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Phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application of phosphorylated Tau pT217 protein monoclonal antibody

A monoclonal antibody and polyclonal antibody technology, applied in the field of biomedicine, can solve the problems of low detection sensitivity and low specificity, and achieve the effect of wide detection range, strong specificity and high sensitivity

Pending Publication Date: 2022-05-27
武汉天德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only a few manufacturers on the market have developed kits for the detection of phosphorylated Tau pT217 protein, but there are problems such as low detection sensitivity and low specificity

Method used

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  • Phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application of phosphorylated Tau pT217 protein monoclonal antibody
  • Phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application of phosphorylated Tau pT217 protein monoclonal antibody
  • Phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application of phosphorylated Tau pT217 protein monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 immune complex preparation

[0036]1) Synthesis of antigen sequences of 217-position threonine (T217) phosphorylated antigens containing tau proteins by solid phase method (amino acid sequences at positions 213 to 227 in tau proteins: PSLPT-pPPTREPKKVA, as shown in SEQ ID NO.5) and antigen sequences containing amino acids at positions 412 to 434 of tau protein (amino acid sequences at positions 412 to 434 in tau proteins: SSTGSIDMVDSPSPlatLADEVSA, As shown in SEQ ID NO.6), performed on ABI's Peptide Synthesizer (431A) in the United States, using Fmoc (9-fluorenemethoxycarbonyl) protocol, the synthesis steps are carried out in accordance with ABI's Polypeptide Synthesis Operation Manual. Purified by high performance liquid chromatography, sequence identification was prepared by mass spectrometry to obtain a phosphorylated antigen sequence containing tau protein 217-position threonine (T217) and an antigen sequence containing tau protein 412~434 amino acids.

[0037...

Embodiment 2

[0039] Example 2 Preparation of monoclonal antibodies

[0040] 2.1 Animal immunity

[0041] Immunized 4 to 6 weeks old BALB / c mice with 217-bit threonine (T217) phosphorylated antigen sequence immune complex containing tau protein (prepared in Example 1) after mixing emulsification with adjuvant, a total of 5 mice, the immunity amount is 0.3 mg / only / time, the immunization mode is abdominal subcutaneous injection, the specific operation steps are shown in Table 1 below:

[0042] Table 1 BALB / c mouse immune processes

[0043] Number of immunizations Immunization time (d) Immune dose Immune pathway First immunization 1 300 μg / pc (equal amount of complete Freund's adjuvant emulsification) Intradermal injection Second immunization 15 300 μg / only (equal amount of incomplete Freud's adjuvant emulsion) subcutaneous injection Third immunization 29 300 μg / only (equal amount of incomplete Freud's adjuvant emulsion) subcutaneous injection Fourth immuni...

Embodiment 3

[0078] Example 3 Identification of monoclonal antibodies

[0079] 3.1 Sequence determination of monoclonal antibody secreted by Tau-6G12 of hybridoma cell line

[0080] The total RNA of the hybridoma cell line Tau-6G12 was extracted, the cDNA was reversed with a universal primer, and then the light and heavy chains of the antibody were amplified, the light and heavy chains were separated, cloned to the standard cloning vector for expression, the single colony PCR identified the light chain and heavy chain, and 5 to 10 single colonies with the correct length of light and heavy chains were selected for sequencing, of which 5 sequencing results were almost the same, which was considered to be the true sequence of the antibody. (The sequencing process and a large amount of antibody expression were entrusted to Pujian Biological (Wuhan) Technology Co., Ltd.)

[0081]The monoclonal antibody heavy chain coding gene sequence secreted by hybridoma cell line Tau-6G12 is 1380bp long, as show...

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Abstract

The invention discloses a phosphorylated Tau pT217 protein monoclonal antibody, an ELISA (Enzyme-Linked Immunosorbent Assay) kit and application of the phosphorylated Tau pT217 protein monoclonal antibody and the ELISA kit. Animal immunization is carried out by using an antigen sequence immune complex containing phosphorylated threonine (T217) at 217 sites of tau protein and an antigen sequence immune complex containing amino acids at 412-434 sites of the tau protein to prepare the phosphorylated Tau pT217 protein monoclonal antibody and a tau (412-434) protein polyclonal antibody; the monoclonal antibody of the phosphorylated Tau pT217 protein is obtained by secreting a hybridoma cell strain Tau-6G12, and has the advantages of high specificity, high binding force with an antigen and the like; then, a monoclonal antibody of phosphorylated Tau pT217 protein is selectively used as a capture antibody, and a tau (412-434) protein polyclonal antibody is used as a detection antibody, so that the established ELISA kit is higher in sensitivity during detection; besides, the detection range of the kit is 1.17-800 pg / mL, the lower detection limit is 1.17 pg / mL, and the kit has the advantages of high sensitivity and wide detection range.

Description

Technical field [0001] The present invention belongs to the field of biomedical technology, specifically relates to a phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application thereof. Background [0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressively exacerbated intellectual decline and is the most important cause of Alzheimer's disease. The main cause of AD pathogenesis is due to excessive phosphorylation of Tau protein in the brain, resulting in neurofibrillary tangles (NFTs) and a large deposition of β amyloid (β Aβ) in the brain to form age spots, which in turn leads to abnormal neuronal function. [0003] Tau protein is a functional protein in the brain, in the mature normal human brain contains 2 to 3 phosphorylation sites, Tau protein gene is located in chromosome 17, due to the different editing methods of tau protein mRNA can express 6 isomers containing 352 ~ 441 amino acids, it is the highest content of mic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N5/20C12N15/13G01N33/577G01N33/68G01N33/58
CPCC07K16/18G01N33/6896G01N33/577G01N33/581G01N2333/47G01N2800/2821
Inventor 王小川王喆曾宽吴刚
Owner 武汉天德生物科技有限公司
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