CN112816684B - Calibrator diluent of serum amyloid A, preparation method and application thereof - Google Patents
Calibrator diluent of serum amyloid A, preparation method and application thereof Download PDFInfo
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- 102000054727 Serum Amyloid A Human genes 0.000 title claims abstract description 45
- 108700028909 Serum Amyloid A Proteins 0.000 title claims abstract description 45
- 239000003085 diluting agent Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims description 8
- 238000010790 dilution Methods 0.000 claims abstract description 29
- 239000012895 dilution Substances 0.000 claims abstract description 29
- 239000000126 substance Substances 0.000 claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000003755 preservative agent Substances 0.000 claims abstract description 18
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 17
- 239000003792 electrolyte Substances 0.000 claims abstract description 17
- 230000002335 preservative effect Effects 0.000 claims abstract description 17
- 239000003223 protective agent Substances 0.000 claims abstract description 15
- 239000004094 surface-active agent Substances 0.000 claims abstract description 15
- 240000006162 Chenopodium quinoa Species 0.000 claims abstract description 8
- 150000004676 glycans Chemical class 0.000 claims abstract description 8
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 8
- 239000005017 polysaccharide Substances 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 36
- 210000002966 serum Anatomy 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 241001494479 Pecora Species 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- 102000007584 Prealbumin Human genes 0.000 claims description 6
- 108010071690 Prealbumin Proteins 0.000 claims description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 102000007327 Protamines Human genes 0.000 claims description 5
- 108010007568 Protamines Proteins 0.000 claims description 5
- 239000004816 latex Substances 0.000 claims description 5
- 229920000126 latex Polymers 0.000 claims description 5
- 229940048914 protamine Drugs 0.000 claims description 5
- 239000002736 nonionic surfactant Substances 0.000 claims description 4
- 102000008186 Collagen Human genes 0.000 claims description 3
- 108010035532 Collagen Proteins 0.000 claims description 3
- 108010082495 Dietary Plant Proteins Proteins 0.000 claims description 3
- 108010033040 Histones Proteins 0.000 claims description 3
- 102000011782 Keratins Human genes 0.000 claims description 3
- 108010076876 Keratins Proteins 0.000 claims description 3
- 108010058846 Ovalbumin Proteins 0.000 claims description 3
- 229920001436 collagen Polymers 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000003607 modifier Substances 0.000 claims description 3
- 229940092253 ovalbumin Drugs 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000011156 evaluation Methods 0.000 abstract description 18
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 abstract description 11
- 239000004386 Erythritol Substances 0.000 abstract description 10
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 abstract description 10
- 229940009714 erythritol Drugs 0.000 abstract description 10
- 235000019414 erythritol Nutrition 0.000 abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 7
- 150000001298 alcohols Chemical class 0.000 abstract description 5
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 abstract description 4
- 230000008569 process Effects 0.000 abstract description 4
- 238000004108 freeze drying Methods 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 27
- 230000000694 effects Effects 0.000 description 21
- 239000011159 matrix material Substances 0.000 description 15
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 239000001257 hydrogen Substances 0.000 description 5
- 229910052739 hydrogen Inorganic materials 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000012946 outsourcing Methods 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 238000005411 Van der Waals force Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000012733 comparative method Methods 0.000 description 1
- 230000004665 defense response Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940100601 interleukin-6 Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
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- 210000004185 liver Anatomy 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- -1 polyoxyethylene Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000004845 protein aggregation Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
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- 230000004044 response Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
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- 102000003390 tumor necrosis factor Human genes 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
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- Cell Biology (AREA)
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- Medicinal Chemistry (AREA)
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- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
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Abstract
The invention relates to a calibrator diluent for serum amyloid A and application thereof. The diluent comprises a protein protecting agent and a preservative, wherein the protein protecting agent is at least one of a saccharide substance, a protein substance, an electrolyte, alcohols and a surfactant, and the saccharide substance is at least one of deoxyribose, proteoglycan, erythritol and quinoa polysaccharide. The diluent can effectively remove the diluent of the calibrator, is favorable for improving the detection accuracy, can realize a liquid transportation mode, and reduces unnecessary freeze-drying process of the calibrator dilution. The calibrator prepared in the embodiment is also applied to daily reagent evaluation, and does not interfere with evaluation works such as precision, sensitivity, detection limit, accuracy and the like.
Description
Technical Field
The invention relates to the technical field of biochemical detection reagents, in particular to a calibrator diluent for serum amyloid A, a preparation method and application thereof.
Background
Human Serum Amyloid A (SAA) is a positive response protein of the acute phase reactive protein species and has a molecular weight of 12000. Its biological functions are lipid metabolism, inflammation defense and immune response. The SAA content in serum of normal people is measured to be present, when the organism is infected by bacteria or viruses, the organism is secreted by liver under the stimulation of interleukin 6, interleukin 1 and tumor necrosis factor, and SAA shows an increasing trend, so that whether the infection occurs and the severity can be judged by detecting the SAA content in blood.
In order to ensure the accuracy of SAA detection results, a reliable matched calibrator is needed, the range of the calibrator should cover 0.0-500mg/L, the average value of SAA concentration of serum of normal people in a reference interval of international general SAA is 2.33mg/L, a method for separating SAA protein from human serum is available in the prior art, but the method is expensive, and is not suitable for industrial production and application, most manufacturers choose to purchase SAA recombinant protein to perform configuration work of the calibrator, but in the configuration process, on one hand, the calibrator has very remarkable difference with serum matrix, so that the determination accuracy deviation is larger; on the other hand, because of the degradation of recombinant proteins and the influence of the stability of the calibrator, the measured value has larger fluctuation, and the requirement of clinical accurate measurement cannot be met. The protective agents on the market are also difficult to meet. Therefore, how to obtain a stable calibrator without a matrix effect, which effectively improves the detection accuracy, is a technical problem to be solved in the prior art for determining SAA by using an immune latex turbidimetric technique.
Disclosure of Invention
In view of the above, it is desirable to provide a calibrator dilution for serum amyloid a to obtain a calibrator with matrix effect and temperature.
The invention provides a calibrator diluent of serum amyloid A, which comprises a protein protecting agent and a preservative, wherein the protein protecting agent is at least one of saccharide substances, protein substances, electrolytes, alcohols and surfactants; wherein the saccharide is at least one selected from deoxyribose, proteoglycan, erythritol and quinoa polysaccharide.
Specifically, the calibrator dilution comprises the following concentration components:
30-50g/l of sugar substances;
5-20g/l of protein substances;
10-15g/l of electrolyte;
Alcohol 10-25g/l;
0.1-1.5ml/l of surfactant;
0.1-2ml/l of preservative;
the calibrator dilution can be used for diluting a calibrator with serum amyloid A with a concentration of 0-500 mg/l.
Specifically, the protein substance is at least one selected from collagen, vegetable protein, protamine, keratin, histone, ovalbumin and sheep serum prealbumin.
Specifically, the electrolyte modifier is at least one selected from sodium chloride, potassium chloride and magnesium chloride.
Specifically, the alcohol substance is at least one of glycerol and ethylene glycol.
Specifically, the surfactant is at least one of PEG and nonionic surfactant.
Specifically, the preservative is at least one selected from sodium azide and PC-300.
The invention also provides a preparation method of the calibrator diluent, which comprises the following steps:
Preparing a solution A: adding preservative and electrolyte into water until the preservative and the electrolyte are completely and uniformly mixed to obtain solution A;
preparing a solution B: completely dissolving a surfactant in water to obtain a solution B;
preparing a solution C: completely dissolving saccharide substances and protein substances in water to obtain a solution C;
preparing a solution D: mixing the solution A, the solution B and the solution C to obtain a blank buffer solution, adding alcohol substances into the blank buffer solution, and completely and uniformly mixing to obtain a solution D;
And (3) after the volume is fixed, filtering by a 0.22um filter membrane to obtain the calibrator diluent.
The invention also provides application of the calibrator diluent in preparation of a SAA detection kit of a latex immunonephelometry detection system.
The beneficial effects are that:
The diluent provided by the invention can effectively remove the diluent of the calibrator, is beneficial to improving the detection accuracy, can realize a liquid transportation mode, and reduces unnecessary freeze-drying process of the calibrator dilution. The calibrator prepared in the embodiment is also applied to daily reagent evaluation, and does not interfere with evaluation works such as precision, sensitivity, detection limit, accuracy and the like.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention aims to obtain a stable calibrator without a matrix effect, effectively improves the detection accuracy, and is a technical problem to be solved in the prior art for determining SAA by using a latex turbidimetric technique.
Factors that have an influence on the matrix effect are: instruments, reagents, measurement methods and samples to be evaluated (treated samples of reference substances, calibrators, quality controls, etc.). In general, the device used for detection and the measurement method are not greatly different, and the sample to be evaluated in the kit becomes a main influencing factor. Wherein, for the sample being evaluated, the ionic strength, pH, temperature, etc. of the diluent contained therein will have an effect on the analyte activity coefficient, thereby affecting the assay, resulting in a matrix effect.
The invention needs to obtain the stability of serum amyloid A calibrator, and the antigen activity of the calibrator needs to be maintained, and the main purpose is to maintain the stability of the structural space of the calibrator, including the stability of peptide bonds in a primary structure, hydrogen bonds in a secondary structure, hydrophobic bonds, ionic bonds, hydrogen bonds and Van der Waals forces in a tertiary structure, and factors influencing the stability of the result bonds are generally pH value, ionic strength, salinity and microorganism action in a system, oxidation of air and the like.
Generally, the amino acid has an antioxidant, can reduce the ionic strength, can effectively maintain the stability of peptide bonds, hydrogen bonds and ionic bonds in the tertiary structure of the calibrator, and has a certain effect on stabilizing the pH value of a solution and the solubility of the calibrator, wherein the effect of arginine and sheep serum preprotein is more prominent, and the structural change of the calibrator cannot be caused under different pH conditions.
In addition, the invention creatively discovers that the saccharides of deoxyribose, proteoglycan, erythritol and quinoa polysaccharide can increase the viscosity of serum amyloid A in a system, prevent the movement of an amino acid chain segment of the serum amyloid A from being unfolded and precipitated in the system, thereby inhibiting the interconversion between a sub-energy level and the structure relaxation of the serum amyloid A, maintaining the stable molecular tertiary structure of the serum amyloid A, and playing a role in protecting the activity of the serum amyloid A, preferably the erythritol and quinoa polysaccharide.
Alcohols not only can keep the surface of the antigen moist and prevent the antigen from being inactivated due to water loss, but also can reduce molecular movement and avoid protein aggregation. The surfactant can reduce intermolecular van der Waals force and inhibit nonspecific binding, and the Polymer (PEG) of other chemically inert high molecular substances can form a protective film on the surface of the antigen due to the large molecular weight, so that the connection position of the hydrogen bond can be protected from being directly exposed in the surrounding environment, and the integrity of the natural structure and the function of the protein is maintained, so that the protein structure is prevented from being damaged; on the other hand, as the shelf life is prolonged, organic substances such as amino acids and saccharides in the antigen and its stabilizer are corroded by microorganisms in the air and water, and therefore, a certain preservative is required to be added.
Calibrator dilution
Therefore, the embodiment of the invention provides a calibrator dilution for serum amyloid A, which comprises a protein protecting agent and a preservative, wherein the protein protecting agent is at least one of saccharide substances, protein substances, electrolytes, alcohols and surfactants; wherein the saccharide is at least one selected from deoxyribose, proteoglycan, erythritol (mE) and quinoa polysaccharide (LM).
Specifically, the calibrator diluent provided by the embodiment of the invention comprises the following concentration components:
30-50g/l of sugar substances;
5-20g/l of protein substances;
10-15g/l of electrolyte;
Alcohol 10-25g/l;
0.1-1.5ml/l of surfactant;
0.1-2ml/l of preservative;
the calibrator dilution can be used to dilute SAA calibrators at concentrations of 0-500 mg/l.
Wherein the protein is at least one selected from collagen, vegetable protein, protamine, keratin, histone, ovalbumin and sheep serum prealbumin. Preferred are protamine (abbreviated as Pro) and sheep serum prealbumin (abbreviated as PA).
Wherein the electrolyte modifier is at least one selected from sodium chloride, potassium chloride and magnesium chloride.
Wherein the alcohol is at least one of glycerol and ethylene glycol.
Wherein the surfactant is at least one of PEG and nonionic surfactant. Wherein the nonionic surfactant is exemplified by alkylphenol ethoxylates (APEO), higher fatty Alcohol Ethoxylates (AEO), fatty acid polyoxyethylene esters (AE) or fatty acid methyl ester ethoxylates (FMEE)
Wherein the preservative is at least one selected from sodium azide and PC-300.
The invention also provides a preparation method of the calibrator diluent provided by the embodiment, which comprises the following steps:
Preparing a solution A: adding preservative and electrolyte into water until the preservative and the electrolyte are completely and uniformly mixed to obtain solution A;
preparing a solution B: completely dissolving a surfactant in water to obtain a solution B;
preparing a solution C: completely dissolving saccharide substances and protein substances in water to obtain a solution C;
preparing a solution D: mixing the solution A, the solution B and the solution C to obtain a blank buffer solution, adding alcohol substances into the blank buffer solution, and completely and uniformly mixing to obtain a solution D;
And (3) after the volume is fixed, filtering by a 0.22um filter membrane to obtain the calibrator diluent.
The calibrator diluent provided by the embodiment of the invention can be applied to preparation of a SAA detection kit of a latex immunonephelometry detection system.
For the purpose of describing specific examples of the above-described calibrator dilutions, they are listed below in tables 1 and 2.
TABLE 1
TABLE 2
Application evaluation
In order to evaluate the influence of the calibrator dilution provided by the embodiment of the present invention on SAA protein, the calibrator dilution provided by the above embodiment of the present invention is used to obtain calibrators (5.0 mg/l,120.0mg/l,250.0 mg/l) with different SAA protein concentrations by gradient dilution according to different ratios; and respectively carrying out a thermal breaking stability test and a transportation stability test, wherein the specific experimental operation and the specific experimental result are as follows: 1. thermal break stability
3 Levels of SAA calibrators were prepared using the dilutions prepared in the examples and comparative examples, respectively, and were placed in a refrigerator at 4℃and an oven at 37℃for 14 days (each tube was provided with a flat tube, and the average value was taken) to sample for a given time, and the rate of change in SAA content of the corresponding calibrators was measured using the outsourcing reagent SAA kit, and the results are shown in Table 3.
Table 3 day 14 different concentration calibration rate of change in product (mg/l) (%)
As can be seen from Table 3, the SAA calibrators prepared in examples 1-13 of the present invention have certain advantages over the calibrators prepared in comparative examples 1-14 and outsourcing dilutions in that they are stored at 37℃and 4℃for 14 days with SAA content fluctuation < 7%.
2. Transportation stability
3 Levels (5.0 mg/l, 120.0mg/l and 250.0 mg/l) of SAA calibrator and 3 levels (5.0 mg/l, 120.0mg/l and 250.0 mg/l) of SAA calibrator prepared from the dilutions prepared in examples 1 to 13 and comparative examples 1 to 14 were prepared, respectively, and placed in a shaker at 42℃for 1, 2, 3,4 and 5 days (simulated transportation conditions, parallel tubes were set for each tube, and average values were taken), and samples were taken until the given time, and the change rates of SAA content of the corresponding calibrator were measured using an outsourcing reagent SAA kit, and the results are shown in Table 4.
Table 4 day 5 different concentration calibration rate of change in product (mg/l) (%)
As is clear from Table 4, the transport stability of the SAA calibrators prepared in examples 1 to 13 was better than that of comparative examples 1 to 14.
3. Matrix effect evaluation:
At least 20 fresh human serum samples were taken, and the concentration of serum should cover a linear range. The treated sample is the evaluated object of the experimental example (the calibration products prepared in examples 1-13 and comparative examples 1-14 respectively), the treated sample is mixed in 20 fresh human serum, the measurement is repeated three times, and calibration is carried out each time; the test was completed in one day, the control method was the self-contained calibration of the kit, and the evaluation method was the treated sample. The data are shown below in table 5 as average deviations for each concentration of calibrator. As can be seen from Table 5, the sample levels in each concentration in each of examples 1 to 13 were significantly lower than those in comparative examples 1 to 14.
TABLE 5
| Examples | 5.0mg/l | 120.0mg/l | 250.0mg/l |
| Example 1 | -7.83 | -4.52 | -4.56 |
| Example 2 | -7.37 | -4.40 | -4.48 |
| Example 3 | -7.29 | -4.13 | -4.05 |
| Example 4 | -7.08 | -3.56 | -3.28 |
| Example 5 | -6.88 | -3.02 | -3.23 |
| Example 6 | -6.58 | -2.47 | -3.07 |
| Example 7 | -8.21 | -4.81 | -4.72 |
| Example 8 | -6.45 | -2.45 | -3.02 |
| Example 9 | -5.36 | -2.03 | -2.92 |
| Example 10 | -4.99 | -1.48 | -1.49 |
| Example 11 | -4.55 | -1.62 | -1.44 |
| Example 12 | -3.87 | -1.21 | -1.14 |
| Example 13 | -3.21 | -1.17 | -1.02 |
| Comparative example 1 | -10.92 | -8.99 | -14.16 |
| Comparative example 2 | -11.42 | -10.81 | -12.62 |
| Comparative example 3 | -11.71 | -12.02 | -11.23 |
| Comparative example 4 | -12.75 | -12.62 | -11.87 |
| Comparative example 5 | -12.42 | -12.42 | -12.27 |
| Comparative example 6 | -12.38 | -12.87 | -12.91 |
| Comparative example 7 | -12.35 | -13.79 | -12.65 |
| Comparative example 8 | -12.86 | -13.82 | -13.02 |
| Comparative example 9 | -14.91 | -13.52 | -14.51 |
| Comparative example 10 | -14.12 | -13.53 | -14.74 |
| Comparative example 11 | -18.21 | -14.35 | -15.14 |
| Comparative example 12 | -19.33 | -18.24 | -16.31 |
| Comparative example 13 | -24.14 | -19.24 | -19.45 |
| Comparative example 14 | -24.09 | -19.18 | -19.83 |
Then, the calibrators prepared in each of examples 1 to 13 and comparative examples 1 to 14 were evaluated for the matrix effect at a level of 120.0mg/l according to WS/T356-2011 matrix effect and interoperability evaluation guidelines, using the following control method: ID-LC/MS/MS (isotope dilution liquid chromatography tandem mass spectrometry method), the evaluation method is as follows: the confidence interval technical formula of zhen ke biological kit is calculated by adopting a formula (1) pointed out by 4.3.4 in the guideline, and is as follows:
Wherein: the X is a comparison method result, and the Y is an evaluation method result; n is the number of samples, S y,x is the result of the evaluation method, the X is the result of the comparison method, linear regression analysis is selected, the regression standard error of the regression equation is constructed, Is the i-th value on the X axis; the overall mean of the mean was determined for all sample comparison methods.
TABLE 5 evaluation of matrix effects (120.0 mg/l), + indicates a matrix effect, -indicates no matrix effect
Using the above method, the 95% confidence interval for the y value of each calibrator (calibrator dilutions of examples 1-13 and comparative examples 1-14 above, respectively) was calculated using the mean of the comparative method measurements as the X axis. If the measured mean value of the evaluation method falls within the interval, the calibrator diluent has no matrix effect on the evaluation method, and the substances on the surfaces have interoperability between the comparison method and the evaluation method. As can be seen from Table 5, the evaluation methods were such that the measurements of the calibrators of examples 1 to 13 all fall within the above confidence interval, while the measurements of the calibrators of comparative examples 1 to 14 all fall within the above confidence interval, and a matrix effect was present. Therefore, the calibrator diluent provided by the invention can effectively remove the calibrator diluent, and is beneficial to improving the detection accuracy.
In summary, the dilutions provided in the present invention are effective in removing calibrator dilutions, especially in examples using erythritol or quinoa polysaccharide as saccharide protectant, which makes the calibrator superior to the comparative examples in heat-break stability and transport stability, and without matrix effect (example 7 versus comparative example 1); erythritol or quinoa polysaccharide not only can increase the system viscosity of SAA, prevent the SAA from being unfolded and precipitated due to the blocked chain segment movement, but also can remove hydroxyl free radicals formed by dissolving oxygen in the air into the solution system, has a certain oxidation resistance, and the erythritol has a protective effect on a calibrator by utilizing the stability of the erythritol to acid and heat. These effects are not provided in existing lactose and other general carbohydrate protectants.
Further, by comparing other examples with comparative examples (comparison of examples 1 to 5 with example 7, comparison of examples 1 to 5 with comparative examples 1 to 5), it was found that protamine can assist erythritol, enhance the antioxidant ability of the system and can reduce the ionic strength, and can effectively maintain the stability of peptide bond, hydrogen bond, and ionic bond in the tertiary structure of the calibrator. The sheep serum prealbumin acting protein protective agent is used, and electrolytes, alcohols, surface active agents and preservatives in a diluent system are optimized, so that the sheep serum prealbumin acting protein protective agent can cooperate with the saccharide protective agent to maintain the structural stability of the calibrator. Therefore, the combined action of the saccharide protective agent, the protein protective agent and other protective agents is beneficial to improving the detection accuracy, can realize a liquid transportation mode and reduces unnecessary freeze-drying process of calibrator dilution. The calibrator prepared in the embodiment is also applied to daily reagent evaluation, and does not interfere with evaluation works such as precision, sensitivity, detection limit, accuracy and the like.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (8)
1. The calibrator diluent for the serum amyloid A is characterized by comprising a protein protecting agent and a preservative, wherein the protein protecting agent is at least one of a saccharide substance, a protein substance, an electrolyte, an alcohol and a surfactant; wherein the saccharide is quinoa polysaccharide;
The calibrator dilution comprises the following concentration components:
30-50g/l of sugar substances;
5-20g/l of protein substances;
10-15g/l of electrolyte;
alcohol 10-25g/l;
0.1-1.5ml/l of surfactant;
0.1-2ml/l of preservative;
the calibrator dilution can be used for diluting a calibrator with serum amyloid A with a concentration of 0-500 mg/l.
2. The calibrator dilution according to claim 1, wherein the proteinaceous material is selected from at least one of collagen, vegetable protein, protamine, keratin, histone, ovalbumin, and sheep serum prealbumin.
3. The calibrator dilution according to claim 1, wherein the electrolyte modifier is selected from at least one of sodium chloride, potassium chloride, and magnesium chloride.
4. The calibrator dilution according to claim 1, wherein the alcohol is at least one of glycerol and ethylene glycol.
5. The calibrator dilution according to claim 1, wherein the surfactant is at least one of PEG and a nonionic surfactant.
6. The calibrator dilution according to claim 1, wherein the preservative is selected from at least one of sodium azide and PC-300.
7. A method of preparing a calibrator dilution according to any one of claims 1-6, comprising the steps of:
Preparing a solution A: adding preservative and electrolyte into water until the preservative and the electrolyte are completely and uniformly mixed to obtain solution A;
preparing a solution B: completely dissolving a surfactant in water to obtain a solution B;
preparing a solution C: completely dissolving saccharide substances and protein substances in water to obtain a solution C;
preparing a solution D: mixing the solution A, the solution B and the solution C to obtain a blank buffer solution, adding alcohol substances into the blank buffer solution, and completely and uniformly mixing to obtain a solution D;
And (3) after the volume is fixed, filtering by a 0.22um filter membrane to obtain the calibrator diluent.
8. Use of a calibrator dilution according to any one of claims 1-6 or prepared by the formulation method of claim 7 in the preparation of a SAA detection kit for a latex immunonephelometry detection system.
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| WO1997006184A1 (en) * | 1995-08-08 | 1997-02-20 | Eiken Kagaku Kabushiki Kaisha | Antibody that recognizes serum amyloid a |
| JPH10282105A (en) * | 1997-04-07 | 1998-10-23 | Eiken Chem Co Ltd | Manufacture of calibrator for measuring serum amyloid a |
| WO2012048854A2 (en) * | 2010-10-12 | 2012-04-19 | Merz Pharma Gmbh & Co. Kgaa | Formulation suitable for stabilizing proteins, which is free of mammalian excipients |
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