An alkaline phosphatase enzyme label buffer and alkaline phosphatase enzyme label reagent

Abstract

The application belongs to the technical field of medical materials, and particularly relates to an alkaline phosphatase enzyme label buffer and an alkaline phosphatase enzyme label reagent. The alkaline phosphatase enzyme label buffer in the technical scheme of the application comprises a first chelate formed by nitrilotriacetic acid (NTA), diethylene triamine pentaacetic acid (DTPA) and magnesium ions and a second chelate formed by methionine, threonine and zinc ions, and further comprises the following components in percentage by mass: 0.2-2.0% reduced glutathione, 1.0-2.5% corn peptide, 0.5-10.0% protein, 0.1-2.0% ginsenoside, 0.1-1.0% surfactant, 1.0-5.0% saccharide compound, 1.0-10.0% polyhydric alcohol, 0.05-0.1% preservative and 0.01-0.05% antibiotic, and the rest is non-ionic amphiphilic buffer. The first chelate formed by nitrilotriacetic acid, diethylene triamine pentaacetic acid and magnesium ions and the second chelate formed by methionine, threonine and zinc ions are used in combination, and glutathione, protein and other substances are combined to obtain the alkaline phosphatase enzyme label buffer with long-term stability.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to an alkaline phosphatase enzyme labeling buffer and an alkaline phosphatase enzyme labeling reagent. Background Technology

[0002] Alkaline phosphatase (ALP) is an enzyme commonly used as a label in immunoassays. It is a homodimeric protein with a molecular weight of 56 kDa. Each monomer consists of 449 amino acids. The complete AKP molecule exhibits a typical α / β topology, and each monomer possesses an active site. The active site region consists of an Asp101-Ser102-Ala103 triplet, Arg166, a water molecule, three metal ions, and their ligand amino acids. AKP is encoded by the phoA gene. Like many secretory proteins, it synthesizes a precursor monomer with a signal peptide at its amino terminus in the cytoplasm. The signal peptide guides the precursor's transmembrane transport, where it is cleaved to form a homodimer. Alkaline phosphatase is an enzyme capable of dephosphorylating its corresponding substrate. It removes the phosphate group from the substrate molecule by hydrolyzing phosphate monoesters, generating phosphate ions and free hydroxyl groups. Substrates include nucleic acids, proteins, and alkaloids. This process of removing the phosphate group is called dephosphorylation. Meanwhile, alkaline phosphatase, as a type of phosphatase, acts in the opposite way to kinases (kinases are phosphorylases that use energy molecules, such as ATP, to add phosphate groups to corresponding substrate molecules). Alkaline phosphatase exhibits maximum activity in alkaline environments. For ALP derived from bacteria, the optimal pH is 8.0, while for ALP derived from cattle, the optimal pH is 8.5. ALP is a zinc-containing glycoprotein that can hydrolyze various natural and synthetic phosphate monoester compounds in an alkaline environment (optimal pH 10).

[0003] Chemiluminescent immunoassay (CLIA) is a type of labeled antibody technology. It uses chemiluminescent agents, catalytic enzymes, or products to indirectly participate in the luminescent reaction to label antigens or antibodies. When the label binds to the corresponding substance, the luminescent substrate undergoes a redox reaction under the action of the luminescent agent, catalytic enzyme, or participating product, releasing a light signal. Finally, a photomultiplier tube is used for detection. The technology has advantages such as high throughput, high specificity, high sensitivity, simple separation, speed, safety, stability, and automation.

[0004] Currently, chemiluminescent enzyme immunoassay is commonly used to detect the concentration of proteins (such as tumor markers, CA125, and HE4). This method combines highly sensitive enzyme-catalyzed chemiluminescence technology with highly specific antigen-antibody reactions for the quantitative detection of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. This technology offers advantages such as high specificity, high sensitivity, simple and rapid separation, and the ability to automate analysis. The enzyme used is primarily alkaline phosphatase, and the alkaline phosphatase label solution is a component of in vitro diagnostic kits. The stability of alkaline phosphatase activity is a crucial factor affecting the quality and performance of these kits. Like other biological enzymes, alkaline phosphatase has a short half-life and is easily inactivated. Changes in acid, alkali, salt ions, and temperature conditions can alter or even completely deactivate the enzyme. Long-term stable preservation technology of alkaline phosphatase in solution is a key technology for in vitro diagnostic kit products. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned technical problems by providing an alkaline phosphatase enzyme labeling buffer solution, which improves the detection efficiency and long-term storage stability of alkaline phosphatase.

[0006] The alkaline phosphatase enzyme-labeled buffer solution of the present invention includes a first chelate formed by NTA, DTPA and magnesium ions, and a second chelate formed by methionine, threonine and zinc ions.

[0007] Magnesium ions are specific "effect molecules" at ALP sites. They can coordinate with relevant groups in alkaline phosphatase, stabilizing the conformation of the enzyme's active site and enabling spontaneous folding to proceed in a near-native state. After chelation with hypozinotriacetic acid and diethylenetriaminepentaacetic acid, they can effectively improve the catalytic efficiency and stability of alkaline phosphatase.

[0008] Furthermore, magnesium ions come from magnesium ion compounds, which can be listed as one or more of magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium acetate, and magnesium lactate.

[0009] Furthermore, zinc ions are derived from magnesium ion compounds, which can be listed as one or more of zinc chloride, zinc sulfate, zinc nitrate, zinc acetate, and zinc lactate.

[0010] Furthermore, the mass ratio of hypotriacetic acid, diethylenetriaminepentaacetic acid to magnesium ions in the first chelate is 0.5–2.5:0.5–2.5:1.0.

[0011] Furthermore, the mass ratio of methionine to threonine to zinc ions in the second chelate is 5–10:5–10:1.

[0012] Furthermore, the mass percentages of the first chelate and the second chelate are 2.0–3.0% and 0.1–0.5%, respectively.

[0013] Furthermore, the pH of the alkaline phosphatase enzyme-labeled buffer solution is 6.0–7.0.

[0014] Furthermore, the above-mentioned alkaline phosphatase enzyme-labeled buffer also includes the following components by mass percentage: 0.2–2.0% reduced glutathione, 1.0–2.5% corn peptide, 0.5–10.0% protein, 0.1–2.0% ginsenosides, 0.1–1.0% surfactant, 1.0–5.0% carbohydrates, 1.0–10.0% polyol, 0.05–0.1% preservative, and 0.01–0.05% antibiotic, with the remainder being nonionic amphoteric buffer.

[0015] Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine. It is the coenzyme of glyceraldehyde dehydrogenase and also a coenzyme of glyoxalase and triose phosphate dehydrogenase. It participates in the tricarboxylic acid cycle and glucose metabolism in the body. Glutathione exists in the body in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Its active component is reduced glutathione, which can participate in the redox process in the body. Under the action of glutathione transferase, reduced glutathione can combine with peroxides and free radicals to counteract the damage of sulfhydryl groups by oxidants, protect sulfhydryl-containing proteins and enzymes from damage, and thus improve the stability of enzyme conjugates. Corn peptides are a mixture of short peptide molecules with small molecular weight but high activity obtained by hydrolysis of corn gluten powder. Compared to amino acids or proteins, corn peptides are soluble in water over a wide pH range without causing turbidity or precipitation. They also exhibit good thermal stability, with no changes in composition, thus improving the stability of enzyme conjugates. Ginsenosides possess antibacterial and antioxidant properties, thereby enhancing the stability of ALP conjugates.

[0016] Further, the protein is one or more of bovine serum albumin (BSA), bovine lactoferrin (LF), and chicken ovalbumin (OVA). A mixture of bovine lactoferrin and chicken ovalbumin in a mass ratio of 2 to 3:1 is preferred.

[0017] Furthermore, the surfactant is a nonionic surfactant, such as one or more of polyoxyethylene polyoxypropylene ether, octylphenol polyoxyethylene ether, polyoxyethylene sorbitan ester, and sorbitan ester.

[0018] Furthermore, the carbohydrate compound is one or more of the following: dextran, fructan, fructose, xylose, and rhamnose.

[0019] Furthermore, the polyol is one or more of mannitol, sorbitol, and xylitol.

[0020] The present invention also provides an alkaline phosphatase enzyme labeling reagent, comprising the above-mentioned alkaline phosphatase enzyme labeling buffer.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0022] (1) By combining the first chelate formed by hyponitrotriacetic acid, diethylenetriaminepentaacetic acid and magnesium ions and the second chelate formed by methionine, threonine and zinc ions, and combining them with glutathione, proteins and other substances, an alkaline phosphatase enzyme-labeled buffer with long-term stability is obtained.

[0023] (2) The alkaline phosphatase enzyme-labeled buffer obtained in this invention can protect the activity of alkaline phosphatase for a long time and maintain the good detection effect of alkaline phosphatase enzyme-labeled reagent. Detailed Implementation

[0024] The technical solution of the present invention will be further described and illustrated below through specific embodiments. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the present invention. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used raw materials in the art, and the methods used in the embodiments are all conventional methods in the art.

[0025] The alkaline phosphatase enzyme-labeled buffers used in the following examples and comparative examples used HEPES nonionic amphoteric buffer as the base buffer, Pluronic F-127 as the surfactant, bronidox as the preservative, and gentamicin sulfate as the antibiotic. The specific formulations are shown in Table 1 below.

[0026] Table 1. Formulation of alkaline phosphatase enzyme-labeled buffer for the examples.

[0027] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 <![CDATA[MgCl2]]> 0.1％ 0.1％ 0.1％ 0.1％ 0.1％ 0.1％ NTA 2.0％ 1.5％ 2.0％ 2.0％ 2.0％ 2.0％ DTPA 0.5％ 1.0％ 0.5％ 0.5％ 0.5％ 0.5％ <![CDATA[ZnCl2]]> 0.05％ 0.05％ 0.05％ 0.05％ 0.05％ 0.05％ Methionine 0.1％ 0.1％ 0.15 0.1％ 0.1％ 0.1％ threonine 0.1％ 0.1％ 0.15 0.1％ 0.1％ 0.1％ Reduced glutathione 1.0％ 1.0％ 1.0％ 1.0％ 1.0％ 0.1％ LF 1.5％ 1.5％ 1.5％ 1.5％ 1.5％ - OVA 1.0％ 1.0％ 1.0％ 1.0％ - - Ginsenosides 0.6％ 0.6％ 0.6％ 0.6％ - - Pluronic F-127 0.5％ 0.5％ 0.5％ - - - Rhamnose 2.0％ 2.0％ 2.0％ - - - Corn peptides 1.5％ 1.5％ 1.5％ 1.5％ 1.5％ - Mannitol 3.0％ 3.0％ - - - - bronidox 0.08％ 0.08％ - - - - Gentamicin sulfate 0.02％ - - - - - pH 6.2 6.5 6.2 6.2 6.2 6.2

[0028] Table 2. Comparative Alkaline Phosphatase Enzyme Label Buffer Formulation Table

[0029]

[0030]

[0031] The alkaline phosphatase label was added to the alkaline phosphatase enzyme-labeled buffer solution described in the above example to prepare an enzyme solution with a final concentration of 2 μg / mL. The solution was then placed in a 37°C constant temperature and humidity incubator for accelerated testing. Using the enzyme solution as a sample, the residual activity ratio of the alkaline phosphatase label in the solution (the ratio of activity after accelerated testing to activity after preparation) was determined using a Haier GV300 chemiluminescence analyzer and its matching assay kit. The results are shown in Table 3 below.

[0032] Table 3. Data on the remaining activity ratio of alkaline phosphatase markers.

[0033] Acceleration Days Day 1 Day 2 Day 5 Day 7 Day 9 Example 1 99.7％ 99.7％ 99.5％ 99.2％ 98.9％ Example 2 99.0％ 98.5％ 97.5％ 97.0％ 96.9％ Example 3 99.0％ 97.0％ 97.0％ 96.0％ 95.2％ Example 4 98.0％ 95.0％ 92.0％ 88.0％ 84.7％ Example 5 97.2％ 90.0％ 85.0％ 76.0％ 74.0％ Example 6 93.5％ 84.0％ 75.0％ 65.0％ 62.3％ Comparative Example 1 92.5％ 81.0％ 74.5％ 62.0％ 59.3％ Comparative Example 2 91.5％ 80.2％ 73.4％ 64.2％ 50.1％ Comparative Example 3 94.5％ 81.2％ 72.8％ 60.1％ 51.4％ Comparative Example 4 92.5％ 79.5％ 71.8％ 65.1％ 59.5％ Comparative Example 5 93.5％ 77.8％ 69.5％ 60.4％ 55.4％ Comparative Example 6 95.5％ 79.2％ 71.4％ 62.4％ 51.8％ Comparative Example 7 92.5％ 79.5％ 70.4％ 60.5％ 51.4％ Comparative Example 8 91.5％ 79.1％ 68.4％ 59.8％ 54.3％

[0034] As shown in Table 3, the alkaline phosphatase enzyme-labeled buffer of the present invention has excellent stability. After acceleration, the alkaline phosphatase activity remained at 98.9% after one week, with a loss of nearly 1.01%, which can protect the activity of alkaline phosphatase for a long time.

[0035] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the spirit of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. An alkaline phosphatase enzyme-labeled buffer solution, characterized in that, This includes the first chelate formed by hypozinotriacetic acid, diethylenetriaminepentaacetic acid and magnesium ions, and the second chelate formed by methionine and threonine and zinc ions; The mass percentages of the first chelate and the second chelate are 2.0–3.0% and 0.1–0.5%, respectively.

2. The alkaline phosphatase enzyme-labeled buffer solution according to claim 1, characterized in that, In the first chelate, the mass ratio of hyponitrotriacetic acid, diethylenetriaminepentaacetic acid to magnesium ions is 0.5~2.5:0.5~2.5:1.

0.

3. The alkaline phosphatase enzyme-labeled buffer solution according to claim 1, characterized in that, The mass ratio of methionine to threonine to zinc ions in the second chelate is 2~5:2~5:

1.

4. The alkaline phosphatase enzyme-labeled buffer solution according to claim 1, characterized in that, The pH of the alkaline phosphatase enzyme-labeled buffer is 6.0~7.

0.

5. The alkaline phosphatase enzyme-labeled buffer solution according to claim 1, characterized in that, The alkaline phosphatase enzyme-labeled buffer further comprises the following components by weight percentage: 0.2–2.0% reduced glutathione, 1.0–2.5% corn peptide, 0.5–10.0% protein, 0.1–2.0% ginsenosides, 0.1–1.0% surfactant, 1.0–5.0% carbohydrates, 1.0–10.0% polyol, 0.05–0.1% preservative, and 0.01–0.05% antibiotic, with the remainder being a nonionic amphoteric buffer.

6. The alkaline phosphatase enzyme-labeled buffer solution according to claim 5, characterized in that, The protein is one or more of bovine serum albumin, bovine lactoferrin, and chicken ovalbumin.

7. The alkaline phosphatase enzyme-labeled buffer solution according to claim 5, characterized in that, The carbohydrate compound is one or more of the following: dextran, fructosaccharide, fructose, xylose, and rhamnose.

8. The alkaline phosphatase enzyme-labeled buffer solution according to claim 5, characterized in that, The polyol is one or more of mannitol, sorbitol, and xylitol.

9. An alkaline phosphatase enzyme-labeled reagent, characterized in that, Includes the alkaline phosphatase enzyme-labeled buffer as described in claim 1.

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