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CN-121995061-A - Method for enhancing detection sensitivity of chemiluminescent system by using tyramine signal amplification technology

CN121995061ACN 121995061 ACN121995061 ACN 121995061ACN-121995061-A

Abstract

The invention relates to a kit and a method for detecting phosphorylated Tau protein. In particular to a detection kit and a detection method of p-Tau217 protein. The kit and the method are based on the p-Tau217 antibody and the total-Tau protein antibody, and are combined with the TSA technology, so that the specificity, sensitivity and accuracy of a detection result are improved, and the background signal is effectively reduced.

Inventors

  • Jin Gaowei
  • CHEN YUAN
  • CHEN JING
  • YE CHENGZHANG
  • ZHOU YUNBO

Assignees

  • 上海伯杰医疗科技股份有限公司

Dates

Publication Date
20260508
Application Date
20260205

Claims (10)

  1. 1. A kit for detecting p-Tau217 protein, comprising: (1) A first container and a first formulation in the first container, the first formulation comprising a p-Tau217 antibody, the p-Tau217 antibody bearing magnetic beads; (2) A second container and a second formulation in the second container, the second formulation comprising a total-Tau protein antibody, the total-Tau protein antibody bearing a peroxidase; (3) A third container and a working solution of biotin tyramine positioned in the third container, wherein the working solution of biotin tyramine comprises biotinylated tyramine and hydrogen peroxide, and (4) And a fourth container and an acridinium ester-labeled streptavidin reagent disposed in the fourth container.
  2. 2. The kit of claim 1, wherein the p-Tau217 antibody has a light chain CDR of the amino acid sequence of SEQ ID NO. 6-8, 14-16 or 22-24 and a heavy chain CDR of the amino acid sequence of SEQ ID NO. 2-4, 10-12 or 18-20.
  3. 3. The kit of claim 1, wherein the concentration of the acridinium ester-labeled streptavidin reagent is from 0.01 to 3 μg/mL.
  4. 4. The kit of claim 1, wherein the concentration of the p-Tau217 antibody is 0.1-5mg/mL and the concentration of the total-Tau protein antibody is 0.1-5 μg/mL.
  5. 5. The kit of claim 1, wherein the concentration of biotinylated tyramine is 1-100 μg/mL and the concentration of hydrogen peroxide is 0.01-3%.
  6. 6. The kit of claim 1, wherein the p-Tau217 antibody has a light chain CDR of the amino acid sequence shown in SEQ ID NOs 14-16 and a heavy chain CDR of the amino acid sequence shown in SEQ ID NOs 10-12; The concentration of the p-Tau217 antibody is 0.3-0.5 mg/mL, and the concentration of the total-Tau protein antibody is 0.2-0.4 mug/mL; the concentration of the biotinylated tyramine is 5-50 mug/mL, and the concentration of the hydrogen peroxide is 0.1-1%.
  7. 7. A formulation, wherein the formulation comprises (Z 1) a first formulation comprising a p-Tau217 antibody, the p-Tau217 antibody bearing magnetic beads; (z 2) a second formulation comprising a total-Tau protein antibody bearing a peroxidase; (z 3) a biotin tyramine working solution comprising biotinylated tyramine and hydrogen peroxide working solution, and (Z 4) acridinium ester-labeled streptavidin reagent.
  8. 8. A method for detecting a p-Tau217 protein, comprising the steps of: (s 1) mixing a sample to be tested with a p-Tau217 antibody and a total-Tau protein antibody to obtain a first mixture, wherein the p-Tau217 antibody is provided with magnetic beads, the total-Tau protein antibody is provided with peroxidase, and when the sample to be tested contains a p-Tau217 antigen, the first mixture contains a magnetic bead-diabody-antigen complex; (s 2) adding a working solution of biotin tyramine comprising biotinylated tyramine and hydrogen peroxide to the first mixture, allowing biotin to deposit through tyramine in the vicinity of the bead-diabody-antigen complex, washing off unreacted biotin tyramine and hydrogen peroxide to obtain a second mixture, and (S 3) adding an acridinium ester-labeled streptavidin reagent into the second mixture, so that streptavidin is combined with biotin, washing away the unbound acridinium ester-labeled streptavidin reagent, adding a pre-excitation solution and an excitation solution, and detecting a luminescence signal to obtain a detection result.
  9. 9. The method of claim 8, wherein the volume ratio of the sample to be tested, the p-Tau217 antibody, and the total-Tau protein antibody is 1-5:1-2:1-2.
  10. 10. The method of claim 8, wherein the p-Tau217 antibody has light chain CDRs of the amino acid sequences shown in SEQ ID NOS.6-8, 14-16 or 22-24, and heavy chain CDRs of the amino acid sequences shown in SEQ ID NOS.2-4, 10-12 or 18-20.

Description

Method for enhancing detection sensitivity of chemiluminescent system by using tyramine signal amplification technology Technical Field The invention relates to the technical field of in-vitro diagnosis and detection, in particular to a method for enhancing detection sensitivity of a chemiluminescent system by using a tyramine signal amplification technology. Background Traditional chemiluminescence methods still face significant challenges in their sensitivity in the face of certain ultra-low abundance biomarkers (e.g., certain early cancer markers, neurological markers, early antigens of infectious disease, etc.). To increase sensitivity, methods of increasing reaction time, optimizing labeling efficiency, or using more efficient optical signal detectors are often employed, but these methods tend to be limited in effectiveness or costly. Thus, there is a strong need in the art for a highly sensitive chemiluminescent-based detection method. Disclosure of Invention The invention aims to provide a high-sensitivity chemiluminescence-based detection method. It is another object of the present invention to provide a high sensitivity chemiluminescent-based detection kit. In a first aspect of the invention, there is provided a kit for detecting p-Tau217 protein comprising: (1) A first container and a first formulation in the first container, the first formulation comprising a p-Tau217 antibody, the p-Tau217 antibody bearing magnetic beads; (2) A second container and a second formulation in the second container, the second formulation comprising a total-Tau protein antibody, the total-Tau protein antibody bearing a peroxidase; (3) A third container and a working solution of biotin tyramine positioned in the third container, wherein the working solution of biotin tyramine comprises biotinylated tyramine and hydrogen peroxide, and (4) And a fourth container and an acridinium ester-labeled streptavidin reagent disposed in the fourth container. In another preferred embodiment, the p-Tau217 antibody has the light chain CDR of the amino acid sequence shown as SEQ ID NO. 6-8, 14-16 or 22-24 and the heavy chain CDR of the amino acid sequence shown as SEQ ID NO. 2-4, 10-12 or 18-20. In another preferred embodiment, the p-Tau217 antibody has a light chain CDR of the amino acid sequence shown in SEQ ID NO:6-8 and a heavy chain CDR of the amino acid sequence shown in SEQ ID NO:2-4, or Light chain CDRs of the amino acid sequences shown in SEQ ID NOS 14-16 and heavy chain CDRs of the amino acid sequences shown in SEQ ID NOS 10-12, or The light chain CDRs of the amino acid sequences shown in SEQ ID NOS.22-24 and the heavy chain CDRs of the amino acid sequences shown in SEQ ID NOS.18-20. In another preferred embodiment, the p-Tau217 antibody has a light chain with the amino acid sequence shown in SEQ ID NO. 5 and a heavy chain with the amino acid sequence shown in SEQ ID NO. 1. In another preferred embodiment, the p-Tau217 antibody has a light chain with the amino acid sequence shown in SEQ ID NO. 13 and a heavy chain with the amino acid sequence shown in SEQ ID NO. 9. In another preferred embodiment, the p-Tau217 antibody has a light chain with the amino acid sequence shown in SEQ ID NO. 21 and a heavy chain with the amino acid sequence shown in SEQ ID NO. 17. In another preferred embodiment, the total-Tau protein antibody specifically binds to all Tau proteins. In another preferred embodiment, the concentration of the p-Tau217 antibody is 0.1-5mg/mL and the concentration of the total-Tau protein antibody is 0.1-5. Mu.g/mL. In another preferred embodiment, the concentration of the p-Tau217 antibody is 0.1-1mg/mL, preferably 0.2-0.5mg/mL, more preferably 0.3-0.5 mg/mL, for example about 0.4 mg/mL. In another preferred embodiment, the total-Tau protein antibody is present at a concentration of 0.1-1. Mu.g/mL, preferably 0.2-0.5. Mu.g/mL, more preferably 0.2-0.4. Mu.g/mL, for example about 0.3. Mu.g/mL. In another preferred embodiment, the concentration of biotinylated tyramine is 1-100. Mu.g/mL and the concentration of hydrogen peroxide is 0.01-3%. In another preferred embodiment, the biotinylated tyramine concentration is from 5 μg/mL to 50 μg/mL. In another preferred embodiment, the concentration of hydrogen peroxide is 0.1-1%, preferably 0.1-0.5%. In another preferred embodiment, the concentration of the acridinium ester-labeled streptavidin reagent is from 0.01 to 3. Mu.g/mL, preferably from 0.05 to 1. Mu.g/mL, more preferably from 0.1 to 0.5. Mu.g/mL. In another preferred embodiment, the buffer of the hydrogen peroxide working solution is 0.1M PBS buffer, ph=8.0. In another preferred embodiment, the p-Tau217 antibody has the light chain CDR of the amino acid sequence shown in SEQ ID NO. 14-16 and the heavy chain CDR of the amino acid sequence shown in SEQ ID NO. 10-12; The concentration of the p-Tau217 antibody is 0.3-0.5 mg/mL, and the concentration of the total-Tau protein antibody is 0.2-0.4 mug/mL; the concentration of th