阅读说明：本技术 一种总蛋白检测试剂及其制备方法 (Total protein detection reagent and preparation method thereof ) 是由 周明先 贾桐煜 孙亚伟 于 2019-09-27 设计创作，主要内容包括：本发明涉及一种总蛋白检测试剂及其制备方法,所述检测试剂中创造性地添加了没食子酰葡萄糖,其与硫酸铜和总蛋白存在强烈的结合力,可以大幅提升铜离子对总蛋白的络合作用,进而提高检测精度,同时还能有效降低铜离子的使用量；另外,本发明所述检测试剂的储存稳定性也明显提升。本发明提供的制备方法优先将没食子酰葡萄糖与非离子表面活性剂混合,使两者分子链充分缠绕后,再与硫酸铜、酒石酸钾钠和部分水混合,没食子酰葡萄糖与铜离子产生络合,这样得到的络合产物既可溶于水,避免了产生氢氧化铜沉淀,又能极大增加络合物在水中的相容性,通过所述制备方法得到的检测试剂中各组分在体系中均能稳定存在,提高了其长时间使用的可靠性。(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.)

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.

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.