阅读说明：本技术 一种高稳定性alp测定试剂盒及其制备方法和用途 (High-stability ALP (alpha-amyloid peptide) determination kit and preparation method and application thereof ) 是由 龚婷 梁艳 吴年芬 舒芹 张雪娇 赵愿安 于 2020-07-30 设计创作，主要内容包括：本发明提供了一种高稳定性ALP测定试剂盒,属于疾病检测试剂领域,包括试剂1和试剂2,所述试剂1的组分包括AMP缓冲液、HEDTA、叠氮钠和第一离子制剂,所述试剂2的组分包括AMP缓冲液、4-NPP、叠氮钠、第二离子制剂和保护剂。ALP试剂开瓶后稳定性好,可做到开瓶30天,偏差不超过10％,大大延长了ALP试剂的开瓶周期,提高了试剂质量,方便客户使用。本发明还提供了一种高稳定性ALP测定试剂盒的制备方法和用途。(The invention provides a high-stability ALP (alpha-amyloid peptide) determination kit, which belongs to the field of disease detection reagents and comprises a reagent 1 and a reagent 2, wherein the component of the reagent 1 comprises AMP buffer solution, HEDTA (ethylene-propylene-diene monomer), sodium azide and a first ionic preparation, and the component of the reagent 2 comprises AMP buffer solution, 4-NPP, sodium azide, a second ionic preparation and a protective agent. The stability of the ALP reagent is good after opening the bottle, the deviation is not more than 10 percent after opening the bottle for 30 days, the bottle opening period of the ALP reagent is greatly prolonged, the quality of the reagent is improved, and the ALP reagent is convenient for customers to use. The invention also provides a preparation method and application of the high-stability ALP determination kit.)

1. A high-stability ALP assay kit, comprising a reagent 1 and a reagent 2, wherein the components of the reagent 1 comprise AMP buffer solution, HEDTA, sodium azide and a first ionic preparation, and the components of the reagent 2 comprise AMP buffer solution, 4-NPP, sodium azide, a second ionic preparation and a protective agent.

2. The high stability ALP assay kit as claimed in claim 1, wherein the concentrations of each component in said reagent 1 are: AMP buffer: 15 ml/L-28 ml/L, HEDTA: 1.5mmol/L-12mmol/L, sodium azide: 0.01-0.1 wt%, first ionic agent: 0.05-50mmol/L, pH 10.4-10.7.

3. The high stability ALP assay kit as claimed in claim 1, wherein the concentration of each component in said reagent 2 is: AMP buffer: 15 ml/L-28 ml/L, 4-NPP: 40-60g/L, sodium azide: 0.01-0.1 wt%, second ionic agent: 0.05-50mmol/L, protective agent: 10-1000mmol/L, pH 10.4-10.7.

4. The high stability ALP assay kit according to claim 1, wherein said first ionic preparation comprises any one or both of:

sodium glutamate, disodium ethylenediaminetetraacetate, potassium bitartrate, sodium sulfate, potassium phosphate, zinc sulfate, magnesium sulfate, sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and potassium carbonate.

5. The high stability ALP assay kit according to claim 1, wherein said second ionic preparation comprises any one or both of:

sodium glutamate, disodium ethylenediaminetetraacetate, potassium bitartrate, sodium sulfate, potassium phosphate, zinc sulfate, magnesium sulfate, sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and potassium carbonate.

6. The high stability ALP assay kit as set forth in claim 1, wherein said protectant comprises any one or two of sucrose, xylitol, mannitol and sorbitol.

7. A method for preparing a high stability ALP assay kit as claimed in any one of claims 1 to 6, comprising:

weighing raw materials required by the reagent 1: AMP buffer, HEDTA, sodium azide and first ionic formulation;

preparing a solution from the raw materials of AMP buffer solution, HEDTA, sodium azide and the first ionic agent, and adjusting the pH by an acid solution or an alkali solution to obtain a reagent 1 having the component concentrations as set forth in claim 2;

the components of the reagent 2 comprise AMP buffer solution, 4-NPP, sodium azide, a second ionic preparation and a protective agent;

preparing a solution of the raw materials AMP buffer solution, 4-NPP, sodium azide, the second ionic agent and the protective agent, and adjusting the pH by an acid solution or an alkali solution to obtain a reagent 2 having the component concentrations as set forth in claim 3.

8. The method of preparing a high stability ALP assay kit according to claim 7, wherein said acid solution is hydrochloric acid and said alkali solution is sodium hydroxide.

9. The method of preparing a high stability ALP assay kit according to claim 7, wherein the effective concentration of AMP in said AMP buffer is 100%.

10. Use of a high stability ALP assay kit as claimed in any one of claims 1-6 in the assay of alkaline phosphatase.

Technical Field

The invention belongs to the field of disease detection reagents, and relates to a high-stability ALP determination kit, and a preparation method and application thereof.

Background

Alkaline Phosphatase (ALP) is an enzyme that is widely distributed in human liver, bone, intestine, kidney, placenta and other tissues and is excreted outside the gallbladder through the liver. Alkaline phosphatase is mainly used for the examination of obstructive jaundice, cholestatic hepatitis, and the like. In these diseases, the hepatic cells overproduce ALP, enter the blood via the lymphatic channel and hepatic sinus, and cause a significant increase in serum alkaline phosphatase due to bile excretion disorder in the hepatic and biliary tracts and reflux into the blood. However, since the enzyme is also active in bone tissue, it should be identified that serum alkaline phosphatase is also elevated in pregnant women, during fracture healing, osteomalacia, rickets, osteoporosis, liver abscess, hepatic tuberculosis, liver cirrhosis, leukemia, and hyperthyroidism.

The alkaline phosphatase is mainly applied to the following aspects in clinic: (1) physiological increase: the alkaline phosphatase activity of the children is 1-2 times higher than that of normal people in the physiological skeletal development stage. (2) Pathological elevation: bone diseases such as rickets, osteomalacia, malignant bone tumor, and malignant tumor bone metastasis; liver and gallbladder diseases such as extrahepatic biliary obstruction, liver cirrhosis, and capillary and biliary hepatitis; other diseases such as hyperparathyroidism. (3) Pathological reduction: it is indicated for severe chronic nephritis, children's thyroid insufficiency, anemia, etc.

The alkaline phosphatase is determined mainly by the following methods: beta-glycerol sodium phosphate method, disodium phenyl phosphate colorimetric method and p-nitrophenol phosphate continuous monitoring method. The disodium phenyl phosphate colorimetric method represents a major advance over the earlier beta-glycerophosphate sodium method. The disodium phenyl phosphate colorimetric method has the advantages of high hydrolysis speed, short heat preservation time, high sensitivity, stable color development, no need of deproteinization, and simple and quick operation. But compared with a continuous monitoring method of the p-nitrophenol phosphate, the method has the advantages of lower accuracy and precision, more complicated operation and low sensitivity; the enzyme unit is not an international unit; is disturbed by bilirubin and hemolysis.

Alkaline phosphatase is a group of enzymes which hydrolyze phosphate monoester compounds or transfer phosphoryl of phosphate monoester to other substances under alkaline conditions, and the elevation of ALP of normal adults is mostly related to bone or liver and gall diseases and the like. For the detection of alkaline phosphatase, a continuous monitoring mode of p-nitrophenol phosphate is more widely used in the market, and p-nitrophenol phosphate (4-NPP) is generally selected as a substrate, and 2-amino-2-methyl-1-propanol (AMP) is selected as an acceptor substance of phosphoryl, so that the enzymatic reaction rate is improved. 4-NPP is colorless in alkaline solution, and under the catalysis of ALP, 4-NPP splits off phosphate group to generate free p-nitrophenol (4-NP), and the latter is converted into quinoid structure in alkaline solution and shows dark yellow. The rate of increase in absorbance was monitored at a wavelength of 405nm and ALP activity units were calculated. The ALP reagent on the market is opened for 14 days generally, the deviation is more than 10 percent, the stability of the reagent is poor, and the storage life is short.

Disclosure of Invention

In order to solve the technical problem of poor stability of the ALP detection reagent after the bottle is opened, the invention provides the high-stability ALP determination kit, the stability of the ALP reagent after the bottle is opened is good, the bottle opening period of the ALP reagent can be prolonged by 30 days, the deviation is not more than 10%, the bottle opening period of the ALP reagent is greatly prolonged, the quality of the reagent is improved, and the use by customers is facilitated.

The invention also provides a preparation method and application of the high-stability ALP determination kit.

The invention is realized by the following technical scheme:

a high stability ALP assay kit comprising a reagent 1 and a reagent 2, wherein the components of said reagent 1 comprise AMP buffer, HEDTA, sodium azide and a first ionic preparation, and the components of said reagent 2 comprise AMP buffer, 4-NPP, sodium azide, a second ionic preparation and a protective agent.

Wherein, the concentration of each component in the reagent 1 is as follows: AMP buffer: 15 ml/L-28 ml/L, HEDTA: 1.5mmol/L-12mmol/L, sodium azide: 0.01-0.1 wt%, first ionic agent: 0.05-50mmol/L, pH 10.4-10.7.

Further, the concentration of each component in the reagent 2 is as follows: AMP buffer: 15 ml/L-28 ml/L, 4-NPP: 40-60g/L, sodium azide: 0.01-0.1 wt%, second ionic agent: 0.05-50mmol/L, protective agent: 10-1000mmol/L, pH 10.4-10.7.

Further, the first ionic agent comprises any one or two of the following substances:

sodium glutamate, disodium ethylenediaminetetraacetate, potassium bitartrate, sodium sulfate, potassium phosphate, zinc sulfate, magnesium sulfate, sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and potassium carbonate.

Further, the second ionic agent comprises any one or two of the following substances:

sodium glutamate, disodium ethylenediaminetetraacetate, potassium bitartrate, sodium sulfate, potassium phosphate, zinc sulfate, magnesium sulfate, sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and potassium carbonate.

Further, the protective agent comprises any one or two of sucrose, xylitol, mannitol and sorbitol.

A method for preparing a high stability ALP assay kit, comprising:

weighing raw materials required by the reagent 1: AMP buffer, HEDTA, sodium azide and first ionic formulation;

preparing a solution from AMP buffer solution, HEDTA, sodium azide and a first ionic preparation, and adjusting the pH value by acid liquor or alkali liquor to obtain a reagent 1 with the concentration of each component as follows:

AMP buffer: 15 ml/L-28 ml/L, HEDTA: 2mmol/L, sodium azide: 0.01 wt%, first ionic agent: 0.05-50mmol/L, pH 10.50.

The components of the reagent 2 comprise AMP buffer solution, 4-NPP, sodium azide, a second ionic preparation and a protective agent;

preparing a solution from the raw materials of AMP buffer solution, 4-NPP, sodium azide, a second ionic preparation and a protective agent, and adjusting the pH value by acid liquor or alkali liquor to obtain a reagent 1 with the concentration of each component as follows:

AMP buffer: 15 ml/L-28 ml/L, 4-NPP:

40g/L, sodium azide: 0.01 wt%, second ionic agent: 0.05-50mmol/L, protective agent: 10-1000mmol/L, pH 10.50.

Further, the acid solution is hydrochloric acid, and the alkali solution is sodium hydroxide.

Further, in the AMP buffer, the effective concentration of AMP is 100%.

Use of a high stability ALP assay kit in the assay of alkaline phosphatase is provided.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

according to the high-stability ALP determination kit, the concentration of the AMP buffer solution in the reagent is controlled, the ionic preparations are added into the reagents 1 and 2, the stability of the reagent after the bottle is opened is improved, the bottle opening period of the ALP reagent can be greatly prolonged by 30 days, the deviation is not more than 10%, the quality of the reagent is improved, and the use is convenient.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a graph showing the ALP test deviation after decapping of the kits for examples and comparative examples of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

for the detection of alkaline phosphatase, a continuous monitoring mode of p-nitrophenol phosphate is more widely used in the market, and p-nitrophenol phosphate (4-NPP) is generally selected as a substrate, and 2-amino-2-methyl-1-propanol (AMP) is selected as an acceptor substance of phosphoryl, so that the enzymatic reaction rate is improved. In the market, the AMP buffer system is also generally adopted, ALP shows higher activity in the AMP buffer solution, and the serum ALP determination reaction needs to be carried out under alkaline conditions. Applicants have found that the pH of the ALP detection reagent decreases upon unsealing due to absorption of carbon dioxide from the air, which affects the accuracy of the measurement results, resulting in a deviation of more than 10% for a period of typically 14 days after the reagent is unsealed, poor reagent stability, and short shelf life.

In the present invention, the HEDT is to control the ion concentration, and most of the ions of the first ionic agent added to the reagent 1 are combined with HEDTA; sodium azide can play a role in corrosion prevention for the reagent to a certain extent.

Based on this, the present application provides a novel ALP assay kit comprising a reagent 1 and a reagent 2, the components of the reagent 1 including AMP buffer, HEDTA, sodium azide and a first ionic agent, and the components of the reagent 2 including AMP buffer, 4-NPP, sodium azide, a second ionic agent and a protective agent.

Wherein, the concentration of each component in the reagent 1 is as follows: AMP buffer: 15 ml/L-28 ml/L, HEDTA: 2mmol/L, sodium azide: 0.01 wt%, first ionic agent: 0.05-50mmol/L, pH 10.50.

Further, the concentration of each component in the reagent 2 is as follows: AMP buffer: 15 ml/L-28 ml/L, 4-NPP: 40g/L, sodium azide: 0.01 wt%, second ionic agent: 0.05-50mmol/L, protective agent: 10-1000mmol/L, pH 10.50.

Further, the first ionic agent comprises any one or two of the following substances:

sodium glutamate, disodium ethylenediaminetetraacetate, potassium bitartrate, sodium sulfate, potassium phosphate, zinc sulfate, magnesium sulfate, sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and potassium carbonate.

Further, the second ionic agent comprises any one or two of the following substances:

sodium glutamate, disodium ethylenediaminetetraacetate, potassium bitartrate, sodium sulfate, potassium phosphate, zinc sulfate, magnesium sulfate, sodium chloride, potassium chloride, calcium chloride, ammonium chloride, magnesium sulfate, lithium sulfate, ammonium sulfate, magnesium acetate, and potassium carbonate.

Further, the protective agent comprises any one or two of sucrose, xylitol, mannitol and sorbitol.

The principle of the invention is as follows:

the AMP buffer solution (15 ml/L-28 ml/L) is used in the reagents 1 and 2, the higher the concentration of the AMP buffer solution is, the more easily the carbon dioxide is absorbed in the process of opening the bottle of the reagents, and the proper reduction of the concentration of the AMP buffer solution is favorable for delaying the condition that the pH value of the reagents is reduced to cause lower test due to the absorption of the carbon dioxide by the AMP buffer solution. And the ion preparation is added in the reagents 1 and 2, and can be cooperated with AMP buffer solution to activate enzyme, accelerate reaction speed and improve reactivity, because zinc ions are structural ions of ALP, magnesium ions and the like are activators of ALP, and the added ions can activate ALP to enhance reaction. The addition of a protectant to reagent 2 can protect the substrate 4-NNP.

Therefore, the ALP detection kit has excellent stability, can realize the bottle opening for 30 days, has the deviation of the ALP detection result not more than 10 percent, greatly prolongs the bottle opening period of an ALP reagent, improves the quality of the reagent and is convenient for customers to use.

A high stability ALP assay kit of the present application will be described in detail below with reference to examples, comparative examples and experimental data.