Multi-protein composition and application and congenital heart disease pulmonary hypertension screening kit
A technology for congenital heart disease and pulmonary hypertension, applied in the field of molecular biology, can solve the problems of not achieving the expected effect, not having many relationships and in-depth and comprehensive research, and achieve good evaluation efficiency and great application value
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Embodiment 1
[0104] 1. Collection of Human Plasma Samples
[0105] Blood samples were collected from all children with congenital heart disease ventricular septal defect (experimental group and disease control group) and normal control children (healthy control group) in the morning before the operation under the condition of fasting>10h. 2ml of whole blood was collected from each patient's peripheral vein, and immediately centrifuged at 2500rpm for 10 minutes to separate the upper layer of plasma. The separated plasma was then divided into several equal parts, placed in plasma collection tubes, and stored in a -80°C refrigerator for testing.
[0106] 2. Proteomic detection
[0107] 1) Plasma proteome detection
[0108] Using iTRAQ combined with multidimensional liquid chromatography-mass spectrometry (LC-MS / MS) (Applied Biosystems, Foster City, CA) to detect proteins in each mixed sample of the two groups.
[0109] 2) Sample preparation
[0110] a) Purchase a high-abundance protein re...
Embodiment 2
[0169] Enzyme-linked immunosorbent assay (ELISA)
[0170] The adiponectin, alanyl membrane aminopeptidase and dopamine β-hydroxylase screened out in the proteomics results were verified in the plasma samples of the verification group by ELISA method. Experimental principle: The antibody of the protein to be tested is pre-embedded at the bottom of the 96-well plate. After the standard and sample are added, the protein to be tested will bind to the antibody. After removal of unbound substrate, a biotin-conjugated antibody to the protein of interest is added. After washing, anti-biotin conjugated horseradish peroxidase-labeled antibody (HRP) was added, and unbound HRP was removed by washing. After adding a chromogenic reagent to terminate the reaction, measure the absorbance of the liquid.
[0171] Validation of the adiponectin protein assay, including:
[0172] Multi-well plate coated with adiponectin antibody
[0173] Pure adiponectin as a standard
[0174] Biotin-labeled ...
Embodiment 3
[0271] The prediction model of congenital heart disease pulmonary hypertension (taking ventricular septal defect as an example) is established, and the specific operation method is as follows:
[0272] 1) In Example 2, stratify and assign values according to the detected plasma concentrations of adiponectin, alanyl membrane aminopeptidase and dopamine β-hydroxylase:
[0273] a) Adiponectin: <111.10ng / ml assigned value 1, 111.10-140ng / ml assigned value 2, ≥140ng / ml assigned value 3;
[0274] b) Alanyl membrane aminopeptidase: <299.80pg / ml assigns value 1, 299.80-500pg / ml assigns value 2, ≥500pg / ml assigns value 3;
[0275] c) Dopamine β-hydroxylase: <28.80ng / ml assigns 1, 28.80-45ng / ml assigns 2, ≥45ng / ml assigns 3;
[0276] 2) Sum the assignments corresponding to the three proteins and name it as a risk score. Risk score results suggest that pulmonary hypertension is very likely to occur in congenital heart disease with ventricular septal defect of 7-9 points; pulmonary hy...
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