A kind of detection kit of alkaline phosphatase and preparation method thereof

Abstract

The present invention relates to the field of medical detection technology, and in particular to an alkaline phosphatase detection kit and a preparation method thereof. The kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a buffer, magnesium acetate, zinc sulfate, HEDTA and sialidase; and the R2 reagent comprises a buffer, pancreatic peptidase and nitrophenol phosphate. Experiments show that the present invention adds a certain amount of sialidase to the R1 reagent and a certain amount of pancreatic peptidase to the R2 reagent, which significantly improves the reactivity of the reagents.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and in particular to an alkaline phosphatase detection kit and a preparation method thereof. Background Art

[0002] Alkaline phosphatase (ALP or AKP) is a phosphomonoesterase that exists mainly in free form and is bound to lipoproteins and immunoglobulins in very small amounts. ALP in serum originates from different tissues (liver, bone, kidney, small intestine, placenta, etc.), mainly from the liver and bones. It is mainly used for the examination of obstructive jaundice, primary liver cancer, secondary liver cancer, cholestatic hepatitis, etc. When suffering from these diseases, liver cells overproduce ALP, which enters the blood through the lymphatic tract and hepatic sinusoids. At the same time, due to the obstruction of bile excretion in the intrahepatic bile duct, it refluxes into the blood, causing a significant increase in serum alkaline phosphatase. Its main physiological function in the body is to hydrolyze phosphate esters during osteogenesis to provide the necessary phosphoric acid for the deposition of hydroxyapatite, and at the same time hydrolyze pyrophosphate to relieve its inhibitory effect on bone salt formation, which is beneficial to osteogenesis. Therefore, serum alkaline phosphatase may also be elevated in pregnant women, during the fracture healing period, in patients with osteomalacia, rickets, osteocellular carcinoma, osteoporosis, liver abscess, liver tuberculosis, cirrhosis, leukemia, and hyperthyroidism, and should be differentiated.

[0003] In the field of basic research, commonly used methods for determining ALP activity include fluorescence determination, chemiluminescence analysis, electrochemical analysis, and surface enhanced resonance Raman scattering. However, these methods are limited in their further application due to the cumbersome detection process and the need for complex and expensive scientific instruments and professional operators (Research Progress in Fluorescence Detection Methods for Alkaline Phosphatase Activity_Zhao Dan). In clinical testing, the main methods for determining alkaline phosphatase are the following: β-glycerophosphate sodium method, disodium phenyl phosphate colorimetric method, and continuous monitoring method for p-nitrophenol phosphate. Disodium phenyl phosphate colorimetric method has made great progress compared with the earlier β-glycerophosphate sodium method. Disodium phenyl phosphate colorimetric method has a fast hydrolysis rate, so the insulation time is short, and the method has high sensitivity, stable color development, no need to remove protein, simple and fast operation. However, compared with the continuous monitoring method for p-nitrophenol phosphate, the accuracy and precision are low, the operation is relatively cumbersome, and the sensitivity is low.

[0004] Prior art: ALP in serum transfers the phosphoryl group of p-nitrophenol phosphate (NPP) to water and 2-amino-2-methyl-1-propanol (AMP) molecules under alkaline conditions to generate p-acylphenol. Under alkaline conditions, p-nitrophenol exists in the form of p-nitrophenoxy ions, which are yellow and have strong absorption at a wavelength of 405nm, while p-nitrophenol phosphate is colorless. Under substrate excess conditions, the generation rate of p-nitrophenoxy ions is proportional to the serum ALP concentration, so the serum ALP activity concentration can be measured by the increase of p-nitrophenoxy ions.

[0005]

[0006]

[0007] The common ALP kit R1 and R2 components are: R1 reagent: AMP buffer (PH 10.5) 0.1-1.0 mol / L, magnesium acetate 1-5 mmol / L, zinc sulfate 1-5 mmol / L, HEDTA 1-5 mmol / L; R2 reagent: p-nitrophenol phosphate 20-100 mmol / L.

[0008] When the above kit is used to continuously monitor p-nitrophenol phosphate, if the reactivity is to be increased, the substrate concentration needs to be increased. Summary of the invention

[0009] In view of this, the present invention provides an alkaline phosphatase detection kit and a preparation method thereof.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] Under alkaline conditions, ALP in serum transfers the phosphoryl group of p-nitrophenol phosphate (NPP) to water and 2-amino-2-methyl-1-propanol (AMP) molecules to generate p-acylphenol. Under alkaline conditions, p-nitrophenol exists in the form of p-nitrophenoxy ion, which is yellow and has strong absorption at a wavelength of 405nm, while p-nitrophenol phosphate is colorless. Under conditions of excess substrate, the generation rate of p-nitrophenoxy ion is proportional to the serum ALP concentration, so the serum ALP activity concentration can be measured by the increase of p-nitrophenoxy ion.

[0012]

[0013]

[0014] An alkaline phosphatase detection kit, consisting of an R1 reagent and an R2 reagent;

[0015] The R1 reagent includes a buffer solution, magnesium acetate, zinc sulfate, HEDTA and sialidase;

[0016] The R2 reagent includes a buffer, trypsin and nitrophenol phosphate.

[0017] In the detection kit of the present invention, the concentration of each component in the R1 reagent is:

[0018]

[0019]

[0020] In the present invention, the concentration of each component in the R2 reagent is:

[0021] Buffer 0.1-1.0mol / L

[0022] p-Nitrophenol phosphate 20-100mmol / L;

[0023] Pancreatic peptidase 2.5-7Ku / L.

[0024] In the present invention, in the R1 reagent and the R2 reagent, the buffer is AMP buffer, glycine-NaOH buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, N-tris(hydroxymethyl)methylamino-2-hydroxypropanesulfonic acid buffer, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer, piperazine-N,N-bis(2-hydroxyethanesulfonic acid) buffer, 3-morpholine-2-hydroxypropanesulfonic acid sodium buffer, 3-(N-morpholine)ethanesulfonic acid sodium buffer, 4-(2-hydroxyethyl) At least one of piperazine-1-2-hydroxypropanesulfonic acid buffer, N-(2-hydroxyethyl)piperazine-N'-4-butanesulfonic acid buffer, 3-bis(2-hydroxyethyl)amino-2-hydroxypropanesulfonic acid buffer, 3-(cyclohexylamine)-2-hydroxy-1-propanesulfonic acid buffer, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid buffer, 3-(cyclohexylamine)-1-propanesulfonic acid buffer, 3-morpholinepropanesulfonic acid buffer, and N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; the pH of the buffer is 10.5.

[0025] In some specific embodiments of the present invention, the kit consists of R1 reagent and R2 reagent;

[0026] R1 reagent includes:

[0027]

[0028] R2 reagents include:

[0029] AMP buffer (pH 10.5) 0.30 mol / L;

[0030] p-Nitrophenol phosphate 81.6mmol / L;

[0031] Pancreatic peptidase 2.5Ku / L.

[0032] In some specific embodiments of the present invention, the kit is composed of R1 reagent and R2 reagent:

[0033] R1 reagent includes:

[0034]

[0035]

[0036] R2 reagents include:

[0037] AMP buffer (pH 10.5) 0.30 mol / L;

[0038] p-Nitrophenol phosphate 81.6mmol / L;

[0039] Pancreatin 2.5Ku / L

[0040] In some specific embodiments of the present invention, the kit is composed of R1 reagent and R2 reagent:

[0041] R1 reagent:

[0042]

[0043] R2 reagent:

[0044] AMP buffer (pH 10.5) 0.30mol / L

[0045] p-Nitrophenol phosphate 81.6mmol / L;

[0046] Pancreatic peptidase 7Ku / L.

[0047] In some specific embodiments of the present invention, the kit is composed of R1 reagent and R2 reagent:

[0048] R1 reagent:

[0049]

[0050] R2 reagent:

[0051] AMP buffer (pH 10.5) 0.30mol / L

[0052] p-Nitrophenol phosphate 81.6mmol / L;

[0053] Pancreatic peptidase 7Ku / L.

[0054] The present invention also provides a method for preparing the detection kit, comprising:

[0055] Mixing pH 10.5 AMP buffer with magnesium acetate, zinc sulfate, HEDTA, and neuraminidase to obtain reagent R1;

[0056] The R2 reagent was obtained by mixing AMP buffer at pH 10.5 with trypsin and nitrophenol phosphate.

[0057] The present invention provides a detection kit for alkaline phosphatase and a preparation method thereof. The kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a buffer, magnesium acetate, zinc sulfate, HEDTA and sialidase; and the R2 reagent comprises a buffer, pancreatic peptidase and nitrophenol phosphate. Experiments show that in the present invention, a certain amount of sialidase is added to the R1 reagent and a certain amount of pancreatic peptidase is added to the R2 reagent, which significantly improves the reactivity of the reagents. DETAILED DESCRIPTION

[0058] The present invention provides a detection kit for alkaline phosphatase and a preparation method thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0059] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0060] The present invention will be further described below in conjunction with embodiments:

[0061] Example 1

[0062] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0063] R1 reagent:

[0064]

[0065] R2 reagent:

[0066] AMP buffer (pH 10.5) 0.30 mol / L;

[0067] p-Nitrophenol phosphate 81.6mmol / L;

[0068] Pancreatic peptidase 2.5Ku / L.

[0069] Preparation method: A small amount of solution of activator magnesium acetate, structural ion zinc sulfate, HEDTA for controlling ion concentration, and sialidase is added to a basic buffer solution of 2-amino-2-methyl-1-propanol (AMP) with a good pH value, thereby obtaining R1 reagent. A small amount of solution of pancreatic peptidase and p-nitrophenol phosphate is added to a basic buffer solution of 2-amino-2-methyl-1-propanol (AMP) with a good pH value, thereby obtaining R2 reagent.

[0070] Example 2

[0071] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0072] R1 reagent:

[0073]

[0074] R2 reagent:

[0075] AMP buffer (pH 10.5) 0.30 mol / L;

[0076] p-Nitrophenol phosphate 81.6mmol / L;

[0077] Pancreatic peptidase 2.5Ku / L.

[0078] The preparation method is the same as Example 1.

[0079] Example 3

[0080] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0081] R1 reagent:

[0082]

[0083] R2 reagent:

[0084] AMP buffer (pH 10.5) 0.30 mol / L;

[0085] p-Nitrophenol phosphate 81.6mmol / L;

[0086] Pancreatic peptidase 7Ku / L.

[0087] The preparation method is the same as Example 1.

[0088] Example 4

[0089] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0090] R1 reagent:

[0091]

[0092] R2 reagent:

[0093] AMP buffer (pH 10.5) 0.30 mol / L;

[0094] p-Nitrophenol phosphate 81.6mmol / L;

[0095] Pancreatic peptidase 7Ku / L.

[0096] The preparation method is the same as Example 1.

[0097] Comparative Example 1

[0098] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0099] R1 reagent:

[0100]

[0101] R2 reagent:

[0102] AMP buffer (pH 10.5) 0.30 mol / L;

[0103] p-Nitrophenol phosphate 81.6mmol / L;

[0104] Comparative Example 2

[0105] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0106] R1 reagent:

[0107]

[0108] R2 reagent:

[0109] AMP buffer (pH 10.5) 0.30 mol / L;

[0110] p-Nitrophenol phosphate 81.6mmol / L;

[0111] The preparation method is the same as Example 1.

[0112] Comparative Example 3

[0113] The components of R1 and R2 are:

[0114] R1 reagent:

[0115]

[0116] R2 reagent:

[0117] AMP buffer (pH 10.5) 0.30 mol / L;

[0118] p-Nitrophenol phosphate 81.6mmol / L;

[0119] The preparation method is the same as Example 1.

[0120] Comparative Example 4

[0121] The components of R1 and R2 are:

[0122] R1 reagent:

[0123]

[0124]

[0125] R2 reagent:

[0126] AMP buffer (PH 10.5) 0.30mol / L

[0127] p-Nitrophenol phosphate 81.6mmol / L;

[0128] The preparation method is the same as Example 1.

[0129] Comparative Example 5

[0130] The components of R1 and R2 are:

[0131] R1 reagent:

[0132]

[0133] R2 reagent:

[0134] AMP buffer (pH 10.5) 0.30 mol / L;

[0135] p-Nitrophenol phosphate 81.6mmol / L.

[0136] The preparation method is the same as Example 1.

[0137] Comparative Example 6

[0138] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0139] R1 reagent:

[0140]

[0141] R2 reagent:

[0142] p-Nitrophenol phosphate 81.6mmol / L;

[0143] Papain 5Ku / L.

[0144] Comparative Example 7

[0145] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0146] R1 reagent:

[0147]

[0148] R2 reagent:

[0149] p-Nitrophenol phosphate 81.6mmol / L;

[0150] Pepsin 5Ku / L.

[0151] Comparative Example 8

[0152] It includes R1 and R2 reagents, wherein the components of R1 and R2 are:

[0153] R1 reagent:

[0154]

[0155] R2 reagent:

[0156] p-Nitrophenol phosphate 81.6mmol / L;

[0157] Central protease 5Ku / L.

[0158] Comparative Example 9

[0159] A highly sensitive ALP kit comprises R1 and R2 reagents, wherein the components of R1 and R2 are:

[0160] R1 reagent:

[0161]

[0162] R2 reagent:

[0163] p-Nitrophenol phosphate 81.6mmol / L;

[0164] Chymotrypsin 5Ku / L.

[0165] Test Case

[0166] Use a fully automatic biochemical analyzer to perform the test according to the test parameters in the following table:

[0167] Table 1. Detection parameters of the kit of the present invention on a fully automatic biochemical analyzer

[0168]

[0169] The above-mentioned Examples 1 to 4 and Comparative Examples 1 to 10 were tested according to the above parameters, and compared with reagents on the market, and calibrated respectively. The test results are shown in Table 2.

[0170] Table 2. Detection data of the kit of the present invention on the fully automatic biochemical analyzer

[0171]

[0172] From the results in Table 2, it can be seen that the reaction degree increases after adding sialidase to R1 reagent and pancreatic peptidase to R2 reagent. The test results of Comparative Example 1 without adding sialidase and pancreatic peptidase and Comparative Examples 2 to 9 with adding papain, pepsin, centrospase and chymotrypsin are obviously inferior to those of Examples 1 to 4 of the present invention.

[0173] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A detection kit for alkaline phosphatase, It is characterized in that It is composed of R1 reagent and R2 reagent; The concentration of each component in the R1 reagent is: In the R2 reagent, the concentration of each component is: Buffer 0.1-1.0mol / L p-Nitrophenol phosphate 20-100mmol / L; Pancreatic peptidase 2.5-7Ku / L; In the R1 reagent and the R2 reagent, the buffer is AMP buffer.

2. The detection kit according to claim 1, It is characterized in that It is composed of R1 reagent and R2 reagent; R1 reagent includes: R2 reagents include: AMP buffer 0.30 mol / L; p-Nitrophenol phosphate 81.6mmol / L; Pancreatic peptidase 2.5Ku / L.

3. The detection kit according to claim 1, It is characterized in that It is composed of R1 reagent and R2 reagent; R1 reagent includes: R2 reagents include: AMP buffer 0.30 mol / L; p-Nitrophenol phosphate 81.6mmol / L; Pancreatic peptidase 2.5Ku / L.

4. The detection kit according to claim 1, It is characterized in that It is composed of R1 reagent and R2 reagent; R1 reagent: R2 reagent: AMP buffer 0.30 mol / L p-Nitrophenol phosphate 81.6mmol / L; Pancreatic peptidase 7Ku / L.

5. The detection kit according to claim 1, It is characterized in that It is composed of R1 reagent and R2 reagent; R1 reagent: R2 reagent: AMP buffer 0.30 mol / L p-Nitrophenol phosphate 81.6mmol / L; Pancreatic peptidase 7Ku / L.

6. A method for preparing the detection kit according to any one of claims 1 to 5, It is characterized in that include: Mixing pH 10.5 AMP buffer with magnesium acetate, zinc sulfate, HEDTA, and neuraminidase to obtain reagent R1; The R2 reagent was obtained by mixing AMP buffer at pH 10.5 with trypsin and nitrophenol phosphate.

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