CN112816684A - Serum amyloid protein A calibrator diluent, and preparation method and application thereof - Google Patents
Serum amyloid protein A calibrator diluent, and preparation method and application thereof Download PDFInfo
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- 239000003085 diluting agent Substances 0.000 title claims abstract description 36
- 102000054727 Serum Amyloid A Human genes 0.000 title claims abstract description 18
- 108700028909 Serum Amyloid A Proteins 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 239000003223 protective agent Substances 0.000 claims abstract description 16
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- 239000004094 surface-active agent Substances 0.000 claims abstract description 15
- 239000003792 electrolyte Substances 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 14
- 230000002335 preservative effect Effects 0.000 claims abstract description 14
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 claims abstract description 11
- 239000004386 Erythritol Substances 0.000 claims abstract description 10
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229940009714 erythritol Drugs 0.000 claims abstract description 10
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- 239000000243 solution Substances 0.000 claims description 47
- 239000000126 substance Substances 0.000 claims description 20
- 238000010790 dilution Methods 0.000 claims description 15
- 239000012895 dilution Substances 0.000 claims description 15
- 210000002966 serum Anatomy 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- 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
- 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
- 102000007584 Prealbumin Human genes 0.000 claims description 5
- 108010071690 Prealbumin Proteins 0.000 claims description 5
- 102000007327 Protamines Human genes 0.000 claims description 5
- 108010007568 Protamines Proteins 0.000 claims description 5
- 150000001298 alcohols Chemical class 0.000 claims description 5
- 229940048914 protamine Drugs 0.000 claims description 5
- 241001494479 Pecora Species 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 239000004816 latex Substances 0.000 claims description 4
- 229920000126 latex Polymers 0.000 claims description 4
- 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
- 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
- 238000003556 assay Methods 0.000 claims 1
- 235000011187 glycerol Nutrition 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 239000003607 modifier Substances 0.000 claims 1
- 238000011156 evaluation Methods 0.000 abstract description 18
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
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- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
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- 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
- 150000001413 amino acids Chemical class 0.000 description 3
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- 241000283707 Capra Species 0.000 description 2
- 238000005411 Van der Waals force Methods 0.000 description 2
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- 238000004879 turbidimetry Methods 0.000 description 2
- 206010048998 Acute phase reaction Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
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- 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
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- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 229930195729 fatty acid Natural products 0.000 description 1
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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)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a serum amyloid A calibrator diluent and application thereof. The diluent comprises a protein protective agent and a preservative, wherein the protein protective agent is at least one of carbohydrate, protein, electrolyte, alcohol and a surfactant, and the carbohydrate is at least one of deoxyribose, proteoglycan, erythritol and quinoa polysaccharide. This diluent can effectively get rid of the calibrator diluent, helps improving detection accuracy, and can carry out liquid transport mode, reduces the calibrator and dilutes unnecessary freeze-drying process. The calibrator configured in the embodiment is also applied to daily reagent evaluation, and does not interfere with evaluation work 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 serum amyloid A calibrator diluent, and a preparation method and application thereof.
Background
Human serum amyloid a (saa) is a positive response protein of the acute phase reaction protein species, with a molecular weight of 12000. Its biological functions are lipid metabolism, inflammation defense and immune response. The content of SAA in the serum of normal people is measured, when the organism is infected by bacteria or viruses and is secreted by the liver under the stimulation of interleukin 6, interleukin 1 and tumor necrosis factor, the SAA shows a rising trend, so whether the infection occurs or not and the severity can be judged by detecting the content of SAA in the blood.
In order to ensure the accuracy of an SAA detection result, a reliable matched calibrator is required, the range of the calibrator is 0.0-500mg/L, the average value of the SAA concentration of normal human serum in a reference interval of international general SAA is 2.33mg/L, a method for separating SAA protein from human serum is used in the prior art, but the method is expensive in cost and is not suitable for industrial production and application, most manufacturers select SAA recombinant protein to carry out configuration work of the calibrator, but in the configuration process, on one hand, the SAA recombinant protein has very obvious difference with a serum matrix, so that the deviation of the determination accuracy is large; on the other hand, due to the degradation of the recombinant protein and the influence of the stability of the calibrator, the measured value has large fluctuation and can not meet the requirement of clinical accurate measurement. The protective agents on the market are also difficult to meet. Therefore, how to obtain a stable calibrator without a matrix effect and effectively improve the detection accuracy is a technical problem to be solved urgently for the current immunolatex turbidimetry technology to measure the SAA.
Disclosure of Invention
Accordingly, there is a need to provide a dilution of serum amyloid a calibrator for obtaining a temperature calibrator with a substrate effect.
The invention provides a serum amyloid A calibrator diluent, which comprises a protein protective agent and a preservative, wherein the protein protective agent is at least one of carbohydrate 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 diluent comprises the following components in concentration:
30-50g/l of carbohydrate;
5-20g/l of protein substances;
10-15g/l of electrolyte;
alcohol substance 10-25 g/l;
0.1-1.5ml/l of surfactant;
0.1-2ml/l of preservative;
the calibrator diluent can be used for diluting a calibrator of serum amyloid A with the concentration of 0-500 mg/l.
Specifically, the protein substances are selected from at least one of collagen, vegetable protein, protamine, keratin, histone, ovalbumin and sheep serum prealbumin.
Specifically, the electrolyte regulator is selected from at least one of 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 a 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:
preparation of solution a: adding a preservative and an electrolyte into water, and completely mixing to obtain a solution A;
preparation of solution B: completely dissolving a surface active agent in water to obtain a solution B;
preparation of solution C: completely dissolving carbohydrate and protein in water to obtain solution C;
preparation of solution D: mixing the solution A, the solution B and the solution C to obtain a blank buffer solution, adding an alcohol substance into the blank buffer solution, and completely mixing the solution A, the solution B and the solution C to obtain a solution D;
and after constant volume, filtering with a 0.22um filter membrane to obtain the calibrator diluent.
The invention also provides application of the calibrator diluent in preparation of a latex immunoturbidimetry detection system SAA detection kit.
Has the advantages that:
the diluent provided by the invention can effectively remove the diluent of the calibrator, is beneficial to improving the detection accuracy, can implement a liquid transportation mode, and reduces unnecessary freeze-drying process for diluting the calibrator. The calibrator configured in the embodiment is also applied to daily reagent evaluation, and does not interfere with evaluation work such as precision, sensitivity, detection limit, accuracy and the like.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention aims to obtain a stable calibrator without matrix effect, effectively improves the detection accuracy, and is a technical problem to be solved urgently in the prior art of latex turbidimetry technology for measuring SAA.
Factors that have an influence on the matrix effect are: instruments, reagents, measurement methods and samples to be evaluated (reference substances, calibrators, quality controls, etc. treated samples). In general, the difference between the apparatus and the measurement method used for the detection is not so great, and the sample to be evaluated in the kit becomes a main influence factor. Among them, the ionic strength, pH, temperature, etc. of the diluent contained in the sample to be evaluated may have an influence on the analyte activity coefficient, thereby affecting the measured value, resulting in a matrix effect.
The invention needs to obtain the stability of the serum amyloid protein A calibrator, needs to keep the antigen activity of the calibrator, and mainly aims to keep 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 and hydrophobic bonds, ionic bonds, hydrogen bonds and Van der Waals forces in a tertiary structure, and factors influencing the stability of the resulting bonds are generally pH value, ionic strength, salt, the action of microorganisms, air oxidation and the like in a system.
Generally, the amino acid has an antioxidant, can reduce ionic strength, can effectively maintain the stability of peptide bonds, hydrogen bonds and ionic bonds in the tertiary structure of the calibrator, and has certain effects on stabilizing the pH value of a solution and the solubility of the calibrator, wherein arginine and sheep serum preproprotein have prominent effects and cannot cause structural change of the calibrator under different pH conditions.
In addition, the invention creatively discovers that several saccharides, namely deoxyribose, proteoglycan, erythritol and quinoa polysaccharide, can increase the viscosity of serum amyloid A in a system, prevent the movement of amino acid chain segments of the serum amyloid A, prevent the serum amyloid A from developing and precipitating in the system, inhibit the mutual conversion between the sublevel and the structure relaxation of the serum amyloid A, maintain the stability of the molecular tertiary structure of the serum amyloid A, and play a role in protecting the activity of the serum amyloid A, and the preferred saccharides are erythritol and quinoa polysaccharide.
The alcohols can not only keep the surface of the antigen moist and prevent the antigen from being inactivated due to water loss, but also reduce molecular movement and avoid protein aggregation. The surfactant can reduce the van der Waals force among molecules and inhibit nonspecific combination, 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 connecting position of a hydrogen bond can be protected from being directly exposed to 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 storage time is prolonged, organic substances such as amino acids and saccharides in the antigen and the stabilizer thereof are corroded by microorganisms in air and water, and therefore, a certain amount of preservative needs to be added.
Dilution of calibrator
Therefore, the embodiment of the invention provides a serum amyloid A calibrator diluent, which comprises a protein protective agent and a preservative, wherein the protein protective agent is at least one of carbohydrate 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 components in concentration:
30-50g/l of carbohydrate;
5-20g/l of protein substances;
10-15g/l of electrolyte;
alcohol substance 10-25 g/l;
0.1-1.5ml/l of surfactant;
0.1-2ml/l of preservative;
the calibrator diluent may be used to dilute SAA calibrators at concentrations of 0-500 mg/l.
Wherein the protein is selected from at least one of collagen, vegetable protein, protamine, keratin, histone, ovalbumin and sheep serum prealbumin. Protamine (Pro for short) and goat serum prealbumin (PA for short) are preferred.
Wherein the electrolyte regulator 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. Among them, nonionic surfactants are 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:
preparation of solution a: adding a preservative and an electrolyte into water, and completely mixing to obtain a solution A;
preparation of solution B: completely dissolving a surface active agent in water to obtain a solution B;
preparation of solution C: completely dissolving carbohydrate and protein in water to obtain solution C;
preparation of solution D: mixing the solution A, the solution B and the solution C to obtain a blank buffer solution, adding an alcohol substance into the blank buffer solution, and completely mixing the solution A, the solution B and the solution C to obtain a solution D;
and after constant volume, filtering with a 0.22um filter membrane to obtain the calibrator diluent.
The calibrator diluent provided by the embodiment of the invention can be applied to the preparation of a latex immunoturbidimetry detection system SAA detection kit.
To illustrate specific examples of the dilution of the calibrator, the following are shown in tables 1 and 2.
TABLE 1
TABLE 2
Application evaluation
In order to evaluate the effect of the calibrator diluent provided by the embodiment of the present invention on SAA protein, the calibrator diluent provided by the embodiment of the present invention is used to obtain calibrators (5.0mg/l, 120.0mg/l, 250.0mg/l) with different concentrations of SAA protein by gradient dilution according to different ratios; and respectively carrying out a heat-breaking stability test and a transportation stability test, wherein the specific experimental operations and results are as follows: 1. heat burst stability
Using the SAA recombinant protein from wuhan huamei, 3 levels of SAA calibrators were prepared using the dilutions prepared in the above examples and comparative examples, respectively, and placed in a 4 ℃ refrigerator and a 37 ℃ oven for 14 days (each tube was equipped with a flat tube, and the average value was taken), and sampled at a predetermined time, and the change rate of the SAA content of the corresponding calibrators was determined using an external reagent SAA kit, and the results are shown in table 3.
TABLE 3 Change rate of calibrator (mg/l) at different concentrations on day 14 (%)
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 commercially available diluents in storage at 37 ℃ and 4 ℃, and the SAA content fluctuation is less than 7% in 14 days.
2. Stability in transport
Using the SAA recombinant protein, SAA calibrators were prepared at 3 levels (5.0mg/l, 120.0mg/l, and 250.0mg/l) from the dilutions prepared in examples 1-13 and comparative examples 1-14, respectively, and were placed in a shaker at 42 ℃ for 1, 2, 3, 4, and 5 days (simulated transport conditions, parallel tubes were placed in each tube, and the average value was taken), samples were taken at a predetermined time, and the change rate of SAA content of the corresponding calibrators was determined using an SAA kit purchased from outsourcing, the results are shown in Table 4.
TABLE 4 Change rate of calibrator (mg/l) at different concentrations on day 5 (%)
As can be seen from Table 4 above, the shipping stability of the SAA calibrators prepared in examples 1-13 above was better than that of comparative examples 1-14.
3. Evaluation of matrix effect:
at least 20 samples of fresh human serum were taken and the serum concentrations should cover a linear range. The treated sample is the evaluated object of the experimental example (adopting the calibrator prepared in examples 1-13 and comparative examples 1-14 respectively), the treated sample is mixed in 20 fresh human serums, the measurement is repeated three times, and the calibration is carried out again each time; the test is completed within one day, the control method is kit self-calibration, and the evaluated method is processed sample. The data are shown below in table 5 below as the mean deviation for each concentration calibrator. As can be seen from Table 5, the sample levels at each concentration are significantly lower for each of examples 1-13 than for comparative examples 1-14.
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 |
And then performing matrix effect evaluation on the calibrators prepared in the embodiments 1-13 and the comparative examples 1-14 at a level of 120.0mg/l according to WS/T356-2011 matrix effect and interoperability evaluation guidelines, wherein the adopted control method comprises the following steps: ID-LC/MS/MS (isotope dilution liquid chromatography tandem mass spectrometry), the evaluation method is: the technical formula of confidence interval in the kit of biological materials for science, which is calculated by using formula (1) indicated by 4.3.4 in the guideline, is as follows:
in the formula:
the value of Y corresponding to the value of X is calculated according to the regression curve, X is the result of the comparison method, and Y is the result of the evaluation method; n is the number of samples, Sy,xSelecting linear regression analysis to construct regression standard error of regression equation with Y axis as evaluation method result and X axis as comparison method result,
is the ith value on the X axis;
the overall mean of the means was determined for all sample alignments.
TABLE 5 evaluation of stromal Effect (120.0mg/l), + indicates stromal Effect, and-indicates no stromal Effect
Using the above method, the 95% confidence intervals for the y-values of each calibrator (calibrators prepared corresponding to the calibrator dilutions of examples 1-13 and comparative examples 1-14 above) were calculated using the comparative mean as the X-axis. If the mean value of the evaluation method falls within this interval, it is indicated that the dilution of the calibrator has no matrix effect on the evaluation method, and the substance is shown to be interactive in the comparison method and the evaluation method. As can be seen from Table 5, the evaluation methods all fall within the above confidence intervals for the calibrators of examples 1-13, while none of the calibrators of comparative examples 1-14, and there is a matrix effect. Therefore, the calibrator diluent provided by the invention can be used for effectively removing the calibrator diluent, and is beneficial to improving the detection accuracy.
In conclusion, the diluent provided by the invention can effectively remove the diluent of the calibrator, particularly erythritol or quinoa polysaccharide is used as a saccharide protective agent in the embodiment, so that the calibrator has better heat crushing stability and transportation stability than the comparative example, and has no matrix effect (as example 7 relative to comparative example 1); the erythritol or quinoa polysaccharide can increase the system viscosity of the SAA, prevent chain segment movement of the SAA, prevent the SAA from unfolding and precipitating, has a certain oxidation resistance when a solution has a scavenging effect on hydroxyl radicals formed by dissolving oxygen in the air in a solution system, and provides a protective effect on a calibrator by utilizing the stability of the erythritol on acid and heat. These effects are not provided by existing lactose and other general carbohydrate protectants.
Further, by comparing other examples with the comparative examples (for example, comparing examples 1 to 5 with example 7, and comparing examples 1 to 5 with comparative examples 1 to 5), it is found that protamine can assist erythritol, enhance the antioxidant ability of the system, reduce the ionic strength, and effectively maintain the stability of peptide bonds, hydrogen bonds, and ionic bonds in the tertiary structure of the calibrator. The goat serum prealbumin acts as a protein protective agent, and electrolytes, alcohols, surface active agents and preservatives in a diluent system are optimized, so that the protein protective agent can act synergistically with the saccharide protective agent to maintain the stable structure of the calibrator. Therefore, the comprehensive effects of the carbohydrate protective agent, the protein protective agent and other protective agents are beneficial to improving the detection accuracy, the liquid transportation mode can be realized, and the unnecessary freeze-drying process for diluting the calibrator is reduced. The calibrator configured in the embodiment is also applied to daily reagent evaluation, and does not interfere with evaluation work such as precision, sensitivity, detection limit, accuracy and the like.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (9)
1. A serum amyloid A calibrator diluent is characterized by comprising a protein protective agent and a preservative, wherein the protein protective agent is at least one of carbohydrate substances, protein substances, electrolytes, alcohols and surfactants; wherein the saccharide is at least one selected from deoxyribose, proteoglycan, erythritol and quinoa polysaccharide.
2. The calibrator dilution of claim 1, comprising the following concentration components:
30-50g/l of carbohydrate;
5-20g/l of protein substances;
10-15g/l of electrolyte;
alcohol substance 10-25 g/l;
0.1-1.5ml/l of surfactant;
0.1-2ml/l of preservative;
the calibrator diluent can be used for diluting a calibrator of serum amyloid A with the concentration of 0-500 mg/l.
3. The calibrator diluent according to claim 1 or 2, wherein the proteinaceous material is at least one selected from the group consisting of collagen, vegetable protein, protamine, keratin, histone, ovalbumin, and sheep serum prealbumin.
4. The calibrator dilution of claim 1 or 2, wherein the electrolyte modifier is selected from at least one of sodium chloride, potassium chloride, and magnesium chloride.
5. The calibrator diluent of claim 1 or 2, wherein the alcohol is at least one of glycerin and ethylene glycol.
6. The calibrator diluent of claim 1 or 2, wherein the surfactant is at least one of PEG and a nonionic surfactant.
7. The calibrator dilution of claim 1 or 2, wherein the preservative is selected from at least one of sodium azide and PC-300.
8. A method of preparing a dilution of a calibrator solution according to any one of claims 1 to 7, comprising the steps of:
preparation of solution a: adding a preservative and an electrolyte into water, and completely mixing to obtain a solution A;
preparation of solution B: completely dissolving a surface active agent in water to obtain a solution B;
preparation of solution C: completely dissolving carbohydrate and protein in water to obtain solution C;
preparation of solution D: mixing the solution A, the solution B and the solution C to obtain a blank buffer solution, adding an alcohol substance into the blank buffer solution, and completely mixing the solution A, the solution B and the solution C to obtain a solution D;
and after constant volume, filtering with a 0.22um filter membrane to obtain the calibrator diluent.
9. Use of a dilution of a calibrator described in any one of claims 1 to 7 or a dilution of a calibrator prepared by the preparation method described in claim 8 in the preparation of a kit for the detection of SAA in a latex immunoturbidimetric assay system.
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