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Serum/plasma protein molecular marker related to auxiliary diagnosis of intrahepatic cholestasis in gestation period and application thereof

A molecular marker, cholestasis technology, applied in the fields of genetic engineering and reproductive medicine, to achieve the effect of rapid and accurate diagnosis, convenient and easy diagnosis

Active Publication Date: 2020-06-05
WUXI MATERNAL & CHILD HEALTH HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports of relatively stable biomarkers for ICP-assisted diagnosis at home and abroad. If ICP-specific or abnormally expressed protein molecules can be screened out as biomarkers through proteomics technology, and corresponding auxiliary diagnostic reagents can be developed The box will greatly improve the diagnosis status of ICP in my country

Method used

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  • Serum/plasma protein molecular marker related to auxiliary diagnosis of intrahepatic cholestasis in gestation period and application thereof
  • Serum/plasma protein molecular marker related to auxiliary diagnosis of intrahepatic cholestasis in gestation period and application thereof
  • Serum/plasma protein molecular marker related to auxiliary diagnosis of intrahepatic cholestasis in gestation period and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The collection of embodiment 1 sample and the arrangement of sample data

[0052] The inventor collected a large number of peripheral blood samples of ICP patients and healthy control pregnant women from October 2016 to September 2017 from the Wuxi Maternal and Child Health Hospital affiliated to Nanjing Medical University (the samples used for research were collected during the same period, sampled, sub-packaged, and stored) The conditions are uniform), and by sorting out the sample data, the inventor selected 100 samples that meet the following criteria as experimental samples for DIA protein quantitative detection and subsequent series of enzyme-linked immunosorbent assays and automatic biochemical analyzer verification:

[0053] 1. The above research objects are second trimester Pregnant women who were confirmed to have ICP during ICP screening (refer to the guidelines for diagnosis and treatment of ICP patients (first edition)) were defined as cases.

[0054] 2. ...

Embodiment 2

[0056] Example 2 DIA protein quantitative detection of protein molecular markers in serum / plasma

[0057] The above-mentioned 10 eligible ICP cases and 10 healthy controls were quantitatively detected by DIA protein to obtain relevant results. The specific steps are:

[0058] 1. Sample Preparation

[0059] 1.1 Add an appropriate amount of SDT lysate to the blood sample, transfer it to Lysing Matrix A tube, and use MP homogenizer for homogenization and crushing (24×2, 6.0M / S, 60s, twice).

[0060] 1.2 After ultrasonication, take a boiling water bath for 10 minutes. Centrifuge at 14000 g for 15 min, take the supernatant and filter it with a 0.22 μm centrifuge tube, and collect the filtrate.

[0061] 1.3 BCA method was used for protein quantification. Aliquot the samples and store at -80°C.

[0062] 2. SDS-PAGE electrophoresis

[0063] 20 μg of protein from each sample was added to 6X loading buffer, boiled in water for 5 minutes, subjected to 12% SDS-PAGE electrophoresis (...

Embodiment 3

[0091] Verification experiment of protein molecules in embodiment 3 serum / plasma

[0092] 1. ELISA

[0093] Select 50 cases of ICP patients and 50 cases of healthy controls who meet the conditions of Example 1, detect the expression levels of protein molecular markers S10A9_HUMAN and ITIH3_HUMAN in the serum / plasma samples of the two groups by enzyme-linked immunosorbent assay, analyze and compare the differences, and draw ROC curve. The specific method is:

[0094] 1.1 Dilution of the standard product: This kit provides a standard product at original times, and the user can dilute it in a small test tube according to the following table.

[0095]

[0096] 1.2 Adding samples: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and sample wells to be tested. Accurately add 50 μl of the standard substance on the enzyme-labeled plate, add 40 μl of the sample diluent to the well of t...

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Abstract

The invention discloses a serum / plasma protein molecular marker related to auxiliary diagnosis of intrahepatic cholestasis in a gestation period and an application thereof. The invention relates to aserum / plasma protein molecular marker related to ICP assisted diagnosis. The serum / plasma protein molecular marker is composed of S10A9_HUMAN (human S100 calcium binding protein A9), CHLE_HUMAN (humancholinesterase) and APOA1_HUMAN (human apolipoprotein A1). The inventor separates and studies ICP cases of initial birth and single pregnancy and protein molecular markers in control serum / plasma ofhealthy pregnant women matched with the ICP cases in age; a group of high-specificity and high-sensitivity protein molecular markers highly related to ICP morbidity are searched, and laboratory support is provided for ICP screening and diagnosis treatment.

Description

[0001] field of invention [0002] The invention belongs to the fields of genetic engineering and reproductive medicine, and relates to a serum / plasma protein molecular marker related to the auxiliary diagnosis of intrahepatic cholestasis during pregnancy and its application. Background technique [0003] Intrahepatic cholestasis of pregnancy (ICP) is a common and severe idiopathic liver disease of pregnancy, usually occurs in the second and third trimesters of pregnancy, clinically manifested by elevated total bile acid (TBA) levels , abnormal liver function and skin itching. The main harm of the disease is to affect the prognosis of perinatal infants, leading to premature birth, meconium amniotic fluid staining, fetal distress, and even unexplained fetal death in utero. The latest estimate of the incidence of ICP in my country is 1.2%, among which the incidence in Chongqing, Chengdu and Shanghai is higher, ranging from 3.2% to 6.3%. A large number of epidemiological and ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/573
CPCG01N33/573G01N33/6893G01N2333/4727G01N2333/775G01N2333/918G01N2800/085
Inventor 张婷董蕊锐罗亮陈道桢李娜王晶周涛陈敏健邹少晗陈月娟邹萍王瑶
Owner WUXI MATERNAL & CHILD HEALTH HOSPITAL
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