Search

CN-121978347-A - Method for detecting phosphorylated Tau-231 protein in urine of Alzheimer disease patient

CN121978347ACN 121978347 ACN121978347 ACN 121978347ACN-121978347-A

Abstract

The invention relates to the field of protein detection, in particular to a method for detecting phosphorylated Tau-231 protein in urine of a patient suffering from Alzheimer's disease, which comprises the steps of fixing a non-phosphorylated anti-Tau-231 antibody on magnetic particle microspheres, fixing the phosphorylated anti-Tau-p-231 antibody on fluorescent microspheres, enabling a detected object to form a sandwich compound of magnetic particles-antigen-fluorescent probes, taking out 100ul urine by using fresh morning urine, adding the 100ul urine into a 5.0ml centrifuge tube which is prefabricated with the phosphorylated anti-Tau-p-231 antibody of fluorescent markers and the non-phosphorylated anti-Tau-p-231 antibody connected with the nano magnetic particle microspheres, adding 4.9 ml urine diluent to dilute the urine, repeatedly mixing, reacting at room temperature for 8 to 12 hours, placing a reaction tube on a separation test tube with a magnet for more than 20 minutes, irradiating a judgment result by using a fluorescent tube rack around 340nm in a dark environment at an early stage, and finally achieving the purposes of screening, intervention and early diagnosis of the Alzheimer's disease.

Inventors

  • WANG ZHIXIN
  • HOU SHUXIA
  • SUN XIAODONG

Assignees

  • 广东朝莱生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260204

Claims (8)

  1. 1. A detection method of phosphorylated Tau-231 protein in urine of a patient suffering from Alzheimer's disease is characterized by comprising the following steps: s1, fixing a non-phosphorylated anti-Tau-231 antibody on a magnetic particle microsphere, and specifically capturing an antigen in a reaction solution; S2, fixing the phosphorylated anti-Tau-p-231 antibody on fluorescent microspheres to enable the detected object to form a sandwich compound of magnetic particles, antigens and fluorescent probes; And S3, finally enriching the reaction complex through a magnetic field, and directly reading the fluorescent signal to judge the negative and positive.
  2. 2. The method for detecting phosphorylated Tau-231 protein in urine of a patient suffering from Alzheimer' S disease according to claim 1, wherein the step of coupling the non-phosphorylated anti-Tau-231 antibody to the carboxyl magnetic particles by a chemical cross-linking method in S1 comprises the following steps: s101, activating magnetic particles; s102, coupling magnetic particles with non-phosphorylated anti-Tau-231 antibodies; s103, closing and storing magnetic particles.
  3. 3. The method for detecting the phosphorylated Tau-231 protein in urine of a patient suffering from Alzheimer' S disease according to claim 1, wherein the step of coupling the phosphorylated anti-Tau-p-231 antibody to the tracer fluorescent microsphere by a chemical crosslinking method in S2 comprises the following steps: S201, activating microspheres; s202, coupling the microsphere with a phosphorylated anti-Tau-p-231 antibody; s203, sealing and preserving the microspheres.
  4. 4. The method for detecting phosphorylated Tau-231 protein in urine of Alzheimer's disease according to claim 1, wherein the anti-Tau antibody and the anti-Tau-p-231 antibody are diluted to 1.0 mg/ml with 25mM phosphate buffer.
  5. 5. The method for detecting phosphorylated Tau-231 protein in urine of a patient suffering from Alzheimer's disease according to claim 2, wherein the magnetic particles are used for enriching and separating microspheres, and the particle size of the enriching and separating magnetic particle microspheres is 100nm-3000nm.
  6. 6. The method for detecting phosphorylated Tau-231 protein in urine of a patient suffering from Alzheimer's disease according to claim 3, wherein the fluorescent microsphere is used for tracing the fluorescent microsphere, and the particle size of the fluorescent microsphere is 100nm-3000nm.
  7. 7. The method for detecting phosphorylated Tau-231 protein in urine of a patient suffering from Alzheimer' S disease according to claim 2, wherein 500. Mu.L of blocking solution is added to the sample S101, wherein the blocking solution comprises Hepes with pH=7.4, BSA glycine with concentration of 5.0% and Proclin with concentration of 0.1%.
  8. 8. The method for detecting the phosphorylated Tau-231 protein in the urine of the patient suffering from Alzheimer' S disease according to claim 2, wherein 2.0ml of a complex solution is added into the S102, and the complex solution comprises Hepes, trehalose with the concentration of 10%, BSA with the concentration of 2%, glycine and Proclin with the concentration of 0.1%.

Description

Method for detecting phosphorylated Tau-231 protein in urine of Alzheimer disease patient Technical Field The invention belongs to the technical field of detection of phosphorylated Tau-231 proteins, and particularly relates to a detection method of phosphorylated Tau-231 proteins in urine of patients with Alzheimer's disease. Background Alzheimer's disease is a degenerative disease of the central nervous system, which occurs mainly in middle-aged or elderly people. The main features of the disease include progressive cognitive dysfunction and impairment of behavior. There are research data showing that some specific biological indicators of Alzheimer's disease are released into blood and excreted from body via urine sequentially 5-10 years before clinical symptoms occur, wherein phosphorylated Tau-231 protein is closely related to the course of Alzheimer's disease, but the concentration of phosphorylated Tau-231 protein is relatively low, and detection is difficult. In the prior art, one of the core neuropathological features of Alzheimer's Disease (AD) is the abnormal accumulation of paired helical filaments in neurofibrillary tangles within neurons, whose main component is hyperphosphorylated microtubule-associated protein Tau. The Tau protein comprises an N-terminal projection domain, a proline-rich domain, a microtubule binding repeat domain, and a C-terminal domain, each of which has multiple potential phosphorylation sites. The marker can accurately distinguish AD from normal control and predict cognitive decline in preclinical and prophase when used alone or in combination with Abeta-42, and the Tau-p-231 can pass through the blood brain barrier to enter blood, the peripheral blood concentration of the Tau-p-231 can also be proved to be used for differential diagnosis of dementia, and the Tau-p-231 in the blood can also be discharged out of the body through urine, thus providing possibility for detecting the Tau-p-231 through urine. In the above technology, tau-p-231 in blood enters the kidney along with physiological metabolism, and finally part of the Tau-p-231 is discharged out of the body through urine, however, the concentration of the Tau-p-231 in urine is only one thousandth or lower than the detection limit of a conventional immunological method, so that false negative is very easy to occur, and the Tau-p-231 in urine is not in a single form but coexists in two forms, namely, a free form and a membrane-bound form wrapped by a nerve-derived exosome, wherein the free form has small molecular weight and is easy to degrade, and the membrane-bound form wrapped by the nerve-derived exosome has higher stability, but is difficult to directly identify by a traditional antibody, so that accurate detection is difficult. Therefore, the invention provides a detection method of phosphorylated Tau-231 protein in urine of patients with Alzheimer's disease. Disclosure of Invention In order to overcome the deficiencies of the prior art, at least one technical problem presented in the background art is solved. The invention solves the technical problems by adopting the technical scheme that the detection method of phosphorylated Tau-231 protein in urine of Alzheimer disease patients comprises the following steps: s1, fixing a non-phosphorylated anti-Tau-231 antibody on a magnetic particle microsphere, and specifically capturing an antigen in a reaction solution; S2, fixing the phosphorylated anti-Tau-p-231 antibody on fluorescent microspheres to enable the detected object to form a sandwich compound of magnetic particles, antigens and fluorescent probes; And S3, finally enriching the reaction complex through a magnetic field, and directly reading the fluorescent signal to judge the negative and positive. Preferably, the step of coupling the anti-Tau-p-231 antibody to the carboxyl magnetic particles in S1 by chemical cross-linking method comprises the following steps: s101, activating magnetic particles; s102, coupling magnetic particles with non-phosphorylated anti-Tau-231 antibodies; s103, closing and storing magnetic particles. Preferably, the step of coupling the anti-Tau antibody to the tracer fluorescent microsphere in S2 by chemical cross-linking method comprises the following steps: S201, activating microspheres; s202, coupling the microsphere with a phosphorylated anti-Tau-p-231 antibody; s203, sealing and preserving the microspheres. Preferably, the anti-Tau antibody and anti-Tau-p-231 antibody are diluted to 1.0 mg/ml with 25mM phosphate buffer. Preferably, the magnetic particles are used for enriching and separating microspheres, and the particle size of the enriching and separating magnetic particle microspheres is 100nm-3000nm. Preferably, the fluorescent microsphere is used for tracing the fluorescent microsphere, and the particle size of the fluorescent microsphere is 100nm-3000nm. Preferably, 500 μl of blocking solution comprising Hepes at ph=7.4, BSA glycine at a concentration of 5.0% and Proc