CN112858678B - Creatine kinase isoenzyme detection kit, preparation method and application - Google Patents

Abstract

The invention relates to a creatine kinase isoenzyme detection kit, preparation method and use. Specifically, the creatine kinase isoenzyme detection kit of the present invention contains a sample diluent, wherein the diluent includes: a pH 7.2-8.5 buffer, an ion chelating agent and a protective agent. In the present invention, the creatine kinase isoenzyme calibrator has long reconstitution stability, long protection period, and stable storage and transportation. At the same time, the detection kit involved in the present invention has better sensitivity and specificity. While saving costs, it reduces the non-specific binding of antibodies to creatine kinase isoenzymes, improves detection sensitivity and detection stability, and It reduces non-specific binding and can quickly detect low-concentration creatine kinase isoenzymes without adding other catalysts, and can be used for early diagnosis of myocardial infarction.

Description

Creatine kinase isoenzyme detection kit, preparation method and use

Technical Field

The invention belongs to the field of immunoassay, and in particular relates to a creatine kinase isoenzyme detection kit, a preparation method and application thereof.

Background Art

In October 2007, the European Society of Cardiology (ESC), the American College of Cardiology (ACC), the American Heart Association (AHA) and the World Heart Federation (WHF) adopted a unified definition of myocardial infarction worldwide, defining acute myocardial infarction (AMI) as myocardial cell death due to myocardial ischemia. This definition has not changed in the new version. AMI is a serious disease of coronary atherosclerotic heart disease, with a dangerous onset and high mortality rate. The best time for treatment is within 120 minutes after onset. Therefore, clinical rapid detection is crucial for the diagnosis and control of AMI. Modern medical research shows that the early, rapid and correct clinical diagnosis of AMI can be improved by jointly detecting myocardial early markers (MYO), determination markers (cTnI) and injury markers (CK-MB). The detection of creatine kinase isoenzyme (Creatinekinase-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, and the diagnosis of brain diseases such as cerebral infarction, meningitis, encephalitis, etc.

Creatine kinase (CK) was formerly known as creatine phosphokinase (CPK). CK has a molecular weight of 81,000 and consists of two subunits. It is usually found in the cytoplasm and mitochondria of animal heart, muscle, brain and other tissues. It is an important kinase that is directly related to intracellular energy flow, muscle contraction, and ATP regeneration. It reversibly catalyzes the transphosphoacyl reaction between creatine and ATP. CK catalyzes the phosphorylation of creatine with the participation of ATP to generate ATP and creatine phosphate. This enzyme-catalyzed reaction is reversible. In 1979, it was first discovered that the content of creatine kinase in the serum of prostate cancer patients was significantly increased, and the same discovery was made in patients with other types of cancer. With the deepening of research, people have begun to search for new anti-tumor drugs by screening creatine kinase inhibitors in recent years. It is precisely because of the important physiological functions of creatine kinase that it has attracted widespread attention and in-depth research.

Each subunit of the creatine kinase isozyme (CK-MB) dimer has a small domain at the N-terminus connected to a large domain at the C-terminus through a linker sequence. In the entire dimer, 98% of the tyrosine residues are wrapped inside the spatial structure, and only 2% are exposed on the surface of the structure; in addition, each subunit surface contains 2 reactive histidine residues and 2 reactive sulfhydryl groups. Its complex spatial structure brings great difficulties to the application of in vitro recombinant technology. Therefore, the difficulty of recombinant is high. At present, the high-quality CK-MB on the market is all blood-derived. However, due to the limited source and high price, it seriously hinders the research and development of CK-MB diagnostic products. Although there are many reports on CK-MB at home and abroad, most of the research is academic research, mainly focusing on the structure and function of the protease; and there are few studies on its product application. In 2001, Yoshiko Sunahara et al. simultaneously expressed three isozymes of MB, MM, and BB, purified them separately, and mixed them in a ratio of 50:40:10. Using the principle of protein dynamics, they completed the late folding of MB and obtained a protein with high CK activity. However, the study ended there, without any research on the stability, specificity, sensitivity and application of the protein as a biological raw material. In addition, in this study, the expression and mixing ratio of MB were complex, and the process stability was difficult to control, so it was impossible to use it for product production. Huang Y et al. (2006) chose to express their isoenzyme MM in vitro, but the spatial structure of MM or BB isoenzyme is very different from MB, and it is far different from the natural structure of MB, so it is impossible to replace CK-MB as a calibration quality control product for clinical testing.

Since creatine kinase isoenzyme is a biological macromolecular protein and exists in the form of dimers, it is extremely unstable. Its harsh storage, transportation, and use conditions bring many inconveniences to the use of proteins. The calibrators of most companies' test kits on the market are stored in the form of lyophilized products. Low-temperature lyophilization is currently the most effective and most commonly used method for drying and preserving biological products such as proteins and microorganisms. At present, commonly used recombinant protein lyophilization protectants include sugars and polymers, polyols, surfactant-free, water-soluble amino acids, and certain salts and amines, but the stability is not good enough. At present, there are many immunoassay methods for creatine kinase isoenzymes, including radioisotope immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography (ICA), and chemiluminescence assay (CLIA) which has been widely used in recent years.

The detection principle of radioisotope immunoassay (RIA) is to competitively react the isotope-labeled antigen and the unlabeled antigen to be tested with insufficient specific antibodies, and then measure the radioactivity of the reaction to obtain the amount of the antigen to be tested. This method is an ultra-microanalysis technology that has been increasingly widely used since the 1980s.

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

As a method of POCT testing, colloidal gold immunochromatography has the advantages of small sample loading, simplicity and speed, and is suitable for bedside testing. Its principle is as follows. Taking the double antibody sandwich method as an example, when the sample containing the antigen is dripped into the absorption well, the antigen first binds to the antibody labeled with colloidal gold to form an antigen-antibody complex, and then another antigen fixed on the sample pad will capture the complex to form a double antibody sandwich complex and produce a color band, which is detected by the immunoassay method.

Chemiluminescence is an immunoassay technology that has gradually emerged in recent years. It is a product of the close combination of the immune response system and chemiluminescence technology. Among them, chemiluminescence technology uses luminescent substances, such as acridinium esters, to form an excited intermediate after oxidation by an oxidant and catalysis by a catalyst, and then uses a corresponding measuring instrument to measure the light quantum yield generated by the intermediate when it returns to the ground state. The immune response system is to mark the luminescent marker substance on the antigen or antibody, and then detect it by forming an antigen-antibody complex. This technology has the advantages of strong specificity, high sensitivity, good precision, wide linear range, high throughput and easy automation.

Direct chemiluminescent immunoassay refers to directly labeling antibodies (antigens) with acridinium esters. After an immune reaction occurs with the corresponding antigens (antibodies) in the sample to be tested, a solid-phase coated antibody-antigen to be tested-acridinium ester labeled antibody complex is formed. Under the action of oxidants (H ₂ O ₂ ) and NaOH, acridinium esters can decompose and emit light without the need for a catalyst. The strong direct luminescence is completed within one second, which is a fast flashing luminescence. The luminescence intensity is proportional to the amount of antigen to be tested, and the content of the antigen to be tested can be calculated from the standard curve.

At present, although there are chemiluminescence kits for detecting creatine kinase isoenzymes on the market, many of them have shortcomings such as insufficient sensitivity and low specificity. At the same time, the calibrator is not stable enough, is unstable in liquid environment, and is easily hydrolyzed, which is not conducive to long-term storage.

For example, CN102435738A, CN107389941A, CN109490549A, and CN109613242A all disclose a kit for detecting creatine kinase isoenzymes by chemiluminescence. However, the stability of the calibrators of the creatine kinase isoenzyme kits disclosed in the aforementioned documents is not good enough.

Radioisotope immunoassay (RIA) has the problems of complicated operation, long time consumption, poor reproducibility of results, poor sensitivity and specificity, and the risk of radioactive contamination. It is not suitable for large-scale testing and is now basically not used.

Enzyme-linked immunosorbent assay (ELISA) has the disadvantages of cumbersome operation, low sensitivity, narrow linear range and long detection cycle. In the early stage of AMI, the content of creatine kinase isoenzyme in serum is often very low. Due to low sensitivity and long detection cycle, this method cannot quickly and accurately detect early AMI, which further limits the use of ELISA in myocardial detection.

As a POCT test method, colloidal gold immunochromatography has the advantages of small sample loading, simplicity and rapidity, and is suitable for bedside testing. However, due to its poor sensitivity, its application in the clinical detection of creatine kinase isoenzymes is further limited.

Enzymatic chemiluminescence is a horseradish peroxidase system and an alkaline phosphatase system. It has the disadvantages of long excitation and luminescence time, slow test speed, enzymes being easily affected by ambient temperature, and reagent stability not as good as direct chemiluminescence.

Electrochemiluminescence is a method in which the antigen-antibody complex labeled with terpyridine ruthenium undergoes a redox reaction under the action of tripropylamine to emit visible light. The excitation process is complex and time-consuming, with each luminescence speed being about 25 seconds.

Liquid CK-MB calibrators have a relatively short storage time, which limits the shelf life of the CK-MB test kit.

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

Therefore, it is necessary to provide a stable CK-MB calibrator diluent so that CK-MB can be dried at low temperature and quickly absorb water to restore to the fresh state before drying. At the same time, it is necessary to overcome the shortcomings of the prior art to solve the problems of short storage time and inconvenient transportation of creatine kinase isoenzyme calibrators and poor resolubility of freeze-dried products.

Summary of the invention

Problem that the invention aims to solve

The object of the present invention is 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 aforementioned creatine kinase isoenzyme detection kit.

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

Solutions for solving problems

Specifically, the present invention records the following technical solutions.

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

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

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

[3] The diluent according to any one of [1] to [2], wherein the protective agent is selected from bovine serum albumin, complex enzyme protective agent AFP and complex enzyme protective agent HPD, and the ion chelating agent is EDTA; wherein,

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

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

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

The concentration of EDTA is 5-15 mM, 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 freeze-dried 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 marker, and a creatine kinase isoenzyme sample diluent as described in any one of [1] to [4]; wherein,

The specific binding sites of the creatine kinase isozymes recognized by the first antibody and the second antibody are different;

Optionally, the detection 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 antibody is 100:0.5-1.25, and the mass ratio of the tracer marker to the second antibody is 15-40:1.

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

[8] A creatine kinase isozyme detection kit according to any one of [5] to [7], wherein the kit further _comprises : a magnetic microsphere cleaning solution, a substrate solution A and a substrate solution B; wherein the magnetic microsphere cleaning solution contains PBST buffer, the substrate solution A is a mixture of _H2O2 and _HNO3 , and the substrate solution B is a mixture of Triton X-100 and NaOH; preferably, _the mass fraction of _H2O2 is 0.01-5.0%, the concentration of _HNO3 is 0.01-1.0 mol/L, the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1 mol/L.

[9] A method for preparing a creatine kinase isoenzyme detection kit according to any one of [5] to [8], wherein the preparation method comprises:

Magnetic microsphere coating step: coating the first antibody on the magnetic microsphere;

Tracing marker labeling step: labeling the tracing marker on the second antibody;

The step of preparing the sample diluent is to mix the components in the sample diluent.

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

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

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

and/or

4-Hydroxyethylpiperazineethanesulfonic acid was used as a buffer in the tracer labeling step.

[11] Use of the creatine kinase isoenzyme sample dilution 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.

Effects of the Invention

In one embodiment, the present invention uses a combination of various types of protective agents to prepare samples for detecting creatine kinase isoenzymes, such as freeze-dried calibrators, which ultimately ensure that the freeze-dried calibrators of creatine kinase isoenzymes have long reconstitution stability, long protection period, and stable storage and transportation.

In another embodiment, the present invention provides a detection kit for creatine kinase isoenzymes, which has better sensitivity and specificity and can be rapidly detected without adding other catalysts, for example, by direct chemiluminescence, to rapidly detect low concentrations of creatine kinase isoenzymes, which can be used for early diagnosis of myocardial infarction.

In another embodiment, the detection kit provided by the present invention reduces the non-specific binding of antibodies to creatine kinase isozymes, improves detection sensitivity and detection stability, and reduces non-specific binding while saving costs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the stability comparison of the calibrators under various buffer systems (where the ordinate represents the decrease in relative light units P at the concentration point, and the abscissa represents the measurement time points of the 3rd day, the 5th day, and the 7th day);

FIG2 shows the effect of different protective agents on the stability of the calibrator (where the ordinate represents the decrease in relative light unit P at the concentration point, and the abscissa represents the measurement time points of the 3rd day, the 5th day, and the 7th day);

FIG3 shows the effect of different concentrations of EDTA on the stability of the calibrator (where the ordinate represents the decrease in relative light units P at the concentration point, and the abscissa represents the measurement time points of the 3rd day, the 5th day, and the 7th day);

FIG4 shows the effect of different concentrations of bovine serum albumin (BSA) on the stability of the calibrator (wherein the ordinate represents the decrease in relative light unit P at the concentration point, and the abscissa represents the measurement time points of the 3rd day, the 5th day, and the 7th day);

FIG5 shows the effect of different concentrations of the composite enzyme protectant AFP on the stability of the calibrator (wherein the ordinate represents the decrease in relative light unit P at the concentration point, and the abscissa represents the measurement time points of the 3rd day, the 5th day, and the 7th day);

FIG6 shows the effect of different concentrations of the composite enzyme protectant HPD on the stability of the calibrator (where the ordinate represents the decrease in the relative light unit P at the concentration point, and the abscissa represents the measurement time points of the 3rd day, the 5th day, and the 7th day);

FIG7 shows the reconstitution stability of the calibrator (wherein the ordinate represents the total decrease, and the abscissa represents the measurement time points of the 3rd day, the 7th day, the 15th day, the 23rd day and the 30th day; FIG7 shows that after the calibrator diluent of the present invention is made into a lyophilized product, the reconstitution stability decreases by about 8% after being reconstituted and stored at 2-8° C. for 30 days, and the stability is good);

FIG8 shows a standard curve prepared using a calibrator prepared with the diluent of the present invention (a standard curve (logX-logY) prepared using a calibrator prepared with the diluent of the present invention after freeze-drying and reconstitution, the correlation ^R2 value can reach 0.9975. FIG8 shows that the calibrator of the present invention has good linearity between 0-300 ng/ml);

FIG9 shows the results of clinical sample determination, indicating that the linear correlation R ² value of the reagent of the present invention can reach 0.9973;

FIG. 10 shows the results of clinical sample determination (wherein the ordinate is the results of the detection method described in Example 4 of the present invention, and the abscissa is the results of the Beckman CK-MB kit determination, and the correlation R ² value can reach 0.9963).

DETAILED DESCRIPTION

Other purposes, 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 (although representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

It should be noted that:

In this specification, the term "magnetic microspheres" and "magnetic beads" have the same meaning, referring to superparamagnetic microspheres with small particle size, generally with super strong paramagnetism. The surface of the microspheres can be coated with specific antibodies, receptors, etc., and used to separate and purify the target in the sample. Magnetic beads have been widely used in many fields such as immunoassay, nucleic acid separation and extraction, cell sorting, enzyme fixation, etc.

In this specification, the references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

<First aspect>

The present invention provides a creatine kinase isozyme sample diluent, wherein the diluent comprises: a buffer solution with a pH 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, complex enzyme protective agent AFP and complex enzyme protective agent HPD.

Furthermore, the present invention also provides 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 marker, and the aforementioned creatine kinase isoenzyme sample diluent;

Optionally, the detection 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 optional embodiment of the present invention, the first antibody and the second antibody in the present invention are both monoclonal antibodies against CK-MB.

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

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

The present invention does not specifically limit the type of tracer markers applicable to the present invention. For example, the tracer markers may be luminol, alkaline phosphatase, horseradish peroxidase, acridinium esters, and adamantane, etc. Preferably, the tracer marker is acridinium ester.

The present invention does not specifically _limit the type of magnetic microspheres applicable to the present invention, and they can be magnetic beads commonly used in the art. As one embodiment of the present invention, the magnetic beads used in the present invention are micron-sized solid-phase microspheres with paramagnetism and a maximum protein adsorption capacity formed by compounding nano-sized _Fe2O3 and _Fe3O4 magnetic particles with _polymer materials. The magnetic microspheres can be rapidly magnetized under the action of an external magnetic field, and the remanence is zero after the magnetic field disappears. In the present invention, there is no specific limitation on the type of polymer material used for compounding with it.

The particle size of the magnetic beads used in the present invention should be 1-5 μm. As one of the embodiments of the present invention, the magnetic beads can be surface modified to add multiple active groups. The types of active groups in the present invention include but are not limited to -OH and -COOH.

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

In the present invention, EDTA in the above diluent is an excellent metal ion complexing agent, detergent, and blood anticoagulant. It is often used as a calcium ion chelating agent in biochemical research to eliminate the inhibitory effect of calcium or other trace heavy metals in enzyme catalysis reactions. After adding EDTA with a final concentration of 5-15mM, the inhibitory effect of other trace heavy metal ions on enzyme reactions can be avoided, thereby increasing the stability of the protein. In some specific embodiments of the present invention, when the concentration of EDTA is 10mM, the stability of the CK-MB calibrator is the highest.

Bovine serum albumin (BSA) has the functions of a cryoprotectant and a dehydration protectant, and can provide good protection for the CK-MB antigen. In some specific embodiments of the present invention, the addition of bovine serum albumin preferably has a final concentration of 1%-10%, and more preferably 2%-5%, and the stability will be significantly improved. In other embodiments of the present invention, when the concentration of bovine serum albumin in the diluent is 3%, the stability of the CK-MB freeze-dried calibrator can be improved to the greatest extent.

The compound enzyme protectant has a protective and stabilizing effect on proteins, enzymes, lipids and other substances in the blood. On the one hand, it maintains the environment required for the physiological activity of enzymes and protein structures; on the other hand, it can improve the efficiency of enzymes and proteins, avoid activity loss during the freeze-drying process and after re-dissolution, and is more conducive to long-term storage. When a final concentration of 5% compound enzyme protectant AFP and 5% compound enzyme protectant HPD are added, the stability of the CK-MB freeze-dried calibrator is the highest. The CK-MB freeze-dried calibrator prepared with the above diluent is used to prepare a standard curve, with a correlation coefficient of R ² =0.99 or above, and the calibrator has good linearity between 0-300ng/ml.

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

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

The magnetic microsphere cleaning solution in the present invention is a PBST buffer solution; preferably, the magnetic microsphere cleaning solution also includes a preservative. The preservative applicable to the present invention may be a preservative 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-isothiazoline-3-one and 5-chloro-2-methyl-4-isothiazoline-3-one, which is a safer preservative and one of the commonly used preservatives in immunodiagnosis) and antibiotics. One or more.

The substrate solution A in the present invention is a mixture of _{H2O2 and HNO3} , preferably, the mass fraction _{of H2O2} is 0.01-5.0%, and the concentration of _HNO3 is 0.01-1.0 mol/L. The substrate solution B is a mixture of Triton X-100 and NaOH, preferably, the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1 mol/L.

In addition to Triton X-100, the surfactant applicable to the present invention can also be a surfactant commonly used in the art, such as one or more of Triton X-405, Tween 20 and Tween 80. In the kit of the present invention, the addition amount of the above surfactant is 0.1-2‰, which is more conducive to the dispersion of magnetic microspheres.

<Second Aspect>

The present invention also provides a method for preparing the creatine kinase isoenzyme CK-MB detection kit according to the first aspect, wherein the preparation method comprises:

Magnetic microsphere coating step: coating the first antibody on the magnetic microsphere;

Tracing marker labeling step: labeling the tracing marker on the second antibody;

The step of preparing the sample diluent is to mix the components in the sample diluent.

As one of the embodiments of the present invention, 2-(N-morpholino)ethanesulfonic acid (ie, MES) buffer is used in the step of coating magnetic microspheres with the first antibody.

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

As one of the embodiments of the present invention, the antibody coupling buffer used in the step of labeling the tracer marker with the second antibody is selected from 4-hydroxyethylpiperazineethanesulfonic acid (ie, HEPES) buffer.

As one of the embodiments of the present invention, the labeling time of the second antibody labeling tracer marker is 0.5-2 hours; optionally, the labeling time of the second antibody labeling tracer marker is 1 hour.

In one embodiment of the present invention, the creatine kinase isoenzyme CK-MB freeze-dried calibrator is a MOPSO buffer containing a certain amount of CK-MB antigen, wherein a certain preservative is added, and then freeze-dried. The calibrator can be stably stored at 4°C.

<Third Aspect>

The present invention also provides a use of the creatine kinase isoenzyme sample diluent or the creatine kinase isoenzyme detection kit according to <the first aspect> in preparing a diagnostic reagent for myocardial infarction.

Example

Embodiment 1

Preparation 1: Magnetic microsphere suspension coated with CK-MB mAb

(1) Take 10 mg of magnetic microsphere solution, add 1 ml of 0.1 M MES buffer, vortex to mix, and adsorb on the magnet for 5 to 10 min until the magnetic beads are completely adsorbed to one side of the tube wall. Discard the supernatant, repeat the above washing steps 3 to 5 times, add 1 ml of 0.1 M MES, and vortex to mix;

(2) Add 100 μg of CK-MB first strain monoclonal antibody (mass ratio of magnetic beads to antibody is 100:1), vortex to mix, and incubate at 37°C for 30 min;

(3) Add 10 μL of 10 mg/ml 1-(3-dimethylaminopropyl)-3-ethyldiimide hydrochloride (EDC), vortex to mix, place in a 37°C incubator, and incubate for 1.5 h;

(4) Add 200 μL of 20% BSA solution and block for 2 h.

(5) Add 1 ml of luminescence recovery solution 2 (5 g of bovine serum albumin + 400 ml of 0.025 M Tris-HCl buffer + 1-2‰ Tween-80 + 100 ml of newborn calf serum) to the blocked magnetic bead suspension, adsorb with a magnet, remove the supernatant, and repeat the above washing steps 3 to 5 times.

(6) Add 1 ml of luminescence recovery solution 2 to the prepared magnetic beads and store them at 2-8°C for a long term.

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

(1) Place 1 mg of CK-MB second monoclonal antibody in a dialysis bag and dialyze for 24 hours using no less than 2 L of 0.05 M HEPES buffer (pH = 8.0), changing the buffer four times. After dialysis, add the antibody to a Millipore Amicon Μltra ultrafiltration centrifuge tube for concentration to a concentration greater than 5 mg/ml.

(2) Place the concentrated CK-MB monoclonal antibody in a 0.5 ml centrifuge tube, add 100 μL of 5 mg/ml NSP-SA-NHS solution dissolved in DMF, so that the molar ratio of NSP-SA-NHS to CK-MB monoclonal antibody is not less than 15:1, mix well, react at room temperature (protected from light) for 2 h, add 100 μL of 100 mg/ml lysine solution as a blocking buffer, react for 30 min, and terminate the reaction.

(3) Place the marker in a dialysis bag and dialyze for 24 h with no less than 2 L of citric acid-sodium citrate buffer (0.01 mol/L, pH = 4.5), changing the solution four times in between to separate free NSP-SA-NHS.

(4) Collect the dialyzed marker, add 5% BSA solution to make the final BSA concentration 1%, then add an equal volume of glycerol and store at -20°C.

Preparation 3: Preparation of Calibrators

Take CK-MB working solution and dilute it to high and low point calibration solutions of 4 ng/ml and 140 ng/ml using MOPSO solution containing 10 mM EDTA, 3% bovine serum albumin, 5% complex enzyme protectant AFP and 5% complex enzyme protectant HPD.

The components of the kit are respectively constituted by using the magnetic microsphere suspension coated with the first anti-CK-MB antibody prepared in Preparation 1, the second CK-MB antibody labeled with acridinium ester in Preparation 2, and the high and low point calibrators in Preparation 3.

Preparation 4: Preparation of chemiluminescent substrate solution

The chemiluminescent substrate solution A is a mixed solution of H ₂ O ₂ and HNO ₃ , wherein the mass fraction of H ₂ O ₂ is 0.01-5.0%, and the concentration of HNO ₃ is 0.01-1.0 mol/L.

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

Preparation 5: Preparation of cleaning solution

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

Embodiment 2

The combination screening of antibodies used in the creatine kinase isoenzyme detection kit

(1) Antibody pairing screening

There are four CK-MB antibodies used in the present invention: antibody 1: CKMB-10-C40A (Fitzgerald, Lot: M9910207), antibody 2: CKMB-03 (Fei Peng, Lot: 20160426-2); antibody 3: CKMB-04 (Fei Peng, Lot: 2071104), antibody 4: CKMB-BB (biospacific, Lot: A5481). Antibodies 1, 3, and 4 are coated with magnetic microspheres to form antibody 1-magnetic microspheres, antibody 3-magnetic microspheres, and antibody 4-magnetic microspheres, and antibodies 2 and 3 are labeled with acridinium ester to form antibody 2-acridinium ester and antibody 3-acridinium ester. The above antibodies are combined and paired, and serum samples are tested to screen the optimal combination.

Table 1 Antibody pairing combination screening results

As shown in Table 1, in combination 4, antibody 4 is combined with antibody 2 and cannot bind to creatine kinase isoenzymes in serum samples; in combination 2 and combination 3, the pairing of antibody 1, antibody 3 and antibody 3, antibody 2, the overall signal value of creatine kinase isoenzymes detected is low, and the discrimination is low; in combination 5, the pairing of antibody 4 and antibody 3, the results of various indicators are better.

Table 2 Serum sample compliance test results

Table 3 Blood homologous sample compliance test results

Referring to the results in Tables 2 and 3, the combined sample consistency rate of Antibody 4 and Antibody 3 and the homologous sample consistency rate are both good.

Embodiment 3

Optimization of the preparation process of magnetic bead acridinium ester

(1) Optimization process of magnetic bead-coated antibody

Table 4 Optimization of magnetic bead-coated antibody coupling buffer

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

After comprehensive comparison of the data from each group, MES buffer was selected as the magnetic bead coating coupling buffer.

Table 5 Screening results of magnetic bead antibody usage 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 Compliance rate 0.9998 0.9997 0.9998 0.9996

As the amount of antibody added increases, the antigen detection signal value also increases, and has no significant effect on the antigen matching rate. When the magnetic beads: antibody ratio reaches 100:1.25, the overall luminescence value does not increase significantly. Therefore, the present invention selects 100:1 as the magnetic beads: antibody addition ratio.

Table 6 Blocking solution screening

Sample No. 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 Compliance rate 0.9999 0.9992 0.9994

The three blocking solutions had no significant effect on the detection compliance rate of antigen dilution solution. The luminescence value of 20% BSA was slightly higher, and the discrimination of low value detection was slightly higher. Therefore, 20% BSA was selected as the blocking solution.

(2) Optimization process of acridinium ester labeled antibodies

Table 7 Optimization of coupling buffer for acridinium ester labeled antibodies

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 Compliance rate 0.9998 0.9996 0.9986

The three coupling buffers have no significant effect on the detection compliance rate of the antigen dilution solution. The detection signal value of the HEPES sample is generally higher, and the low value distinction range is larger. The present invention selects HEPES as the coupling buffer for the acridinium ester-labeled antibody.

Table 8 Optimization of coupling time of acridinium ester labeled antibodies

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 Compliance rate 0.9998 0.9998 0.9997

The antibody was coupled to the acridinium ester for 1 h, and the reaction reached a plateau.

Table 9 Screening of the ratio of acridinium ester antibodies

Acridinium 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 Compliance rate 0.9998 0.9998 0.9999 0.9998

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

Embodiment 4

Selection of pH value for CK-MB calibrator dilution

1. Preparation of CK-MB calibrator dilutions in different buffer systems

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

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

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

Weigh 1.19156 g HEPES, 0.292 g EDTA, 3 g bovine serum albumin (BSA), 5 g complex enzyme protectant AFP, and 5 g complex enzyme protectant HPD. Add 60 ml purified water, adjust the pH to 7.5 after complete dissolution, and make up to 100 ml.

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

Weigh 0.60566 g of Tris-HCl (Sinopharm, 20170216), 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of complex enzyme protectant AFP, and 5 g of complex enzyme protectant HPD. Add 60 ml of purified water, adjust the pH to 8.5 after complete dissolution, and make up to 100 ml.

2. Lyophilization experiment of CK-MB calibrator

The calibrant was packaged in brown glass bottles at 500 μl per bottle, quickly frozen, placed in a freeze dryer for 24-30 hours, and sealed for storage.

3. Stability test of CK-MB calibrator

Prepare 300 ng/ml freeze-dried calibrators using the three dilutions prepared in step 1, place them in a 37°C incubator for 3, 5, and 7 days, respectively, and use 2-8°C as a control for the following experiments:

(1) Mix the CK-MB antibody solution coated with magnetic microspheres as reagent 1 and the CK-MB antibody solution labeled with acridinium ester as reagent 2 at room temperature for 30 minutes;

(2) Add 20 μl of the prepared antigen solution into the reaction cup;

(3) Add 50 μl of reagent 1 (magnetic bead suspension) and 50 μl of reagent 2 (acridinium ester solution) into the reaction cup in sequence;

(4) After the solution in the reaction cup is fully mixed, incubate at 37°C for 6 min;

(5) placing under magnetic conditions and washing multiple times with a magnetic bead washing solution;

(6) Add 100 μl of substrate solution A and substrate solution B to the reaction cup and detect the luminescence value. The luminescence intensity is expressed in relative light units (RLU).

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

In this experiment, different buffer systems were prepared to study the stability of the CK-MB project under the conditions of pH 7.2-8.5. The calibration products were prepared with three buffers: MOPSO weak base system at pH 7.2, HEPES weak base system at pH 7.5, and Tris-HCL alkaline system at pH 8.5. They were placed in a 37°C constant temperature incubator for stability experiments. As shown in Table 1-3, point P is a 300ng/ml calibrator, and point O is a calibrator buffer. The change in relative light units (RLU) at point P was verified under different buffer systems. The smaller the change, the more stable the calibrator. The results showed that under the weak base conditions of MOPOS at pH 7.2, the decrease in point P was the lowest, below 15%, and the stability of the calibrator was relatively high. (See Tables 10-12)

Table 10 MOPSO buffer

Table 11 HEPES buffer

Table 12 Tris-HCL buffer

Embodiment 5

Selection of protective agent for CK-MB calibrator diluent

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

(1) 100 ml CK-MB calibration buffer (protective agent is bovine serum albumin)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, and 5 g of bovine serum albumin (BSA). Add 60 ml of purified water, adjust the pH to 7.2, and make up to 100 ml after the mixture is completely dissolved.

(2) 100 ml CK-MB calibrator buffer (protective agent is casein)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, and 5 g of Casein. Add 60 ml of purified water, and adjust the pH to 7.2 after the mixture is completely dissolved, and make up to 100 ml.

(3) 100 ml CK-MB calibration buffer (protective agent is complex enzyme protective agent AFP)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, and 5 g of complex enzyme protectant AFP. Add 60 ml of purified water, and adjust the pH value to 7.2 after they are completely dissolved, and make up to 100 ml.

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

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, and 5 g of compound enzyme protective agent HPD. Add 60 ml of purified water, and adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

2. Lyophilization experiment of CK-MB calibrator

The calibrant was packaged in brown glass bottles at 500 μl per bottle, quickly frozen, placed in a freeze dryer for 24-30 hours, and sealed for storage.

3. Stability test of CK-MB calibrator

The four dilutions prepared in step 1 were used to prepare 300 ng/ml CK-MB freeze-dried calibrators, which were placed in a 37°C constant temperature incubator for 3 days, 5 days and 7 days, respectively, and 2-8°C conditions were used as controls. The test method was the same as in Example 1.

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

This experiment used different protective agents in the MOPSO buffer system at pH 7.2. 5% bovine serum albumin (BSA), 5% Caesin, 5% complex enzyme protective agent AFP, and 5% complex enzyme protective agent HPD were prepared into calibration diluents respectively. The calibration products were prepared with these four calibration diluents and stability experiments were carried out. As shown in Table 13-16, point P is the 300ng/ml calibration product, and point O is the calibration buffer. The relative light unit (RLU) of point P changes, and the smaller the change, the more stable the calibration product. The results show that the P point of the calibration product diluents with the addition of 5% bovine serum albumin, 5% complex enzyme protective agent AFP and 5% complex enzyme protective agent HPD has a lower decrease, and the stability of the calibration product is higher. (See Table 13-16).

Table 13 Effect of 5% bovine serum albumin on the stability of calibrators

Table 14 Effect of 5% Casein on the stability of calibrators

Table 15 Effect of 5% compound enzyme protective agent AFP on the stability of calibrators

Table 16 Effect of 5% compound enzyme protective agent HPD on the stability of calibrators

Embodiment 6

Screening of the most suitable concentration of each component in the CK-MB calibrator dilution solution

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

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

Weigh 1.1263 g of MOPSO, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, and adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

(2) 100 ml CK-MB Calibrator Buffer (5 mM EDTA)

Weigh 1.1263 g of MOPSO, 0.146 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

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

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

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

Weigh 1.1263 g of MOPSO, 0.438 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

2. Preparation of CK-MB calibrator dilutions containing different concentrations of bovine serum albumin (BSA)

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

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, and adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

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

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 1 g of bovine serum albumin (BSA), 5 g of complex enzyme protectant AFP, and 5 g of complex enzyme protectant HPD. Add 60 ml of purified water, adjust the pH to 7.2 after complete dissolution, and make up to 100 ml.

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

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

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

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 5 g of bovine serum albumin (BSA), 5 g of complex enzyme protectant AFP, and 5 g of complex enzyme protectant HPD. Add 60 ml of purified water, adjust the pH to 7.2 after complete dissolution, and make up to 100 ml.

3. Preparation of CK-MB calibrator dilutions containing different concentrations of complex enzyme protectant AFP

(1) 100 ml CK-MB calibration buffer (without AFP)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), and 5 g of compound enzyme protective agent HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

(2) 100 ml CK-MB calibration buffer (3% complex enzyme protectant AFP)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 3 g of complex enzyme protectant AFP, and 5 g of complex enzyme protectant HPD. Add 60 ml of purified water, adjust the pH to 7.2 after complete dissolution, and make up to 100 ml.

(3) 100 ml CK-MB calibration buffer (5% complex enzyme protectant AFP)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

(4) 100 ml CK-MB calibration buffer (10% complex enzyme protectant AFP)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 10 g of complex enzyme protectant AFP, and 5 g of complex enzyme protectant HPD. Add 60 ml of purified water, adjust the pH to 7.2 after complete dissolution, and make up to 100 ml.

4. Preparation of CK-MB calibrator dilutions containing different concentrations of HPD compound enzyme protectant

(1) 100 ml CK-MB calibration buffer (without HPD)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), and 5 g of complex enzyme protectant AFP. Add 60 ml of purified water, adjust the pH to 7.2, and make up to 100 ml after the mixture is completely dissolved.

(2) 100 ml CK-MB calibration buffer (3% complex enzyme protective agent HPD)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of complex enzyme protectant AFP, and 3 g of complex enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

(3) 100 ml CK-MB calibration buffer (5% complex enzyme protective agent HPD)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

(4) 100 ml CK-MB calibration buffer (10% complex enzyme protective agent HPD)

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of complex enzyme protectant AFP, and 10 g of complex enzyme protectant HPD. Add 60 ml of purified water, adjust the pH to 7.2 after complete dissolution, and make up to 100 ml.

5. Lyophilization experiment of CK-MB calibrator

The calibrant was packaged in brown glass bottles at 500 μl per bottle, quickly frozen, placed in a freeze dryer for 24-30 hours, and sealed for storage.

6. Stability test of CK-MB calibrator

The 16 dilutions prepared in steps 1-4 were used to prepare 300 ng/ml CK-MB freeze-dried calibrators, which were placed in a 37°C constant temperature incubator for 3 days, 5 days and 7 days, respectively, and 2-8°C conditions were used as controls. The test method was the same as in Example 1.

7. Effects of different concentrations of diluent components on the stability of CK-MB calibrators

(1) By comparing the stability of the calibrators under different EDTA concentrations (0 mM, 5 mM, 10 mM, 15 mM), the results showed that the stability of the calibrators was the best when 10 mM EDTA was added to the calibrator diluent (see Table 17).

Table 17 EDTA concentration screening

(2) By comparing the stability of the calibrators under different concentrations of bovine serum albumin (0%, 1%, 3%, 5%), the results showed that the stability of the calibrators was the best when 3% bovine serum albumin was added to the calibrator diluent (see Table 18).

Table 18 Bovine serum albumin (BSA) concentration screening

(3) By comparing the stability of the calibrator under different concentrations of the complex enzyme protectant AFP (0%, 3%, 5%, 10%), the results showed that the stability of the calibrator was the best when 5% of the complex enzyme protectant AFP was added to the calibrator diluent (see Table 19).

Table 19 Screening of AFP concentration of compound enzyme protective agent

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

Table 20 Composite enzyme protective agent HPD concentration screening

Embodiment 7

Stability test of reconstitution of CK-MB calibrator

1. Determination steps:

(1) Prepare the optimal dilution according to the following formula:

Weigh 1.1263g MOPSO, 0.292g EDTA, 3g bovine serum albumin (BSA), 5g complex enzyme protectant AFP, and 5g complex enzyme protectant HPD. Add 60ml purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100ml. Use brown glass bottles to pack the calibrant at 500μl per bottle, freeze quickly, place in a freeze dryer for 24-30h, and seal for storage.

(2) The diluted solution obtained in step (1) was used to prepare lyophilized calibrators at concentrations of 300 ng/ml, 140 ng/ml, 35 ng/ml, 4 ng/ml, and 1 ng/ml. After reconstitution, the calibrators were stored at 2-8°C for 30 days. The reconstitution stability of the calibrators was tested by comparison with the lyophilized calibrators stored at 2-8°C.

(3) Determination method:

a. The CK-MB antibody solution coated with magnetic microspheres as reagent 1 and the CK-MB antibody solution labeled with acridinium ester as reagent 2 were placed at room temperature and mixed thoroughly for 30 minutes;

b. Add 20 μl of the reconstituted calibrator solution into the reaction cup;

c. Add 50 μl of reagent 1 (magnetic bead suspension) and 50 μl of reagent 2 (acridinium ester solution) into the reaction cup in sequence;

d. After the solution in the reaction cup is fully mixed, incubate at 37°C for 6 minutes;

e. Place under magnetic conditions and use magnetic bead cleaning solution to wash multiple times;

f. Add 100 μl of substrate solution A and substrate solution B to the reaction cup and detect the luminescence value. The luminescence intensity is expressed in relative light units (RLU).

2. Measurement results

The reconstitution stability of the lyophilized calibrators using the diluent of the present invention was tested through experiments. The results showed that the stability of the calibrators was good within 30 days of reconstitution (see Table 21).

Table 21 Reconstitution stability of calibrators

Embodiment 8

Limit of blank detection

The detection method is carried out with reference to the test method of YY/T 1233-2014 "Cardiac Troponin-I Quantitative Determination Reagent (Kit) (Chemiluminescent Immunoassay)" issued by the State Food and Drug Administration.

The blank sample (sample dilution) was tested repeatedly 20 times, and then the analytical sensitivity was calculated using the formula based on the test results. The results are shown in Table 23.

Table 22 Detection results of initial calibration curve

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

Table 23 Blank limit test results

A linear equation is obtained by performing two-point regression fitting based on the results of the concentration-chemiluminescence (RLU) value between the zero-concentration calibrator (S0) and the adjacent calibrator (S1), and then the RLU value corresponding to the Mean+2SD obtained by detecting the 20-well blank sample (S0) is substituted into the above equation. The obtained concentration value is the analytical sensitivity. The analytical sensitivity of the present invention can reach 0.06 ng/ml, which is of great significance for the early diagnosis of AMI.

Embodiment 9

Precision testing within a day

Repeat the test for 10 times for high and low standards, and then calculate the CV based on the test results. The results are shown in Table 24.

Table 24 days precision test results

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 present invention can reach 5.15% and 3.21%, which is of great significance for the diagnostic accuracy of AMI.

Embodiment 10

Detection of linear correlation

Linear samples were tested in duplicate, and the linear correlation coefficient between the theoretical value and the measured value at each point was calculated. The results are shown in Table 25.

Table 25 Test results of linear correlation

Luminous value 1 Luminous value 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 R of the present invention is greater than 0.990. The linear samples (7 points) were tested in duplicate wells, and the linear correlation coefficients of the theoretical values and the measured values of each point were calculated. The theoretical concentration was not the concentration calculated using the calibration curve, and the measured value was the sample value after the comparison machine Roche was used for testing. FIG9 shows that the linear correlation is good.

Embodiment 11

Accuracy test

Serum was diluted in a ratio of 1:9 and tested

A recovery test was performed to detect the accuracy of the test kit. The test results are shown in Table 26.

Table 26 Test results of recovery test

A liquid concentration 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 221.6615 0.724593 22.0985 Theoretical concentration 22.81828 96.85%

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

Embodiment 12

Clinical application of CK-MB calibrator

Twenty-eight clinical samples were collected, and Roche CK-MB in vitro diagnostic kit was used for methodological comparison.

Determination steps:

(1) Prepare the optimal dilution according to the following formula:

Weigh 1.1263 g of MOPSO, 0.292 g of EDTA, 3 g of bovine serum albumin (BSA), 5 g of compound enzyme protectant AFP, and 5 g of compound enzyme protectant HPD. Add 60 ml of purified water, adjust the pH value to 7.2 after complete dissolution, and make up to 100 ml.

(2) using the diluent obtained in step (1) above to prepare calibrators at 300 ng/ml, 140 ng/ml, 35 ng/ml, 4 ng/ml, and 1 ng/ml, freeze-drying them, re-dissolving them, and making a standard curve;

(3) Determination method:

a. The CK-MB antibody solution coated with magnetic microspheres as reagent 1 and the CK-MB antibody solution labeled with acridinium ester as reagent 2 were placed at room temperature and mixed thoroughly for 30 minutes;

b. Add 20 μl of the reconstituted calibrator solution into the reaction cup;

c. Add 50 μl of reagent 1 (magnetic bead suspension) and 50 μl of reagent 2 (acridinium ester solution) into the reaction cup in sequence;

d. After the solution in the reaction cup is fully mixed, incubate at 37°C for 6 minutes;

e. Place under magnetic conditions and use magnetic bead cleaning solution to wash multiple times;

f. Add 100 μl of substrate solution A and substrate solution B to the reaction cup and detect the luminescence value. The luminescence intensity is expressed in relative light units (RLU).

1. Measurement results

The standard curve (logX-logY) prepared by using the calibrator prepared by the diluent of the present invention after freeze-drying and reconstitution is shown in Figure 8, and the correlation coefficient ^R2 = 0.9975, indicating that the calibrator has good linearity between 0-300ng/ml. The determination results of the calibrator prepared by the diluent of the present invention after freeze-drying and reconstitution were compared with the Roche CK-MB in vitro diagnostic kit. The results are shown in Figure 10, and the correlation coefficient ^R2 = 0.9963, indicating that the determination results of the calibrator prepared by the diluent of the present invention are highly correlated with the determination results of the Roche CK-MB kit, and the consistency is high.

The above embodiments 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. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A creatine kinase isoenzyme assay kit, wherein the kit comprises: a primary antibody coated on the magnetic microsphere, a secondary antibody marked with a tracer marker and creatine kinase isoenzyme sample diluent; wherein,,

the specific binding sites of creatine kinase isozymes recognized by the first antibody and the second antibody are different, the first antibody is CKBB-BB, and the second antibody is CKBB-04;

the mass ratio of the magnetic microsphere to the first antibody is 100:0.5-1.25, and the mass ratio of the tracer marker to the second antibody is 15-40:1;

the creatine kinase isoenzyme sample diluent comprises: buffer solution, ion chelating agent and protective agent, wherein,

the protective agent comprises bovine serum albumin, complex enzyme protective agent AFP and complex enzyme protective agent HPD;

the buffer solution is MOPSO buffer solution, and the pH value is 7.2;

the ion chelating agent is EDTA, and the concentration is 10 mM;

the content of the bovine serum albumin is 3%; the content of the AFP of the complex enzyme protective agent is 5%; the content of the complex enzyme protective agent HPD is 5%;

the sample is a calibrator; the calibrator is a freeze-dried calibrator.

2. The creatine kinase isoenzyme assay kit of claim 1, wherein the assay kit further comprises a calibrator.

3. The creatine kinase isoenzyme detection kit according to claim 1 or 2, wherein the tracer label is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane.

4. The creatine kinase isozyme detection kit according to claim 1 or 2, wherein the tracer label is an acridinium ester.

5. The creatine kinase isoenzyme detection kit according to claim 1 or 2, wherein the kit further comprises: magnetic microsphere cleaning liquid, substrate liquid A and substrate liquid B; wherein the magnetic microsphere cleaning liquid contains PBST buffer solution, and the substrate solution A is H ₂ O ₂ And HNO ₃ The substrate solution B is a mixed solution of Triton X-100 and NaOH.

6. The creatine kinase isozyme detection kit according to claim 5, wherein the H ₂ O ₂ Is 0.01-5.0% by mass, the HNO is ₃ The concentration of the Triton X-100 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 0.05-1mol/L.

7. The method for preparing a creatine kinase isoenzyme detection kit according to any one of claims 1 to 6, wherein the method for preparing comprises:

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

labeling a tracer marker: labeling the tracer label on the second antibody;

preparing a sample diluent: the components of the sample dilutions were mixed.

8. The method for preparing a creatine kinase isoenzyme assay kit according to claim 7, wherein,

the magnetic microsphere coating step uses 2- (N-morpholino) ethanesulfonic acid as a buffer solution.

9. The method for preparing a creatine kinase isoenzyme detection kit according to claim 7 or 8, wherein a blocking liquid is further used in the magnetic microsphere coating step, and the blocking liquid is BSA;

and/or

The tracer labeling step uses 4-hydroxyethyl piperazine ethane sulfonic acid as a buffer.

10. Use of the creatine kinase isozyme detection kit according to any one of claims 1-6 in the preparation of a reagent for diagnosing myocardial infarction.