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Three kits for detecting human phosphorylated Tau protein and preparation method thereof

A detection kit and phosphorylation technology, which is applied in the field of immunoassay, can solve the problems of unknown antigenic determinants, and achieve the effect of high-efficiency enrichment

Pending Publication Date: 2022-02-01
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Further, although this previous study verified that the hybridoma cell lines Hustaumab-3G5 and Hustaumab-4B1 can be used to prepare and obtain specific recognition of human p-Tau 396,404 monoclonal antibodies to their aggregates, but whether these two antibodies have different epitopes on the same antigen remains unknown

Method used

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  • Three kits for detecting human phosphorylated Tau protein and preparation method thereof
  • Three kits for detecting human phosphorylated Tau protein and preparation method thereof
  • Three kits for detecting human phosphorylated Tau protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Screening and detection of p-hTau 396,404 protein antibody pair

[0063] 1. Epitope identification of monoclonal antibodies 3C1, 3G5, 4B1, 4C6

[0064] 1.1. Design of polypeptides truncated at different positions: amino acid 379 to amino acid 385 of Tau protein (Tau 379-385 ): RENAKAK, the 386th amino acid to the 392nd amino acid of Tau protein (Tau 386-392 ): TDHGAEI, 396-site serine phosphorylated Tau protein (p-Tau 396 ): VYK-[Ser(P)]PVVS, serine phosphorylated Tau protein at position 404 (p-Tau 404 ): GDT[Ser(P)]PRHL, non-396 serine phosphorylated Tau protein (np-Tau 396 ): VYKSPVVS, non-404-site serine phosphorylated Tau protein (np-Tau 404): GDTSPRHL. All designed peptides were synthesized by existing peptide synthesis techniques.

[0065] 1.2. Dot blot verification

[0066] ① Coating with different truncated polypeptides: Dilute different truncated polypeptides with CBS buffer to a final concentration of 10 μg / mL, then add 20 μL successively on...

Embodiment 2

[0080] Example 2: Sensitivity and specificity evaluation of double-antibody sandwich ELISA

[0081] 2.1 Specific detection of double-antibody sandwich ELISA

[0082] ① Coating with capture antibody: Dilute the 3G5 antibody to 10 μg / mL with CBS, then add 100 μL of antibody to each well and coat at 4°C for 12 hours;

[0083] ②Blocking: After the coating is completed, discard the liquid in the well, wash with PBS-T for 3 times, add 200 μL of blocking solution to each well, and block at 37°C for 2 hours;

[0084] ③Add antigen: add 100μL 0.1mg / mL p-hTau 396,404 , Cis-hTau, Trans-hTau, p-hTau 231 , np-hTau 231 , hAβ 42 , hAβ 40 Incubate at room temperature for 2 hours; discard the supernatant, wash each well with PBS-T 3 times;

[0085] ④ Add enzyme-labeled antibody: add 100 μL HRP-labeled 4B1 to each well, and react at room temperature for 1 hour;

[0086] ⑤ Color development: After the reaction, discard the liquid in the well, wash with PBS-T for 3 times, add 100 μL TMB to ...

Embodiment 3

[0097] Example 3: Construction of an immune nano-magnetic bead detection kit

[0098] 3.1. Preparation of immune nanomagnetic beads

[0099] 1) Mix the magnetic beads evenly, take 10mg of magnetic beads and add them to a 2mL centrifuge tube, magnetically separate and remove the supernatant;

[0100] 2) Add 1mL deionized water, mix well, and remove the supernatant from the magnetic separation zone (repeat twice);

[0101] 3) Add 500-1000μL of reaction buffer (100mM MES, pH 5.0), mix and resuspend the magnetic beads, remove the supernatant by magnetic separation (repeat twice);

[0102] 4) Add 200 μL of reaction buffer, mix and resuspend the magnetic beads;

[0103] 5) Add 500 μg of 3G5 antibody (dissolved in 200 μL of reaction buffer in advance), add 100 μL of 10 mg / mL EDC-HCl and 100 μL of 10 mg / mL NHS and mix for 30 min at room temperature;

[0104] 6) Add the activated antibody to the pretreated magnetic beads and rotate and mix at room temperature for 2-6 hours;

[0105...

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Abstract

The invention belongs to the field of immunodetection, and particularly discloses three kits for detecting human phosphorylated Tau protein and a preparation method thereof. According to the three detection kits disclosed by the invention, the monoclonal antibody 3G5 is used as a capture antibody, and the monoclonal antibody 4B1 is used as a detection antibody, or the monoclonal antibody 4B1 is used as a capture antibody, and the monoclonal antibody 3G5 is used as a detection antibody, wherein p-hTau396, 404 protein can be detected by the kit. The three detection kits constructed by using the monoclonal antibodies 3G5 and 4B1 are respectively suitable for detection in different scenes. The method can rapidly and accurately detect the human phosphorylated Tau, integrates the characteristics of specificity of the antibody, high sensitivity of ELISA, efficient enrichment of the immune nano magnetic beads, convenience and rapidness of the colloidal gold test strip and the like, and has the characteristics of accuracy, rapidness, high efficiency, specificity and sensitivity. Therefore, the method is suitable for rapid and sensitive early diagnosis of Alzheimer's disease, and is of great significance to timely and accurate guidance of medicine use for patients.

Description

technical field [0001] The invention belongs to the field of immunoassay, and more specifically, relates to three kinds of kits for detecting human phosphorylated Tau protein and preparation methods thereof, which are respectively: a double-antibody sandwich ELISA kit for detecting human phosphorylated Tau protein and its Preparation, human phosphorylated Tau protein immune nano-magnetic bead kit and its preparation, human phosphorylated Tau protein colloidal gold immunochromatographic test strip and its preparation. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease with insidious onset and progressive development. Clinically, it is characterized by generalized dementia such as memory impairment, aphasia, apraxia, agnosia, impairment of visuospatial skills, executive dysfunction, and personality and behavior changes. As of 2020, there will be more than 1 million new cases of Alzheimer's disease in China every year, and the total number of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/58G01N33/558G01N33/543G01N33/52C07K16/18C07K16/44C12N5/20C12R1/91
CPCG01N33/6896G01N33/6842G01N33/577G01N33/587G01N33/54346G01N33/54326G01N33/558G01N33/526C07K16/18C07K16/44G01N2800/2821G01N2333/4706
Inventor 骆海明张立定李艳青牛是琦
Owner HUAZHONG UNIV OF SCI & TECH
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