CN114487420A - Creatine kinase isoenzyme detection kit - Google Patents
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Abstract
The invention discloses a creatine kinase isoenzyme detection kit, and belongs to the technical field of biological detection. The creatine kinase isoenzyme detection kit comprises a reagent R1 and a reagent R2; wherein the reagent R1 comprises the following components: buffers, coagulants, chelating agents, blockers, surfactants, stabilizers, and preservatives; the reagent R2 comprises the following components: buffer solution, latex particles coated by creatine kinase isoenzyme antibody, a blocking agent, a stabilizing agent and a preservative. The creatine kinase isoenzyme detection kit overcomes the defects of the existing creatine kinase isoenzyme detection kit, has the advantages of strong anti-interference performance, high sensitivity, high specificity, high accuracy, wide linear range, good stability and the like, and provides a safe, quick, accurate and pollution-free detection means for the detection of creatine kinase isoenzyme.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a creatine kinase isoenzyme detection kit.
Background
Creatine Kinase (CK) is a dimeric enzyme that has four different forms: mitochondrial isozyme, cytoplasmic isozyme CK-MM (muscular type), creatine kinase brain isozyme CK-BB (brain type), creatine kinase isozyme (CK-MB), wherein the creatine kinase isozyme is mainly located in cardiac muscle.
Creatine kinase isoenzyme diagnosis transmural myocardial infarction is extremely high in sensitivity and specificity, the expression level of the creatine kinase isoenzyme is low under normal conditions, the creatine kinase isoenzyme is discharged from damaged cells and rapidly begins to increase in blood 4-8 hours after onset of disease, reaches the maximum level after 12-24 hours, and returns to normal within 24-48 hours. If the creatine kinase isoenzyme still keeps high level after acute myocardial infarction, myocardial necrosis is still performed; if the normal state is recovered, the increase is again shown, which indicates that the original infarction part is expanded or a new infarction part appears. Therefore, the quantitative detection of the creatine kinase isoenzyme level in blood has very important value for the early diagnosis of Acute Myocardial Infarction (AMI).
At present, the creatine kinase isoenzyme is measured by electrophoresis, immunosuppression, radioimmunoassay, immunoenzyme labeling, gold labeling, ion exchange column chromatography, and the like. The more applied methods are mainly electrophoresis and immunosuppression, but the electrophoresis cannot be applied to a full-automatic biochemical analyzer, and the used instruments are expensive and cannot be popularized; immunosuppression has the advantages of rapidness, time saving, high sensitivity, etc., but has many factors. Other commonly used detection methods include immunoturbidimetry, enzyme-linked immunosorbent assay and the like, the enzyme-linked immunosorbent assay has the defects of complex operation, low sensitivity, narrow linear range, long detection period and the like, and cannot meet the requirements of quick quantitative and simple detection in large hospitals, and the immunoturbidimetry develops various quick immunoturbidimetry detection technologies along with the popularization of full-automatic biochemical instruments.
The latex enhanced immunoturbidimetry is an antigen-antibody combination dynamic detection method, and an antibody corresponding to a target antigen to be detected is coated on a latex microsphere, so that the volume of an antigen-antibody combination is increased, and the intensity change of transmitted light and scattered light in a detection light path is more obvious, thereby improving the detection sensitivity.
Although creatine kinase isoenzyme detection kits based on a latex enhanced immunoturbidimetry method exist in the market at present, the kits have the problems of low detection linear range, low sensitivity, poor anti-interference capability, unstable reagent storage and the like.
Based on the above, the research and development of a creatine kinase isoenzyme detection kit with strong anti-interference performance, high sensitivity, high specificity, high accuracy, wide detection range and good stability is needed in the field, so that the creatine kinase isoenzyme can be popularized and applied in large scale on the clinical diagnosis of acute myocardial infarction.
Disclosure of Invention
Aiming at the problems, the invention provides the creatine kinase isoenzyme detection kit which has the advantages of strong anti-interference performance, high sensitivity, high specificity, high accuracy, wide linear range and good stability.
The invention is realized by the following technical scheme:
a creatine kinase isoenzyme detection kit, which comprises a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in percentage by weight: 10-50mM of buffer solution, 2-5g/L of coagulant, 1-2g/L of chelating agent, 0.5-2g/L of blocking agent, 1-5g/L of surfactant, 0.5-1g/L of stabilizer and 1-2g/L of preservative;
the reagent R2 comprises the following components in percentage by weight: 10-50mM of buffer solution, 0.05-0.4% w/v of latex particles coated by creatine kinase isoenzyme antibody, 1-10g/L of blocking agent, 5-20g/L of stabilizing agent and 1-2g/L of preservative.
Further, the surfactant in the reagent R1 is prepared by compounding TritonX-100 and styrene polyoxyethylene ether according to the mass ratio of 2-5: 1.
Further, the blocking agent in the reagent R2 is prepared by compounding triethanolamine, glycine and casein according to the mass ratio of 0.5-2:1: 1-4.
Further, the blocking agent in the reagent R1 is mouse IgG; the chelating agent is EGTA.
Further, the coagulant in the reagent R1 is any one of PEG-4000, PEG-6000, PEG-8000 and dextran.
Further, the buffer solution in the reagent R1 and the reagent R2 is independently selected from one of phosphate buffer solution, glycine buffer solution, Tris buffer solution, HEPES buffer solution and MES buffer solution.
Further, the stabilizing agent in the reagent R1 and the reagent R2 is independently selected from one of bovine serum albumin, trehalose, chitosan and glycerol.
Further, the preservatives in the reagent R1 and the reagent R2 are respectively and independently selected from ProClin300, ProClin950, phenol, p-hydroxybenzoic acid and ethyl p-hydroxybenzaldehyde.
Further, the creatine kinase isoenzyme antibody in the reagent R2 is a murine monoclonal antibody; the latex particles are polystyrene latex microspheres with the diameter of 250-400nm, the surfaces of the microspheres are modified with functional groups, and the modified functional groups are selected from carboxyl, amino or hydroxyl.
Further, the creatine kinase isoenzyme antibody in the reagent R2 is coated on the surface of the latex microsphere by a chemical crosslinking method, the chemical crosslinking agent is selected from one of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride, maleimide, carbodiimide, mercaptonicotinamide and isocyanate, the crosslinking buffer is selected from one of HEPES buffer, MOPS buffer, MES buffer and MOPSO buffer, and the pH value is 6.0-8.0.
Further, the method for preparing the creatine kinase isoenzyme detection kit comprises the following steps:
(1) preparation of reagent R1: adding each component of the reagent R1 into purified water, mixing uniformly, adjusting the pH value to 6.5-8.0, filtering through a filter membrane, and fixing the volume to obtain a reagent R1;
(2) preparation of reagent R2:
activating microspheres: adding polystyrene latex microspheres with the diameter of 250-400nm and the surface modified with carboxyl, amino or hydroxyl into a buffer solution with the pH value of 6.0-8.0, adding an activator, reacting at 25-30 ℃ for 30-40min, centrifuging, removing the redundant activator in the supernatant, taking the precipitate, and then using the buffer solution for heavy suspension to obtain the activated polystyrene latex microspheres;
② crosslinking of the antibody: adding creatine kinase isoenzyme antibody into the activated polystyrene latex microspheres, and reacting at 25-30 ℃ for 1.5-2h to cross-link the antibody on the surfaces of the latex microspheres;
sealing the microspheres: adding a sealing agent into the latex microsphere reaction solution after the antibody is crosslinked, sealing for 1-2h, and sealing the region which is not adhered by the antibody on the surface of the latex microsphere;
removing impurities: centrifuging the mixed solution of the sealed latex microspheres to remove redundant cross-linking agent, cross-linking by-products, antibody and sealing agent, wherein the bottom precipitate is latex particles coated by creatine kinase isoenzyme antibody;
mixing and storing: adding the buffer solution, the stabilizer and the preservative in the reagent R2 into purified water, uniformly mixing, adding latex particles coated by creatine kinase isoenzyme antibody, fully stirring, uniformly mixing, and performing constant volume to obtain a reagent R2.
The creatine kinase isoenzyme detection kit of the invention adopts latex enhanced immunoturbidimetry to quantitatively detect the content of creatine kinase isoenzyme in blood, and the principle is as follows: the creatine kinase isoenzyme antigen in the sample to be detected and the creatine kinase isoenzyme antibody coated on the latex particles are subjected to antigen-antibody reaction to form an antigen-antibody-latex particle compound and generate agglutination to cause turbidity rise, so that the light scattering or light transmission performance of the reaction liquid is changed, a standard curve is drawn by using a standard product, and the content of the substance to be detected can be determined according to the change of the absorbance value of the sample.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. the creatine kinase isoenzyme detection kit overcomes the defects of the existing creatine kinase isoenzyme detection kit, has the advantages of strong anti-interference performance, high sensitivity, high specificity, high accuracy, wide linear range, good stability and the like, provides a safe, quick, accurate and pollution-free detection means for the detection of creatine kinase isoenzyme, and thus provides a powerful test diagnosis basis for the clinical diagnosis of acute myocardial infarction.
2. The surfactant in the reagent R1 is formed by compounding TritonX-100 and styrene polyoxyethylene ether, and can well promote the uniform dispersion of each component in a reagent system and each substance in a detection sample, so that the detection precision is improved, the repeatability of a detection result is ensured, the stability of the detection result is good, the influence of the turbidity of the sample on the detection result can be reduced, and the anti-interference capability of the reagent is improved.
3. The sealant in the reagent R2 is compounded by triethanolamine, glycine and casein, can be combined with free-COOH sites in latex particles, avoids adverse effects of the free carboxyl sites on detection results, and effectively improves the detection sensitivity and the stability of the kit.
4. According to the invention, the blocker mouse IgG is added into the reagent R1, so that the nonspecific adsorption in the antigen-antibody reaction process can be effectively reduced, the interference of nonspecific reaction on the detection result is avoided, the sensitivity of sample detection is remarkably improved, and the kit has a lower detection lower limit. In the invention, chelating agent EGTA is added into reagent R1, thus effectively improving the upper limit of the detection range of the kit. Compared with the existing method, the linear range of the kit is wider, and the detection range of the kit is 3.0-180.0 ng/mL.
5. The surface functional group of the polystyrene latex microsphere in the reagent R2 is carboxyl, amino or hydroxyl, and the surface functional group can be combined with amino on the surface of an antibody to form a covalent coupling structure, so that the creatine kinase isoenzyme antibody is firmly combined on the surface of the latex microsphere, the reagent R2 is ensured to have good stability, and the effective period of the reagent is effectively prolonged.
6. The particle size of the polystyrene latex particles adopted by the invention is 250-400nm, and the polystyrene latex particles have larger particle size, so that the turbidity of the creatine kinase isoenzyme and creatine kinase isoenzyme antibody in blood during reaction can be increased, the reaction sensitivity in the detection process is increased, and the reaction time is effectively shortened.
Drawings
FIG. 1 is a comparison of the creatine kinase isoenzyme detection kit prepared in example 1 and the Beijing Jiuqiang kit.
FIG. 2 is a comparison of the creatine kinase isoenzyme detection kit prepared in comparative example 1 and the Beijing Jiuqiang kit.
FIG. 3 is a graph showing the trend of the stability of the reagents of example 1 and comparative example 2 during 14 months of storage at 2-8 ℃.
Detailed Description
The present invention is further illustrated by the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
The creatine kinase isoenzyme detection kit provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 are composed of the following components:
reagent R1:
in the table: the mass ratio of TritonX-100 to styrene polyoxyethylene ether is 3.2: 1.
Preparation of reagent R1: according to the components and the content of the reagent R1 in the table, the components of the reagent R1 are taken and added into purified water, the mixture is uniformly mixed, the pH value is adjusted to 7.0, and the reagent R1 is obtained through filtering by a filter membrane and volume fixing.
Reagent R2:
in the table: the mass ratio of the triethanolamine to the glycine to the casein is 0.5:1: 2.5.
Preparation of reagent R2:
activating microspheres: adding polystyrene latex microspheres with the diameter of 300nm and modified with carboxyl on the surface into MOPS buffer solution with the pH value of 7.2, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, reacting for 30min at 25 ℃, removing redundant activating agent in supernatant after centrifugation, taking precipitate, and then using MOPS buffer solution for heavy suspension to obtain the activated polystyrene latex microspheres;
② crosslinking of the antibody: adding creatine kinase isoenzyme antibody into the activated polystyrene latex microspheres, and reacting at 25 ℃ for 1.5h to cross-link the antibody on the surfaces of the latex microspheres;
sealing the microspheres: adding triethanolamine, glycine and casein into the latex microsphere reaction solution after the antibody is crosslinked, sealing for 1.5h, and sealing the region which is not adhered by the antibody on the surface of the latex microsphere;
removing impurities: centrifuging the mixed solution of the sealed latex microspheres to remove redundant crosslinking byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein, wherein bottom sediment is latex particles coated by the creatine kinase isoenzyme antibodies;
mixing and storing: adding phosphate buffer solution, chitosan and ProClin950 into purified water, mixing uniformly, adding latex particles coated by creatine kinase isoenzyme antibody, fully stirring, mixing uniformly, and fixing the volume to obtain a reagent R2.
Example 2
The creatine kinase isoenzyme detection kit provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 are composed of the following components:
reagent R1:
in the table: the mass ratio of TritonX-100 to styrene polyoxyethylene ether is 2.5: 1.
Preparation of reagent R1: according to the components and the content of the reagent R1 in the table, the components of the reagent R1 are taken and added into purified water, the mixture is uniformly mixed, the pH value is adjusted to 6.5, and the reagent R1 is obtained through filtering by a filter membrane and volume fixing.
Reagent R2:
in the table: the mass ratio of the triethanolamine to the glycine to the casein is 1:1:3.
Preparation of reagent R2:
activating microspheres: adding polystyrene latex microspheres with 400nm diameter and modified with amino on the surface into HEPES buffer solution with pH of 6.0, adding mercaptonicotinamide, reacting at 30 ℃ for 35min, centrifuging, removing redundant activator in supernatant, taking precipitate, and then using HEPES buffer solution for heavy suspension to obtain activated polystyrene latex microspheres;
② crosslinking of the antibody: adding creatine kinase isoenzyme antibody into activated polystyrene latex microspheres, and reacting at 30 ℃ for 2h to cross-link the antibody on the surfaces of the latex microspheres;
sealing the microspheres: adding triethanolamine, glycine and casein into the latex microsphere reaction solution after the antibody is crosslinked, sealing for 1h, and sealing the region which is not adhered by the antibody on the surface of the latex microsphere;
removing impurities: centrifuging the mixed solution of the sealed latex microspheres, removing redundant crosslinking byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein, and obtaining bottom sediment, namely latex particles coated by the creatine kinase isoenzyme antibodies;
mixing and storing: adding glycine buffer solution, chitosan and phenol into purified water, uniformly mixing, adding latex particles coated with creatine kinase isoenzyme antibody, fully stirring, uniformly mixing, and performing constant volume to obtain a reagent R2.
Example 3
The creatine kinase isoenzyme detection kit provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 are composed of the following components:
reagent R1:
in the table: the mass ratio of TritonX-100 to styrene polyoxyethylene ether is 4.5: 1.
Preparation of reagent R1: according to the components and the content of the reagent R1 in the table, the components of the reagent R1 are taken and added into purified water, the mixture is uniformly mixed, the pH value is adjusted to 8.0, and the reagent R1 is obtained through filtering by a filter membrane and volume fixing.
Reagent R2:
in the table: the mass ratio of the triethanolamine to the glycine to the casein is 2:1: 4.
Preparation of reagent R2:
activating microspheres: adding polystyrene latex microspheres with the diameter of 250nm and the surface modified with hydroxyl into MOPSO buffer solution with the pH value of 7.5, adding isocyanate, reacting for 40min at 25 ℃, removing redundant activating agent in supernatant after centrifugation, taking precipitate, and then using MOPSO buffer solution for heavy suspension to obtain activated polystyrene latex microspheres;
② crosslinking of the antibody: adding creatine kinase isoenzyme antibody into activated polystyrene latex microspheres, and reacting for 2 hours at 25 ℃ to crosslink the antibody on the surfaces of the latex microspheres;
sealing the microspheres: adding triethanolamine, glycine and casein into the latex microsphere reaction solution after the antibody is crosslinked, sealing for 1h, and sealing the region which is not adhered by the antibody on the surface of the latex microsphere;
removing impurities: centrifuging the mixed solution of the sealed latex microspheres, removing redundant crosslinking byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein, and obtaining bottom sediment, namely latex particles coated by the creatine kinase isoenzyme antibodies;
mixing and storing: adding Tris buffer solution, bovine serum albumin and p-hydroxybenzoic acid into purified water, mixing uniformly, adding latex particles coated by creatine kinase isoenzyme antibody, fully stirring, mixing uniformly, and obtaining reagent R2 after constant volume.
Example 4
The creatine kinase isoenzyme detection kit provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 are composed of the following components:
reagent R1:
in the table: the mass ratio of TritonX-100 to styrene polyoxyethylene ether is 5: 1.
Preparation of reagent R1: according to the components and the content of the reagent R1 in the table, the components of the reagent R1 are taken and added into purified water, the mixture is uniformly mixed, the pH value is adjusted to 6.5, and the reagent R1 is obtained through filtering by a filter membrane and volume fixing.
Reagent R2:
in the table: the mass ratio of the triethanolamine to the glycine to the casein is 1:1: 3.5.
Preparation of reagent R2:
activating microspheres: adding 400 nm-diameter polystyrene latex microspheres with carboxyl modified on the surface into MES buffer solution with the pH value of 6.0, adding carbodiimide, reacting for 40min at 30 ℃, centrifuging, removing redundant activating agent in supernatant, taking precipitate, and then using MES buffer solution for heavy suspension to obtain activated polystyrene latex microspheres;
② crosslinking of the antibody: adding creatine kinase isoenzyme antibody into activated polystyrene latex microspheres, and reacting at 30 ℃ for 2h to cross-link the antibody on the surfaces of the latex microspheres;
sealing the microspheres: adding triethanolamine, glycine and casein into the latex microsphere reaction solution after the antibody is crosslinked, sealing for 1h, and sealing the region which is not adhered by the antibody on the surface of the latex microsphere;
removing impurities: centrifuging the mixed solution of the sealed latex microspheres to remove redundant crosslinking byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein, wherein bottom sediment is latex particles coated by the creatine kinase isoenzyme antibodies;
mixing and storing: adding MES buffer solution, glycerol and ethyl p-hydroxybenzaldehyde into purified water, mixing uniformly, adding latex particles coated with creatine kinase isoenzyme antibody, stirring fully, mixing uniformly, and diluting to constant volume to obtain reagent R2.
Example 5
The creatine kinase isoenzyme detection kit provided by the embodiment comprises a reagent R1 and a reagent R2, wherein the reagent R1 and the reagent R2 are composed of the following components:
reagent R1:
in the table: the mass ratio of TritonX-100 to styrene polyoxyethylene ether is 2: 1.
Preparation of reagent R1: according to the components and the content of the reagent R1 in the table, the components of the reagent R1 are taken and added into purified water, the mixture is uniformly mixed, the pH value is adjusted to 7.0, and the reagent R1 is obtained through filtering by a filter membrane and volume fixing.
Reagent R2:
in the table: the mass ratio of the triethanolamine to the glycine to the casein is 2:1: 1.
Preparation of reagent R2:
activating microspheres: adding polystyrene latex microspheres with the diameter of 300nm and modified with carboxyl on the surface into MES buffer solution with the pH of 8.0, adding maleimide, reacting for 40min at 25 ℃, centrifuging, removing redundant activating agent in supernatant, taking precipitate, and then using MES buffer solution for heavy suspension to obtain activated polystyrene latex microspheres;
antibody cross-linking: adding creatine kinase isoenzyme antibody into activated polystyrene latex microspheres, and reacting for 2 hours at 25 ℃ to crosslink the antibody on the surfaces of the latex microspheres;
sealing the microspheres: adding triethanolamine, glycine and casein into the latex microsphere reaction solution after the antibody is crosslinked, sealing for 2h, and sealing the region which is not adhered by the antibody on the surface of the latex microsphere;
removing impurities: centrifuging the mixed solution of the sealed latex microspheres to remove redundant crosslinking byproducts, creatine kinase isoenzyme antibodies, triethanolamine, glycine and casein, wherein bottom sediment is latex particles coated by the creatine kinase isoenzyme antibodies;
mixing and storing: adding HEPES buffer solution, bovine serum albumin and ProClin300 into purified water, uniformly mixing, adding latex particles coated by creatine kinase isoenzyme antibody, fully stirring, uniformly mixing, and performing constant volume to obtain a reagent R2.
Comparative example 1
Comparative example 1 is different from example 1 in that the surfactant in the reagent R1 of comparative example 1 is only triton x-100, and the rest is the same as that of homogeneous example 1.
Comparative example 2
Comparative example 2 differs from example 1 in that the blocking agent in reagent R2 of comparative example 2 is casein only, the rest being the same as in homogeneous example 1.
Comparative example 3
Comparative example 3 differs from example 1 in that the reagent R1 of comparative example 3 has no blocking agent mouse IgG, and is otherwise identical to that of homogeneous example 1.
Evaluation of Performance
Test example 1 Linear Range test of creatine kinase isoenzyme detection kit
1. Test sample
(1) A test sample: creatine kinase isoenzyme detection kits prepared in examples 1-5;
(2) control sample: the creatine kinase isoenzyme detection kit prepared in comparative examples 1-3;
2. test method
Obtained by dilution of high concentration samples with low concentration samples in a multiple ratio, the formulation scheme is given in the following table. The concentration and volume unit of each sample are unified, and a liquid transfer device with good precision and accuracy is selected when liquid is sucked; the sample is mixed completely during preparation and evaporation or other deterioration of the sample is avoided
| Sample number | 1 | 2 | 3 | 4 | 5 | 6 |
| Low-concentration serum 3.2ng/ml | 1.00 | 0.80 | 0.60 | 0.40 | 0.20 | 0.00 |
| High concentration serum 180ng/ml | 0.00 | 0.20 | 0.40 | 0.60 | 0.80 | 1.00 |
The creatine kinase isoenzyme detection kits prepared in examples 1-5 and comparative examples 1-3 are respectively adopted to detect the creatine kinase isoenzyme, and standard working curves of the detection kits are drawn.
The detection method is a two-point end point method and comprises the following steps: adding 150 mu L of reagent R1 into 10 mu L of samples to be detected (a calibration tube takes a calibrator as a sample, and distilled water as a blank sample), fully mixing, keeping the temperature at 37 ℃ for 5min, then adding 50 mu L of reagent R2, mixing, keeping the temperature at 37 ℃ for 40sec, measuring the absorbance Al of each tube at the wavelength of 660nm, reading the absorbance A2 after 260sec, and obtaining the reaction absorbance OA ═ A2-A1. And determining a working curve by adopting a multipoint nonlinear calibration mode according to the concentration of the multipoint calibrator and the corresponding absorbance change value OA, wherein the concentration value of the absorbance change of the sample on the working curve is the measured concentration.
3. Test results
The results of the analysis of the creatine kinase isoenzyme detection kit of examples 1 to 5 and comparative examples 1 to 3 are as follows: (the technical index is that the linear correlation coefficient r is more than or equal to 0.990 within the range of [3, 180] ng/mL, the absolute deviation of linearity should not exceed plus or minus 3ng/mL within the range of [3, 20] ng/mL, and the relative deviation of linearity should not exceed plus or minus 15 percent within the range of (20, 180] ng/mL)
And (4) analyzing results: from the experimental data in the above table, it can be seen that the linear range indexes of examples 1-5 all meet the specification requirements, while comparative example 1 and comparative example 2 do not meet the requirements at low concentrations, indicating that the surfactant in reagent R1 and the blocking agent in reagent R2 have an effect on the dispersibility of the reagent latex and the background value, while the linear range data of comparative example 3 are very cluttered, indicating that mouse IgG without blocking agent in reagent R1 will cause strong interference.
Test example 2 lowest detection limit test of creatine kinase isoenzyme detection kit
1. Test sample
(1) A sample to be tested: the creatine kinase isoenzyme detection kit prepared in example 1;
(2) control sample: the creatine kinase isoenzyme detection kit prepared in comparative example 1;
2. test method
Distilled water was used as a blank sample, and the creatine kinase isoenzyme detection kits prepared in example 1 and comparative example 1 were used to detect the blank sample for 10 times. The mean and standard deviation of the results were calculated 10 times, and the blank mean plus two times the standard deviation was taken as the lowest detection limit (+2 SD).
3. Test results
(technical index: blank sample test kit is used, the blank limit of the reagent is less than or equal to 2ng/mL)
Test results of creatine kinase isoenzyme detection kit of example 1
Test results of creatine kinase isoenzyme detection kit of comparative example 1
And (4) analyzing results: the minimum detection limit results of example 1 and comparative example 1 show that the minimum detection limit of example 1 reaches 0.43, whereas the background value of the comparative example 1 reagent is very high, resulting in a disqualified minimum detection limit.
Test example 3 accuracy test of creatine kinase isoenzyme detection kit
1. Test sample
(1) A sample to be tested: the creatine kinase isoenzyme detection kit prepared in example 1;
(2) control sample: the creatine kinase isoenzyme detection kit prepared in comparative example 1;
2. test method
The creatine kinase isoenzyme detection kit prepared in example 1 and comparative example 1 and the marketed creatine kinase isoenzyme detection kit of Jiuqiang biology are respectively adopted to carry out clinical detection on 40 creatine kinase isoenzyme samples with different contents, and correlation analysis is carried out on the measured values.
3. Test results
The measurement results of the creatine kinase isoenzyme detection kit of example 1 and comparative example 1 are shown in fig. 1 and 2.
And (4) analyzing results: from the results, it can be seen that, when compared with the product on the market of Jiuqiang, Beijing, the serum clinical alignment shows that example 1 has better correlation with comparative example 1 (the correlation r of example 1 is 0.99, and the correlation r of comparative example 1 is 0.92), and that comparative example 1 has a great amount of interference in serum measurement, and has a great deviation at low concentration level.
Test example 4 stability test of creatine kinase isoenzyme assay kit
1. Test sample
(1) A sample to be tested: the creatine kinase isoenzyme detection kit prepared in example 1;
(2) control sample: the creatine kinase isoenzyme detection kit prepared in comparative example 2;
2. test method
Creatine kinase isoenzyme samples with high concentration of 120ng/mL and low concentration of 50ng/mL were tested 10 times per sample after 0 month, 3 months, 6 months, 9 months, 12 months and 14 months using the creatine kinase isoenzyme test kits prepared in example 1 and comparative example 2, respectively, under storage conditions of 2-8 deg.C, and an average value was taken.
3. Test results
And (4) analyzing results: as can be seen from the analysis of variance of the stability data in example 1, the trend was not significant when the reagent in example 1 was stored at 2 to 8 ℃ for 14 months, with p being 0.868 at a high concentration, p being 0.252 at a low concentration, and p being > 0.05 at a high concentration and a low concentration, i.e., when the reagent in example 1 was stored at 2 to 8 ℃ for 14 months, the trend of the detection result was not significant, and the reagent was in a stable state.
And (4) analyzing results: the analysis of variance of the stability data of the comparative example 2 can be used for obtaining that the test value of the reagent is obviously reduced when the reagent of the comparative example 2 is stored for 14 months at the temperature of 2-8 ℃, and the p of high and low concentrations is less than 0.05, namely the reagent of the comparative example 2 is stored for 14 months at the temperature of 2-8 ℃, the change trend of the detection result is obvious, and the detection result is in an extremely unstable state.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
Claims (10)
1. The creatine kinase isoenzyme detection kit is characterized by comprising a reagent R1 and a reagent R2;
the reagent R1 comprises the following components in percentage by weight: 10-50mM of buffer solution, 2-5g/L of coagulant, 1-2g/L of chelating agent, 0.5-2g/L of blocking agent, 1-5g/L of surfactant, 0.5-1g/L of stabilizer and 1-2g/L of preservative;
the reagent R2 comprises the following components in percentage by weight: 10-50mM of buffer solution, 0.05-0.4% w/v of latex particles coated by creatine kinase isoenzyme antibody, 1-10g/L of blocking agent, 5-20g/L of stabilizing agent and 1-2g/L of preservative.
2. The creatine kinase isoenzyme detection kit according to claim 1, characterized in that a surfactant in the reagent R1 is compounded by TritonX-100 and styrene polyoxyethylene ether according to a mass ratio of 2-5: 1.
3. The creatine kinase isoenzyme detection kit according to claim 1, characterized in that a blocking agent in the reagent R2 is compounded by triethanolamine, glycine and casein according to a mass ratio of 0.5-2:1: 1-4.
4. The creatine kinase isoenzyme detection kit according to claim 1, wherein the blocking agent in the reagent R1 is mouse IgG; the chelating agent is EGTA.
5. The creatine kinase isoenzyme detection kit according to claim 1, wherein the coagulant in the reagent R1 is any one of PEG-4000, PEG-6000, PEG-8000 and dextran.
6. The creatine kinase isoenzyme detection kit according to claim 1, wherein the buffer solution in the reagent R1 and the reagent R2 is independently selected from one of phosphate buffer solution, glycine buffer solution, Tris buffer solution, HEPES buffer solution and MES buffer solution.
7. The creatine kinase isoenzyme detection kit according to claim 1, wherein the stabilizer in the reagent R1 and the stabilizer in the reagent R2 are independently selected from one of bovine serum albumin, trehalose, chitosan and glycerol.
8. The creatine kinase isoenzyme detection kit according to claim 1, wherein the preservatives in reagent R1 and reagent R2 are independently selected from ProClin300, ProClin950, phenol, p-hydroxybenzoic acid and ethyl p-hydroxybenzaldehyde.
9. The creatine kinase isoenzyme detection kit according to claim 1, wherein the creatine kinase isoenzyme antibody in the reagent R2 is a murine monoclonal antibody; the latex particles are polystyrene latex microspheres with the diameter of 250-400nm, and the surfaces of the microspheres are modified with functional groups selected from carboxyl, amino or hydroxyl.
10. The creatine kinase isoenzyme detection kit according to claim 1, wherein the creatine kinase isoenzyme antibody in the reagent R2 is coated on the surface of the latex microsphere by a chemical crosslinking method, the chemical crosslinking agent is selected from one of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride, maleimide, carbodiimide, mercaptonicotinamide and isocyanate, the crosslinking buffer is selected from one of HEPES buffer, MOPS buffer, MES buffer and MOPSO buffer, and the pH is 6.0-8.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261354A (en) * | 2022-07-27 | 2022-11-01 | 东方伊诺(苏州)医疗科技有限公司 | Creatine kinase isoenzyme quality control product, preparation method thereof and detection kit |
| CN116593701A (en) * | 2023-07-19 | 2023-08-15 | 北京豪迈生物工程股份有限公司 | Creatine kinase isoenzyme detection kit containing biological blocker |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261354A (en) * | 2022-07-27 | 2022-11-01 | 东方伊诺(苏州)医疗科技有限公司 | Creatine kinase isoenzyme quality control product, preparation method thereof and detection kit |
| CN115261354B (en) * | 2022-07-27 | 2023-12-15 | 东方伊诺(苏州)医疗科技有限公司 | Creatine kinase isoenzyme quality control product, preparation method thereof and detection kit |
| CN116593701A (en) * | 2023-07-19 | 2023-08-15 | 北京豪迈生物工程股份有限公司 | Creatine kinase isoenzyme detection kit containing biological blocker |
| CN116593701B (en) * | 2023-07-19 | 2024-01-23 | 北京豪迈生物工程股份有限公司 | Creatine kinase isoenzyme detection kit containing biological blocker |
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