CN110632325A - Total protein detection reagent and preparation method thereof - Google Patents
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Abstract
The invention relates to a total protein detection reagent and a preparation method thereof, galloylglucose is creatively added into the detection reagent, and has strong binding force with copper sulfate and total protein, so that the complexation of copper ions to the total protein can be greatly improved, the detection precision is further improved, and the usage amount of the copper ions can be effectively reduced; in addition, the storage stability of the detection reagent is also obviously improved. The preparation method provided by the invention has the advantages that the galloyl glucose and the nonionic surfactant are mixed firstly, molecular chains of the galloyl glucose and the nonionic surfactant are fully wound, then the galloyl glucose and the nonionic surfactant are mixed with the copper sulfate, the potassium sodium tartrate and part of water, and the galloyl glucose and the copper ions are complexed, so that the obtained complex product is soluble in water, the generation of copper hydroxide precipitate is avoided, the compatibility of the complex in water is greatly increased, all components in the detection reagent obtained by the preparation method can stably exist in a system, and the reliability of the detection reagent in long-time use is improved.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a total protein detection reagent and a preparation method thereof.
Background
Total Protein (TP) is an important index for testing the metabolic capacity of liver function and reflects the reserve capacity of the liver. The high TP can bring certain harm to people, the TP of normal people is between 60 and 80g/L, and the condition that the TP exceeds the range indicates that the liver is damaged to a certain extent.
The clinically common methods for detecting total protein include turbidimetry, dye binding and biuret colorimetry.
The turbidimetry is to add sulfosalicylic acid (SSA), trichloroacetic acid (TCA) and tungstic acid into urine to precipitate protein to form certain turbidity, and then to detect the protein content by turbidimetry. The method has poor accuracy and poor anti-interference capability.
Dye binding methods are classified into direct methods and precipitation methods. The direct method is to detect the maximum absorption peak shift of the protein when the dye is combined with the protein, and the dyes used in the method include Coomassie brilliant blue G-250, pyrogallol red, and phenylmethyl violet. CN105572117A discloses a urine total protein detection kit suitable for a full-automatic biochemical analyzer, which contains pyrogallol red, methanol, sodium molybdate, succinic acid, sodium oxalate, sodium benzoate, sodium dodecyl sulfate and bovine serum albumin.
The biuret colorimetric method is to utilize Cu and protein in alkaline solution2+A purple compound is formed, and the protein concentration is in direct proportion to the shade of the color of the purple compound in a certain concentration range. The method is simple and convenient, has good preparation and repeatability, and presents good linear relation in a higher concentration range, but when the concentration is lower, the accuracy of total protein detection is lower, and the condition of lower concentration of total protein cannot be accurately reflected.
In view of this, it is a difficult task to develop a reagent for detecting total protein with higher accuracy.
Disclosure of Invention
The invention aims to provide a total protein detection reagent and a preparation method thereof, wherein galloylglucose is creatively added into the detection reagent, and has strong binding capacity with copper sulfate and total protein, so that the complexing capacity of copper ions to the total protein can be greatly improved, the detection precision is further improved, and the using amount of the copper ions can be effectively reduced. In addition, the storage stability of the detection reagent is also obviously improved.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a total protein detection reagent comprising;
the total protein detection reagent is obtained by compounding the components according to a specific proportion for the first time, and due to the fact that galloylglucose is creatively added into the detection reagent, strong binding force exists between the galloylglucose and copper sulfate and the total protein, the complexation of copper ions to the total protein can be greatly improved, the detection precision is further improved, and meanwhile, the usage amount of the copper ions can be effectively reduced. In addition, the storage stability of the detection reagent is also obviously improved.
The total protein detection reagent adopts the nonionic surfactant, and mainly considers that the nonionic surfactant can help galloyl glucose to stably exist in alkali liquor, so that galloyl glucose is prevented from being hydrolyzed under alkaline conditions, and the complexation of the galloyl glucose to protein is not hindered.
The amount of the above-mentioned 0.5 to 1.5g/L is, for example, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L or 1.5 g/L.
The concentration of the above-mentioned 10 to 15g/L may be, for example, 10g/L, 10.5g/L, 11g/L, 11.5g/L, 12g/L, 12.5g/L, 13g/L, 13.5g/L, 14g/L, 14.5g/L or 15 g/L.
The concentration of the above-mentioned compound is, for example, 5 to 10g/L, 5.5g/L, 6g/L, 6.5g/L, 7g/L, 7.5g/L, 8g/L, 8.5g/L, 9g/L, 9.5g/L or 10 g/L.
The concentration of the above-mentioned 10-20g/L may be, for example, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L or 20 g/L.
The concentration of the above-mentioned 5 to 15g/L may be, for example, 5g/L, 5.4g/L, 6g/L, 6.6g/L, 7g/L, 7.5g/L, 8g/L, 8.5g/L, 9g/L, 9.5g/L, 10g/L, 10.5g/L, 11g/L, 11.5g/L, 12g/L, 12.5g/L, 13g/L, 13.5g/L, 14g/L, 14.5g/L or 15 g/L.
Preferably, the nonionic surfactant is selected from alkylphenol polyoxyethylene ether surfactants including octylphenol polyoxyethylene ether and/or nonylphenol polyoxyethylene ether, and further preferably octylphenol polyoxyethylene ether.
The alkylphenol polyoxyethylene ether surfactant is preferably selected in the invention, because the molecular chain of the alkylphenol polyoxyethylene ether surfactant has benzene ring and alkoxy which can interact with benzene ring and phenolic hydroxyl in galloyl glucose, the compatibility of the surfactant and the galloyl glucose is good, and the molecular chains of the surfactant and the phenolic hydroxyl are intertwined, the galloyl glucose can be prevented from being hydrolyzed in alkaline environment, and the stable existence of the galloyl glucose in a reagent system can be ensured.
Preferably, the nonionic surfactant is octylphenol polyoxyethylene ether, and the mass ratio of the galloylglucose to the octylphenol polyoxyethylene ether is 1 (1-1.3), and may be, for example, 1:1, 1:1.02, 1:1.05, 1:1.08, 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, or the like, preferably 1: 1.1.
The invention preferably selects the nonionic surfactant with the content slightly higher than that of galloyl glucose, so that the molecular chain of the nonionic surfactant can be fully wound with the molecular chain of the galloyl glucose, the galloyl glucose can stably exist in an alkaline environment, the galloyl glucose is not completely wrapped by the nonionic surfactant, and the reduction of the complexing ability of the galloyl glucose on total protein is avoided.
Preferably, the mass ratio of the galloylglucose to the copper sulfate is (8-12) to 1, and can be 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1 or 12:1, for example.
The invention preferably selects the mass ratio range of galloyl glucose and copper sulfate, because when the ratio of galloyl glucose to copper sulfate is lower than 8:1, the content of galloyl glucose is too small, the complexation action on total protein and copper ions is possibly weakened, the detection precision is reduced, and when the ratio of galloyl glucose to copper ions is higher than 12:1, the content of copper ions is too small, the content of purple compounds generated by complexation with protein is small, the color development is not obvious, and the detection precision is also reduced.
Preferably, the total protein detection reagent further comprises disodium edetate.
The total protein detection reagent can also comprise disodium ethylene diamine tetraacetate, and because the disodium ethylene diamine tetraacetate is added, the interference of calcium ions and magnesium ions in a test sample can be avoided.
Preferably, the concentration of the disodium edetate in the total protein detection reagent is 1-3g/L, and may be, for example, 1g/L, 1.2g/L, 1.5g/L, 1.8g/L, 2g/L, 2.3g/L, 2.5g/L, 2.8g/L or 3g/L, etc.
Preferably, the alkaline substance is potassium hydroxide and/or sodium hydroxide, preferably potassium hydroxide.
Preferably, potassium iodide is further included in the total protein detection reagent.
Preferably, the concentration of potassium iodide in the total protein detection reagent is 15-20g/L, and may be, for example, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L, 20g/L, or the like.
In a second aspect, the present invention also provides a method for preparing the total protein detection reagent according to the first aspect, the method comprising the steps of:
(1) mixing galloylglucose with a nonionic surfactant, and then mixing with copper sulfate, sodium potassium tartrate and part of water to obtain a mixed solution A;
(2) mixing the alkaline substance and the rest part of water to obtain a mixed solution B;
(3) and mixing the mixed liquor A and the mixed liquor B to obtain the total protein detection reagent.
The preparation method of the invention preferentially mixes the galloyl glucose with the nonionic surfactant, so that molecular chains of the galloyl glucose and the nonionic surfactant are fully wound, and then the galloyl glucose and the copper ions are mixed with the copper sulfate, the potassium sodium tartrate and part of water, and the galloyl glucose and the copper ions can be complexed, so that the obtained complex product can be dissolved in water, copper hydroxide precipitation is avoided, and the compatibility of the complex in water can be greatly improved.
Preferably, the preparation method further comprises adding disodium ethylene diamine tetraacetate and/or potassium iodide into the mixed solution B for mixing.
Compared with the prior art, the invention has the following beneficial effects:
(1) the detection reagent is creatively added with galloyl glucose, and the galloyl glucose has strong binding force with copper sulfate and total protein, so that the complexing effect of copper ions on the total protein can be greatly improved, the detection precision is further improved, and the usage amount of the copper ions can be effectively reduced; in addition, the storage stability of the detection reagent is also obviously improved.
(2) According to the preparation method, galloyl glucose and a nonionic surfactant are mixed preferentially, molecular chains of the galloyl glucose and the nonionic surfactant are fully wound, then the galloyl glucose and the nonionic surfactant are mixed with copper sulfate, potassium sodium tartrate and part of water, and the galloyl glucose and copper ions are complexed, so that an obtained complex product is soluble in water, copper hydroxide precipitation is avoided, the compatibility of the complex in water is greatly improved, all components in the detection reagent obtained by the preparation method can stably exist in a system, and the reliability of the detection reagent in long-term use is improved.
Drawings
FIG. 1 is a graph showing the open bottle stability test of example 1 and comparative example 10.
Detailed Description
The present invention is described in further detail below. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.
Example 1
This example provides a total protein detection reagent and a method for preparing the same.
Wherein, the total protein detection reagent comprises the following components: 1g/L of copper sulfate, 12g/L of potassium hydroxide, 8g/L of galloylglucose, 15g/L of sodium potassium tartrate, 10g/L of octyl phenol polyoxyethylene ether, 2g/L of disodium ethylene diamine tetraacetate, 16g/L of potassium iodide and the balance of water.
The preparation method comprises the following steps:
(1) mixing galloyl glucose with octylphenol polyoxyethylene ether, and then mixing with copper sulfate, sodium potassium tartrate and 50% of water to obtain a mixed solution A;
(2) mixing potassium hydroxide, disodium ethylene diamine tetraacetate, potassium iodide and the rest part of water to obtain a mixed solution B;
(3) and mixing the mixed liquor A and the mixed liquor B to obtain the total protein detection reagent.
Example 2
This example provides a total protein detection reagent and a method for preparing the same.
Wherein, the total protein detection reagent comprises the following components: 0.6g/L copper sulfate, 10g/L potassium hydroxide, 6g/L galloyl glucose, 10g/L potassium sodium tartrate, 7g/L nonylphenol polyoxyethylene ether, 3g/L disodium ethylenediamine tetraacetate, 15g/L potassium iodide and the balance of water.
The preparation method comprises the following steps:
(1) mixing galloyl glucose with octylphenol polyoxyethylene ether, and then mixing with copper sulfate, sodium potassium tartrate and 50% of water to obtain a mixed solution A;
(2) mixing potassium hydroxide, disodium ethylene diamine tetraacetate, potassium iodide and the rest part of water to obtain a mixed solution B;
(3) and mixing the mixed liquor A and the mixed liquor B to obtain the total protein detection reagent.
Example 3
This example provides a total protein detection reagent and a method for preparing the same.
Wherein, the total protein detection reagent comprises the following components: 0.9g/L of copper sulfate, 15g/L of potassium hydroxide, 10g/L of galloylglucose, 20g/L of sodium potassium tartrate, 12g/L of octylphenol polyoxyethylene ether, 2g/L of disodium ethylene diamine tetraacetate, 20g/L of potassium iodide and the balance of water.
The preparation method comprises the following steps:
(1) mixing galloyl glucose with octylphenol polyoxyethylene ether, and then mixing with copper sulfate, sodium potassium tartrate and 60% of water to obtain a mixed solution A;
(2) mixing potassium hydroxide, disodium ethylene diamine tetraacetate, potassium iodide and the rest part of water to obtain a mixed solution B;
(3) and mixing the mixed liquor A and the mixed liquor B to obtain the total protein detection reagent.
Example 4
This example provides a total protein detection reagent and a method for preparing the same.
Wherein, the total protein detection reagent comprises the following components: 0.5g/L copper sulfate, 12g/L potassium hydroxide, 5g/L galloyl glucose, 18g/L potassium sodium tartrate, 5g/L octyl phenol polyoxyethylene ether, 1g/L disodium ethylene diamine tetraacetate, 18g/L potassium iodide and the balance of water.
The preparation method comprises the following steps:
(1) mixing galloyl glucose with octylphenol polyoxyethylene ether, and then mixing with copper sulfate, sodium potassium tartrate and 80% of water to obtain a mixed solution A;
(2) mixing potassium hydroxide, disodium ethylene diamine tetraacetate, potassium iodide and the rest part of water to obtain a mixed solution B;
(3) and mixing the mixed liquor A and the mixed liquor B to obtain the total protein detection reagent.
Example 5
This example provides a total protein detection reagent and a method for preparing the same.
Wherein, the total protein detection reagent comprises the following components: 1g/L of copper sulfate, 12g/L of potassium hydroxide, 8g/L of galloylglucose, 18g/L of sodium potassium tartrate, 10g/L of octylphenol polyoxyethylene ether and the balance of water.
The preparation method comprises the following steps:
(1) mixing galloyl glucose with octylphenol polyoxyethylene ether, and then mixing with copper sulfate, sodium potassium tartrate and 50% of water to obtain a mixed solution A;
(2) mixing potassium hydroxide and the rest part of water to obtain a mixed solution B;
(3) and mixing the mixed liquor A and the mixed liquor B to obtain the total protein detection reagent.
Example 6
The only difference from example 1 is that disodium edetate was not added.
Example 7
The only difference from example 1 is that no potassium iodide was added.
Example 8
The only difference from example 1 is that the octylphenol polyoxyethylene ether concentration is 12 g/L.
Example 9
The only difference from example 1 is that the octylphenol polyoxyethylene ether concentration is 7 g/L.
Example 10
The only difference from example 1 is that the copper sulfate concentration is 0.6 g/L.
Example 11
The only difference from example 1 is that the copper sulfate concentration is 1.2 g/L.
Comparative example 1
The only difference from example 1 is that no galloylglucose was added.
Comparative example 2
The only difference from example 1 is that trehalose was used instead of galloylglucose.
Comparative example 3
The only difference from example 1 is that sucrose was used instead of galloylglucose.
Comparative example 4
The only difference from example 1 is that the concentration of galloylglucose was 12 g/L.
Comparative example 5
The only difference from example 1 is that the concentration of galloylglucose was 3 g/L.
Comparative example 6
The only difference from example 1 is that the concentration of octylphenol polyoxyethylene ether was 18 g/L.
Comparative example 7
The only difference from example 1 is that the concentration of octylphenol polyoxyethylene ether was 2 g/L.
Comparative example 8
The only difference from example 1 is that the copper sulfate concentration is 1.8 g/L.
Comparative example 9
The only difference from example 1 is that the copper sulfate concentration is 0.2 g/L.
Comparative example 10
This comparative example provides a total protein quantification test kit (with standard, dual-urea method) purchased from Shanghai Tong Wei Kogyo, product code: TWp 016898.
Performance testing
1. Precision: a sample having a serum total protein concentration of 60mg/mL was diluted with deionized water to 0.001mg/mL, 0.002mg/mL, 0.003mg/mL, 0.005mg/mL, 0.007mg/mL, 0.008mg/mL, 0.009mg/mL, 0.01mg/mL, 0.015mg/mL, 0.018mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.09mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL, 1mg/mL, respectively. 21 parts of each sample with the above concentration were prepared, and total protein detection was performed at each of the above concentrations using examples according to the present invention and comparative examples, and the results of the detectable lowest concentration (detection accuracy) are shown in Table 1:
TABLE 1
From the test results, the lowest concentration detectable for total protein in the examples can reach 0.01mg/mL, which is far lower than the lowest concentration detectable in comparative example 10, which shows that the total protein detection reagent of the present invention can effectively improve the detection precision for total protein, because galloylglucose in the detection reagent of the present invention is cooperated with a nonionic surfactant to generate strong complexation with total protein, the detection precision is greatly improved.
2. And (3) testing the bottle opening stability:
the test reagent obtained in example 1 of the present invention was used as a test group, and the test reagent obtained in comparative example 10 was used as a control group, and a 15-day decapping stability test was carried out, in which the test reagent was placed in a 2-8 ℃ refrigerator (without being removed for 15 days) to test the 15-day decapping stability. Taking the freeze-dried powder quality control product, evenly dissolving, evenly dividing into 15 parts, storing at-20 ℃, controlling the quality of one part on a Hitachi 7180 full-automatic biochemical analyzer every day, tracking the detection result, and tracking and monitoring the trend of opening the bottle as shown in figure 1.
As can be seen from FIG. 1, the results of the detection of total serum protein by the detection reagent of the present invention are substantially stable and unchanged as compared with comparative example 10, which indicates that the detection reagent of the present invention is highly stable, and each component thereof can stably exist in the system, ensuring the reliability of long-term use thereof.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A total protein detection reagent, wherein said total protein detection reagent comprises;
2. the total protein detection reagent according to claim 1, wherein said nonionic surfactant is selected from alkylphenol ethoxylate surfactants including octylphenol polyoxyethylene ether and/or nonylphenol polyoxyethylene ether, further preferably octylphenol polyoxyethylene ether.
3. The total protein detection reagent according to claim 1 or 2, wherein the nonionic surfactant is octylphenol polyoxyethylene ether, and the mass ratio of the galloylglucose to the octylphenol polyoxyethylene ether is 1 (1-1.3), preferably 1: 1.1;
preferably, the mass ratio of the galloylglucose to the copper sulfate is (8-12): 1.
4. The total protein detection reagent of any one of claims 1-3, further comprising disodium edetate in the total protein detection reagent.
5. The total protein detection reagent of claim 4, wherein said disodium edetate is present in said total protein detection reagent at a concentration of 1-3 g/L.
6. The total protein detection reagent of any one of claims 1-5, wherein the basic substance is potassium hydroxide and/or sodium hydroxide, preferably potassium hydroxide.
7. The total protein detection reagent of any one of claims 1-6, further comprising potassium iodide in the total protein detection reagent.
8. The total protein detection reagent of claim 7, wherein said potassium iodide is present in a concentration of 15-20g/L in said total protein detection reagent.
9. The method for preparing a total protein detection reagent according to any one of claims 1-8, wherein said method for preparing comprises the steps of:
(1) mixing galloylglucose with a nonionic surfactant, and then mixing with copper sulfate, sodium potassium tartrate and part of water to obtain a mixed solution A;
(2) mixing the alkaline substance and the rest part of water to obtain a mixed solution B;
(3) and mixing the mixed liquor A and the mixed liquor B to obtain the total protein detection reagent.
10. The method according to claim 9, further comprising adding disodium ethylenediaminetetraacetate and/or potassium iodide to the mixed solution B and mixing.
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| CN110824174A (en) * | 2019-11-28 | 2020-02-21 | 南京迪安医学检验所有限公司 | Reagent for detecting total serum protein and preparation method thereof |
| CN113281520A (en) * | 2021-04-26 | 2021-08-20 | 深圳市锦瑞生物科技有限公司 | Preparation method of reagent ball for determining total serum protein, reagent ball and microfluidic chip |
| CN114755195A (en) * | 2022-06-14 | 2022-07-15 | 浙江省立同德医院(浙江省精神卫生研究院) | Preparation method of reagent for quickly detecting urine protein |
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| CN105004589A (en) * | 2015-07-21 | 2015-10-28 | 中国科学技术大学 | Method for accelerating protein precipitation through saccharose |
| CN105486682A (en) * | 2015-12-28 | 2016-04-13 | 东北农业大学 | Rapid protein detection kit as well as detection method and application using same |
| CN107525774A (en) * | 2016-06-21 | 2017-12-29 | 山东博科生物产业有限公司 | Potassium hydroxide method total protein diagnostic test kits |
| CN106442352A (en) * | 2016-09-24 | 2017-02-22 | 济南中安生物技术服务有限公司 | Total serum protein detection kit with strong anti-interference capability |
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| CN110824174A (en) * | 2019-11-28 | 2020-02-21 | 南京迪安医学检验所有限公司 | Reagent for detecting total serum protein and preparation method thereof |
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