CN109781991B - C-reactive protein preserving fluid, calibrator, quality control product and detection kit - Google Patents
C-reactive protein preserving fluid, calibrator, quality control product and detection kit Download PDFInfo
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- CN109781991B CN109781991B CN201811597268.2A CN201811597268A CN109781991B CN 109781991 B CN109781991 B CN 109781991B CN 201811597268 A CN201811597268 A CN 201811597268A CN 109781991 B CN109781991 B CN 109781991B
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Abstract
The invention discloses a C-reactive protein preserving fluid, a calibrator, a quality control product and a detection kit, wherein the C-reactive protein preserving fluid comprises a buffer solution, 0.9-9.0 wt% of sodium chloride, 10-30 wt% of sorbitol, 0.5-5 wt% of cane sugar, 0.05-0.2 wt% of a surfactant, 0.05-0.1 wt% of a preservative and 0.5-5 wt% of a protein protective agent. The C-reactive protein preserving fluid can prolong the effective period of low-temperature preservation of the C-reactive protein, and can achieve the effect of normal-temperature preservation through the mutual matching of the components, and the waste caused by improper low-temperature preservation can be avoided and the detection cost is reduced by using the C-reactive protein preserving fluid.
Description
Technical Field
The invention relates to the technical field of protein preservation, in particular to a C-reactive protein preserving fluid, a calibrator, a quality control material and a detection kit.
Background
C-reactive protein (CRP) is a substance which is found in 1930 by Tillett and Francis in the serum of pneumonia patients and has precipitation reaction with C-polysaccharide (CPS), a nonspecific type of mycopolysaccharide component of pneumococcus (now called streptococcus pneumoniae). Macleod and Avery, in 1941, demonstrated that this protein appears during acute infection, and that the flocculent precipitation reaction with C-polysaccharide is Ca-dependent2+Is present. CRP is mainly synthesized in the liver as an acute phase-responsive protein when it is induced by cytokines such as interleukin 6(IL-6) and the like at the time of tissue damage, inflammation or infection. CRP is very low in the serum of healthy people, but is very sensitive to inflammatory reaction, and the concentration of CRP is obviously increased within 4-7 h from the beginning of inflammation, so that the CRP is a non-specific inflammation indicator. CRP level can reflect the potential inflammation activity of organism, and is widely applied in clinic. CRP is a sensitive inflammatory reaction marker, the elevation of which can indicate the occurrence of a plurality of inflammatory reactions, and the elevation degree is always in positive correlation with the inflammatory reactions and the degree of tissue damage, and is widely applied to various fields of medicine, such as cardiovascular, digestive, respiratory, urinary, endocrine and reproductive system inflammatory reaction diseases, various inflammatory reaction processes and tissue necrosis, damage and recovery period thereofThe screening, monitoring, disease condition assessment and curative effect judgment of the traditional Chinese medicine have important values. With the continuous updating and improvement of the CRP detection technology, the clinical application prospect of CRP is wider.
CRP is non-covalently linked by 5 identical non-glycosylated subunits, each subunit consisting of 206 amino acid residues with a relative molecular mass of 23017. This discotic pentamer signature of CRP has led to its classification into the pentamer (pentraxins) family. The pentamer structure is easily denatured by external conditions, is easily degraded and inactivated at low concentrations, and is easily adsorbed to the tube wall to cause loss at low concentrations. For C-reactive proteins, they are typically frozen at-20 ℃ or-80 ℃, but repeated freezing and thawing can reduce their activity.
At present, the C-reactive protein calibrator and the quality control material are liquid calibrators stored at 2-8 ℃, and the general validity period is 1 year. The degradation is already carried out after the material is placed for 7 days at normal temperature, and the material is almost completely degraded after being placed for one month. Therefore, great care is required during the storage process, especially during the transportation process, cold chain transportation must be ensured, and the temperature is monitored in real time, which may cause deterioration of the calibrator or quality control product and further cause wrong test results. The C-reactive protein calibrator and the quality control product in the prior art have the advantages of high transportation cost, complex packaging, time and labor consumption, and serious waste because only the C-reactive protein calibrator and the quality control product can be discarded once the deterioration phenomenon occurs.
In the prior art, some preserving solutions for maintaining the stability and the activity of the protein exist, but some preserving solutions are not suitable for preserving a C-reactive protein reagent or have unsatisfactory preserving effect on the C-reactive protein. For example, chinese patent document CN 101324589 discloses a chemically synthesized highly efficient protein stabilizer for maintaining the stability of immunodiagnostic reagents, wherein an aqueous solution prepared from the stabilizer is used for diluting protein, and the activity of protein can be preserved by more than 80% at 4 ℃ for 10 days, and the activity retention time is still not ideal. Therefore, there is a need to develop a better preservation solution for preserving C-reactive protein.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a C-reactive protein preserving fluid, a calibrator, a quality control material and a detection kit.
The technical scheme adopted by the invention is as follows:
the invention provides a C-reactive protein preserving fluid which comprises a buffer solution, 0.9-9.0 wt% of sodium chloride, 10-30 wt% of sorbitol, 0.5-5 wt% of cane sugar, 0.05-0.2 wt% of a surfactant, 0.05-0.1 wt% of a preservative and 0.5-5 wt% of a protein protective agent.
The buffer solution in the C-reactive protein preservation solution provides necessary conditions for maintaining the activity and the structural stability of the C-reactive protein, and the addition amount of the buffer solution can be adjusted according to actual requirements.
Preferably, the protein protectant is the complex protein protectant HPD.
Further preferably, the content of the protein protective agent is 2-5 wt%.
Preferably, the pH value of the buffer solution is 7.5-9.0.
Further, the buffer solution is one of a Tris buffer solution of 10-50 mmol/L and a phosphate buffer solution of 0.05-0.2 mol/L.
Preferably, the surfactant is one or more of Triton X-405, Tween 20, cetyl ammonium bromide and cetyl-3-methyl ammonium chloride.
Preferably, the preservative is one of KroVin400, sodium azide and Proclin-300.
The present invention also provides a C-reactive protein calibrator comprising the C-reactive protein preservative solution and C-reactive protein at a concentration required for calibration.
The invention also provides a C-reactive protein quality control product, which comprises the C-reactive protein preserving fluid and C-reactive protein with the concentration required by quality control.
The C-reactive protein calibrator and the C-reactive protein quality control product can be obtained by adding the C-reactive protein with the concentration required by the calibrator or the quality control product into the prepared C-reactive protein preservation solution. The prepared C-reactive protein calibrator and the C-reactive protein quality control product are generally subjected to filtering and packaging under an aseptic environment, and can be stored at low temperature and normal temperature, and the preferred low-temperature storage temperature is 2-8 ℃.
As another object of the invention, the invention also provides a C-reactive protein detection kit, which comprises the C-reactive protein calibrator and/or the C-reactive protein quality control.
The C-reactive protein calibrator with a plurality of concentration gradients can be used for calibrating the C-reactive protein detection kit, for example, the C-reactive protein calibrator with the C-reactive protein concentration of 0mg/L, 5mg/L, 20mg/L, 45mg/L, 90mg/L and 180mg/L can be prepared for calibration. The C-reactive protein quality control material with single or two concentration gradients is prepared for indoor quality control of the C-reactive protein detection kit, for example, the C-reactive protein quality control material with the C-reactive protein concentration of 10mg/L and 50mg/L can be prepared for quality control.
The invention has the beneficial effects that:
the invention provides a C-reactive protein preserving fluid aiming at the problem of unsatisfactory preserving effect of C-reactive protein in the prior art according to the structural characteristics of the C-reactive protein. The preservation solution for the C-reactive protein prolongs the effective period of the preservation of the C-reactive protein at low temperature by adding the components such as the protein protective agent, sorbitol and sucrose, and achieves the effect of preservation at normal temperature through the combined action of the components.
Drawings
FIG. 1 is a graph comparing the calibration standard of C-reactive protein in example 3 with the CRP (II) standard.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
In the following examples, the concentration of C-reactive protein was measured using a C-reactive protein assay kit (latex-enhanced immunoturbidimetry), the assay apparatus used was an SC-5280CRP series full-automatic hematology analyzer manufactured by Shenzhen Kaili Biotechnology, Inc., and the C-reactive protein item was calibrated using a CRP (II) standard solution manufactured by Nissan research, Inc., having a product number of 612014.
Example 1
20mmoL/L, pH 8.0.0 Tris buffer solution, 0.9% NaCl, 15% sorbitol, 3% sucrose, 0.1% Triton X-405 and 0.1% KroVin400 are used as a basic formula A, and a stabilizer is added on the basis of the basic formula A to prepare the C reaction protein preservative solution.
C-reactive protein is added into the prepared C-reactive protein preservation solution to prepare samples to be tested with protein concentrations of 5mg/L, 20mg/L, 80mg/L and 180mg/L, the samples are respectively numbered as samples 1, 2, 3 and 4, the change conditions of the C-reactive protein concentration measured value and the C-reactive protein concentration value of the original sample to be tested in each sample preserved for 6 months at normal temperature are considered, and the experimental results are shown in Table 1.
TABLE 1 stability of C-reactive protein in C-reactive protein preservative fluid at room temperature for 1 month, 2 months, 4 months and 6 months
In this example, the effect of PMSF, benzamidine, EDTA, TPCK, and a protein protectant (in this example, the composite protein protectant HPD) as a stabilizer on the stability of C-reactive protein was examined. PMSF (benzoylsulfonyl fluoride) is a very common protease inhibitor, which irreversibly inhibits serine hydrolase and some cysteine hydrolase, Benzamidine (Benzamidine) is a serine inhibitor, EDTA is a metalloproteinase inhibitor, TPCK is often used as a inhibitor of serine hydrolase and cystine hydrolase, but these inhibitors are not ideal for the preservation of C-reactive protein and are cumbersome to handle. In the embodiment, the compound protein protectant HPD is matched with each component in the basic formula A, so that the C-reactive protein can be stored for a long time at normal temperature, and particularly, the C-reactive protein can be stably stored for 6 months at normal temperature under the condition that the addition amount of the compound protein protectant HPD is 2% or 5%, which shows that the C-reactive protein preservation solution can enhance the stability of the C-reactive protein.
Example 2
The C-reactive protein preservative fluid is prepared by 20mmoL/L, pH 8.0.0 Tris buffer solution, 0.9% NaCl, 15% sorbitol, 3% sucrose, 0.1% Triton X-405, 0.1% KroVin400 and 2% HPD (composite protein protectant).
Comparative solution: CRP (II) standard solution manufactured by Nissan research.
Taking the prepared C-reactive protein preservation solution, preparing 5mg/L, 20mg/L, 80mg/L and 180mg/L C-reactive protein samples 1, 2, 3 and 4 to be detected according to the concentration range of the samples to be detected, taking the comparison solution to prepare a solution corresponding to the concentration of the C-reactive protein as a comparison example, and inspecting the bottle opening stability and the long-term stability of two groups of C-reactive protein solutions, wherein the specific experimental operation is as follows: and respectively packaging the two groups of C-reactive protein solutions into 8 parts, wherein after three parts of C-reactive protein solutions are unpacked for 4 hours, covering the three parts of C-reactive protein solutions, storing the three parts of C-reactive protein solutions at 2-8 ℃ for an unpacked stability test, and taking out the two groups of C-reactive protein solutions at 1 month and 2 months respectively and measuring the concentration of the C-reactive protein. An additional 5 aliquots were stored at 2-8 ℃ for long term stability testing, and both sets of solutions were removed and assayed for C-reactive protein concentration at months 6, 12, 18, 24 and 30, respectively. Each sample was measured 10 times, averaged, and the relative deviation calculated, with the test results for open vial stability shown in table 2 and long term stability shown in table 3.
TABLE 2 open bottle stability test results
The results in Table 2 show that the concentration of C-reactive protein in the comparative solution begins to decrease after 1 month of decapping, while the concentration of C-reactive protein in the C-reactive protein preservative solution of the present example remains stable, with small relative deviation and good accuracy, regardless of the concentration values of 5mg/L, 20mg/L, 80mg/L and 180mg/L among the multiple measurements of each sample after 2 months of decapping.
TABLE 3 Long term stability test results
The results in Table 3 show that the C-reactive protein preserved in the preservation solution of the C-reactive protein of the embodiment shows good stability within two years, and the relative deviation of the samples of the concentration values of 5mg/L, 20mg/L, 80mg/L and 180mg/L all meet the technical requirements of the C-reactive protein determination kit (the accuracy requirement is that the relative deviation is +/-8%).
Example 3: accuracy analysis of C-reactive protein calibrators
Preparing a C-reactive protein preservation solution from 20mmoL/L of 8.0Tris buffer solution with pH value, 0.9% NaCl, 15% sorbitol, 3% sucrose, 0.1% Triton X-405, 0.1% KroVin400 and 2% HPD (composite protein protectant) to prepare a C-reactive protein preservation solution, and adding C-reactive protein with a concentration required by calibration into the C-reactive protein preservation solution to prepare a C-reactive protein calibrator. 100 clinical specimens were tested by using the above C-reactive protein calibrator as a calibration standard, and the 100 specimens were tested by calibrating the test system with CRP (II) standard solution manufactured by Nippon university Co., Ltd. The two groups of results were compared, and the comparison results are shown in FIG. 1. The experimental result shows that the C-reactive protein calibrator has the same accuracy as CRP (II) standard solution produced by Nippon research.
The C-reactive protein with the concentration required by quality control or calibration is added into the C-reactive protein preserving fluid prepared by the invention to prepare a C-reactive protein quality control product or a calibrator.
Claims (9)
1. The C-reactive protein calibrator is characterized by comprising C-reactive protein preserving fluid and C-reactive protein with a concentration required by calibration, wherein the C-reactive protein preserving fluid comprises a buffer solution, 0.9-9.0 wt% of sodium chloride, 10-30 wt% of sorbitol, 0.5-5 wt% of sucrose, 0.05-0.2 wt% of a surfactant, 0.05-0.1 wt% of a preservative and 2-5 wt% of a protein protective agent, and the protein protective agent is a compound protein protective agent HPD.
2. The C-reactive protein calibrator according to claim 1, wherein the pH of the buffer is 7.5-9.0, and the buffer is one of 10-50 mmol/L Tris buffer and 0.05-0.2mol/L phosphate buffer.
3. The C-reactive protein calibrator according to claim 1, wherein said surfactant is one or more of Triton X-405, Tween 20, cetyl ammonium bromide, and cetyl-3-methylammonium chloride.
4. The C-reactive protein calibrator according to claim 1, wherein said preservative is one of KroVin400, sodium azide and Proclin-300.
5. The C-reactive protein quality control product is characterized by comprising a C-reactive protein preserving fluid and C-reactive protein with the concentration required by quality control, wherein the C-reactive protein preserving fluid comprises a buffer solution, 0.9-9.0 wt% of sodium chloride, 10-30 wt% of sorbitol, 0.5-5 wt% of sucrose, 0.05-0.2 wt% of a surfactant, 0.05-0.1 wt% of a preservative and 2-5 wt% of a protein protective agent, and the protein protective agent is a compound protein protective agent HPD.
6. The C-reactive protein quality control product of claim 5, wherein the pH of the buffer solution is 7.5-9.0, and the buffer solution is one of 10-50 mmol/L Tris buffer solution and 0.05-0.2mol/L phosphate buffer solution.
7. The C-reactive protein quality control material according to claim 5, wherein the surfactant is one or more of Triton X-405, Tween 20, cetyl ammonium bromide and cetyl-3-methyl ammonium chloride.
8. The C-reactive protein quality control material of claim 5, wherein the preservative is one of KroVin400, sodium azide and Proclin-300.
9. A C-reactive protein detection kit comprising the C-reactive protein calibrator of any one of claims 1 to 4 and/or the C-reactive protein quality controller of any one of claims 5 to 8.
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| CN110331186A (en) * | 2019-07-18 | 2019-10-15 | 迪瑞医疗科技股份有限公司 | A kind of 5'-NT quality-control product |
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| CN110988367B (en) * | 2019-11-18 | 2024-02-09 | 迈克生物股份有限公司 | Storage agent, calibrator for detecting LH and detection kit |
| CN111041021A (en) * | 2019-12-27 | 2020-04-21 | 桂林优利特医疗电子有限公司 | Preparation method of angiotensin converting enzyme calibrator |
| CN111175515B (en) * | 2019-12-30 | 2024-04-26 | 上海奥普生物医药股份有限公司 | Two-in-one quality control substance of serum amyloid A and serum amyloid C reactive protein and preparation method thereof |
| CN112904028B (en) * | 2021-01-21 | 2023-11-24 | 宁波职业技术学院 | Serum amyloid A quality control product and preparation method thereof |
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| CA2333217A1 (en) * | 2001-01-31 | 2002-07-31 | Hisahide Hiura | Quality control material |
| CN102128917A (en) * | 2010-12-07 | 2011-07-20 | 江西特康科技有限公司 | Clinical biochemical quality control products and preparation process thereof |
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