CN113358869A - Stable and sensitive myeloperoxidase detection kit - Google Patents
Stable and sensitive myeloperoxidase detection kit Download PDFInfo
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Abstract
The invention relates to the technical field of biochemical detection, in particular to a stable and sensitive myeloperoxidase detection kit. The detection kit comprises reagents R1 and R2, wherein the reagent R1 contains buffer solution, lithium chloride, D-idose, erythritol, PEG-10000, triisopropanolamine polyoxypropylene polyoxyethylene ether and a preservative; the reagent R2 contains buffer solution, D-idose, erythritol, triisopropanolamine polyoxypropylene polyoxyethylene ether, carboxylated ferrimagnetic trioxide nanoparticles, 83nm rabbit anti-human myeloperoxidase antibody coated latex particles, 185nm rabbit anti-human myeloperoxidase antibody coated latex particles and preservative. The method can effectively detect the content of the myeloperoxidase, and has the advantages of high sensitivity, good stability, wide linear range and the like.
Description
Technical Field
The invention belongs to the technical field of biochemical detection, and particularly relates to a stable and sensitive myeloperoxidase detection kit.
Background
Myeloperoxidase (MPO) is a heme protease of the heme prosthetic group, one of the members of the heme peroxidase superfamily, and is present in the azurophil granules of myeloid lineage cells (mainly neutrophils and monocytes), a marker specific for myeloid cells. With the progress of research on MPO, people find that the difference of individual susceptibility to some diseases caused by MPO gene polymorphism is closely related to the occurrence and development of various human diseases, so that the MPO gene polymorphism is more and more valued by scholars at home and abroad.
MPO exists in medullary cells in 3 subtypes, MPO I, II and III. The 3 subtypes mainly have differences of heavy chains and light chains, so that the light chains are different in relative molecular mass, hydrophobicity and the like, and the differences of the 3 subtypes in functions are not clear and need to be further researched.
At present, flow cytometry, ELISA, continuous monitoring, and the like are commonly used as methods for measuring myeloperoxidase. The technical procedure of the flow cytometry measurement method is complex, and a special instrument is required to be used; the ELISA method has the disadvantages of complex operation, low sensitivity and qualitative detection result; the continuous monitoring method has the advantages of simple operation and convenient use, but has poor sensitivity and stability and narrow linear range.
Disclosure of Invention
In order to solve the problems, the invention provides a stable and sensitive myeloperoxidase detection kit. The kit can effectively detect the content of the myeloperoxidase, and has the advantages of high sensitivity, good stability, wide linear range and the like.
In order to solve the technical problems, the invention provides the following technical scheme:
a stable and sensitive myeloperoxidase detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight:
the reagent R2 comprises the following components in percentage by weight:
further, the buffer solutions of the reagents R1 and R2 were DIPSO buffer solutions having a pH of 6 to 8 at 25 ℃.
Further, the preservative is o-cymene-5-ol.
Further, the preparation steps of the rabbit anti-human myeloperoxidase antibody coated latex particle are as follows:
step 1: respectively adding 1mg of carboxyl latex particles with the particle size of 83nm and 185nm into MOPS-HCL buffer solution, oscillating, uniformly mixing, centrifuging, discarding supernatant, and then respectively adding the MOPS-HCL buffer solution for ultrasonic resuspension to obtain an 83nm carboxyl latex particle buffer system and a 185nm carboxyl latex particle buffer system;
step 2: adding EDC and NHS into the 83nm carboxyl latex particle buffer system and the 185nm carboxyl latex particle buffer system obtained in the step 1 respectively, supplementing MOPS-HCL buffer solution to 5mL, and carrying out oscillation reaction for a period of time at a certain temperature and a certain rotating speed for activation;
and step 3: after activation, centrifuging, removing supernatant, respectively adding MOPS-HCL buffer solution for ultrasonic resuspension, repeating once, and then adding PIPES for ultrasonic resuspension for later use;
and 4, step 4: respectively adding myeloperoxidase antibodies into the liquid obtained in the step 3, uniformly mixing, and reacting in a constant-temperature oscillation box;
and 5: after the reaction is finished, adding BSA, and sealing for a period of time at 2-8 ℃;
step 6: centrifuging, and discarding the supernatant to obtain precipitate, i.e. 83nm rabbit anti-human myeloperoxidase antibody coated latex particles and 185nm rabbit anti-human myeloperoxidase antibody coated latex particles.
Further, the centrifugal speed corresponding to the carboxyl latex particles with the particle size of 83nm is 20000rpm, and the time is 45 min; the centrifugation speed corresponding to the carboxyl latex particles with the particle size of 185nm is 12000rpm, and the time is 45 min.
Further, the kit is measured by an endpoint method using a full-automatic biochemical analyzer, the detection dominant wavelength is 570nm, and the ratio of the R1 reagent to the R2 reagent is 4: 1.
The basic principle of the detection of the invention is as follows: the antigen myeloperoxidase in the sample and the hypersensitized rabbit anti-human myeloperoxidase antibody latex particles in the reagent form insoluble immune complexes, and the turbidity change of the insoluble immune complexes is detected at the wavelength of 570nm, and the change degree is in direct proportion to the concentration of the myeloperoxidase in the sample.
The invention has the beneficial effects that:
(1) according to the invention, a latex enhanced immunoturbidimetry method is adopted, and stabilizers such as D-idose, erythritol and the like are added into a buffer solution by optimizing a reaction system and adding a reagent R1 and a reagent R2, so that the proportion of each stabilizer is optimized, and the stability of the reagent is obviously improved.
(2) The optimized novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether can promote and maintain the stability of an antibody, prevent a system from being turbid and remarkably enhance the stability and the anti-interference capability of the reagent.
(3) The carboxylated ferrimagnetic nanoparticles are added, and the reaction sensitivity and the linear range of the reagent are greatly enhanced through scientific proportioning, so that the repeatability and the anti-interference capability of the reagent are stronger.
(4) The latex enhanced immunoturbidimetry is adopted, and the 83nm and 185nm carboxyl latex microspheres with different particle sizes are combined, so that the proportion is scientific, the reaction sensitivity and the linear range of the reagent are greatly enhanced, and the repeatability and the anti-interference capability of the reagent are stronger.
(5) The reagent is simple and quick to operate, is suitable for automatic analysis, is a myeloperoxidase reagent which is more stable, sensitive and wide in linear range, has good accuracy and stability, strong anti-interference performance and convenient use, and can completely meet clinical requirements.
Drawings
FIG. 1 is a graph showing the calibration of the reagents of the stable and sensitive myeloperoxidase detection kit of the present invention.
FIG. 2 is a graph comparing the stability of reagents of the stable and sensitive myeloperoxidase detection kit of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will explain in further detail with reference to specific comparative examples and examples.
Example 1
The myeloperoxidase detection kit provided by the embodiment comprises a reagent R1 and a reagent R2:
(1) the reagent R1 comprises the following components in percentage by weight:
(2) the reagent R2 comprises the following components in percentage by weight:
(3) the DIPSO buffer solution of the reagent R1 is a buffer solution with the temperature of 25 ℃ and the pH value of 6.35, and the DIPSO buffer solution of the reagent R2 is a buffer solution with the temperature of 25 ℃ and the pH value of 7.88.
(4) The preparation steps of the 83nm and 185nm rabbit anti-human myeloperoxidase antibody coated latex particles are as follows:
step 1: respectively taking 1mg of carboxyl latex particles with the particle size of 83nm and 185nm, respectively adding MOPS-HCL buffer solution with the pH value of 5.8 of 5 mLcarboxyl latex particles, uniformly mixing by oscillation, then centrifuging (the carboxyl latex particles with the particle size of 83nm, the centrifugal speed of 20000rpm, the time of 45 min; the carboxyl latex particles with the particle size of 185nm, the centrifugal speed of 12000rpm, the time of 45min), discarding supernatant, adding MOPS-HCL buffer solution with the pH value of 3mLpH value of 5.8, and carrying out ultrasonic resuspension to obtain a buffer system of 83nm carboxyl latex particles and a buffer system of 185nm carboxyl latex particles;
step 2: respectively adding 0.6mg of EDC and 0.4mg of NHS into each 1mg of carboxyl latex particles in the 83nm carboxyl latex particle buffer system and the 185nm carboxyl latex particle buffer system obtained in the step 1, then supplementing MOPS-HCL buffer solution to 5mL, carrying out oscillation reaction at the temperature of 32 ℃ and the rotating speed of 110rpm for 25min, and activating;
and step 3: after activation, centrifuging (carboxyl latex particles with the particle size of 83nm, the centrifugation speed of 20000rpm for 45 min; carboxyl latex particles with the particle size of 185nm, the centrifugation speed of 12000rpm for 45min), removing supernatant, adding 3mL of MOPS-HCL buffer solution for ultrasonic resuspension, repeating once, adding 5 mLPIPES with the pH value of 7.15 for ultrasonic resuspension, and standing by;
and 4, step 4: adding 3mg of myeloperoxidase antibody into the solution obtained in the step 3, uniformly mixing, and reacting for 3 hours at the temperature of 40 ℃ in a constant-temperature oscillation box;
and 5: after the reaction is finished, adding 200 mu L of 50mg/mL BSA, and blocking for 12 hours at 2-8 ℃;
step 6: centrifuging (83 nm carboxyl latex particle with centrifuging speed of 20000rpm for 45 min; 185nm carboxyl latex particle with centrifuging speed of 12000rpm for 45min), discarding the supernatant to obtain precipitate as 83nm rabbit anti-human myeloperoxidase antibody-coated latex particle and 185nm rabbit anti-human myeloperoxidase antibody-coated latex particle.
(5) The kit of the embodiment is used as follows:
the myeloperoxidase detection kit described in this example uses a full-automatic biochemical analyzer with a double reagent function, such as Hitachi 7180 full-automatic analyzer, to perform the measurement by an end-point method. R1 and R2 were placed at the corresponding reagent sites in a ratio of 4:1, and distilled water, standards and specimens were placed at the corresponding positions on the sample tray, as shown in Table 1.
TABLE 1 method of reagent detection
And (3) calculating: myeloperoxidase content (ng/mL) ═ Δ a assay ÷ Δ a standard) × C standard
Example 2
The myeloperoxidase detection kit described in this example comprises reagent R1 and reagent R2:
(1) the components of the reagent R1 comprise:
the reagent R1 comprises the following components in percentage by weight:
(2) the reagent R2 comprises the following components in percentage by weight:
the specific preparation process and reagent application method are the same as in example 1.
Comparative example 1
The comparative example adopts a Myeloperoxidase (MPO) detection kit which is approved by the national food and drug administration and is common in the market. The kit does not adopt 2 latex particles, and does not add a novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether and carboxylated ferrimagnetic nanoparticles (DMSA-Fe2O 3).
Comparative tests will be conducted on example 1, example 2 and comparative example 1.
Test one precision test
1 part of each of the traceable high-value quality control substance (target value 320ng/mL) and the traceable low-value quality control substance (target value 80ng/mL) is taken, the reagents are prepared by the formulas of example 1, example 2 and comparative example 1 respectively to prepare the myeloperoxidase detection kit for contrast detection, each quality control substance is detected for 20 times, and the average value, the standard deviation and the variation coefficient of the 20 detection results are calculated. The results are shown in tables 2-1 and 2-2.
TABLE 2-1 high-value quality control substance precision test data table
| Example 1 | Example 2 | Comparative example 1 | |
| Mean value (X) | 319.7 | 291.7 | 287.4 |
| Standard deviation (S) | 2.621 | 2.246 | 10.318 |
| Coefficient of variation CV | 0.82％ | 0.77％ | 3.59％ |
TABLE 2-2 Low-value quality control substance precision test data table
| Example 1 | Example 2 | Comparative example 1 | |
| Mean value (X) | 79.6 | 79.2 | 78.1 |
| Standard deviation (S) | 1.425 | 1.433 | 3.836 |
| Coefficient of variation CV | 1.79％ | 1.81％ | 4.91％ |
As can be seen from the variation coefficients in tables 2-1 and 2-2, compared with comparative example 1, the detection values of the kit prepared in the formulas of examples 1 and 2 are closer to the target value, the standard deviation and the variation coefficient are smaller, and the kit has higher batch precision, which indicates that the invention optimizes the reaction system and greatly improves the batch precision of the reagent by adopting a latex enhanced immunoturbidimetry (combining 2 carboxyl nano latex particles with different particle sizes), adopting a novel surfactant of triisopropanolamine polyoxypropylene polyoxyethylene ether and carboxylated ferrimagnetism trioxide nano particles in scientific proportioning.
Test two-accuracy comparison test
1 part of each of the traceable high-value quality control substance (target value 320ng/mL) and the traceable low-value quality control substance (target value 80ng/mL) is taken, the reagents are prepared by the formulas of example 1, example 2 and comparative example 1 respectively, the myeloperoxidase detection kit is prepared for contrast detection, each detection is carried out for 5 times, the average value is calculated, and the control is carried out with the target value of the quality control substance. The results are shown in tables 3-1 and 3-2.
TABLE 3-1 high-value quality control substance accuracy test data table
| Example 1 | Example 2 | Comparative example 1 | |
| Maximum value | 321.0 | 320.8 | 321.6 |
| Minimum value | 319.0 | 319.4 | 310.6 |
| Mean value of | 320.3 | 320.6 | 313.6 |
TABLE 3-2 Low-value quality control substance accuracy test data table
| Example 1 | Example 2 | Comparative example 1 | |
| Maximum value | 80.0 | 79.2 | 79.8 |
| Minimum value | 77.0 | 76.3 | 74.6 |
| Mean value of | 79.1 | 79.6 | 76.3 |
As can be seen from the detection results in tables 3-1 and 3-2, compared with comparative example 1, the detection values of the kit prepared in the formulas of examples 1 and 2 are closer to the target value and have higher accuracy, which indicates that the invention optimizes the reaction system and greatly improves the accuracy of the reagent by adopting a latex enhanced immunoturbidimetry (2 types of carboxyl nano latex particles with different particle sizes are used together), simultaneously adopting novel surfactants of triisopropanolamine polyoxypropylene polyoxyethylene ether, carboxyl ferric oxide magnetic nano particles and scientifically proportioning stabilizers of D-idose, erythritol and the like.
Comparative test for testing sensitivity of three reagents
Taking 7 concentration samples of traceable calibrator diluted from low to high, preparing reagents by using the formulas of example 1, example 2 and comparative example 1 respectively to prepare a myeloperoxidase detection kit for contrast detection, and comparing the detection result with the theoretical concentration. The results are shown in Table 4.
TABLE 4 table of sensitivity comparison test data
| Theoretical concentration | Example 1 | Example 2 | Comparative example 1 |
| 2.5ng/mL | 2.8 | 2.3 | 0.5 |
| 12.5ng/mL | 12.8 | 12.3 | 9.8 |
| 25ng/mL | 25.5 | 26.3 | 23.5 |
| 400ng/mL | 401.0 | 401.4 | 400.4 |
| 600ng/mL | 602.4 | 602.6 | 601.4 |
| 800ng/mL | 800.4 | 800.5 | 751.5 |
| 1300ng/mL | 1304.8 | 1302.6 | 1108.2 |
As can be seen from the results of the tests in Table 4, when the sample concentration is as low as 2.5ng/mL, the test value of comparative example 1 is 0.5, while the reagent prepared by the formulations of examples 1 and 2 can still detect the accurate value of the sample; and the accuracy of the reagent formulated in examples 1 and 2 was higher in detecting low value samples (2.5-25ng/mL) near the lower limit of linearity than in comparative example 1. In addition, when the sample concentration is at the upper linear limit of 1300ng/mL, the detection value of the comparative example is 1108.2ng/mL, the test value is obviously lower, and the reagent formulated in the formulas of examples 1 and 2 can still detect the accurate value of the sample, which shows that the reagent formulated in the formulas of examples 1 and 2 has higher analysis sensitivity and accuracy and wider linear range. The invention adopts latex enhanced immunoturbidimetry (2 carboxyl nano latex particles with different particle diameters are used together), adopts a novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether and carboxylated ferrimagnetic oxide nano particles, scientifically mixes stabilizers such as D-idose, erythritol and the like, optimizes a reaction system and greatly improves the analysis sensitivity of the reagent.
Test four-reagent calibration curve contrast test
Reagents are prepared by the formulas of example 1, example 2 and comparative example 1, and a myeloperoxidase detection kit is prepared for calibration detection. The absorbance results for each concentration point are shown in Table 5, and the calibration curve is shown in FIG. 1.
TABLE 5 calibration and comparison test data sheet for each kit
| Theoretical concentration | Example 1 | Example 2 | Comparative example 1 |
| 0ng/mL | 0.12 | 0.54 | 1.14 |
| 300ng/mL | 852.21 | 785.13 | 421.02 |
| 600ng/mL | 1822.15 | 1752.32 | 1225.21 |
| 900ng/mL | 3200.33 | 3001.23 | 2450.1 |
| 1300ng/mL | 5200.25 | 5012.45 | 3785.36 |
As can be seen from the results in Table 5, when the concentration of the calibrator was 300ng/mL, the absorbance values of comparative example 1 were 421.02, while the absorbance values of the formulated reagents of examples 1 and 2 were 852.21 and 785.13; in addition, when the sample concentration is 1300ng/mL at the upper linear limit, the absorbance value of the comparative example 1 is 3785.36 and is far lower than that of the formulation reagent of the examples 1 and 2, which shows that the formulation reagent kit of the examples 1 and 2 has higher analysis sensitivity and wider linear range. Therefore, the latex enhanced immunoturbidimetry (combining 2 carboxyl nano latex particles with different particle sizes) is adopted, a novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether and carboxylated ferrimagnetic oxide nano particles are adopted, and stabilizers such as D-idose, erythritol and the like are scientifically proportioned, so that a reaction system is optimized, and the analysis sensitivity and the linear range of the reagent are greatly improved.
Stability control test for test five reagents
The reagents of example 1, example 2 and comparative example 1 were uniformly distributed into 13 groups, and the reagent amount of each group was 20mL for R1 and 5mL for R2. The test sample is placed in a refrigerator at 2-8 ℃, a group of reagents are taken out on the same day of each month for testing the quality control of the myeloperoxidase (the target value is 320ng/mL), the test result is shown in figure 2, and the reagents of example 1-2 are more stable than the comparison sample under the storage condition at 2-8 ℃.
According to the detection result, the detection value of the comparative example 1 is 257.7-326.5ng/mL within 13 months of storage, the difference with the target value is 6.5-62.3ng/mL, and the detection value shows obvious reduction trend along with the prolonging of the storage time; the detection value of the reagent in the embodiment 1 and the embodiment 2 is 316.4-330.9ng/mL, and the difference with the target value is 3.6-10.9; this indicates that the reagents of example 1 and example 2 possess higher stability. The invention adopts latex enhanced immunoturbidimetry (2 kinds of carboxyl nano latex particles with different particle diameters are combined), adopts a novel surfactant triisopropanolamine polyoxypropylene polyoxyethylene ether and scientifically mixes stabilizers such as D-idose, erythritol and the like, optimizes a reaction system and greatly improves the stability of the reagent.
In conclusion, the kit provided by the invention has high sensitivity, good repeatability and wide linear range compared with the kit of the comparative example 1, can meet the application requirements of the market on products, has high accuracy, and is a more stable and sensitive myeloperoxidase detection kit.
Claims (6)
1. A stable and sensitive myeloperoxidase detection kit is characterized by comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight:
the reagent R2 comprises the following components in percentage by weight:
2. the myeloperoxidase detection kit according to claim 1, characterized in that the buffer solution of the reagents R1, R2 is DIPSO buffer solution with pH 6-8 at 25 ℃.
3. The myeloperoxidase detection kit of claim 1, where in the preservative is o-cymene-5-ol.
4. The myeloperoxidase detection kit according to claim 1, wherein the rabbit anti-human myeloperoxidase antibody coated latex particle is prepared by the following steps:
step 1: respectively adding 1mg of carboxyl latex particles with the particle size of 83nm and 185nm into MOPS-HCL buffer solution, oscillating, uniformly mixing, centrifuging, discarding supernatant, and then respectively adding the MOPS-HCL buffer solution for ultrasonic resuspension to obtain an 83nm carboxyl latex particle buffer system and a 185nm carboxyl latex particle buffer system;
step 2: adding EDC and NHS into the 83nm carboxyl latex particle buffer system and the 185nm carboxyl latex particle buffer system obtained in the step 1 respectively, supplementing MOPS-HCL buffer solution to 5mL, and carrying out oscillation reaction for a period of time at a certain temperature and a certain rotating speed for activation;
and step 3: after activation, centrifuging, removing supernatant, respectively adding MOPS-HCL buffer solution for ultrasonic resuspension, repeating once, and then adding PIPES for ultrasonic resuspension for later use;
and 4, step 4: respectively adding myeloperoxidase antibodies into the liquid obtained in the step 3, uniformly mixing, and reacting in a constant-temperature oscillation box;
and 5: after the reaction is finished, adding BSA, and sealing for a period of time at 2-8 ℃;
step 6: centrifuging, and discarding the supernatant to obtain precipitate, i.e. 83nm rabbit anti-human myeloperoxidase antibody coated latex particles and 185nm rabbit anti-human myeloperoxidase antibody coated latex particles.
5. The myeloperoxidase detection kit according to claim 4, characterized in that the centrifugation speed corresponding to the carboxyl latex particles with the particle size of 83nm is 20000rpm for 45 min; the centrifugation speed corresponding to the carboxyl latex particles with the particle size of 185nm is 12000rpm, and the time is 45 min.
6. The myeloperoxidase detection kit of claim 1, where in the kit is measured using an endpoint method using a fully automated biochemical analyzer with a detection dominant wavelength of 570nm and a ratio of R1 reagent to R2 reagent of 4: 1.
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|---|---|---|---|---|
| CN114966022A (en) * | 2022-05-24 | 2022-08-30 | 山东博科生物产业有限公司 | Myeloperoxidase detection kit |
| WO2022252241A1 (en) * | 2021-06-04 | 2022-12-08 | 山东博科生物产业有限公司 | Stable and sensitive myeloperoxidase detection kit |
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| CN113358869B (en) * | 2021-06-04 | 2022-10-14 | 山东博科生物产业有限公司 | Stable and sensitive myeloperoxidase detection kit |
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| WO2022252241A1 (en) * | 2021-06-04 | 2022-12-08 | 山东博科生物产业有限公司 | Stable and sensitive myeloperoxidase detection kit |
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| WO2022252241A1 (en) | 2022-12-08 |
| CN113358869B (en) | 2022-10-14 |
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