KR102554138B1 - Use of Omega-3 metabolites as a biomarker for determining persistancy of food allergy - Google Patents

Abstract

The present invention relates to the use of an omega-3 metabolite as a marker for determining the persistence of food allergy, and more particularly, the use of an omega-3 metabolite as a marker for determining the persistence of future food allergy by quantifying the level at the time of diagnosis. It is about. According to the present invention, it is possible to evaluate the persistence of food allergy from the difference in metabolism of omega-3 metabolites at the time of diagnosis, providing accurate and objective information on whether or not food allergy persists to patients suffering from food allergy. However, it has the advantage of being able to establish an appropriate clinical treatment plan based on such information.

Description

Use of Omega-3 metabolites as a biomarker for determining persistancy of food allergy}

The present invention relates to the use of an omega-3 metabolite as a marker for determining the persistence of food allergy, and more particularly, the use of an omega-3 metabolite as a marker for determining the persistence of future food allergy by quantifying the level at the time of diagnosis. It is about.

The persistence of childhood-onset food allergy (FA) varies considerably from patient to patient. Some children overcome symptoms at an early age, while others persist with food allergy for life. Although extensive research has been conducted on food allergy, strict dietary avoidance is still the dominant method for coping with food allergy, placing a significant burden on people with persistent symptoms. The exact mechanism for treating food allergy has not yet been fully elucidated, and therefore, methods for accurately predicting and treating food allergy are still limited. A number of factors affect food allergy treatment, including the type and number of causative allergens and the severity of symptoms. Several attempts have been made to predict food allergy treatment through the number of food allergies and allergen-specific IgE levels (Foong RX et al., Pediatr Allergy Immunol 2021; 32:223-33), but their accuracy is controversial. Therefore, research on the mechanisms underlying food allergy is essential for understanding and better predicting the disease.

Recently, omics research, which has been in the limelight, has shown great potential in revealing the correlation between genomes and human diseases. In particular, metabolomics provides great insight because it reflects both the genetic basis and environmental changes that contribute to disease. Several metabolomic approaches to food allergy allow the identification of metabolites in a variety of biological samples in relation to disease state and severity. For example, previous studies have identified differences in baseline metabolite levels between patients with and without peanut allergy. Another study reported lower sphingolipid and lysophospholipid levels in children with food allergy than in healthy controls. However, no metabolomic studies have yet provided guidance for the treatment of food allergy.

The present inventors carried out metabolomic profiling in serum samples of children with food allergy and healthy control children to identify metabolites related to the onset and treatment of food allergy, and quantified effector molecules in the metabolic pathway to determine the level of metabolites. The present invention was completed by confirming that the persistence of food allergy in children can be evaluated from the difference.

Foong RX et al., Pediatr Allergy Immunol 2021; 32:223-33

The present invention provides the use of an omega-3 metabolite as a novel marker capable of determining the persistence of food allergy.

In order to achieve the above object, the present invention provides a composition for determining the persistence of food allergy, including an agent for measuring the level of omega-3 metabolites.

In the present invention, the omega-3 metabolite is one or more selected from the group consisting of 7-HDoHE, 10-HDoHE, 13-HDoHE, 17-HDoHE, Resolvin D1 (RvD1) and Resolvin D2 (RvD2) can be characterized.

In the present invention, the agent may be characterized in that it is an antibody or an antigen-binding fragment thereof, or an aptamer that specifically binds to an omega-3 metabolite.

In the present invention, the preparation may be characterized in that it is a preparation for chromatography and/or mass spectrometry for measuring the level of the omega-3 metabolite.

The present invention also provides a kit for determining the persistence of food allergy, including the composition.

The present invention also provides a method for providing information for predicting whether food allergy persists, comprising measuring the level of omega-3 metabolites in a sample isolated from a subject having food allergy.

In the present invention, the omega-3 metabolite is one or more selected from the group consisting of 7-HDoHE, 10-HDoHE, 13-HDoHE, 17-HDoHE, Resolvin D1 (RvD1) and Resolvin D2 (RvD2) can be characterized.

In the present invention, the level of resolvin D1 and / or resolvin D2 is measured by immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, protein chip, immunoprecipitation, mass spectrometry , chromatography, or a combination thereof.

In the present invention, the sample is blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary tract fluid, breast milk, lymphatic body fluid, semen, It may be characterized as cerebrospinal fluid, intraorgan system fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof.

In the present invention, when the resolvin D1 is 280 pg/ml or more, the subject may be characterized as predicting that the allergy will persist.

In the present invention, when the resolvin D2 is 134 pg/ml or more, the subject may be characterized as predicting that the allergy will persist.

According to the present invention, it is possible to evaluate the persistence of food allergy from the difference in metabolism of omega-3 metabolites at the time of diagnosis, providing accurate and objective information on whether or not food allergy persists to patients suffering from food allergy. However, it has the advantage of being able to establish an appropriate clinical treatment plan based on such information.

1 shows the sPLS-DA of subjects in an identification cohort based on food allergy and resolution of food allergy. Figure 1a shows a score plot of the sPLS-DA model for a healthy control group (red) and a subject with food allergy (green), and Figure 1b shows a subject with continued food allergy (red) and a subject with resolved food allergy (green). ), Figure 1c shows a score plot of the sPLS-DA model for healthy controls (blue), subjects with continued food allergy (green), and subjects with resolved food allergy (red) indicates Plots were drawn with central scale using MetaboAnalyst 5.0, sPLS-DA for sparse partial least squares discriminant analysis and FA for food allergy.
Figure 2 shows metabolomes associated with food allergy and resolution of food allergy identified in the identification cohort. Figure 2a shows the levels of metabolites with statistically significant differences between subjects with food allergy and controls. Figure 2b shows the levels of metabolites with statistically significant differences between subjects whose food allergy was resolved and subjects whose food allergy persisted. Metabolites were grouped according to biochemical class, and only biochemical classes containing two or more metabolites significantly associated with phenotypes per class or those previously associated with allergic disease were indicated. FA, food allergy; AC, acylcarnitine; LPA, lysophosphatidic acid; HDoHE, hydroxydocosahexaenoic acid; PAF, platelet activating factor; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine.
Figure 3 shows the ROC curve of the random forest model for food allergy and food allergy resolution. A random forest model was built with metabolomes highly associated with (a) food allergy (16 traits) and (b) food allergy resolution (13 traits). CI, confidence interval.
Figure 4 shows the trajectory of omega-3 metabolite levels from diagnosis to follow-up in an identification cohort of subjects. Figure 4a shows the change in 7-HDoHE, 10-HDoHE, 13-HDoHE, 17-HDoHE levels between diagnosis and follow-up between food allergy resolution and food allergy persistence subjects, and Figure 4b shows the standard calculated in the linear mixed model analysis. Average profile plots are shown for each omega-3 metabolite with error bars showing error and p-value. Blue color represents food allergy resolution, and red color represents food allergy continuation. HDoHE, hydroxydocosahexaenoic acid; FA, food allergy
5 is a result of confirming the level of resolvin in the subjects of the quantitative cohort. Figure 5a shows the level of RvD1 in subjects with food allergy and a healthy control group, Figure 5b shows the level of RvD1 in subjects with food allergy resolved and subjects with persistent food allergy, Figure 5c shows the level of food allergy in subjects and The level of RvD2 in a healthy control group, FIG. 5d shows the level of RvD2 in a subject whose food allergy has resolved and a subject whose food allergy persists. Resolvin levels were quantified using ELISA and natural log transformed for box plots. *t-test or ANOVA p-value <0.05; FA, food allergy; RvD1, Resolvin D1; RvD2, Resolvin D2
Figure 6 shows the correlation between total serum IgE and resolvin levels in the quantification cohort. Scatter plots were drawn with (Fig. 5a) RvD1 and (Fig. 5b) RvD2 natural logarithm values. R values and p-values were calculated using Pearson's correlation analysis or Spearman's correlation analysis according to data distribution. The shaded area represents the 95% confidence interval. IgE, immunoglobulin E; RvD1, Resolvin D1; RvD2, resolvin D2.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, metabolites showing quantitative differences at the time of diagnosis in children diagnosed with food allergy were identified, and these children were followed up to confirm whether these metabolites showed a significant correlation with the resolution or persistence of food allergy. As a result, it was confirmed that there was a significant difference in the levels of omega-3 metabolites in serum metabolomic analysis at the time of diagnosis between subjects whose childhood food allergy was resolved and subjects whose childhood food allergy persisted. Specifically, it was confirmed that the level of HDoHE was relatively low and the level of resolvin was relatively high in subjects whose childhood food allergy was resolved. It was found that it can act as a biomarker for

Therefore, in one aspect, the present invention relates to a composition for determining the persistence of food allergy, including an agent for measuring the level of omega-3 metabolites.

In the present invention, the omega-3 metabolite is characterized in that at least one selected from the group consisting of 7-HDoHE, 10-HDoHE, 13-HDoHE, 17-HDoHE, Resolvin D1 (RvD1) and Resolvin D2 (RvD2) can be done with

In the present invention, 7-HDoHE is a compound of PubChem CID: 16061142, specifically (4Z,8E,10Z,13Z,16Z,19Z)-7-hydroxydocosa-4,8,10,13,16,19 -hexaenoic acid ((4Z,8E,10Z,13Z,16Z,19Z)-7-hydroxydocosa-4,8,10,13,16,19-hexaenoic acid), represented by the following formula (1) that can be characterized.

Formula (1)

In the present invention, 10-HDoHE is a compound of PubChem CID: 11537494, specifically, (4Z,7Z,11E,13Z,16Z,19Z)-10-hydroxydocosa-4,7,11,13,16, It may be 19-hexaenoic acid ((4Z,7Z,11E,13Z,16Z,19Z)-10-hydroxydocosa-4,7,11,13,16,19-hexaenoic acid), represented by the following formula (2) can be characterized as being

Formula (2)

In the present invention, 13-HDoHE is a compound of PubChem CID: 11559259, specifically (4Z,7Z,10Z,14E,16Z,19Z)-13-hydroxydocosa-4,7,10,14,16,19 -hexaenoic acid ((4Z,7Z,10Z,14E,16Z,19Z)-13-hydroxydocosa-4,7,10,14,16,19-hexaenoic acid), represented by the following formula (3) that can be characterized.

Formula (3)

In the present invention, 17-HDoHE is a compound of PubChem CID: 6439179, specifically, (4Z,7Z,10Z,13Z,15E,19Z)-17-hydroxydocosa-4,7,10,13,15, It may be 19-hexaenoic acid ((4Z,7Z,10Z,13Z,15E,19Z)-17-hydroxydocosa-4,7,10,13,15,19-hexaenoic acid), represented by the following formula (4) can be characterized as being

Formula (4)

In the present invention, Resolvin D1 is a compound of PubChem CID: 44251266, specifically (4Z,7S,8R,9E,11E,13Z,15E,17S,19Z)-7,8,17-trihydroxydocosa- 4,9,11,13,15,19-hexaenoate ((4Z,7S,8R,9E,11E,13Z,15E,17S,19Z)-7,8,17-trihydroxydocosa-4,9,11 ,13,15,19-hexaenoate), and may be characterized by being represented by the following formula (5).

Formula (5)

In the present invention, Resolvin D2 is a compound of PubChem CID: 11383310, specifically (4Z,7S,8E,10Z,12E,14E,16R,17S,19Z)-7,16,17-trihydroxydocosa- 4,8,10,12,14,19-hexaenoic acid ((4Z,7S,8E,10Z,12E,14E,16R,17S,19Z)-7,16,17-trihydroxydocosa-4,8,10, 12,14,19-hexaenoic acid), and may be characterized by being represented by the following formula (6).

formula (6)

In the present invention, the agent may be an antibody that specifically binds to an omega-3 metabolite, an antigen-binding fragment thereof, or an aptamer, but is not limited thereto.

In another aspect, the preparation may be characterized in that it is a competitive enzyme-linked immuno-sorbent assay preparation, but is not limited thereto.

In the present invention, the preparation is coated with an antibody on a well plate and a competitive inhibition reaction between a biotin-labeled omega-3 metabolite and a biotin-unlabeled omega-3 metabolite is performed to omega-3 in the test sample. 3 can be quantified.

In the present invention, an antigen-binding fragment or aptamer may be used instead of the antibody.

In another aspect, the agent may be an antibody or an antigen-binding fragment thereof that specifically binds to omega-3, or an aptamer, but is not limited thereto.

In the present invention, the preparation may be characterized in that it is a preparation for chromatography and/or mass spectrometry for measuring the level of the omega-3 metabolite, but is not limited thereto.

On the other hand, in the present invention, four SNPs significantly associated with metabolite levels were identified in the subjects of the identification cohort for the four omega-3 metabolites.

Therefore, the present invention from another point of view p21 (RAC1) activated kinase 5 (p21 (RAC1) activated kinase 5, PAK5 ) SNP (rs7274775); SNP (rs6957108) of distal-less homeobox 6 antisense RNA 1 (DLX6-AS1 ); SNP (rs235907 ) of proline-rich coiled-coil 2C ( PRRC2C ); And a SNP (rs8039336) of gamma-aminobutyric acid type A receptor subunit beta3 ( GABRB3 ); It relates to a composition for determining the persistence of allergy.

In the present invention, the Gene ID of the gene encoding the PAK5 protein is 57144. , XP_016883450.1, XM_017027962.1, XP_016883451.1, XM_017027964.1, XP_016883453.1, XM_017027965.1, XP_016883454.1, XM_017027963.1, XP_01 GeneBank Accession No. 6883452.1 etc. Alternatively, it may be a protein identifiable by a nucleotide or amino acid sequence according to Uniprot Accession No.

In the present invention, the Gene ID of the gene encoding the DLX6-AS1 protein is 285987, and may be a protein identifiable by a nucleotide sequence according to GeneBank Accession No. such as NR_015448.1, for example.

In the present invention, the Gene ID of the gene encoding the PRRC2C protein is 23215, for example, NM_001387844.1, NP_001374773.1, NM_015172.4, NP_055987.2, XM_005245020.2, XP_005245077.1, XM_00524 5015.2 , XP_005245072.1, XM_005245025.2, XP_005245082.1, XM_005245023.2, XP_005245080.1, XM_005245028.2, XP_005245085.1, XM_005245027.2, XP_00 5245084.1, XM_017000761.1, XP_016856250.1, XM_005245026.2, XP_005245083 .1, XM_005245024.2, XP_005245081.1, XM_005245029.2, XP_005245086.1, XM_005245019.2, XP_005245076.1, XM_017000762.1, XP_016856251.1, XM _005245018.3, XP_005245075.1, XM_005245016.5, XP_005245073.1 , XM_006711238.4, XP_006711301.1, XM_017000760.2, XP_016856249.1, etc. GeneBank Accession No. Alternatively, it may be a protein identifiable by a nucleotide or amino acid sequence according to Uniprot Accession No.

In the present invention, the Gene ID of the gene encoding the GABRB3 protein is 2562. 8631.2 , NP_001265560.1, NM_021912.5, NP_068712.1, XM_011521428.3, XP_011519730.1, etc. GeneBank Accession No. Alternatively, it may be a protein identifiable by a nucleotide or amino acid sequence according to Uniprot Accession No.

In this specification, the term "marker" is a substance that can distinguish whether or not food allergy persists, and since it shows a difference in whether food allergy persists or resolves food allergy in the future depending on the quantitative difference at the time of diagnosis in food allergy patients, food allergy continues or disappears in the future. It refers to a substance that can determine whether or not it resolves, and can judge or evaluate the persistence of food allergy by evaluating its quantitative level.

In the present invention, prediction means to judge or evaluate in advance whether a food allergy will persist or be resolved, and includes both the meaning of determining a prognosis or diagnosing a treatment effect.

Preferably, the present invention can predict in advance whether or not the child's food allergy persists through the diagnosis of the pediatric food allergy patient using the biomarker according to the present invention.

In the present invention, children may mean 3 months or more and less than 18 years of age, preferably 6 months or more and less than 12 years of age, but are not limited thereto. Therefore, the present invention may be characterized by diagnosing children with food allergies aged 3 months or more and younger than 18 years of age to predict whether food allergy persists, but this is because food allergies mainly occur in childhood, and also in adults The present invention may be applied to determine whether or not food allergy persists.

In the present invention, the marker may be characterized in predicting the sustainability of food allergy when 1 year or more elapses from the time of diagnosis, but is not limited thereto, and the biomarker is 1 year to 10 years after diagnosis, It can be characterized in predicting the sustainability of food allergy, preferably at 1 year to 5 years, and in some embodiments, the biomarker is 12 months, 13 months, 14 months, 15 months, 16 months, 17 months from diagnosis. months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, It can be characterized by predicting the likelihood of food allergy persistence at 34, 35, or 36 months.

In the present invention, the omega-3 metabolite may be bound to an immunogenic fragment. That is, the omega-3 metabolite may be bound to a fragment having at least one epitope that can be recognized by an antibody.

As used herein, the term "antibody" is a term known in the art and may refer to a specific protein molecule directed against an antigenic site. The type of antibody according to one aspect is not particularly limited, and polyclonal antibodies, monoclonal antibodies, or any immunoglobulin antibody may be included as long as it has antigen binding properties, and furthermore, special antibodies such as humanized antibodies may be included. Antibodies may also be included. Antibodies according to one embodiment may include functional fragments of the antibody molecule as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be, for example, Fab, F(ab'), F(ab')2, Fv, scFv, and the like.

In this specification, chromatography is gas chromatography (Gas Chromatography), liquid-solid chromatography (Liquid-Solid Chromatography, LSC), paper chromatography (Paper Chromatography, PC), thin-layer chromatography (Thin-Layer Chromatography, TLC) , Gas-Solid Chromatography (GSC), Liquid-Liquid Chromatography (LLC), Foam Chromatography (FC), Emulsion Chromatography (EC) , including Gas-Liquid Chromatography (GLC), Ion Chromatography (IC), Gel Filtration Chromatography (GFC) or Gel Permeation Chromatography (GPC) However, it is not limited thereto, and all quantitative chromatography commonly used in the art may be used. Preferably, the chromatography used in the present invention is gas chromatography. Preferably, the mass spectrometer used in the present invention is MALDI-TOF MS or TOF MS, more preferably TOF MS.

As used herein, the term "SNP (single nucleotide polymorphism)" refers to a single nucleotide polymorphism, which is a common mutation that appears in one of several DNA bases in a single site of a chromosome. SNPs are frequent, stable, and distributed throughout the genome. and thereby individual genetic diversity occurs. In the present application, the "marker" may be named in combination with "".

As used herein, the term "allele" or "allele" refers to several types of a gene present at the same locus on a homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two or more types, for example, two types of alleles.

As used throughout this specification, the term "agent for determining a SNP" refers to an agent that can specifically bind to and recognize an SNP marker or SNP included in the SNP marker composition, or can detect and amplify the SNP. And, “agent capable of detecting and amplifying” means an agent capable of increasing the number by repeating the replication of the SNP marker or SNP included in the SNP marker composition, for example, a polyol containing the SNP It may mean a primer capable of specifically amplifying a nucleotide or a probe capable of specifically binding, but is not limited thereto.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3' hydroxyl group, capable of base pairing with a complementary template, and serving as a starting point for template strand copying. refers to the sequence of nucleic acids. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. In addition, primers are sense and antisense nucleic acids with a sequence of 7 to 50 nucleotides, which can incorporate additional features that do not change the basic properties of the primers that serve as starting points for DNA synthesis. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, as long as it is sufficiently complementary to allow hybridization with the template. The position of the primer or the binding site of the primer may mean a target DNA fragment to which the primer hybridizes. According to one embodiment of the present invention, PCR amplification is performed using sense and antisense primers of mRNA of the gene, and diagnosis can be made by measuring the production amount of a desired product. PCR conditions and lengths of sense and antisense primers can be appropriately selected according to techniques known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundred bases in length that can specifically bind to mRNA and is labeled, so that the presence or absence of a specific mRNA, expression quantity can be checked.

The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. By performing hybridization using a probe complementary to the polynucleotide of the gene according to one aspect, the presence and degree of radiation exposure can be determined through the presence and degree of hybridization. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.

The primers or probes can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologues of the native nucleotide, and internucleotide modifications, e.g., uncharged uncharged compounds such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, and the like. There is a transformation into a linker or a charged linker such as phosphorothioate, phosphorodithioate, and the like.

In another aspect, the present invention relates to a kit for determining the persistence of food allergy, including the composition.

In the present invention, the kit may be a competitive enzyme-linked immuno-sorbent assay kit in one aspect, and a quantitative sandwich ELISA kit in another aspect. .

The kit may be by RvD1 antibody-RvD1 antigen or RvD2 antibody-RvD2 antigen reaction.

In the present invention, the kit may include an antibody, a substrate for immunological detection of the antibody, a suitable buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, or a chromogenic substrate. The kit may include an aptamer, primer, probe or oligonucleotide.

For example, in the present invention, the kit for competitive enzyme-linked immuno-sorbent assay is in the group consisting of an antibody-coated well plate, a biotin-labeled omega-3 metabolite, an HRP-conjugated reagent, and a TMB substrate. It may include one or more selected, but is not limited thereto.

In another aspect, the kit comprises p21 (RAC1) activated kinase 5 ( PAK5 ) SNP (rs7274775), distal-less homeobox 6 antisense RNA 1 , DLX6-AS1 ) SNP (rs6957108 ), proline-rich coiled-coil 2C (proline-rich coiled-coil 2C, PRRC2C ) SNP (rs235907 ), and gamma-aminobutyric acid type A receptor subunit beta3 (gamma-aminobutyric acid type A receptor subunit beta3, GABRB3 SNP (rs8039336); may be any one or more SNP analysis kits selected from the group consisting of.

In the present invention, the kit may include not only the SNP, polynucleotide, cDNA, etc. of the present application, but also other component compositions, solutions or devices suitable for the analysis method.

In the present invention, the kit may be a PCR kit, RT-PCR kit or DNA chip kit, but is not limited thereto.

The kit may further include a lysis buffer. The buffer solution may include various materials such as commercially available products. The buffer may include HEPES, MgCl ₂ , NaCl, imidazole, DNAse, and β, and a protease inhibitor PMSF (phenylmethanesulfonylfluoride) may be added to prevent protein degradation, but is not limited thereto.

The kit may further include a reagent for digesting genes or proteins. The gene degradation reagent may include a restriction enzyme or the like. The proteolytic reagent may include trypsin, trypsin/EDTA, or TrypLE, but the gene or proteolytic reagent is not limited thereto.

The kit may further include a mixture of isotope labeled standard peptides.

The kit may further include a program capable of data analysis or statistical processing. The program may analyze the expression level of a gene or protein. The data analysis or statistical processing program may be one or more selected from Skyline Software, ProteoWizard Software, SCIEX OS Software, SPSS Statistics Software, MedCalc Software, MultiQuant Software, MasterView Software, or Cliquid Software, but is not limited thereto.

The kit may further include a sample required for diagnosis. The kit may include an antibody, a substrate for immunological detection of the antibody, a suitable buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, or a chromogenic substrate. The kit may include an aptamer, primer, probe or oligonucleotide.

The kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.

As one aspect of the present invention, the PCR kit may be a kit including essential elements required to perform PCR. The PCR kit contains, in addition to polynucleotides, primers or probes specific for the SNP, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase enzymes such as, DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.

As one aspect of the present invention, the DNA chip kit may be a kit including essential elements required to perform DNA chip, and the DNA chip kit may include a substrate to which a polynucleotide, primer or probe specific for the SNP is attached. And the substrate may include a nucleic acid corresponding to a quantitative control gene or a fragment thereof.

As one aspect of the present invention, the RT-PCR kit may be a kit including essential elements required to perform RT-PCR, and the RT-PCR kit, in addition to each primer specific for the SNP, a test tube or Other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), dideoxynucleotides (ddNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water ( DEPC-water), sterile water, and the like.

In another aspect, the present invention relates to a method for providing information for predicting the persistence of food allergy, comprising measuring the level of an omega-3 metabolite in a sample isolated from a subject having food allergy.

In the present invention, the omega-3 metabolite is one or more selected from the group consisting of 7-HDoHE, 10-HDoHE, 13-HDoHE, 17-HDoHE, Resolvin D1 (RvD1) and Resolvin D2 (RvD2) It may be characterized, but is not limited thereto.

In the present invention, the level of the omega-3 metabolite is measured by immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, protein chip, immunoprecipitation, mass spectrometry, chromatography or It may be characterized in that it is performed in a combination thereof, but is not limited thereto.

In another aspect, the present invention is a SNP (rs7274775) of p21 (RAC1) activated kinase 5 ( PAK5 ), homeobox 6 antisense RNA 1 without end in a sample isolated from a subject with food allergy SNP (rs6957108) of (distal-less homeobox 6 antisense RNA 1, DLX6-AS1 ) , SNP (rs235907) of proline-rich coiled-coil 2C ( PRRC2C ) , and gamma-aminobutyric acid type A Determining one or more SNPs selected from the group consisting of; It relates to a method for providing information for predicting whether food allergy persists, including the step of doing.

In the present invention, the SNP is sequencing analysis, pyrosequencing, hybridization by microarray, PCR_RELP (restriction fragment length polymorphism) method, PCR-SSCP (single strand conformation polymorphism) method, PCR-SSO (specific sequence oligonucleotide) method, ASO (allele specific oligonucleotide) hybridization method combining PCR-SSO method and dot hybridization method, TaqMan-PCR method, MALDI-TOF/MS method, RCA (rolling circle amplification) method, HRM (high resolution melting) method, primer extension method, Southern blot hybridization method, dot hybridization method, or a combination thereof, but is not limited thereto.

In the present invention, the sample is blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary tract fluid, breast milk, lymphatic body fluid, semen, It may be characterized as cerebrospinal fluid, organ system fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof, but is not limited thereto.

In the present invention, when at least one omega-3 metabolite selected from the group consisting of 7-HDoHE, 10-HDoHE, 13-HDoHE and 17-HDoHE is 0.001 or more, the subject is predicted to be relieved of allergy. can be done with

In the present invention, when the resolvin D1 is 265 pg/ml or more, more preferably 275 pg/ml or more, and most preferably 280 pg/ml or more, the subject predicts that the allergy will last can be done with

For example, when the resolvin D1 is 280.2 pg/ml or higher, the subject may predict that the allergy will persist.

In the present invention, when the resolvin D2 is 126 pg/ml or more, more preferably 130 pg/ml or more, and most preferably 134 pg/ml or more, the subject predicts that the allergy will last can be done with

For example, when the resolvin D2 is 134.3 pg/ml or more, the subject may predict that the allergy will persist.

In the present invention, the level of Resolvin D1 may be measured using a Resolvin D1 (RvD1) ELISA Kit (Cat. No.: MBS053145, Mybiosource, San Diego, CA, USA)), but is not limited thereto. don't

In the present invention, the level of Resolvin D2 may be measured using Resolvin D2 (RvD2), ELISA Kit (Cat. No.: MBS051498, Mybiosource, San Diego, CA, USA)), but is not limited thereto. does not

In the present invention,

Alleles of the SNP (rs7274775) include T;

Alleles of the SNP (rs6957108) include T;

Alleles of the SNP (rs235907) include C; and

The allele of the SNP (rs8039336) includes T; If at least one selected from the group consisting of, the subject may be characterized as predicting that the food allergy will be resolved.

In the information providing method according to the present invention, the results can be statistically processed using statistical analysis methods commonly used in the art, for example, Student's t-test, chi- Continuous variables and absolute variables obtained through chi-square test, linear regression line analysis, multiple logistic regression analysis, meta-analysis, etc. It can be analyzed using variables such as categorical variables, odds ratio (OR), and 95% confidence interval (CI).

As used herein, the term "subject" refers to any organism having a food allergy, and may include mammals, including mice, monkeys, cows, pigs, mini-pigs, livestock, humans, etc. as specific examples, but is limited thereto. it is not going to be

In the present invention, the subject may be a human, specifically Korean or Asian, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Study Population and Sample Collection

1-1. study population

Two independent cohorts, an identification cohort and a quantification cohort, were used to study the metabolic profiles of children with food allergy.

The identification cohort consisted of 20 children with food allergy and 20 healthy controls. Food allergy was diagnosed by a physician based on a combination of identified allergic hypersensitivity reactions, including a specific IgE level >0.7 kU/L to one or more specific food allergens and a history of IgE-mediated symptoms occurring within 2 hours of ingesting the food . Resolution of food allergy was defined as the loss of local and systemic symptoms from any food allergen that had previously caused a food allergy, and resolution was confirmed by attempting to eat the food or observation by a caregiver. Serum was collected at diagnosis and at follow-up for children with food allergy. Follow-up over the same time was conducted for subjects whose food allergy was resolved and subjects whose food allergy persisted. Persistence was confirmed in the clinic using the same criteria as for food allergy diagnosis. Samples were stored at -20 °C until metabolomic profiling was performed. The severity of food allergy and atopic dermatitis for subjects with persistent food allergy and subjects with resolved food allergy were evaluated by B. Niggemann et al (Allergy 2016; 71:135-6) and SCORing Atopic Dermatitis (SCORAD) (Dermatology 1993; 186:23-31). The control group was selected as a subject without a history of allergic disease or symptoms of allergic hypersensitivity. Additional whole blood samples were collected from these 40 subjects for genotyping.

The quantification cohort consisted of 48 subjects with food allergy and 30 healthy controls. Subjects (i.e., subjects) were recruited with the same inclusion criteria as the identification cohort, and serum samples were used to quantify selected metabolites. All subjects were unrelated, and the experiment was conducted after providing written informed consent. All studies were conducted with the approval of the Institutional Review Board of Severance Hospital (Seoul, Korea, IRB No. 4-2019-1271).

1-2. clinical evaluation

Food allergy (FA) symptoms identified for diagnosis of food allergy include tingling or itching in the mouth; hives; swelling of the face, mouth, or other body parts; appointment; shortness of breath; sickness; throw up; colic; Diarrhea was included. Some subjects with food allergies had allergies to multiple foods, and all subjects had egg allergies. Allergies to milk and nuts were most common in subjects with multiple allergies. Subjects with a history of non-IgE mediated or chronic inflammatory gastrointestinal disorders and the use of immunosuppressants within the past 6 weeks were excluded from subjects.

1-3. Sample preparation and subject analysis

20 paired serum samples from subjects with food allergy and 20 unpaired serum samples from controls were collected. Information on identification cohorts is summarized in Table 1.

Food allergy (n = 20) control group (n = 20) p-value** age at diagnosis 2.74 ± 2.30 2.70 ± 2.28 0.95 male 11 (55) 10 (50) 0.75 Total serum IgE, kU/L 676.49 ± 1059.48 17.72 ± 14.73 0.01 Food Allergy Relief* 8 (44.4) - -

Data are given as n (%) or mean ± standard deviation

* Rate calculated for a total of 18 people with food allergy, excluding 2 people with insufficient information on food allergy relief

** p-value calculated by chi-square test for categorical values and Welch's t-test for continuous values

The average age of the subjects was 2.72 years (standard deviation: 2.29), and there was no significant age difference between subjects with food allergies and controls. Total serum IgE levels were higher on average in subjects with food allergy than in controls, as expected. At the time of diagnosis, food allergy patients were on a diet avoiding the causative allergen (Table 2).

Patient No. food allergy group food allergy One food allergy persists eggs, milk, peanuts 2 food allergy persists egg 3 food allergy persists eggs, milk 4 food allergy persists Eggs, Milk, Peanuts, Pork, Wheat 5 food allergy persists egg 6 food allergy persists eggs, wheat 7 food allergy persists eggs, wheat 8 food allergy persists eggs, milk 9 food allergy persists eggs, peanuts 10 food allergy persists egg 11 food allergy relief eggs, peanuts 12 food allergy relief eggs, milk 13 food allergy relief egg 14 food allergy relief egg 15 food allergy relief eggs, milk 16 food allergy relief eggs, milk 17 food allergy relief eggs, milk 18 food allergy relief egg

Ten of the food allergy patients showed persistent symptoms, and food allergy resolved in eight. Two subjects were excluded from the analysis of food allergy resolution due to insufficient prognostic information. The follow-up period and disease attributes for these subjects are summarized in Table 3.

food allergy persists
(n = 10) food allergy relief
(n = 8) p-value* age at diagnosis 4.06 ± 2.63 1.38 ± 0.44 0.02 male 5 (50) 6 (75) 0.28 Total serum IgE, kU/L 761.90 ± 567.64 345.73 ± 275.93 0.09 Time span between sample collections, months 18.39 ± 9.06 15.45 ± 4.52 0.44 multiple food allergies 8 (80) 4 (50) 0.18 with atopic dermatitis 4 (40) 4 (50) 0.67 SCORAD 18.5 ± 15.04 23.75 ± 6.22 0.62 food related symptoms hives 10 (100) 8 (100) - angioedema 6 (60) 4 (50) 0.67 Anaphylaxis 4 (40) 2 (25) 0.50

SCORAD, SCORing Atopic DermatitisData are given as n (%) or mean ± standard deviation

*p-value calculated by chi-square test for categorical values and Welch's t-test for continuous values

The average time interval between diagnostic sample collection and follow-up was 16.5 months, and the children were between 4 and 5 years of age at follow-up. Subjects showed a similar distribution in severity of food allergy. Seven subjects with persistent food allergy and six subjects with resolution of food allergy presented Grade 2A (mild to moderate systemic reaction with cutaneous or gastrointestinal involvement). Two subjects with persistent food allergy and one subject whose food allergy resolved experienced severe systemic reactions. Subjects with atopic dermatitis all experienced mild or moderate symptoms of atopic dermatitis, and there was no significant difference in SCORAD between groups.

The quantitative cohort consisted of 25 subjects with persistent food allergy, 23 subjects with resolution of food allergy, and 30 subjects in the control group (Table 4). Mean age and total serum IgE levels were similar to those of the same population. The average follow-up period for food allergy resolution was 18.5 months.

food allergy persists
(n = 25) food allergy relief
(n = 23) control group
(n = 30) age at diagnosis 3.07 ± 3.02 1.38 ± 1.40 3.53 ± 2.98 male 14 (56) 19 (83) 15 (50) Total serum IgE, kU/L 719.73 ± 1222.67 368.20 ± 538.80 76.26 ± 80.70 multiple food allergies 22 (88) 5 (22) - Follow-up period*, months 68.74 ± 32.18 81.21 ± 47.21 - Time to first confirmation of resolution, months - 19.9 ± 13.5 -

*Time between diagnosis and most recent resolution or persistence check

Data are given as n (%) or mean ± standard deviation

Example 2. Metabolite profiling

Metabolomic profiling was performed from serum samples from a discrete cohort using liquid chromatography-tandem mass spectrometry (LC-MS) in two modes (cationic and anionic metabolites).

For LC-MS analysis, each 100 μL sample was mixed with 300 μL of methanol (with 0.1% formic acid) containing the internal standard and centrifuged (9,100 x g, 4° C., 10 min). Next, 250 μL of the supernatant and 550 μL of 0.1% formic acid aqueous solution were mixed and filtered using an SPE column (MonoSpin C18, 5010-2170, GL Sciences Inc., Tokyo, Japan). The filtrate was purified with a 0.1% formic acid solution and a 0.1% formic acid-25% methanol solution. The purified lipid metabolite was dissolved in 200 μL of methanol (including 0.1% formic acid) immediately before measurement. Peaks detected in the LC-MS analysis were extracted using the automatic integration software MultiQuant (AB Sciex, Framingham, MA, USA) to obtain information including m/z, retention time and peak area. Peak areas were then converted to relative peak areas and annotated using a standard library (Human Metabolome Technologies, Inc., Tsuruoka, Japan) as follows: [Biochemical classification] [Number of acyl chain carbon atoms]:[ number of double bonds in the fatty acid moiety].

Free fatty acid, acylcarnitine, oxylipin, lysophosphatidylcholine, and lysophosphatidylethanola MINE), Lysophosphatidyrinositol, Lysophosphatidylserine, Lysopos Party Deal Glycerol (lysophosphatidylglycerol), lysophosphatidic acid, platelet-activating factor, acylethanolamine, sphinganine, sphingosine, ganglioside, gluco A total of 344 metabolites from several classes were identified, including glucosylceramide, lactosylceramide, ceramide-1P and steroids.

Metabolite levels were subjected to missing value imputation and general quality control procedures. Correlations between each metabolite and subject age were determined by calculating Pearson's correlation using log-transformed metabolome data from healthy controls and food allergy subjects at diagnosis. Metabolites significantly associated with age (p-value < 0.05) (see Table 5) were excluded from further analysis.

correlation coefficient Correlation p-value Lactosylceramide (d18:1/16:0) 0.572128 0.000115 LPS (22:4) -0.56264 0.000157 GM3 (d18:1/14:0) -0.55314 0.000214 TXB2 -0.53315 0.000397 LPS (20:4) -0.49013 0.001326 Glucosylceramide (d18:1/14:0) -0.48004 0.001721 LPS (20:3) -0.47532 0.001938 Sphingosine-1P (d16:1) -0.45419 0.003241 LPS (22:6) -0.43521 0.005006 GM3 (d18:1/12:0) -0.42841 0.005816 LPS (18:1) -0.41726 0.007391 FFA(12:0) -0.41384 0.007942 Lactosylceramide (d18:1/24:1) 0.411492 0.00834 Lactosylceramide (d18:1/24:2) 0.407275 0.009099 FFA(10:0) -0.40572 0.009393 LPS (22:5) -0.39489 0.011679 LPE(24:0) -0.37923 0.015808 LPS (20:1) -0.37432 0.017334 12-HETE -0.37101 0.018430 LPE(20:0) -0.36771 0.019580 Sphinganine-1P (d18:0) -0.36602 0.020191 LPS(22:0) -0.36423 0.020857 LPA(12:0) -0.35242 0.025727 PAF (22:4) -0.35082 0.026453 LPG(22:4) -0.34367 0.029913 LPS(18:0) -0.33690 0.033526 AC(24:1) -0.33543 0.034350 LPG(20:2) -0.33520 0.034487 FFA (22:3) -0.33423 0.035040 LPS(17:0) -0.33421 0.035051 LPC(14:0) -0.33278 0.035888 LPA (20:2) -0.31999 0.044125 FFA(14:0) -0.31841 0.045239 Sphingosine (d16:1) -0.31581 0.047128 LPA (22:4) -0.31386 0.048579 GD3 (d18:1/16:0) -0.31328 0.049023

LPS, lysophosphatidylserine; GM3, monosialodihexosylganglioside; TXB2, thromboxane B2; FFA, free fatty acid; LPE, lysophosphatidylethanolamine; HETE, hydroxyeicosatetraenoic acid; LPA, lysophosphatidic acid; PAF, platelet activating factor; LPG, lysophosphatidylglycerol; AC, acylcarnitine; LPC, lysophosphatidylcholine

For quality control of metabolome data, metabolites that were not quantified in more than 10% of the samples were excluded. Missing values for metabolites were replaced using the k-nearest neighbor algorithm. The median of the 10 nearest neighbors was used for the calculation. A probabilistic quotient normalization method was used to normalize the metabolomic data.

In order to observe data patterns, sparse partial least-squares discriminant analysis (sPLS-DA) was performed using MetaboAnalyst on the metabolomic data obtained from subjects with food allergy at the time of diagnosis and controls. Five components were used to control the sparsity of the sPLS-DA model, and five-fold cross-validation was used to evaluate the predictive ability. The first five components of the sPLS-DA model were tested for correlation with age to evaluate the effect of age on group segregation.

To identify metabolites that are differentially expressed between a group with food allergy and a control group, or a group in which food allergy persists or resolves, omega-3 metabolites significantly associated with resolution of food allergy are tracked at diagnosis. Paired t-test or Wilcoxon signed rank test was performed until observation. A p-value of less than 0.05 was considered statistically significant. Using MetaboAnalyst, metabolites with significantly different expression levels were constructed with a Random Forest model in which the number of trees was set to 500 and the number of variables tried in each partition was set to 7. Receiver operating characteristic (ROC) curves were generated by Monte Carlo cross-validation using balanced sub-sampling.

In addition, a linear mixed model (LMM) was used to evaluate changes in trends between subjects with persistent food allergy and subjects with resolved food allergy at the average metabolite level. An unstructured covariance matrix was applied to the LMM.

A total of 275 metabolites were identified according to quality control results or after removing metabolites related to subject age. Figure 1 shows the sPLS-DA plot with data patterns in the supervised model of food allergy and food allergy resolved groups. In the sPLS-DA model, the weights of the first two components were 16.5% and 13% for the food allergy group and 9.5% and 8.4% for the food allergy resolved group. The clustering of the subjects for the food allergy group and the food allergy resolved group showed a tendency to separate between the groups, and in particular, the boundary between the food allergy resolved group and the food allergy sustained group overlapped only slightly and was clearly distinguished. Figure 1c shows the score plot for the sPLS-DA model identified using all three groups. Compared to the relatively good separation between the control group and the group with continued food allergy, the group with resolved food allergy was located within the boundaries of the other groups, so that the case in which food allergy was resolved was the case with continued food allergy and the metabolism in the control group. It was found that it overlapped with all of the features. In all plots, the distribution of subjects did not show clustering by age and none of the first 5 components were significantly correlated with age (p-value > 0.05).

As a result of comparing the subjects with food allergy and the control group, 8 metabolites with significantly high levels and 8 metabolites with significantly low levels were identified in subjects with food allergies. Figure 2a shows 12 of these metabolites with classification according to biochemical class. Levels of all sphingolipid metabolites were consistently higher in subjects with food allergies than in controls. Six acylcarnitine metabolites with high carbon numbers and low double bonds tended to have lower levels in subjects with food allergies than in controls. Levels of both lysophosphatidic acids were higher in subjects with food allergies.

Figure 2b shows that 8 metabolites were significantly higher and 4 metabolites were significantly lower in subjects with resolving food allergy than in subjects with persistent food allergy. Consistently higher levels of the four omega-3 metabolites, particularly hydroxydocosahexaenoic acid (HDoHE), were observed in subjects whose food allergy resolved. The levels of omega-3 metabolites in subjects whose food allergy resolved were on average 1.66 times higher than those in subjects whose food allergy persisted. Levels of the two lysophosphatidylcholines (LPCs) were also higher in subjects whose food allergy resolved. A full list of metabolites related to food allergy and resolution of food allergy is shown in Table 6.

average metabolite level p-value* Associated with food allergy food allergy group control group LPG(22:5) 0.001017 0.000696 0.007995 AC(18:0) 0.000703 0.001035 0.009484 Glucosylceramide (d18:1/24:2) 0.000972 0.000663 0.009484 AC(22:0) 0.000824 0.001456 0.021077 LPA (20:5) 0.001491 0.00104 0.032643 PGH2 0.000724 0.001013 0.03493 5,15-diHETE 0.000651 0.000965 0.035011 AC(20:0) 0.00072 0.001017 0.035011 AC(19:0) 0.000696 0.000917 0.036855 LPA (22:5) 0.001128 0.000809 0.040175 Glucosylceramide (d18:1/24:1) 0.000937 0.000713 0.042985 Sphinganine (d18:0) 0.001156 0.000762 0.042985 Glucosylceramide (d18:1/22:2) 0.001077 0.000777 0.045954 LPC(24:1) 0.000966 0.000792 0.045954 AC(22:1) 0.000671 0.001067 0.04909 AC(24:0) 0.00082 0.001098 0.04909 Associated with resolution of food allergy food allergy persists food allergy relief p-value* LPE(22:5) 0.001254 0.000725 0.006216 17-HDoHE 0.000714 0.00126 0.007518 10-HDoHE 0.000776 0.00125 0.011286 13-HDoHE 0.000741 0.001194 0.013186 7-HDoHE 0.000773 0.001269 0.015457 LPE(20:3) 0.000861 0.000578 0.035942 PAF (16:1) 0.000849 0.000695 0.036515 LPA (20:1) 0.000722 0.00117 0.041768 Glucosylceramide (d18:1/14:0) 0.000906 0.001153 0.043421 LPC(12:0) 0.000757 0.002272 0.043421 LPC(22:3) 0.000873 0.000941 0.043421 LPE(16:1) 0.001337 0.00065 0.043421 LPS (20:0) 0.0008 0.001083 0.043421 LPS (20:4) 0.000747 0.001134 0.043421 LPA (20:4) 0.000697 0.000975 0.048788

FA, food allergy; LPG, lysophosphatidylglycerol; AC, acylcarnitine; LPA, lysophosphatidic acid; PGH2, prostaglandin H2; diHETE, dihydroxyicosatetraenoic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; HDoHE, hydroxydocosahexaenoic acid; PAF, platelet activating factor; LPS, lysophosphatidylserine

*p-value calculated by Welch's t-test for normally distributed data and Mann-Whitney U test for nonparametric data.

Next, a random forest model was constructed using significantly related metabolites to evaluate the accuracy of classification (FIG. 3). The area under the ROC curve of the food allergy model was 0.708 (95% CI: 0.483 - 0.926) and the area under the food allergy resolution model was 0.947 (95% CI: 0.748 - 1). The food allergy resolution model showed higher sensitivity and overall better performance than the food allergy model. It suggests that the metabolic profile of food allergy resolution may be more unique and phenotype-specific than food allergy models.

Example 3. Differences in omega-3 metabolite levels according to persistence of food allergy

In order to closely examine changes in omega-3 metabolite levels in the identification cohort subjects, follow-up observations were conducted for changes in metabolite levels after food allergy was diagnosed.

Paired t-tests or Wilcoxon signed-rank tests were performed to test changes in metabolite levels from diagnosis to follow-up of omega-3 metabolites significantly associated with resolution of food allergy, and linear mixed models (LMMs) were used to evaluate mean metabolism. At the body level, changes in trends between subjects with persistent food allergy and subjects with resolved food allergy were evaluated. An unstructured covariance matrix was applied to the LMM.

Figure 4 shows four omega-3 metabolites significantly associated with resolution of food allergy at the time of diagnosis. In contrast to the trend toward a decrease in the levels of the four omega-3 metabolites in subjects whose food allergy resolved, the levels of the four omega-3 metabolites increased in subjects with persistent food allergy. Mean fold change from diagnosis to follow-up was found to be -1.47, -1.49, -1.44 and -1.40 in subjects with resolved food allergy for 7-HDoHE, 10-HDoHE, 13-HDoHE, and 17-HDoHE, respectively , 1.38, 1.42, 1.77 and 1.34 in subjects with persistent food allergy. Although metabolite levels did not change significantly from diagnosis to follow-up (unpaired t-test or Wilcoxon signed-rank test p-value < 0.05), the trend between subjects with persistent food allergy and subjects with resolving food allergy was significant. significantly different (linear mixed model p-value < 0.05), suggesting that the trajectories of omega-3 levels show opposite trends.

Example 4. SNP analysis affecting omega-3 metabolite levels

Considering the genetic influences affecting the levels of omega-3 metabolites, quantitative trait association analyzes of genotypic variation and levels of four omega-3 metabolites were conducted using data derived from the identification cohort.

Genotyping was performed with an Infinium Omni5Exome-4 BeadChip (Illumina Inc., San Diego, CA, USA) using whole blood samples from an identified cohort. Quantitative trait association analysis was performed using the Wald test for four omega-3 metabolites using PLINK v.1.0712 in 1,841,897 SNPs obtained after quality control. A Bonferroni corrected p-value of less than 0.05 was considered statistically significant. For quality control of genotyping data, if the missing rate is higher than 5%, or the Hardy-Weinberg equilibrium p-value is less than 10 ^-5 , the missing rate is significantly different between cases and controls (p-value < 0.001), or a small number SNPs were excluded if the allele frequency was less than 1%. Data were analyzed using R version 3.3 (R Foundation for Statistical Computing, Vienna, Austria) and SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

SNPs significantly associated with metabolite levels were identified in subjects in the identification cohort for the four omega-3 metabolites. Four of the 1,841,897 SNPs were significantly associated with levels of one or more omega-3 metabolites (Bonferroni corrected p-value < 0.05) (Table 7).

Quantitative trait association analysis of SNP and omega-3 metabolite levels SNPs Location Gene Minor allele MAF Metabolites Bonferroni
corrected
p-value Mean metabolite level by genotype mm mm MM rs7274775 20:9742727 PAK5 T 0.10 7-HDoHE 0.00155 0.004243 0.001725 0.0008027 10-HDoHE 0.005272 0.00412 0.001638 0.0008121 13-HDoHE 0.002903 0.004271 0.001689 0.000777 17-HDoHE 0.03749 0.004284 0.001628 0.0007791 rs6957108 7:96996807 DLX6-AS1 T 0.18 7-HDoHE 0.03427 0.004243 0.00146 0.0007158 17-HDoHE 0.02822 0.004284 0.001442 0.000673 rs235907 1:171592086 PRRC2C C 0.08 13-HDoHE 0.03533 0.004271 0.001712 0.0008265 rs8039336 15:26680254 GABRB3 T 0.08 13-HDoHE 0.01411 0.004271 0.001753 0.008218

SNP, single nucleotide polymorphism; HDoHE, hydroxydocosahexaenoic acid; MAF, minor allele frequency; mm, minor allele homozygous; Mn heterozygotes; MM, major allele homozygous SNP position corresponds to the GRCh38/hg38 genome assembly

SNP rs7274775 located in PAK5 was identified to be associated with all four omega-3 metabolites. SNPs in DLX6-AS1 were associated with 7-HDoHE and 17-HDoHE, and SNPs in PRRC2C and GABRB3 were associated with 13-HDoHE. All four SNPs were located at different loci.

Example 5. Resolvin Quantitative Analysis

Within biological systems, HDoHE metabolites are known to react with arachidonate 15-lipoxygenase (ALOX15) and arachidonate 5-lipoxygenase (ALOX5) to generate a group of effector molecules called D-series resolvins. . Therefore, in order to further verify the above results on the association between omega-3 metabolites and resolution of food allergy, the levels of RvD1 and RvD2 were quantified in the subjects of the quantification cohort.

Enzyme-linked immunosorbent assay (ELISA) was performed using serum samples from the quantification cohort to measure levels of resolvin D1 (RvD1) and resolvin D2 (RvD2) (Mybiosource, San Diego, CA, USA) according to the manufacturer's instructions. measured. A negative assay result in one sample was excluded from further analysis. Welch's two-sample t-test or Mann-Whitney U test was used according to the normality of the data to evaluate the differences in metabolite levels and enzyme activity between groups. Total serum IgE levels were evaluated using Pearson's correlation analysis or Spearman's correlation analysis according to data distribution to investigate the correlation between metabolite levels and food allergy central phenotypes. The normality of the data was confirmed using the Shapiro-Wilk test.

As a result, as shown in FIG. 4, the levels of RvD1 and RvD2 were not only significantly different between subjects whose food allergy was resolved and subjects whose food allergy persisted, but also between subjects with food allergy and a healthy control group. Both resolvins were found to be high in subjects with food allergies, particularly in subjects with persistent food allergies.

Median RvD1 was 295.7 pg/mL (IQR: 248.0-334.4) in subjects with food allergy and 250.0 pg/mL (IQR: 225.3-259.0) in controls. In addition, the median value of RvD1 in subjects whose food allergy was resolved was 260.1 pg/mL (IQR: 234.0-326.0), and the median value of RvD1 in subjects whose food allergy persisted was 300.2 pg/mL (IQR: 259.9-334.9).

Median RvD2 was 137.9 pg/mL (IQR: 122.1-161.8) in subjects with food allergy and 122.0 pg/mL (IQR: 114.7-134.7) in controls. In addition, RvD2 levels were significantly higher in subjects with persistent food allergy (149.3pg/mL (IQR: 130.5-176.7)) compared to subjects with resolving food allergy (125.9pg/mL (IQR: 116.0-152.5)). Both RvD1 and RvD2 levels were significantly higher in subjects with persistent food allergy than in controls.

No correlation was observed between total serum IgE levels and RvD1 or RvD2 (Fig. 5).

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

FA: food allergy
IgE: Immunoglobulin E
SNP: single nucleotide polymorphism
LC-MS: liquid chromatography-mass spectrometry
sPLS-DA: sparse partial least squares-discriminant analysis
ROC: receiver operating characteristic
LMM: linear mixed model
ELISA: enzyme-linked immunosorbent assay
SD: standard deviation
HDoHE: hydroxydocosahexaenoic acid
LPC: lysophosphatidylcholine
CI: confidence interval
PAK5: p21 (RAC1) activated kinase 5 (p21 (RAC1) activated kinase 5)
DLX6-AS1: distal-less homeobox 6 antisense RNA 1
PRRC2C: proline-rich coiled-coil 2C
GABRB3: gamma-aminobutyric acid type A receptor subunit beta3
ALOX15: arachidonate 15-lipoxygenase
ALOX5: arachidonate 5-lipoxygenase
IQR: interquartile range
EPA: eicosapentaenoic acid
DHA: docosahexanoic acid
IFN: interferon
Th2: T helper 2
ORMDL3: ORMDL sphingolipid biosynthesis regulator 3 (ORMDL sphingolipid biosynthesis regulator 3)
AD: atopic dermatitis

Claims (11)

A composition for determining the persistence of food allergy, comprising an agent for measuring the level of omega-3 metabolites.
The method of claim 1, wherein the omega-3 metabolite is at least one selected from the group consisting of 7-HDoHE, 10-HDoHE, 13-HDoHE, 17-HDoHE, Resolvin D1 (RvD1) and Resolvin D2 (RvD2). Characterized in that, the composition for determining the persistence of food allergy.
The composition according to claim 1, wherein the agent is an antibody or an antigen-binding fragment thereof that specifically binds to an omega-3 metabolite, or an aptamer.
The composition according to claim 1, wherein the preparation is a preparation for chromatography and/or mass spectrometry for measuring the level of the omega-3 metabolite.
A kit for determining the persistence of food allergy, comprising the composition of any one of claims 1 to 4.
A method of providing information for predicting whether food allergy persists, comprising measuring the level of an omega-3 metabolite in a sample isolated from a subject having food allergy.
The method of claim 6, wherein the omega-3 metabolite is at least one selected from the group consisting of 7-HDoHE, 10-HDoHE, 13-HDoHE, 17-HDoHE, Resolvin D1 (RvD1) and Resolvin D2 (RvD2). Characterized in that, the method.
According to claim 6,
The level of the omega-3 metabolite is measured by immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, protein chip, immunoprecipitation, mass spectrometry, chromatography, or a combination thereof. Characterized in that, the method.
According to claim 6,
The sample is blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary tract fluid, breast milk, lymphatic system fluid, semen, cerebrospinal fluid, intratracheal fluid , ascites, cystic tumor fluid, amniotic fluid, or a combination thereof.
According to claim 7,
When the resolvin D1 is 280 pg/ml or more, the subject predicts that the allergy will persist.
According to claim 7,
When the resolvin D2 is 134 pg/ml or more, the subject predicts that the allergy will persist.

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