a calibrator.

【6】 The creatine kinase isoenzyme detection kit according to [ 5 ], wherein the first antibody is CKMB-BB, and the second antibody is CKMB-04; preferably, the mass ratio of the magnetic microspheres to the first antibodies is 100:0.5-1.25, and the mass ratio of the tracer labels to the second antibodies is 15-40: 1.

【7】 The creatine kinase isoenzyme detection kit according to any one of [ 5 ] to [ 6 ], wherein the tracer marker is one or more selected from the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer label is an acridinium ester.

【8】 The creatine kinase isoenzyme detection kit according to any one of [ 5 ] to [ 7 ], wherein the kit further comprises: magnetic microsphere cleaning solution, substrate solution A and substrate solution B; wherein the magnetic microsphere cleaning solution contains PBST buffer solution, and the substrate solution A is H₂O₂And HNO₃The substrate solution B is a mixed solution of Triton X-100 and NaOH; preferably, said H₂O₂Is 0.01-5.0 percent by mass, and the HNO₃The concentration of the compound is 0.01-1.0mol/L, the mass fraction of the Triton X-100 is 0.01-2.0%, and the concentration of the NaOH is 0.05-1 mol/L.

【9】 The preparation method of the creatine kinase isoenzyme detection kit according to any one of [ 5 ] to [ 8 ], wherein the preparation method comprises the following steps:

coating magnetic microspheres: coating the first antibody on the magnetic microsphere;

tracer marker marking step: labeling the tracer label on the second antibody;

preparing a sample diluent: mixing the components of the sample dilution.

【10】 The method for preparing a creatine kinase isoenzyme detection kit according to [ 9 ], wherein,

2- (N-morpholino) ethanesulfonic acid is used as a buffer solution in the magnetic microsphere coating step;

optionally, a blocking solution is also used in the magnetic microsphere coating step, and the blocking solution is BSA;

and/or

In the tracer marker marking step, 4-hydroxyethyl piperazine ethanesulfonic acid is used as a buffer solution.

【11】 Use of the creatine kinase isoenzyme sample diluent according to any one of [ 1 ] to [ 4 ] or the creatine kinase isoenzyme detection kit according to any one of [ 5 ] to [ 8 ] in the preparation of a reagent for diagnosing myocardial infarction.

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment, the present invention employs a combination of multiple types of protectants for preparing a sample, such as a lyophilized calibrator, for detecting creatine kinase isoenzyme, which ultimately results in a lyophilized calibrator of creatine kinase isoenzyme with long reconstitution stability, long shelf life, storage stability, and stable transportation.

In another embodiment, the present invention provides a kit for detecting creatine kinase isoenzyme, which has better sensitivity and specificity, can rapidly detect creatine kinase isoenzyme with low concentration without adding other catalysts, such as by direct chemiluminescence, and can be used for early diagnosis of myocardial infarction.

In another embodiment, the detection kit provided by the invention reduces the nonspecific binding of the antibody to creatine kinase isoenzyme, improves the detection sensitivity and detection stability, and reduces the nonspecific binding while saving the cost.

Drawings

FIG. 1 shows a comparison of the stability of the calibrators under each buffer system (where the ordinate indicates the decrease of the concentration point relative to the light unit P and the abscissa indicates the day 3, day 5 and day 7 of the measurement time point);

FIG. 2 shows the effect of different protective agents on the stability of the calibrator (where the ordinate indicates the decrease of the concentration point in relation to the light units P and the abscissa indicates the day 3, day 5 and day 7 of the measurement time points);

FIG. 3 shows the effect of different concentrations of EDTA on the stability of the calibrator (where the ordinate indicates the decrease of the concentration point in relation to the light units P and the abscissa indicates the day 3, day 5 and day 7 of the measurement time points);

FIG. 4 shows the effect of different concentrations of Bovine Serum Albumin (BSA) on the stability of the calibrator (where the ordinate indicates the decrease of the concentration points in relation to the light units P and the abscissa indicates the day 3, day 5 and day 7 of the measurement time points);

FIG. 5 shows the effect of different concentrations of complex enzyme protective agent AFP on the stability of the calibrator (where the ordinate indicates the decrease in concentration points versus light units P and the abscissa indicates the day 3, day 5 and day 7 of the measurement time points);

FIG. 6 shows the effect of different concentrations of the complexed enzyme protectant HPD on the stability of the calibrator (where the ordinate indicates the decrease in concentration points versus light units P and the abscissa indicates day 3, day 5 and day 7 at the time points of measurement);

FIG. 7 shows the reconstitution stability of the calibrator (wherein the ordinate represents the total decrease and the abscissa represents the measurement time points at day 3, day 7, day 15, day 23 and day 30; FIG. 7 shows that the reconstitution stability decreases by about 8% after reconstitution and storage at 2-8 ℃ for 30 days, and the stability is better) after the calibrator diluent is prepared into a freeze-dried calibrator;

FIG. 8 shows the correlation R, the standard curve (logX-logY) for a calibrator prepared with a diluent according to the present invention and reconstituted after lyophilization²The value can reach 0.9975. FIG. 8 shows that the calibrator of the present invention is linear well between 0-300 ng/ml);

FIG. 9 shows the results of a clinical sample assay showing the linear dependence R of the reagents of the invention²The value can reach 0.9973;

FIG. 10 shows the results of a clinical sample assay (where the ordinate is an embodiment of the invention)4, the abscissa represents the result of the assay with the Beckmann CK-MB kit, and the correlation R²A value of 0.9963).

Detailed Description

Other objects, features and advantages of the present application will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.

It should be noted that:

in the present specification, the term "magnetic microsphere" has the same meaning as "magnetic bead", and means a superparamagnetic microsphere having a fine particle size, generally having a super-strong paramagnetism. The surface of the sample can be coated with specific antibodies, receptors and the like for separating and purifying target bodies in the sample. Magnetic beads have been widely used in various fields such as immunoassay, nucleic acid separation and extraction, cell sorting, and enzyme immobilization.

In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, means that a particular element (e.g., feature, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

< first aspect >

The invention provides a creatine kinase isoenzyme sample diluent, wherein the diluent comprises: a buffer solution with pH value of 7.2-8.5, an ion chelating agent and a protective agent, wherein,

Further, the invention also provides a creatine kinase isoenzyme detection kit, wherein the kit comprises: a first antibody coated on the magnetic microsphere, a second antibody marked with a tracer marker and the creatine kinase isoenzyme sample diluent;

optionally, the test kit further comprises a calibrator.

Meanwhile, the CK-MB specific binding sites recognized by the first antibody and the second antibody are different.

As an alternative embodiment of the invention, the first antibody and the second antibody of the invention are both monoclonal antibodies against CK-MB.

In a preferred embodiment of the invention, the first antibody is CKMB-BB and the second antibody is CKMB-04.

The method for detecting CK-MB is a double-antibody sandwich method, and mainly uses two different specific monoclonal antibodies, wherein the binding site of the antibody marked on a tracer marker is different from the binding site of the antibody coated on a magnetic microsphere. The selection of these binding sites not only facilitates the labeling of the tracer or the coating of the magnetic microspheres, but also does not prevent the binding of the antibody to the antigen to form a sandwich complex, thus improving the specificity and sensitivity of the reaction.

The present invention is not particularly limited with respect to the kind of tracer labels suitable for use in the present invention. For example, the tracer label can be luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester, adamantane, and the like. Preferably, the tracer label is an acridinium ester.

The present invention is not particularly limited with respect to the kind of magnetic microspheres suitable for use in the present invention, and may be magnetic beads commonly used in the art. As an embodiment of the present inventionFirstly, the magnetic beads used in the invention are nanoscale Fe₂O₃And Fe₃O₄The magnetic particles and the high molecular material are compounded to form the micron-sized solid phase microsphere with paramagnetism and extremely large protein adsorption capacity, the magnetic microsphere can be quickly magnetized under the action of an external magnetic field, and the remanence is zero after the magnetic field disappears. In the present invention, the kind of the polymer material to be used for the composite is not particularly limited.

The particle size of the magnetic beads used in the present invention should be 1-5 μm, and as one embodiment of the present invention, the magnetic beads may be surface modified to add a plurality of active groups, and the types of active groups in the present invention include, but are not limited to-OH and-COOH.

In the invention, the mass ratio of the magnetic microspheres to the first strain antibody is 100: 0.5-1.25; the mass ratio of the tracer marker to the second strain antibody is not less than 15: 1; preferably, the mass ratio of the magnetic microspheres to the first strain antibody is 100: 1; the mass ratio of the tracer marker to the second strain antibody is 15-40: 1.

In the invention, EDTA in the diluent is an excellent metal ion complexing agent, detergent and blood anticoagulant. The calcium ion chelating agent is commonly used as a calcium ion chelating agent in biochemical research and is used for eliminating the inhibition effect in enzyme catalytic reaction caused by calcium or other trace heavy metals. After EDTA with the final concentration of 5-15mM is added, the inhibition effect of other trace heavy metal ions on enzyme reaction can be avoided, thereby increasing the stability of the protein. In some embodiments of the invention, the stability of the CK-MB calibrator is highest at an EDTA concentration of 10 mM.

Bovine Serum Albumin (BSA) has the functions of a low-temperature protective agent and a dehydration protective agent, and can play a good protection role on CK-MB antigens. In some embodiments of the present invention, the addition of bovine serum albumin preferably results in a final concentration of 1% to 10%, more preferably 2% to 5%, and the stability is significantly improved. In other embodiments of the invention, bovine serum albumin is present in the diluent at a concentration of 3% to maximize the stability of the lyophilized CK-MB calibrator.

Compound enzyme protective agent for bloodThe medium proteins, enzymes, lipids and other substances have protection and stabilization effects, so that the environment required by the physiological activity of the enzymes and the protein structures is maintained; on the other hand, the efficiency of the enzyme and the protein can be improved, the activity loss in the freeze-drying process and after re-dissolution is avoided, and the long-term storage is facilitated. When a compound enzyme protective agent AFP with the final concentration of 5% and a compound enzyme protective agent HPD with the final concentration of 5% are added, the stability of the CK-MB freeze-dried calibration product is highest. The CK-MB freeze-dried calibrator prepared by the diluent is used for preparing a standard curve and a correlation coefficient R²The calibrator was linear well between 0-300ng/ml, above 0.99.

In the invention, the buffer system in the creatine kinase isoenzyme sample diluent is an MOPSO buffer system; preferably, the MOPSO buffer system has a pH value of 7.2.

In the present invention, the kit further comprises: magnetic microsphere cleaning solution, substrate solution A and substrate solution B.

The magnetic microsphere cleaning solution is PBST buffer solution; preferably, the magnetic microsphere cleaning solution further comprises a preservative. The preservative suitable for use in the present invention may be one or more of preservatives commonly used in the art, such as potassium sorbate, sodium benzoate, sodium nitrite, sodium azide, proclin-300 (the main active ingredients are 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one, which is a safer preservative and one of the commonly used preservatives in immunodiagnostics), and antibiotics.

In the invention, the substrate solution A is H₂O₂And HNO₃Preferably, H₂O₂0.01-5.0% of HNO₃The concentration is 0.01-1.0 mol/L. The substrate solution B is a mixed solution of Triton X-100 and NaOH, preferably, the mass fraction of the Triton X-100 is 0.01-2.0%, and the concentration of the NaOH is 0.05-1 mol/L.

Surfactants suitable for use in the present invention may be, in addition to Triton X-100, surfactants commonly used in the art, such as one or more of Triton X-405, Tween 20 and Tween 80. The addition amount of the surfactant in the kit is 0.1-2 per mill, so that the dispersion of the magnetic microspheres is facilitated.

< second aspect >

The invention also provides a preparation method of the creatine kinase isoenzyme CK-MB detection kit according to the < first aspect >, wherein the preparation method comprises the following steps:

tracer marker marking step: labeling the tracer label on the second antibody;

preparing a sample diluent: mixing the components of the sample dilution.

As one embodiment of the present invention, 2- (N-morpholino) ethanesulfonic acid (i.e., MES) buffer is used in the first antibody-coated magnetic microsphere step.

As one embodiment of the present invention, a blocking solution, which is 20% BSA, is also used in the step of coating the magnetic microspheres with the first antibody.

As one embodiment of the present invention, the antibody coupling buffer used in the second antibody labeling tracer label step is selected from 4-hydroxyethylpiperazine ethanesulfonic acid (i.e., HEPES) buffer.

As one embodiment of the present invention, the labeling time of the second antibody labeled with the tracer label is 0.5 to 2 hours; alternatively, the second antibody labels the tracer label for a labeling time of 1 hour.

In one embodiment of the invention, the creatine kinase isoenzyme CK-MB freeze-dried calibrator is MOPSO buffer solution containing a certain amount of CK-MB antigen, and a certain preservative is added. Then, lyophilization was performed. The calibrator can be stored stably at 4 deg.C.

< third aspect >

The invention also provides application of the creatine kinase isoenzyme sample diluent or the creatine kinase isoenzyme detection kit in preparing a myocardial infarction diagnosis reagent according to the first aspect.

Examples

Example one

Preparation 1: magnetic microsphere suspension coated with CK-MB monoclonal antibody

(1) Measuring 10mg of magnetic microsphere solution, adding 1ml of 0.1M MES buffer solution, carrying out vortex mixing, carrying out magnet adsorption for 5-10 min until magnetic beads are completely adsorbed to one side of the tube wall, discarding the supernatant, repeating the cleaning step for 3-5 times, adding 1ml of 0.1M MES, and carrying out vortex mixing;

(2) adding 100 mu g of CK-MB first monoclonal antibody (the mass ratio of magnetic beads to antibody is 100:1) and mixing uniformly by vortex, and incubating for 30min at 37 ℃;

(3) adding 10 μ L10 mg/ml 1- (3-dimethylaminopropyl) -3-ethyldiimine hydrochloride (EDC), mixing by vortex, placing in an incubator at 37 ℃, and incubating for 1.5 h;

(4) add 200. mu.L of 20% BSA solution and block for 2 h.

(5) And adding 1ml of luminescence recovery solution 2(5g of bovine serum albumin, 400ml of 0.025M Tris-HCl buffer solution for dissolving, 1-2 per thousand of Tween-80 and 100ml of newborn bovine serum) into the closed magnetic bead suspension, carrying out magnetic adsorption, removing supernatant, and repeating the cleaning step for 3-5 times.

(6) And adding 1ml of luminescence recovery solution 2 into the prepared magnetic beads, and storing for a long time at the temperature of 2-8 ℃.

Preparation 2: preparation of CK-MB monoclonal antibody solution labeled with acridinium ester

(1) 1mg of the second monoclonal antibody against CK-MB was placed in a dialysis bag, and then dialyzed with not less than 2L of 0.05M HEPES buffer (pH 8.0) for 24 hours, followed by four changes in the middle. After dialysis, the antibody was concentrated in Millipore Amicon μm ultra-filtration tubes to a concentration of greater than 5 mg/ml.

(2) Putting the concentrated CK-MB monoclonal antibody into a 0.5ml centrifugal tube, adding 100 mu L of 5mg/ml NSP-SA-NHS solution dissolved in DMF to ensure that the molar ratio of the NSP-SA-NHS to the CK-MB monoclonal antibody is not less than 15:1, uniformly mixing, reacting for 2 hours at room temperature (in a dark place), adding 100 mu L of lysine solution with the concentration of 100mg/ml as a blocking buffer solution, reacting for 30min, and stopping the reaction.

(3) The label was placed in a dialysis bag and dialyzed with not less than 2L citric acid-sodium citrate buffer (0.01mol/L, pH 4.5) for 24h, with four intermediate changes, to isolate free NSP-SA-NHS.

(4) The dialyzed label was collected, 5% BSA solution was added to make the BSA concentration 1% and then glycerol was added in equal volume and stored at-20 ℃.

Preparation 3: preparation of calibrator

CK-MB working solution is taken and diluted to 4ng/ml and 140ng/ml high-low point calibrator solution by using MOPSO solution containing 10mM EDTA, 3% bovine serum albumin, 5% composite enzyme protective agent AFP and 5% composite enzyme protective agent HPD.

Preparing a second CK-MB antibody marked with acridinium ester in step 2 by using the magnetic microsphere suspension coated with the first CK-MB antibody prepared in step 1, and preparing 3 high-low point calibrators which respectively form the components of the kit.

Preparation 4: preparation of chemiluminescent substrate solution

The chemiluminescent substrate solution A is H₂O₂And HNO₃In which H is₂O₂0.01-5.0% of HNO₃The concentration is 0.01-1.0 mol/L.

The chemiluminescence substrate solution B is a mixed solution of Triton X-100 and NaOH, wherein the mass fraction of the Triton X-100 is 0.01-2.0%, and the concentration of the NaOH is 0.05-1 mol/L.

Preparation 5: preparation of cleaning solution

The cleaning solution is a PBST solution with the pH value of 7.0-9.0 and the concentration of 0.02mol/L, and contains 0.5% of Tween-20 by mass fraction.

Example two

Matched screening of antibody used in creatine kinase isoenzyme detection kit

(1) Antibody panel screening

The CK-MB antibodies used in the present invention are four in total: antibody 1: CKMB-10-C40A (Fitzgerald, Lot: M9910207), antibody 2: CKMB-03 (Roc, Lot: 20160426-2); antibody 3: CKMB-04 (Roc, Lot: 2071104), antibody 4: CKMB-BB (biospacific, Lot: A5481). Respectively coating the

antibodies

1, 3 and 4 with magnetic microspheres to form an antibody 1-magnetic microsphere, an antibody 3-magnetic microsphere and an antibody 4-magnetic microsphere, and labeling the

antibodies

2 and 3 with acridinium ester to form an antibody 2-acridinium ester and an antibody 3-acridinium ester. And (3) carrying out combination pairing on the antibodies, detecting a serum sample, and screening the optimal combination.

TABLE 1 results of antibody pairwise combinatorial screening

As shown in table 1, in combination 4, antibody 4, in combination with antibody 2, failed to bind to creatine kinase isoenzyme in the serum sample; in the combination 2 and the combination 3, the antibody 1 and the antibody 3 are matched with the antibody 3 and the antibody 2, so that the overall signal value of the creatine kinase isoenzyme is low and the discrimination is low; and the combination 5 is matched with the antibody 4 and the antibody 3, so that the results of all indexes are better.

TABLE 2 serum sample compliance rates test results

TABLE 3 results of the test of the coincidence rate of blood homologous samples

Referring to the results of tables 2 and 3, the combined sample coincidence rate and the homologous sample coincidence rate of antibody 4 and antibody 3 were both good.

EXAMPLE III

Magnetic bead acridinium ester preparation process optimization

(1) Optimization process for magnetic bead coated antibody

TABLE 4 optimization of magnetic bead coated antibody coupling buffer

	MES	MOPSO	Boric acid buffer solution
				S0	694	943	1693
S1	2950	2787	2326
				S2	11358	8967	9411
S3	168634	172850	181064
				S4	619634	599228	649621
S5	1406569	1133330	1265588
				Rate of agreement	0.9996	0.9977	0.9987

Comprehensively comparing each group of data, and selecting MES buffer as magnetic bead coating coupling buffer.

TABLE 5 screening results of magnetic bead antibody use ratio

Magnetic beads: antibodies	ng/ml	100:0.5	100:0.75	100:1	100:1.25
						S0	0	792	672	629	821
S1	1	2011	2281	2615	2622
						S2	4	7240	8778	9936	8976
S3	35	109681	129741	148826	140178
						S4	140	396271	469700	533583	518082
S5	300	838337	1049198	1176216	1173205
						Rate of agreement		0.9998	0.9997	0.9998	0.9996

The addition of the antibody is increased, the signal value of the antigen detection is increased, and the coincidence rate of the antigen is not obviously influenced, when the magnetic bead: when the antibody reaches 100:1.25, the overall luminous value is not obviously increased, so that the invention selects 100:1 as magnetic beads: antibody addition ratio.

TABLE 6 confining liquid screening

Sample numbering	1M Gly	5％BSA	20％BSA
				S0	335	255	507
S1	1494	1917	2677
				S2	6721	7899	10596
S3	105807	140221	169422
				S4	421702	490714	620613
S5	923364	1143301	1437048
				Rate of agreement	0.9999	0.9992	0.9994

The three confining liquids have no obvious influence on the detection coincidence rate of the antigen diluent, the luminescence value of 20% BSA is higher, and the detection low value discrimination degree is slightly higher, so 20% BSA is selected as the confining liquid.

(2) Optimization process of acridinium ester labeled antibody

TABLE 7 optimization of acridinium ester-labeled antibody coupling buffer

	CB	HEPES	TAPS
				S0	812	504	355
S1	2711	4235	2375
				S2	10155	11522	10072
S3	145591	172682	153894
				S4	570396	618728	554005
S5	1183146	1403262	1342447
				Rate of agreement	0.9998	0.9996	0.9986

The three coupling buffer solutions have no obvious influence on the detection coincidence rate of the antigen diluent, the detection signal value of the HEPES sample is integrally higher, the low value distinguishing range is larger, and the HEPES is selected as the acridine ester labeled antibody coupling buffer solution.

TABLE 8 acridinium ester-labeled antibody coupling time optimization

	0.5h	1h	2h
				S0	540	402	376
S1	2749	2776	2911
				S2	10997	10903	11624
S3	165327	177902	183645
				S4	609391	651064	645744
S5	1272180	1449572	1447030
				Rate of agreement	0.9998	0.9998	0.9997

The antibody is coupled with acridine ester for 1h, and the reaction reaches a plateau phase.

TABLE 9 acridinium ester antibody use ratio screening

Acridine ester: antibodies	40:1	30:1	20:1	15:1
					S0	565	792	759	537
S1	1181	984	2015	2759
					S2	3250	4415	8569	10449
S3	46529	73314	132378	151619
					S4	160012	281253	473106	564721
S5	346510	633329	1033244	1174164
					Rate of agreement	0.9998	0.9998	0.9999	0.9998

According to the experimental data above, acridinium ester: the antibody should not be less than 15: 1.

example four

Selection of pH value of CK-MB calibrator diluent

1. Preparation of CK-MB calibrator diluent with different buffer systems

(1)100ml CK-MB calibrator buffer (0.05M MOPSO)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g, a complex enzyme protective agent AFP 5g and a complex enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(2)100ml CK-MB calibrator buffer (0.05M HEPES)

Weighing HEPES1.19156g, EDTA0.292g, Bovine Serum Albumin (BSA)3g, a complex enzyme protective agent AFP 5g and a complex enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.5 after complete dissolution, and fixing the volume to 100 ml.

(3)100ml CK-MB calibrator buffer (0.05M Tris-HCL)

Weighing 0.60566g of Tris-HCl (Chinese medicine, 20170216), 3g of EDTA0.292g of Bovine Serum Albumin (BSA), 5g of composite enzyme protective agent AFP and 5g of composite enzyme protective agent HPD. Adding 60ml of purified water, adjusting the pH value to 8.5 after complete dissolution, and fixing the volume to 100 ml.

2. Freeze-drying experiment of CK-MB calibrator

Subpackaging the calibrator with 500 μ l per bottle by brown glass bottle, quickly freezing, freeze-drying in a freeze-dryer for 24-30h, sealing, and storing.

3. Stability test of CK-MB calibrator

Using the three dilutions prepared in step 1 to prepare 300ng/ml of lyophilized calibrator, placing in a constant temperature incubator at 37 ℃ for 3 days, 5 days and 7 days, respectively, and using 2-8 ℃ as a control to perform the following experiments:

(1) placing the CK-MB antibody solution coated with the magnetic microspheres as a reagent 1 and the CK-MB antibody solution marked with acridinium ester as a reagent 2 at room temperature, and fully and uniformly mixing for 30 min;

(2) adding 20 mul of prepared antigen solution into a reaction cup;

(3) mu.l of reagent 1 (magnetic bead suspension) and 50. mu.l of reagent 2 (acridinium ester solution) were added to the reaction cuvette in this order;

(4) after the solution in the reaction cup is fully and uniformly mixed, the mixture is incubated for 6min at 37 ℃;

(5) placing the magnetic beads under a magnetic condition, and washing the magnetic beads for multiple times by using a magnetic bead washing liquid;

(6) to the reaction cuvette, 100. mu.l of substrate solution A and substrate solution B were added, and the luminescence value was measured, and the luminescence intensity was expressed in Relative Light Units (RLU).

4. Stability analysis of CK-MB calibrator under different pH values

Different buffer systems are prepared in the experiment, so that the stability of the CK-MB project under the condition of pH7.2-8.5 is researched. Three buffers, namely MOPSO weak base system with pH7.2, HEPES weak base system with pH7.5 and Tris-HCL alkaline system with pH8.5 are used for preparing a calibrator and are respectively placed in a constant-temperature incubator at 37 ℃ for stability experiments. As shown in tables 1-3, point P is 300ng/ml calibrator and point O is calibrator buffer. The P-point versus light unit (RLU) variation under different buffer systems was verified, with smaller variations indicating more stable calibrators. The result shows that the reduction of the P point is the lowest under the MOPOS weak base condition of pH7.2, the P point is less than 15%, and the stability of the calibrator is higher. (see tables 10-12)

TABLE 10 MOPSO buffer

TABLE 11 HEPES buffer

TABLE 12 Tris-HCL buffer

EXAMPLE five

Protectant selection for CK-MB calibrator dilutions

1. Preparation of CK-MB calibrator diluent containing different protective agents

(1)100ml CK-MB calibrator buffer (bovine serum albumin as protective agent)

MOPSO1.1263g, EDTA0.292g and Bovine Serum Albumin (BSA)5g were weighed. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(2)100ml CK-MB calibrator buffer (Casein as protective agent)

Weighing MOPSO1.1263g, EDTA0.292g and Casein 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(3)100ml CK-MB calibrator buffer (protective agent is compound enzyme protective agent AFP)

Weighing MOPSO1.1263g, EDTA0.292g and 5g of composite enzyme protective agent AFP. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(4)100ml CK-MB calibrator buffer (protective agent is compound enzyme protective agent HPD)

Weighing MOPSO1.1263g, EDTA0.292g and 5g of compound enzyme protective agent HPD. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

2. Freeze-drying experiment of CK-MB calibrator

3. Stability test of CK-MB calibrator

The four dilutions prepared in step 1 were used to prepare 300ng/ml CK-MB lyophilized calibrator, which was incubated at 37 ℃ for 3 days, 5 days and 7 days, respectively, and the assay was performed at 2-8 ℃ as control, as in example 1.

4. Stability analysis of CK-MB calibrator under different protective agents

In the test, different protective agents, namely 5% Bovine Serum Albumin (BSA), 5% Caesin, 5% composite enzyme protective agent AFP and 5% composite enzyme protective agent HPD are adopted to prepare calibrator diluents under an MOPSO buffer system with the pH value of 7.2, and the calibrator diluents are prepared by the four calibrator diluents to perform a stability test. As shown in tables 13-16, point P is 300ng/ml calibrator and point O is calibrator buffer. The P-point changes relative to the light units (RLU), with less change indicating more stable calibrators. The result shows that the point P reduction amplitude of the calibrator diluent added with 5 percent of bovine serum albumin, 5 percent of composite enzyme protective agent AFP and 5 percent of composite enzyme protective agent HPD is lower, and the calibrator has higher stability. (see tables 13-16).

TABLE 135% bovine serum Albumin Effect on calibrator stability

TABLE 145% Casein Effect on calibrator stability

TABLE 155% Effect of Complex enzyme protectant AFP on calibrator stability

Table 165% Effect of Complex enzyme protectant HPD on calibrator stability

EXAMPLE six

Screening of optimum concentration of each component in CK-MB calibrator diluent

1. Preparation of CK-MB calibrator diluent containing EDTA with different concentrations

(1)100ml CK-MB calibrator buffer (without EDTA)

Weighing MOPSO1.1263g, Bovine Serum Albumin (BSA)3g, a compound enzyme protective agent AFP 5g and a compound enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(2)100ml CK-MB calibrator buffer (5mM EDTA)

Weighing MOPSO1.1263g, EDTA0.146g, Bovine Serum Albumin (BSA)3g, a compound enzyme protective agent AFP 5g and a compound enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(3)100ml CK-MB calibrator buffer (10mM EDTA)

(4)100ml CK-MB calibrator buffer (15mM EDTA)

Weighing MOPSO1.1263g, EDTA0.438g, Bovine Serum Albumin (BSA)3g, composite enzyme protective agent AFP 5g and composite enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

2. Preparation of CK-MB calibrator diluent containing Bovine Serum Albumin (BSA) with different concentrations

(1)100ml CK-MB calibrator buffer (without bovine serum albumin)

Weighing MOPSO1.1263g, EDTA0.292g, 5g of compound enzyme protective agent AFP and 5g of compound enzyme protective agent HPD. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(2)100ml CK-MB calibrator buffer (1% bovine serum albumin)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)1g, a complex enzyme protective agent AFP 5g and a complex enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(3)100ml CK-MB calibrator buffer (3% bovine serum albumin)

(4)100ml CK-MB calibrator buffer (5% bovine serum albumin)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)5g, a complex enzyme protective agent AFP 5g and a complex enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

3. Preparation of CK-MB calibrator diluent containing composite enzyme protective agent AFP with different concentrations

(1)100ml CK-MB calibrator buffer (AFP without compound enzyme protective agent)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g and a compound enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(2)100ml CK-MB calibrator buffer (3% Complex enzyme protective agent AFP)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g, a complex enzyme protective agent AFP 3g and a complex enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(3)100ml CK-MB calibrator buffer (5% Complex enzyme protective agent AFP)

(4)100ml CK-MB calibrator buffer (10% Complex enzyme protective agent AFP)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g, a complex enzyme protective agent AFP 10g and a complex enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

4. Preparation of CK-MB calibrator diluent containing different concentrations of compound enzyme protective agent HPD

(1)100ml CK-MB calibrator buffer (without adding compound enzyme protective agent HPD)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g and a compound enzyme protective agent AFP 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(2)100ml CK-MB calibrator buffer (3% composite enzyme protectant HPD)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g, a compound enzyme protective agent AFP 5g and a compound enzyme protective agent HPD 3 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

(3)100ml CK-MB calibrator buffer (5% composite enzyme protectant HPD)

(4)100ml CK-MB calibrator buffer (10% composite enzyme protectant HPD)

Weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g, a complex enzyme protective agent AFP 5g and a complex enzyme protective agent HPD 10 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml.

5. Freeze-drying experiment of CK-MB calibrator

6. Stability testing of CK-MB calibrators

The 16 dilutions prepared in steps 1-4 were used to prepare 300ng/ml CK-MB lyophilized calibrators, which were placed in a 37 ℃ incubator for 3 days, 5 days, and 7 days, respectively, and the assay was performed as in example 1 with 2-8 ℃ control.

7. Effect of Diluent Components at different concentrations on the stability of CK-MB calibrators

(1) The stability of the calibrators was best when 10mM EDTA was added to the calibrant dilution as shown by comparing the stability of the calibrators at different concentrations of EDTA (0mM, 5mM, 10mM, 15mM) (see Table 17).

TABLE 17 EDTA concentration screening

(2) The stability of the calibrators was best when 3% bovine serum albumin was added to the calibrant dilution as shown by comparing the stability of the calibrators at different concentrations (0%, 1%, 3%, 5%) (see table 18).

TABLE 18 Bovine Serum Albumin (BSA) concentration screening

(3) By comparing the stability of the calibrators at different concentrations of the complex enzyme protectant AFP (0%, 3%, 5%, 10%), the results show that the stability of the calibrators was best when 5% of the complex enzyme protectant AFP was added to the calibrators dilution (see Table 19).

TABLE 19 Complex enzyme protectant AFP concentration Screen

(4) By comparing the stability of the calibrator under different concentrations of the complex enzyme protectant HPD (0%, 3%, 5%, 10%), the results show that the stability of the calibrator was the best when 5% of the complex enzyme protectant HPD was added to the calibrator dilution (see Table 20).

TABLE 20 Complex enzyme protectant HPD concentration screening

EXAMPLE seven

CK-MB calibrator redissolution stability test

1. The determination step comprises:

(1) the optimal diluent is prepared according to the following formula:

weighing MOPSO1.1263g, EDTA0.292g, Bovine Serum Albumin (BSA)3g, a complex enzyme protective agent AFP 5g and a complex enzyme protective agent HPD 5 g. Adding 60ml of purified water, adjusting the pH value to 7.2 after complete dissolution, and fixing the volume to 100 ml. Subpackaging the calibrator with 500 μ l per bottle by brown glass bottle, quickly freezing, freeze-drying in a freeze-dryer for 24-30h, sealing, and storing.

(2) And (2) preparing freeze-dried calibrators of 300ng/ml, 140ng/ml, 35ng/ml, 4ng/ml and 1ng/ml by using the diluent obtained in the step (1), after redissolving, storing for 30 days at the temperature of 2-8 ℃, comparing with the freeze-dried calibrators at the temperature of 2-8 ℃, and testing the redissolving stability of the calibrators.

(3) The determination method comprises the following steps:

a. placing the CK-MB antibody solution coated with the magnetic microspheres as a reagent 1 and the CK-MB antibody solution marked with acridinium ester as a reagent 2 at room temperature, and fully and uniformly mixing for 30 min;

b. adding 20 mul of redissolved calibrator solution into a reaction cup;

c. mu.l of reagent 1 (magnetic bead suspension) and 50. mu.l of reagent 2 (acridinium ester solution) were added to the reaction cuvette in this order;

d. after the solution in the reaction cup is fully and uniformly mixed, the mixture is incubated for 6min at 37 ℃;

e. placing the magnetic beads under a magnetic condition, and washing the magnetic beads for multiple times by using a magnetic bead washing liquid;

f. to the reaction cuvette, 100. mu.l of substrate solution A and substrate solution B were added, and the luminescence value was measured, and the luminescence intensity was expressed in Relative Light Units (RLU).

2. Measurement results

Through tests, the reconstitution stability of the lyophilized calibrator of the diluent of the present invention was tested, and the results show that the calibrator has good stability within 30 days of reconstitution (see table 21).

TABLE 21 reconstitution stability of calibrator

Example eight

Detection of white space limit

The detection method is carried out by referring to the test method of YY/T1233-.

The blank sample (sample diluent) was repeatedly tested 20 times, and the analytical sensitivity was calculated according to the test results using the formula, the results are shown in Table 23.

TABLE 22 examination results of initial calibration curves

	RLU	Concentration of
			S0	369	0
S1	2736	1
			S2	10795	4
S3	178105	35
			S4	661426	140
S5	1449379	300

TABLE 23 detection of blank limits

Two-point regression fitting is carried out according to the results of concentration-chemiluminescence (RLU) values between a zero-concentration calibrator (S0) and an adjacent calibrator (S1) to obtain a linear equation, then the RLU value corresponding to Mean +2SD obtained by detecting a blank sample (S0) with 20 holes is substituted into the equation, and the obtained concentration value is the analysis sensitivity, and the analysis sensitivity of the invention can reach 0.06ng/ml, which has great significance for the early diagnosis of AMI.

Example nine

Detection of precision within a day

The high and low standards were repeatedly tested 10 times each, and then CV was calculated based on the test results, which are shown in Table 24.

TABLE 24 measurement results of precision within days

Number of tests	QCL signal	QCH signal	QCL concentration	QCH concentration
						1	3635	524847	0.990285715	159.3409027
2	3302	508537	0.897685624	154.2831433
					3	4047	495755	1.105108612	150.3218863
4	3755	505908	1.02370137	153.4682135
					5	3746	511162	1.021194379	155.0969247
6	3758	537151	1.024537062	163.1586667
					7	3857	502976	1.052123002	152.5594695
8	3708	477823	1.010610758	144.7683599
					9	3752	500464	1.022865692	151.780992
10	3829	508045	1.044319337	154.1306276
					MEAN			1.019243155	153.8909186
CV			5.15％	3.21％

The precision of the invention can reach 5.15 percent and 3.21 percent, which has great significance for the diagnosis accuracy of AMI.

Example ten

Detection of linear correlation

The linear samples were examined in duplicate wells and the linear correlation coefficient between the theoretical and measured values was calculated for each point, and the results are shown in table 25.

TABLE 25 detection results of Linear dependence

	Luminous value 1	Luminous value of 2	Measured value (ng/ml)	Theoretical value (ng/ml)
					Sample 1	752782	779120	340.20	340.20
Sample 2	546884	551542	241.35	226.86
					Sample 3	292373	305655	128.87	113.52
Sample 4	152830	162028	66.47	56.85
					Sample 5	96318	92556	39.23	34.19
Sample 6	20341	19828	7.94	6.98
					Sample 7	579	466	0.18	0.18
Linear dependence				0.9973

The linear correlation of the present invention, R > 0.990. The complex aperture detects a linear sample (7 points), and calculates a linear correlation coefficient of a theoretical value and an actually measured value of each point. The theoretical concentration is not calculated by using a calibration curve, and the measured value is a sample value detected by using a Roche comparison machine. Figure 9 shows that the linear dependence is good.

EXAMPLE eleven

Accuracy detection

Diluting serum according to the ratio of 1:9, and detecting

The accuracy of the recovery test detection kit is adopted, and the detection results are shown in table 26.

TABLE 26 test results of the recovery test

	Concentration of solution A	Concentration of solution B	Mixed concentration	Recovery rate
						226.094	0.681158	22.27394
	228.0779	0.76603	22.39977
						210.8127	0.726591	21.62178
Mean value	221.6615	0.724593	22.0985
					Theoretical concentration			22.81828	96.85％

The recovery test result can reach more than 90 percent, which shows that the CK-MB detection method established by the invention has good accuracy and basically has no matrix effect.

Example twelve

Clinical detection application of CK-MB calibrator

28 clinical samples were collected and compared methodologically using the Roche CK-MB in vitro diagnostic kit.

The determination step comprises:

(1) the optimal diluent is prepared according to the following formula:

(2) Preparing calibrators of 300ng/ml, 140ng/ml, 35ng/ml, 4ng/ml and 1ng/ml by using the diluent obtained in the step (1), freeze-drying, and performing redissolution to obtain a standard curve;

(3) the determination method comprises the following steps:

b. adding 20 mul of redissolved calibrator solution into a reaction cup;

1. Measurement results

The diluent is prepared by adopting the inventionFIG. 8 shows a standard curve (logX-logY) of the lyophilized reconstituted calibrator²0.9975, indicating that the calibrator was linear well between 0 and 300 ng/ml. The determination result of the calibrator reconstituted after the dilution liquid is prepared and freeze-dried is compared with the methodology of the Rockwell CK-MB in-vitro diagnostic kit, the result is shown as figure 10, and the correlation coefficient R²0.9963, the results of the assay of the calibrator prepared with the diluent of the present invention are highly correlated with the results of the assay of the rockwell CK-MB kit, and the consistency is high.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A creatine kinase isoenzyme sample diluent, wherein the diluent comprises: a buffer solution with pH value of 7.2-8.5, an ion chelating agent and a protective agent, wherein,

2. The diluent of claim 1, wherein the buffer is a MOPSO buffer; preferably, the MOPSO buffer has a pH value of 7.2.

3. The diluent of any one of claims 1-2, wherein the protectant is selected from the group consisting of bovine serum albumin, complex enzyme protectant, AFP, and complex enzyme protectant, HPD, and the ion chelating agent is EDTA; wherein the content of the first and second substances,

the concentration of EDTA is 5-15mM, preferably 10 mM.

4. The diluent of any one of claims 1 to 3, wherein the sample is a calibrator; preferably, the calibrator is a lyophilized calibrator.

5. A creatine kinase isoenzyme detection kit, wherein the kit comprises: a first antibody coated on a magnetic microsphere, a second antibody labeled with a tracer label, and a creatine kinase isoenzyme sample diluent according to any one of claims 1 to 4; wherein the content of the first and second substances,

optionally, the test kit further comprises a calibrator.

6. The creatine kinase isoenzyme detection kit of claim 5, wherein the first antibody is CKMB-BB and the second antibody is CKMB-04; preferably, the mass ratio of the magnetic microspheres to the first antibodies is 100:0.5-1.25, and the mass ratio of the tracer labels to the second antibodies is 15-40: 1.

7. The creatine kinase isoenzyme detection kit of any one of claims 5-6, wherein the tracer label is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer label is an acridinium ester.

8. The creatine kinase isoenzyme detection kit of any one of claims 5-7, wherein the kit further comprises: magnetic microsphere cleaning solution, substrate solution A and substrate solution B; wherein the magnetic microsphere cleaning solution contains PBST (poly-p-phenylene benzobisoxazole) bufferThe substrate liquid A is H₂O₂And HNO₃The substrate solution B is a mixed solution of Triton X-100 and NaOH; preferably, said H₂O₂Is 0.01-5.0 percent by mass, and the HNO₃The concentration of the compound is 0.01-1.0mol/L, the mass fraction of the Triton X-100 is 0.01-2.0%, and the concentration of the NaOH is 0.05-1 mol/L.

9. The preparation method of the creatine kinase isoenzyme detection kit according to any one of claims 5 to 8, wherein the preparation method comprises:

tracer marker marking step: labeling the tracer label on the second antibody;

preparing a sample diluent: mixing the components of the sample dilution.

10. The method for preparing a creatine kinase isoenzyme detection kit according to claim 9,

and/or

11. Use of the creatine kinase isoenzyme sample diluent according to any one of claims 1 to 4 or the creatine kinase isoenzyme detection kit according to any one of claims 5 to 8 in the preparation of a reagent for diagnosing myocardial infarction.