CN-116735857-B - Alkaline phosphatase enzyme-labeled buffer solution and alkaline phosphatase enzyme-labeled reagent

Abstract

The invention belongs to the technical field of medical materials, and particularly relates to an alkaline phosphatase enzyme-labeled buffer solution and an alkaline phosphatase enzyme-labeled reagent. The alkaline phosphatase enzyme-labeled buffer solution comprises, by mass, 0.2-2.0% of reduced glutathione, 1.0-2.5% of corn peptide, 0.5-10.0% of protein, 0.1-2.0% of ginsenoside, 0.1-1.0% of surfactant, 1.0-5.0% of carbohydrate compound, 1.0-10.0% of polyol, 0.05-0.1% of preservative and 0.01-0.05% of antibiotic, and the balance of nonionic amphoteric buffer solution. The alkaline phosphatase enzyme-labeled buffer solution with long-term stability is obtained by combining substances such as glutathione, protein and the like through the complex use of a first chelate formed by nitrilotriacetic acid, diethylenetriamine pentaacetic acid and magnesium ions and a second chelate formed by methionine, threonine and zinc ions.

Inventors

• YAN JUNJIE
• GUO JUNMEI
• WANG YUNFENG
• ZHOU GUOCHAO
• LIU JIANMIN

Assignees

• 宁波海尔施智造有限公司

Dates

Publication Date

20260512

Application Date

20230308

Claims (9)

1. 1. An alkaline phosphatase enzyme-labeled buffer solution is characterized by comprising a first chelate formed by nitrilotriacetic acid, diethylenetriamine pentaacetic acid and magnesium ions and a second chelate formed by methionine, threonine and zinc ions; The mass percentages of the first chelate and the second chelate are respectively 2.0-3.0% and 0.1-0.5%.
2. 2. The alkaline phosphatase enzyme-labeled buffer according to claim 1, wherein the mass ratio of nitrilotriacetic acid, diethylenetriamine pentaacetic acid and magnesium ions in the first chelate is 0.5-2.5:0.5-2.5:1.0.
3. 3. The alkaline phosphatase enzyme-labeled buffer according to claim 1, wherein the mass ratio of methionine to threonine to zinc ions in the second chelate is 2-5:2-5:1.
4. 4. The alkaline phosphatase enzyme-labeled buffer according to claim 1, wherein the pH of the alkaline phosphatase enzyme-labeled buffer is 6.0-7.0.
5. 5. The alkaline phosphatase enzyme-labeled buffer solution according to claim 1, further comprising, by mass, 0.2-2.0% of reduced glutathione, 1.0-2.5% of corn peptide, 0.5-10.0% of protein, 0.1-2.0% of ginsenoside, 0.1-1.0% of surfactant, 1.0-5.0% of saccharide compound, 1.0-10.0% of polyol, 0.05-0.1% of preservative and 0.01-0.05% of antibiotic, and the balance being nonionic amphoteric buffer solution.
6. 6. The alkaline phosphatase enzyme-labeled buffer according to claim 5, wherein the protein is one or more of bovine serum albumin, bovine lactoferrin, and chicken ovalbumin.
7. 7. The alkaline phosphatase enzyme-labeled buffer according to claim 5, wherein the saccharide compound is one or more of dextran, polyfructose, fructose, xylose, rhamnose.
8. 8. The alkaline phosphatase enzyme-labeled buffer according to claim 5, wherein the polyol is one or more of mannitol, sorbitol, xylitol.
9. 9. An alkaline phosphatase enzyme-labeled reagent, comprising the alkaline phosphatase enzyme-labeled buffer according to claim 1.

Description

Alkaline phosphatase enzyme-labeled buffer solution and alkaline phosphatase enzyme-labeled reagent Technical Field The invention belongs to the technical field of medical materials, and particularly relates to an alkaline phosphatase enzyme-labeled buffer solution and an alkaline phosphatase enzyme-labeled reagent. Background Alkaline phosphatase (hereinafter also referred to as ALP) is an enzyme commonly used as a label in immunoassays, belonging to a homodimer protein, having a molecular weight of 56kDa, each monomer consisting of 449 amino acids, the complete AKP molecule exhibiting a typical alpha/beta topology, each monomer having an active center consisting of Asp101-Ser102-Ala103 triplex, arg166, a water molecule, three metal ions and its ligand amino acids, AKP being encoded by the phoA gene, and, like many secreted proteins, synthesizing a monomeric precursor with a signal peptide at the amino terminus in the cytoplasm, which signal peptide directs the precursor to be cleaved off after trans-endomembrane transport, forming a homodimer. Alkaline phosphatase is an enzyme capable of dephosphorylating a corresponding substrate, by hydrolyzing a phosphomonoester to remove phosphate groups on the substrate molecule and generate phosphate ions and free hydroxyl groups, and the substrate includes nucleic acids, proteins, alkaloids, etc., the dephosphorylation process is called dephosphorylation or dephosphorylation. While alkaline phosphatase acts as a phosphatase in direct opposition to the action of a kinase (a kinase is a phosphorylase that can use energy molecules such as ATP to add phosphate groups to corresponding substrate molecules), alkaline phosphatase has maximum activity in alkaline environments and has an optimum pH of 8.0 for ALP from bacteria and 8.5 for ALP from cattle. ALP is a zinc-containing glycoprotein that hydrolyzes a variety of natural and synthetic phosphomonoester compound substrates in alkaline environments (pH optimum of 10). The chemiluminescent immunoassay (CLIA) belongs to a labeled antibody technology, and is characterized by that the antigen or antibody is labeled by using a chemiluminescent agent, a catalytic luminescent enzyme or a substance with indirect participation of a product in luminescent reaction, after the marker is combined with a corresponding substance, the luminescent substrate is acted by the luminescent agent, the catalytic enzyme or the participation of the product to produce redox reaction, and the light signal is released in the reaction, and finally the photomultiplier is used for detection. Currently, chemiluminescent enzyme immunoassay methods are generally used to detect the concentration of proteins (such as tumor markers, CA125, HE 4), i.e., enzyme-catalyzed chemiluminescent techniques with high sensitivity are combined with highly specific antigen-antibody reactions for quantitative detection of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, etc. The technology has the advantages of high specificity, high sensitivity, simple and quick separation, automatic analysis and the like. Wherein the enzyme is mainly alkaline phosphatase, the alkaline phosphatase marker solution is one of the components of the in vitro diagnostic kit, and the enzyme activity stability of the alkaline phosphatase becomes an important influencing factor of the quality performance of the kit. As with other biological enzymes, alkaline phosphatase has a short half-life and is easily inactivated. The change in acid, base, salt ion, temperature conditions will change or even deactivate the enzyme completely. The long-term stable preservation technology of alkaline phosphatase in solution is a key technology of in vitro diagnostic kit products. Disclosure of Invention The invention aims at solving the technical problems and provides an alkaline phosphatase enzyme-labeled buffer solution which improves the detection efficiency and long-term storage stability of alkaline phosphatase. The alkaline phosphatase enzyme-labeled buffer solution in the technical scheme of the invention comprises a first chelate formed by nitrilotriacetic acid (NTA), diethylenetriamine pentaacetic acid (DTPA) and magnesium ions and a second chelate formed by methionine, threonine and zinc ions. Magnesium ion is a specific 'effector molecule' of ALP site, can coordinate with related groups in alkaline phosphatase, stabilizes the conformation of enzyme active site, enables spontaneous folding process to proceed to natural-like direction, and can effectively improve catalytic efficiency and stability of alkaline phosphatase after chelating with nitrilotriacetic acid and diethylenetriamine pentaacetic acid. Further, the magnesium ion is derived from a magnesium ion compound, and the magnesium ion compound may be one or more of magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium acetate, and magnesium lactate. Further, the zinc ion is derived from a magn