BIOMARKER COMPOSITION FOR DIAGNOSING PRE-ECLAMPSIA AND USE THEREOF
20230035339 · 2023-02-02
Assignee
- SUNGKWANG MEDICAL FOUNDATION (Seoul, KR)
- UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE UNIVERSITY (Yongin-si, KR)
Inventors
Cpc classification
G01N2800/368
PHYSICS
International classification
Abstract
Provided are: a composition for diagnosing pre-eclampsia; a kit; a method of diagnosing pre-eclampsia using the same; and a method of providing information on diagnosis of pre-eclampsia. The composition, the kit, and the methods provide the effect of enabling the accurate and sensitive diagnosis of pre-eclampsia in a subject.
Claims
1. A composition for diagnosing pre-eclampsia comprising: a formulation for measuring an expression level of at least one protein selected from the group consisting of ANXA3, A2M, APOB, PZP, FETUB, FNI, LCN2, APOM, QSOXI, TGOLN2, FIO, SERPINAII, PRG2, SHBG, TNC, HBD, AGT, CP, HEXB, SERPINA4, and VWF, or a fragment thereof.
2. The composition for diagnosing pre-eclampsia of claim 1, wherein the formulation for measuring a level of the protein or the fragment thereof is an antibody or an antigen-binding fragment thereof that specifically binds to the protein or the fragment thereof.
3. The composition for diagnosing pre-eclampsia of claim 1, wherein the pre-eclampsia is at least one selected from the group consisting of chronic hypertension, gestational hypertension, pre-eclampsia, eclampsia and complex pre-eclampsia.
4. A kit for diagnosing pre-eclampsia comprising the composition of claim 1.
5. The kit for diagnosing pre-eclampsia of claim 4, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.
6. A method of providing information on diagnosis of pre-eclampsia, the method comprising: (a) measuring an expression level of at least one protein selected from the group consisting of ANXA3, A2M, APOB, PZP, FETUB, FNI, LCN2, APOM, QSOXI, TGOLN2, FIO, SERPINAII, PRG2, SHBG, TNC, HBD, AGT, CP, HEXB, SERPINA4, and VWF, or a fragment thereof in a biological sample isolated from a subject; (b) comparing the expression level of the measured protein or the fragment thereof with an expression level of the protein or the fragment thereof in a control group sample.
7. The method of providing information on diagnosis of pre-eclampsia of claim 6, further comprising: determining the subject as pre-eclampsia, when the expression level of the at least one protein selected from the group consisting of ANXA3, A2M, APOB, PZP, FETUB, FNI, LCN2, APOM, QSOXI, TGOLN2, FIO, SERPINAII, PRG2, SHBG, and TNC, or the fragment thereof, measured from the biological sample isolated from the subject in step (b), is increased compared to the control group.
8. The method of providing information on diagnosis of pre-eclampsia of claim 6, further comprising: determining the subject as pre-eclampsia, when the expression level of the at least one protein selected from the group consisting of HBD, AGT, CP, HEXB, SERPINA4, and VWF, or the fragment thereof, measured from the biological sample isolated from the subject in step (b), is decreased compared to the control group.
9. The method of providing information on diagnosis of pre-eclampsia of claim 6, wherein the expression level of the protein is measured by any one selected from the group consisting of western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, fluorescenceactivated cell sorting (FACS) analysis, and protein chip technology assay.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0038]
[0039]
MODE OF DISCLOSURE
[0040] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these examples.
Example 1: Isolation of Protein from Patient's Blood Sample and Hydrolysis into Peptides
[0041] The patient selection criteria are as follows. 25 pregnant women with hypertension (≥140/90 mmHg) accompanied by proteinuria (≥300 mg/day) after 20 weeks of pregnancy, the representative symptom of pre-eclampsia, were selected as the disease group, and 29 pregnant women who delivered normally at the end of the pregnancy without clinically specific symptoms for both the fetus and mother were selected as the normal group.
[0042] The process of collecting a blood sample, a biological sample from the pregnant women, was as follows. The pregnant woman's blood collected in a 10 cc EDTA tube was subjected to a first centrifugation process (8000 rpm, 15 minutes), and then the isolated supernatant (plasma) was again subjected to a second centrifugation process (13,000 rpm, 10 minutes), and finally, plasma to be used in the experiment was obtained.
[0043] Protein isolation and peptide experiments were carried out as follows. 14 high-abundant proteins leading to reduced sensitivity of LC-MS/MS analysis (albumin, [0075] IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein Al, apolipoprotein All, complement C3, transthyretin) were removed by using the IGY Column (P/N SEP030-1KT, Sigma). After removal, only proteins less than 5% of the total blood proteins were analyzed.
[0044] For the multiple reaction monitoring (MRM), an assay method using a mass spectrometer, a process of cutting proteins into peptide units was required, and fragmentation was carried out by using an enzyme (trypsin). 50 μl of 6 M urea buffer (0.1 M DTT, 50 mM ABC) was added to the remaining 50 μg of protein after removing the abundant proteins, and incubated at 37° C. for 1 hour. The 50 μl sample was treated with 5 μl of 0.5 M IAA and reacted in the dark for 30 minutes. After the reaction, the urea concentration was made less than 1 M by using 400 μl of 50 mM ABC, and then 2 μg of trypsin was added and incubated at 37° C. for 12 hours. After incubation, salts were removed by using pierce c-18 spin columns (P/N: 89870, Theromo) to remove salts used in the reaction and to obtain only peptides.
Example 2: Identification of Biomarker by Using Multiple Reaction Monitoring (MRM)
[0045] Verification of biomarker candidates was carried out as follows. The MRM transition of the candidate group was selected for verification of the biomarker candidates. For the selection of MRM transition, SRM Atlas (www.srmatlas.org) with MS-based verification data for all peptides was used. In this regard, the selection criteria are as follows.
[0046] 1. Exclude peptides containing methionine (M) and histidine (H).
[0047] 2. Exclude peptides including a miscleavage
[0048] 3. Exclude peptides having Arginine (R) or Lysine (K) in the sequence before protein (p).
[0049] 4. Peptide length is 5<x<20.
[0050] Using the selected candidate transition, a primary target selection was preceded and the suitability of the transition was identified. The transition identification conditions are as follows.
[0051] 1. Whether 3 types of fragment ions derived from the same precursor ion have the same dissolution time
[0052] 2. Whether to have the same retention time (RT) in the experiments repeated 3 or more times.
[0053] 3. Whether signa to noise (S/N)>10 that meets the limit of quantification (LOQ) condition is satisfied
[0054] The results of an MRM analysis were obtained through the following process. After selecting a transition that satisfied the above conditions, an MSM analysis was performed on the prepared peptide samples. As shown in