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CN-121978354-A - Magnetic particle chemiluminescence detection kit for insulin and preparation method thereof

CN121978354ACN 121978354 ACN121978354 ACN 121978354ACN-121978354-A

Abstract

The invention relates to the technical field of kits, in particular to a magnetic particle chemiluminescence detection kit for insulin and a preparation method thereof, and particularly relates to a kit composed of a reagent M, a reagent R2 and a reagent R1, wherein the reagent M is a magnetic particle working solution containing streptavidin magnetic beads, the reagent R2 is an enzyme-labeled working solution containing alkaline phosphatase-labeled insulin antibodies, and the reagent R1 is a working solution containing biotin-labeled insulin monoclonal antibodies. Proved by verification, the chemiluminescent kit for insulin provided by the invention has the test sensitivity reaching 2.00 mu IU/mL, the test repeatability is less than 3% for high and low value samples, and the linear correlation with Roche reaches 0.98 in the concentration range of 2.00-450 mu IU/mL. The invention also provides a preparation method of the chemiluminescent kit of insulin, which greatly shortens the production period, is easy to control the batch difference and is beneficial to enterprises to carry out production work.

Inventors

  • WANG HUIHONG
  • CHEN TING
  • LIU HONGPING

Assignees

  • 桂林优利特医疗电子有限公司

Dates

Publication Date
20260505
Application Date
20260113

Claims (8)

  1. 1. A magnetic particle chemiluminescence detection kit for insulin is characterized in that, The reagent comprises a reagent M, a reagent R2 and a reagent R1, wherein the reagent M is a magnetic particle working solution containing streptavidin magnetic beads, the reagent R2 is an enzyme-labeled working solution containing alkaline phosphatase-labeled insulin antibody, and the reagent R1 is a working solution containing biotin-labeled insulin monoclonal antibody; The reagent M comprises 0.01-0.1 mol/L of PBS buffer solution, 0.1-10 g/L of protein stabilizer, 5-25 g/L of saccharide molecule, 0.5-5 g/L of nonionic surfactant, 0.5-1 g/L of preservative, and 0.1-0.5 mg/mL of blocker and streptavidin magnetic beads; The reagent R2 comprises 0.05-0.1 mol/L of MES buffer solution, 0.1-10 g/L of zinc chloride, 0.5-5 g/L of magnesium chloride and protein stabilizer, 0.5-1 g/L of non-ionic surfactant and 0.5-2 mu g/mL of alkaline phosphatase-labeled insulin antibody; The reagent R1 comprises the components of PBS buffer solution, 0.1-10 g/L of protein stabilizer, 5-25 g/L of saccharide molecule, 0.5-5 g/L of nonionic surfactant, 0.5-1 g/L of preservative and 0.5-2 mu g/mL of biotin-labeled insulin monoclonal antibody.
  2. 2. The magnetic particle chemiluminescence detection kit of insulin according to claim 1, The pH value of the PBS buffer solution is within the range of 7.0+/-8.0, 0.1-0.2 mol/L sodium chloride is contained, and the pH value of the MES buffer solution is within the range of 6.0+/-7.0, and 0.1-0.2 mol/L sodium chloride is contained.
  3. 3. A method for preparing a magnetic particle chemiluminescence detection kit for insulin, which is used for preparing the magnetic particle chemiluminescence detection kit for insulin according to any one of claims 1 and 2, and is characterized by comprising the following steps: step 1, preparing a reagent M; Step 2, preparing a reagent R2; and 3, preparing a reagent R1.
  4. 4. The method for preparing the magnetic particle chemiluminescence detection kit of insulin according to claim 3, The execution process of the step 1 comprises the following steps: Step 1.1, taking out magnetic bead stock solution according to the proportion of 0.1 mg/mL-0.5 mg/mL of streptavidin magnetic beads; Step 1.2, carrying out liquid exchange pretreatment operation on the extracted magnetic bead stock solution, taking a washing liquid Buffer B which is 3 times the volume of the magnetic bead stock solution according to calculation, adding the washing liquid Buffer B into magnetic particles of which the supernatant is discarded in a magnetic separator, and uniformly mixing the magnetic particles on a uniformly mixing instrument for 30 min-1 h; Step 1.3, weighing a washing solution Buffer B which is 3 times the volume of the magnetic bead stock solution according to the calculation, adding the washing solution Buffer B into the magnetic particles of which the supernatant is removed after pretreatment, cleaning for 3 times, and placing the magnetic particles on a magnetic separator, and removing the supernatant after the liquid is clear and transparent; And 1.4, adding the magnetic particle working solution Buffer A into the magnetic particles with the supernatant removed, fixing the volume to the target concentration, and adding the blocker specific interference elimination protein of 10-100 mu g/mL to obtain the reagent M.
  5. 5. The magnetic particle chemiluminescence detection kit of insulin according to claim 4, The magnetic particle working solution Buffer A in the step 1 is PBS Buffer with pH value of 7.40+/-0.05, 0.15mol of NaCl, 5g/L of casein, 1g/L of bovine serum albumin, 20g/L of mannitol, 0.1g/L of 4 aminopyrine, 0.01g/L of biotin, 20g/L of Tween-20, 0.5g/L of TX-100 and 0.5g/L of PC-300 are added; the washing solution Buffer B is PBS Buffer solution with pH of 7.40+/-0.05, 0.15mol of NaCl, 5g/L of casein, 1g/L of bovine serum albumin, 10g/L of sucrose, 1g/L of Tween-20, 0.5g/L of TX-100 and 0.2g/L of PC are added; all buffers were prepared and filtered through a 0.22 μm pore size nylon filter.
  6. 6. The method for preparing the magnetic particle chemiluminescence detection kit of insulin according to claim 5, The execution process of the step 2 comprises the following steps: 2.1, dissolving and diluting the heteroleptic bifunctional crosslinking agent by using dimethyl sulfoxide, and carrying out light-shielding reaction on the heteroleptic bifunctional crosslinking agent and an antigen derivative diluted by using a buffer solution 1 for 0.5-2 hours at the temperature of 20-37 ℃ according to the molar ratio of 1:10-1:40; 2.2, dissolving and diluting the sulfhydryl modified cross-linking agent by using a buffer solution 2, and carrying out light-shielding reaction on the cross-linking agent and alkaline phosphatase diluted by using the buffer solution 2 for 0.5-2 hours at the temperature of 20-37 ℃ according to the molar ratio of 1:10-1:30; 2.3, performing photoreaction on the activated antigen derivative and the ALP modified with the sulfhydryl group for 1-3 hours at the temperature of 20-37 ℃ according to the mass ratio of 1:0.5-1:2 to obtain an alkaline phosphatase-labeled insulin antibody; Step 2.4, purifying the alkaline phosphatase-labeled insulin antibody obtained by the above method by using a buffer solution 1; Step 2.5, preparing enzyme-labeled working solution by using 0.05-0.1 mol/L MES buffer solution, 0.1-10 g/L zinc chloride, magnesium chloride, 0.5-5 g/L nonionic surfactant and 0.5-1 g/L preservative, and 0.5-2 mu g/mL purified alkaline phosphatase-labeled insulin antibody.
  7. 7. The method for preparing the magnetic particle chemiluminescence detection kit of insulin of claim 6, wherein, The buffer solution 1 in the step 2 is prepared from 10mM phosphate buffer salt ions, 9g/L sodium chloride and the pH value of which is within the range of 7.40+/-0.05, and the buffer solution 2 is prepared from 100mM phosphate buffer salt ions, 9g/L sodium chloride, 10mM disodium ethylenediamine tetraacetate and the pH value of which is within the range of 8.00+/-0.05.
  8. 8. The method for preparing the magnetic particle chemiluminescence detection kit of insulin as defined in claim 7, wherein, The execution process of the step 3 comprises the following steps: step 3.1, taking out the antibody according to the proportion of 0.5 mu g/mL-2 mu g/mL of the biotin-labeled insulin monoclonal antibody; Step 3.2, adding the biotin-labeled insulin monoclonal antibody into a working solution Buffer A to prepare a target concentration; The working solution Buffer A is PBS Buffer solution with pH of 7.40+/-0.05, 0.15mol of NaCl, 5g/L of casein, 1g/L of bovine serum albumin, 20g/L of mannitol, 0.1g/L of 4-aminopyrine, 0.01g/L of biotin, 1g/L of Tween-20, 0.5g/L of TX-100 and 0.5g/L of PC-300 are added.

Description

Magnetic particle chemiluminescence detection kit for insulin and preparation method thereof Technical Field The invention relates to the technical field of kits, in particular to a magnetic particle chemiluminescence detection kit for insulin and a preparation method thereof. Background Insulin (INS) is a hormone secreted by the beta cells of the pancreas, consisting of 51 amino acids. Insulin regulates glucose uptake and use and is also involved in protein synthesis and triglyceride storage. An increase in the amount of glucose in the circulation stimulates insulin secretion. In turn, insulin stimulates uptake of glucose by tissues while inhibiting hepatic glycogen breakdown in the liver. Diabetes is an increase in blood glucose due to absolute or relative inadequate insulin secretion, a disorder of tissue utilization of glucose. Diabetes may be accompanied by serious complications such as renal failure, heart disease, nerve damage, blindness and gangrene. Severe hyperglycemic episodes can also cause ketoacidosis and coma. Early diabetes manifests itself only as a reduced insulin response to glucose stimulation, and detection of insulin levels under basal conditions or after glucose treatment aids in diagnosing early diabetes and assessing the ability of the pancreas to secrete insulin. Currently, INS detection kits on the market are classified into Radioimmunoassay (RIA), enzyme-linked immunosorbent assay, chemiluminescent immunoassay, and the like. The method has the advantages of strong radioimmunoassay pollution, low sensitivity, great influence of personal factors, low automation degree and long detection time, and is unfavorable for higher and higher test requirements and full-automatic detection. The chemiluminescent immunoassay method is used for detecting INS, and products imported by Roche company are market dominant at present, so that the price is high, and the purchasing period is long. At present, INS detection kits are researched and developed in China, such as carboxylated nano magnetic beads adopted by Chinese patent with application publication number of CN106596524A, have poor dispersibility, are easy to agglomerate, cause high luminous intensity, cause non-specific adsorption of antibodies, and reduce specific capture capacity. The Chinese patent with the application publication number of CN110146692A amplifies the detection signal and improves the detection sensitivity by introducing a streptavidin-biotin system, but simultaneously has long production and manufacturing period and is easy to cause the product batch-to-batch difference. Disclosure of Invention The invention aims to provide a magnetic particle chemiluminescence detection kit for insulin and a preparation method thereof, and aims to solve the technical problems that the existing insulin detection kit is insufficient in detection sensitivity or long in production and manufacturing period and is easy to cause product batch-to-batch difference. The invention provides a magnetic particle chemiluminescence detection kit for insulin, which comprises a reagent M, a reagent R2 and a reagent R1, wherein the reagent M is magnetic particle working solution containing streptavidin magnetic beads, the reagent R2 is enzyme-labeled working solution containing alkaline phosphatase-labeled insulin antibody, and the reagent R1 is working solution containing biotin-labeled insulin monoclonal antibody; The reagent M comprises 0.01-0.1 mol/L of PBS buffer solution, 0.1-10 g/L of protein stabilizer, 5-25 g/L of saccharide molecule, 0.5-5 g/L of nonionic surfactant, 0.5-1 g/L of preservative, and 0.1-0.5 mg/mL of blocker and streptavidin magnetic beads; The reagent R2 comprises 0.05-0.1 mol/L of MES buffer solution, 0.1-10 g/L of zinc chloride, 0.5-5 g/L of magnesium chloride and protein stabilizer, 0.5-1 g/L of non-ionic surfactant and 0.5-2 mu g/mL of alkaline phosphatase-labeled insulin antibody; The reagent R1 comprises the components of PBS buffer solution, 0.1-10 g/L of protein stabilizer, 5-25 g/L of saccharide molecule, 0.5-5 g/L of nonionic surfactant, 0.5-1 g/L of preservative and 0.5-2 mu g/mL of biotin-labeled insulin monoclonal antibody. Wherein the pH value of the PBS buffer solution is within the range of 7.0+/-8.0, 0.1-0.2 mol/L sodium chloride is contained, and the pH value of the MES buffer solution is within the range of 6.0+/-7.0, and 0.1-0.2 mol/L sodium chloride is contained. The invention also provides a preparation method of the magnetic particle chemiluminescence detection kit for insulin, which is used for preparing the magnetic particle chemiluminescence detection kit for insulin and comprises the following steps of: step 1, preparing a reagent M; Step 2, preparing a reagent R2; and 3, preparing a reagent R1. Optionally, the performing process of step 1 includes the following steps: Step 1.1, taking out magnetic bead stock solution according to the proportion of 0.1 mg/mL-0.5 mg/mL of streptavidin magnetic beads; Step