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Human AD7C-NTP epitope polypeptide, human AD7C-NTP antibody and human AD7C-NTP in-vitro diagnosis reagent kit with human AD7C-NTP antibody

An antigen epitope, in vitro diagnostic technology, applied in the biological field

Active Publication Date: 2015-11-25
ZHEJIANG JUKANG BIOENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] AD7c-NTP expression was detectable with AD7c-NTP probes in neurons of normal adult temporal lobe and frontal cortex, and low-level AD7c-NTP transcription was also detected in glial cells of cerebral cortex and white matter products, but not in lung, pancreas, kidney, spleen, gastrointestinal tract, liver, fallopian tubes, uterus, ovary, skeletal muscle, thyroid, testis, and thymus

Method used

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  • Human AD7C-NTP epitope polypeptide, human AD7C-NTP antibody and human AD7C-NTP in-vitro diagnosis reagent kit with human AD7C-NTP antibody
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Screening and synthesis of antigenic epitopes in human AD7C-NTP protein

[0028] (1) Selection of the polypeptide sequence: the recognition epitope was screened from the human AD7c-NTP protein sequence (as described in SEQ ID NO: 3) obtained from Genebank (AAC08737.1) by computer software. The main basis for epitope recognition is: hydrophilicity, surface degree, flexibility of protein or polypeptide amino acid sequence, Chou-Fasman's helix, sheet and turn, Robson-Garnier's secondary structure and C- and N-terminus Predicted antigen comprehensive data (AntigenicIndex, AI). According to human AD7c-NTP protein sequence, utilize network resources (such as: http: / / www.cbs.dtu.dk / services / BepiPred / ) and biological software, such as DNAStar (Madison, Wisconsin, USA), to predict its antigenic region (AI).

[0029] After analysis, the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 was selected as the human AD7C-NTP epitope.

[0030] The above-menti...

Embodiment 2

[0031] Example 2: Preparation of human AD7C-NTP monoclonal antibody

[0032] Any human AD7C-NTP antigen epitope polypeptide prepared above is cross-linked to hemocyanin (KLH) by BDB (Bis-diazotizedbenzidinedichloride) as an antigen to prepare an antibody. The specific process is as follows:

[0033] (1) Intraperitoneal injection of immunized mice

[0034] BALB / c female mice aged 6-8 weeks were selected, and six mice were planned to be injected. For the first intraperitoneal injection, the AD7c-NTP antigen polypeptide and Freund's complete adjuvant (CFA) were mixed according to the volume ratio of 1:1, and the injection dose was 100ug / head. The second immunization was carried out 14 days later, and the AD7c-NTP antigen and incomplete Freund's adjuvant (IFA) were mixed according to the volume ratio of 1:1, and the injection dose was 100ug / mouse. The third immunization was carried out 14 days after the second immunization, and the AD7c-NTP antigen and Freund's incomplete adjuva...

Embodiment 3

[0042] Embodiment 3: human AD7C-NTP in vitro diagnostic kit

[0043] (1) Antibody pair screening

[0044] Screening antibody pairs: choose a cell line, biotin or HRP labeled antibody secreted by the cell line as a detection antibody, detect human AD7c-NTP antigen by ELISA, and analyze whether antibodies secreted by other cell lines can be used as capture antibodies. If the result is unsatisfactory, biotin or HRP-labeled second antibody is used for the next round of screening until the ideal antibody pair is screened.

[0045] Antibody class and subclass identification: identify the heavy chain and light chain type of monoclonal antibody immunoglobulin by ELISA, and analyze the physicochemical properties of monoclonal antibody, that is, the sensitivity of monoclonal antibody to temperature and pH changes and the affinity of monoclonal antibody

[0046] Monoclonal antibody preparation and purification: After screening the ideal antibody pair, the antibody was produced by intrap...

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Abstract

The invention relates to a human AD7C-NTP epitope polypeptide. The amino acid sequence of the human AD7C-NTP epitope polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2. The invention further relates to a human AD7C-NTP antibody specifically bound with the human AD7C-NTP epitope polypeptide and a human AD7C-NTP in-vitro diagnosis reagent kit with the human AD7C-NTP antibody. The prepared human AD7C-NTP epitope polypeptide has excellent antigenicity; the antibody produced by an antigen immune animal prepared from the human AD7C-NTP epitope polypeptide can be highly specifically bound with AD7C-NTP in a urine sample; the prepared human AD7C-NTP in-vitro diagnosis reagent kit can effectively detect the AD7C-NTP level in urine and has great significance in early diagnosis and clinic treatment of a patient with the Alzheimer disease.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human AD7C-NTP antigen epitope polypeptide, an antibody and an in vitro diagnostic kit. Background technique [0002] Alzheimer's disease (Alzheimer's disease, AD), also known as senile dementia, is a primary chronic degenerative disease of the central nervous system, characterized by neuronal cell loss, diffuse cerebral cortical atrophy, extraneuron senile plaques, deposition , Neurofibrillary tangle feet, formation, and synaptic degeneration in neurons are a common disease in the elderly. The clinical syndrome of AD is the late stage of its long course of disease. Various pathogenic factors and protective factors play their respective roles in different periods, and eventually lead to neuropathological changes and corresponding clinical manifestations, such as memory impairment, aphasia, apraxia, etc. , agnosia, impairment of visuospatial skills, executive dysfunction, and perso...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K14/46C07K16/18G01N33/577
Inventor 杨永芳张守涛郭亚楠陶新博
Owner ZHEJIANG JUKANG BIOENG CO LTD
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