Enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide and application thereof in CTD-PAH (connective tissue diseases-pulmonary arterial hypertension)

An immunodetection method and endothelin receptor technology are applied in the application field of anti-endothelin receptor A antibody enzyme-linked immunosorbent assay and connective tissue disease complicated with pulmonary arterial hypertension, which can solve the problems of high cost and achieve cost reduction and improvement. The effect of practicality

Inactive Publication Date: 2014-04-16
吴庄民
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the deficiencies in the prior art, one of the purposes of the present invention is to provide an anti-ENRA antibody ELISA method based on the endothelin receptor A epitope antigen peptide, so as to overcome the expensive defects in the prior art

Method used

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  • Enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide and application thereof in CTD-PAH (connective tissue diseases-pulmonary arterial hypertension)
  • Enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide and application thereof in CTD-PAH (connective tissue diseases-pulmonary arterial hypertension)
  • Enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide and application thereof in CTD-PAH (connective tissue diseases-pulmonary arterial hypertension)

Examples

Experimental program
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Effect test

Embodiment 1

[0031] The establishment of the anti-endothelin receptor A antibody ELISA method based on the epitope antigen peptide of the present invention:

[0032] 1.15ug / ml of antigen was coated in carbonic acid buffer solution for 96-well plate overnight, and then washed three times with PBS-T.

[0033] 1.23% bovine serum albumin (or 0.3% gelatin, 10% fetal bovine serum) was blocked for 2 hours, and washed three times with PBS-T.

[0034] 1.3 After the patient's serum was diluted 1:100-1:300, it was added to the microplate plate to react at room temperature for 1 hour, and washed three times with PBS-T.

[0035] 1.4 Add horseradish peroxidase-anti-human IgG secondary antibody diluted in an appropriate ratio, and react at room temperature for 1 hour.

[0036] 1.5 After adding TMB (Tetramethyl benzidine) substrate and reacting for 10 minutes, stop the reaction with 2M sulfuric acid.

[0037] 1.6 Read the plate with a microplate reader at an absorbance of 450.

Embodiment 2

[0039] Effect of the anti-ENRA IgG obtained by the present invention on the proliferation of vascular smooth muscle cells

[0040] 2.1 For the SLE-PAH positive serum detected and screened, anti-ENRA IgG was obtained by IgG affinity chromatography

[0041] 1) Take out the EP tube containing the peptide-linked protein magnetic beads of the cell supernatant in the previous step and put it on the EP tube rack;

[0042] 2) Put the protein G magnetic beads containing 200ul cell supernatant on the magnetic stand;

[0043]3) After the magnetic beads are collected on one side of the EP tube, aspirate and discard the supernatant;

[0044] 4) After suction is clean, take out the EP tube and place it on the EP tube plate;

[0045] 5) Add 100ul PBS buffer solution to the above EP tube to wash the immobilized magnetic beads-IgG protein complex 2 times;

[0046] 6) During the third wash, elute in a non-denaturing manner;

[0047] 7) Non-denaturing eluent: add 50ul Tris-glycine (pH=2.5) t...

Embodiment 3

[0059] Kit preparation for detection of anti-DT-56 polypeptide antibody

[0060] Unless otherwise specified, all are conventional methods

[0061] Reagents required for kit preparation

[0062] 1. DT-56 polypeptide, coupled with KLH, coated with 5ug / ml high-affinity microtiter plate;

[0063] 2. The concentration of horseradish peroxidase-labeled goat anti-human IgG working solution is 1:10000, 12mL / bottle, 1 bottle. Contains 1% calf serum, 0.1% thimerosal.

[0064] 3. Chromogenic solution is tetramethylethylenediamine (TMB) solution, 10mL / bottle.

[0065] 4. The washing solution is 10XPBS-T containing 0.1% sodium azide (NaN3), 100mL.

[0066] 5. The stop solution is 2M sulfuric acid, 10mL / bottle.

[0067] 6. Coating buffer (PH9.6 0.05M carbonate buffer), consisting of 1.59 grams of NaHCO3, 2.93 grams of NaHCO3 plus distilled water to 1000ml;

[0068] 7. The blocking solution was 0.5% bovine serum albumin in PBS.

[0069] Microplate preparation

[0070] 1. DT-5...

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Abstract

The invention relates to an enzyme-linked immunosorbent assay (ELISA) based on anti-ENRA (anti-endothelin receptor A) antibody of epitope antigen peptide; the enzyme-linked immunosorbent assay (ELISA) can be used in clinical tests of CTD-PAH (connective tissue diseases-pulmonary arterial hypertension); four extracellular peptide fragments with different lengths are synthesized in vitro, an epitope peptide fragment in good consistency with full-length ENRA is screened, the epitope peptide fragment is artificially synthesized to use as an antigen peptide package board to establish the enzyme-linked immunosorbent assay (ELISA) based on the anti-ENRA (anti-endothelin receptor A) antibody of the epitope antigen peptide; the epitope antigen peptide is a peptide fragment comprising the following amino acid sequence: DNPERYSTNLSNHVDDFTTFRGTELSFLVTTHQPTNLVLPSNGSMHNYCPQQTKIT; the enzyme-linked immunosorbent assay reduces the cost using full-length eukaryotic-expression endothelin receptor as a substrate, improves the clinical detection practicality, becomes a biomarker of CTD-PAH (especially SLE(systemic lupus erythematosus)-PAH), and provides valuable information for clinical diagnosis and treatment decisions.

Description

technical field [0001] The invention belongs to the field of clinical autoantibody detection, and in particular relates to an ELISA detection method for an anti-endothelin receptor A antibody based on an epitope antigen peptide and its application in connective tissue disease complicated with pulmonary hypertension. Background technique [0002] Pulmonary arterial hypertension (PAH) is a pulmonary circulation disease caused by different reasons, which eventually causes irreversible pulmonary vascular remodeling and right heart failure. The median survival time of the natural course is only 2-3 years. PAH caused by connective tissue disease (CTD) is an important part of the spectrum of pulmonary hypertension. According to foreign reports, CTD-PAH accounts for 10% of all PAH, while domestic data shows that the figure is close to 20%. Western data show that systematic Sclerosis (SSc) is absolutely dominant in CTD-PAH, while systemic lupus erythematosus (SLE) is the primary cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6872G01N2800/10G01N2800/12
Inventor 陈晓翔叶霜吴庄民张瑱
Owner 吴庄民
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