JP7448929B2 - How to collect data for diagnosis of neonatal-infant gastrointestinal allergies - Google Patents

Description

The present invention relates to a method for collecting data for diagnosing neonatal-infant gastrointestinal allergy, and a kit for diagnosing neonatal-infant gastrointestinal allergy.

Neonatal-infant gastrointestinal allergy is an IgE-independent food allergy, and the main symptoms are repeated vomiting, diarrhea, and other gastrointestinal symptoms, and it is a disease in which the entire body becomes limp due to dehydration caused by vomiting and diarrhea. be. Such neonatal/infant gastrointestinal allergy is thought to be caused by cell-mediated immunity, that is, antigen-presenting cells, allergens, T lymphocytes, eosinophils, local epithelial cells, etc., but the details of its pathology are still unknown. It's not clear.

This neonatal/infant gastrointestinal allergy requires early diagnosis and intervention, but non-specific symptoms such as inactivity and poor weight gain are also observed, and in clinical practice it is difficult to differentiate it from other diseases that cause vomiting and diarrhea. It is often difficult. Furthermore, in the pediatric medical field, diseases that present with gastrointestinal symptoms such as vomiting and diarrhea are relatively common, such as viral gastroenteritis caused by norovirus, rotavirus, etc., and bacterial enteritis caused by campylobacter, pathogenic E. coli, salmonella, etc. There are multiple cases, and neonatal/infant gastrointestinal allergy is often suspected after the above-mentioned diseases have been excluded, so diagnosis is often delayed.

Tests used to diagnose gastrointestinal allergies in newborns and infants include allergen lymphocyte stimulation tests, gastrointestinal endoscopy, and food challenge tests. Allergen lymphocyte stimulation tests are not covered by insurance, and false positives and false negatives are also observed, so a definitive diagnosis cannot be made. Gastrointestinal endoscopy and food challenge tests are useful for definitive diagnosis, but there are problems such as the physical burden and risks, the need for hospitalization, and the limited number of facilities that can handle such tests. There is.

In recent years, research has been conducted on the relationship between neonatal and infant gastrointestinal allergies and cytokines. It has been disclosed that cytokine levels are high (see Non-Patent Documents 1 and 2).

Cytokine profile after oral food challenge in infants with food protein-induced enterocolitis syndrome. Kimura M. et al., Allergol Int. 2017 Humoral and cellular responses to casein in patients with food protein-induced enterocolitis to cow's milk. Caubet JC. et al., J Allergy Clin Immunol. 2017

As mentioned above, the food challenge test is currently the only definitive diagnosis method, but the facilities that can perform the diagnosis are limited. There are many diseases that exhibit symptoms similar to neonatal/infant gastrointestinal allergies, and there is a need for a simple method that can be quickly differentiated from these and that can be used in acute stage diagnosis in general practice. Therefore, an object of the present invention is to provide a non-invasive and simple method for collecting data for diagnosing neonatal/infant gastrointestinal allergy.

To date, the present inventors have measured cytokines in children with neonatal-infant gastrointestinal allergy in order to elucidate the pathology of neonatal-infant gastrointestinal allergy. As a result, it was discovered that the concentrations of IL-2 in serum and IL-10 in serum were significantly increased when patients had gastrointestinal symptoms, and the present invention was completed.

That is, the present invention is as follows.
[1] A method for treating neonatal-infant gastrointestinal allergies, comprising the step of measuring interleukin-2 (IL-2) and interleukin-10 (IL-10) protein levels in a biological sample collected from a subject. How to collect data for diagnosis.
[2] Obtaining an indicator that neonatal-infant gastrointestinal allergy has developed when the IL-2 protein level in the biological sample and the IL-10 protein level in the biological sample are each higher than a predetermined threshold value. The method for collecting data for diagnosing neonatal/infant gastrointestinal allergy according to [1] above, comprising:
[3] The method for collecting data for diagnosis of neonatal/infant gastrointestinal allergy according to [1] or [2] above, wherein the biological sample is serum.
[4] If the serum IL-2 protein level is 4 pg/ml or higher, or the serum IL-10 protein level is 7 pg/ml or higher, the subject is diagnosed as having neonatal-infant gastrointestinal allergy. The method for collecting data for diagnosing neonatal/infant gastrointestinal allergy according to [3] above, comprising the step of obtaining an index of.
[5] If the serum IL-2 protein level is 4 pg/ml or higher and the serum IL-10 protein level is 7 pg/ml or higher, the subject is diagnosed with neonatal-infant gastrointestinal allergy. The method for collecting data for diagnosing neonatal/infant gastrointestinal allergy according to [3] above, which comprises the step of obtaining an index.
[6] A diagnostic kit for neonatal/infant gastrointestinal allergy, containing an anti-IL-2 antibody and an anti-IL-10 antibody.

By using the present invention, it becomes possible to collect data for non-invasively diagnosing whether a newborn or infant has a gastrointestinal allergy without conducting a food challenge test. Furthermore, since it is a simple method, rapid diagnosis is possible even in facilities other than allergy specialized facilities.

FIG. 2 is a diagram showing the results of measuring and graphing the serum IL-2 concentration (pg/ml) in FPIES, viral gastroenteritis, and bacterial gastroenteritis in Example 1. FIG. 2 is a diagram showing the results of measuring and graphing the serum IL-10 concentration (pg/ml) in FPIES, viral gastroenteritis, and bacterial gastroenteritis in Example 1. FIG. 2 is a diagram showing the results of measuring and graphing the IL-2 concentration (pg/ml) in serum during FPIES and anaphylaxis in Example 1. FIG. 2 is a diagram showing the results of measuring and graphing the serum IL-10 concentration (pg/ml) in FPIES and anaphylaxis cases in Example 1. FIG. 2 is a diagram showing the results of measuring serum IL-2 and serum IL-10 concentrations over time in one episode (No. 5) of the FPIES patients in Example 1.

The method of collecting data for diagnosis of neonatal-infant gastrointestinal allergy of the present invention is characterized by comprising the step of measuring IL-2 and IL-10 protein levels in a biological sample collected from a subject. There is no particular restriction here, as long as it is a method for collecting data for diagnosing neonatal-infant gastrointestinal allergy (hereinafter also referred to as "method for collecting data for diagnosing neonatal-infant gastrointestinal allergy"). Neonatal-infant gastrointestinal allergy means gastrointestinal allergy in infants (within 1 month of age) or newborns (within 1 year of age). Specific examples include neonatal-infant food protein-induced gastroenteritis (FPIES), food protein-induced rectal colitis (FPIAP), and food protein-induced gastroenteropathy (FPE).

The kit for diagnosing neonatal-infant gastrointestinal allergy of the present invention is not particularly limited as long as it contains an anti-IL-2 antibody and an anti-IL-10 antibody. It is also referred to as the "diagnosis kit for neonatal/infant gastrointestinal allergy."

The above-mentioned "diagnosis of neonatal-infant gastrointestinal allergy" refers to the determination of the presence or absence of neonatal-infant gastrointestinal allergy, especially within 48 hours of ingesting the food that causes neonatal-infant gastrointestinal allergy. This refers to the determination of the presence or absence of gastrointestinal allergy.

In this specification, "subject" refers to a person who has not developed neonatal-infant gastrointestinal allergy, a person who may be suffering from neonatal-infant gastrointestinal allergy, or a person who has actually developed neonatal-infant gastrointestinal allergy. I can name people who have done so. Furthermore, the "subject" is not limited to humans, but may also be mammals such as mice, rats, rabbits, cats, dogs, monkeys, pigs, sheep, cows, and horses.

Biological samples include serum, blood, plasma, saliva, and urine, with serum being preferred. Such biological samples are collected within 48 hours, preferably within 36 hours, more preferably within 24 hours, even more preferably within 12 hours, and most preferably within 6 hours after ingesting the food causing the neonatal-infant gastrointestinal allergy. Examples include those collected from subjects.

The method for measuring the level of IL-2 protein or IL-10 protein in a biological sample is not particularly limited as long as it can specifically detect part or all of IL-2 protein or IL-10 protein. , epidemiological measurement methods and mass spectrometry.

Examples of the immunoassay methods include ELISA (Enzyme-Linked Immuno Sorbent Assay), immunochromatography, immunoblotting, CBA (Cytometric Bead Assay), Western blotting, flow cytometry, and the like.

The antibodies used in the above epidemiological measurement method or the antibodies included in the diagnostic kit for neonatal/infant gastrointestinal allergy may be monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, or humanized antibodies. monoclonal antibodies are preferred. The above-mentioned antibodies also include antibody fragments consisting of a portion of an antibody such as F(ab') ₂ , Fab, diabody, Fv, ScFv, Sc(Fv) ₂ and the like.

Moreover, the above-mentioned antibody may be labeled with a labeling substance. Such labeling substances include peroxidase (for example, horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, Enzymes such as apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothiocyanate, and green fluorescent protein (GFP). ), Cyan Fluorescence Protein (CFP), Blue Fluorescence Protein (BFP), Yellow Fluorescence Protein (YFP), Red Fluorescence Protein (RFP), Luciferase fluorescent proteins such as 3H, 14C, 125I or 131I, radioisotopes such as 3H, 14C, 125I or 131I, metal colloids, non-metal colloids, dye particles such as dye sols or disperse dyes, latex particles or colored microparticles, biotin, avidin, or chemiluminescent substances. can be mentioned.

Although commercial products may be used as the above antibodies, the gene sequence of IL-2 or IL-10 can be obtained from a database such as NCBI (National Center for Biotechnology Information), and recombinant IL-2 or IL-10 protein can be obtained by cloning. It may also be produced by synthesizing and immunizing mice with the synthesized recombinant IL-2 or IL-10 protein.

An indicator that the subject has developed neonatal-infant gastrointestinal allergy when the IL-2 protein level in the biological sample collected from the subject and the IL-10 protein level in the biological sample are above a predetermined threshold. can be obtained.

The method for determining the above predetermined threshold value is not particularly limited, but for example, patients who have developed neonatal-infant gastrointestinal allergy, patients who have not developed neonatal-infant gastrointestinal allergy, and patients who have developed viral gastroenteritis. List methods for determining IL-2 protein levels in biological samples and IL-10 protein levels in biological samples in patients, patients with bacterial gastroenteritis, and/or patients with anaphylaxis. be able to. In this case, the predetermined threshold value may be determined with reference to the average value or median value of the IL-2 protein level in the biological sample and the IL-10 protein level in the biological sample for each patient. In addition, the predetermined threshold value may be determined using an ROC curve (Receiver Operating Characteristic curve).

Specifically, the above predetermined threshold value is when the concentration of IL-2 protein in the biological sample is 4 pg/ml, 5 pg/ml, 10 pg/ml, 15 pg/ml, 50 pg/ml, or 100 pg/ml. Examples include a concentration of IL-10 protein in the biological sample of 7 pg/ml, 10 pg/ml, 20 pg/ml, 50 pg/ml, or 80 pg/ml.

The neonatal/infant gastrointestinal allergy diagnostic kit may further collect data for diagnosis of newborn/infant gastrointestinal allergy patients, in addition to diluents, washing buffers, standards, substrate reagents, etc. It is preferable to include an instruction manual that describes this, and it is preferable to have a structure that allows for easy inspection.

In the neonatal/infant gastrointestinal allergy diagnostic kit, the anti-IL-2 antibody and the anti-IL-10 antibody may be immobilized on a carrier, and the levels of IL-2 and IL-10 proteins can be measured by immunochromatography, etc. becomes easier. Examples of the carrier include nitrocellulose, cellulose acetate, cellulose, nylon, polyethersulfone, polyvinyl alcohol, polyester, and polyolefin. By reacting anti-IL-2 antibodies and anti-IL-10 antibodies labeled with the above-mentioned labeling substances using immunochromatography with biological samples collected from subjects, IL-2 and/or Alternatively, it can be determined whether the IL-10 protein level is equal to or higher than the above-described predetermined threshold. Such discrimination results can be used as data for diagnosing neonatal/infant gastrointestinal allergy.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] Measurement of cytokines
Cytokine concentrations in serum in cases of FPIES, viral gastroenteritis, bacterial gastroenteritis, and anaphylaxis were measured by the following method.

(Target patients for FPIES)
The subjects were 5 patients with FPIES (6 episodes). Five of the six patients were diagnosed with FPIES because of repeated episodes of vomiting and bloody stool after ingesting milk or egg yolk, and one patient was diagnosed with FPIES based on a positive oral food challenge test. Blood samples were collected within 3 hours of vomiting. The diagnostic criteria for FPIES were in accordance with the following literature (J Allergy Clin Immunol. 2017 Apr;139(4):1111-1126).

(targets for comparison)
For comparison, 10 cases of anaphylaxis met the definition of Food Allergy Treatment Guidelines 2016 (prepared by the Japanese Society of Pediatric Allergy, edited by Kyowa Kikaku), 7 cases of viral gastroenteritis caused by norovirus and rotavirus, Yersinia, Salmonella, Campylobacter, and pathogenic Escherichia coli. Blood samples were collected from nine patients with bacterial gastroenteritis at the time of their visit.

(Cytokine measurement method)
The collected blood sample was centrifuged at 3000 rpm for 5 minutes, and the supernatant after centrifugation was separated as serum. Serum IL-2, IL-4, IL-6, IL-10, IFN, and TNF-α concentrations were measured using a Cytometric Bead Assay (CBA) Kit (BD Biosciences) containing antibodies against each of the above cytokines. It was measured. IL-8 concentration in serum was measured using Quantikine (registered trademark) ELISA Human IL-8/CXCL8 Immunoassay (R&D Systems).

(Cytokine measurement results)
Figure 1 shows the results of measuring and graphing the serum IL-2 concentration (pg/ml) in FPIES, viral gastroenteritis, and bacterial gastroenteritis. Figure 2 shows the results of measuring and graphing the serum IL-10 concentration (pg/ml) in FPIES and anaphylaxis cases. The results are shown in FIG. 3, and FIG. 4 shows the results of measuring and graphing the IL-10 concentration (pg/ml) in serum in FPIES and anaphylaxis cases. It is clear that serum IL-2 and serum IL-10 concentrations are significantly elevated in FPIES compared to cases of viral gastroenteritis, bacterial gastroenteritis, and anaphylaxis. became.

Furthermore, Table 1 summarizes the serum IL-2 concentration and serum IL-10 concentration for each case (No. 1 to 5) in the six FPIES cases.

Nos. 2-1 and 2-2 are the same case with two episodes, and 2-2 is a case in which the disease developed again 4 months after the first onset. In addition, serum cytokine concentrations (pg/ml) in 9 cases of gastroenteritis (7 cases of viral gastroenteritis, 2 cases of bacterial gastroenteritis) are shown in Table 2, and serum cytokine concentrations (pg/ml) in 10 cases of anaphylaxis are shown in Table 3. Shown below.

From Table 1, it was revealed that in FPIES, the serum IL-2 concentration was 4 or higher, and the serum IL-10 concentration was 7 or higher. Furthermore, Table 2 revealed that in cases of gastroenteritis, the serum IL-2 concentration was less than 4, and the serum IL-10 concentration was less than 10. Furthermore, Table 3 revealed that in anaphylaxis, the serum IL-2 concentration was less than 4, the serum IL-10 concentration was less than 20 in all but one case, and less than 10 in all but four cases.

Based on these results, the serum IL-2 and serum IL-10 concentrations were measured, and based on the obtained serum IL-2 and serum IL-10 concentrations, a diagnosis of FPIES was made. It became clear that the data could be provided. Note that IL-2 is mainly produced from activated T cells, and has the effect of proliferating T cells that have reacted with antigens and the effect of activating macrophages. On the other hand, IL-10 is mainly produced by helper T cells, and has the effect of suppressing the production of IL-2, TNF, interferon γ, etc., and the expression of MHC class II molecules and costimulatory molecules on macrophages and dendritic cells. It has the effect of decreasing antigen presenting ability by decreasing That is, it was extremely unexpected that in the diagnosis of neonatal/infant gastrointestinal allergy, specific diagnosis could be made by measuring two cytokines that have contradictory effects as indicators.

As shown in Table 1, there are some variations in serum IL-2 concentration and serum IL-10 concentration depending on the case. This is due to the fact that the half-life of IL-2 in serum is different from that of IL-10 in serum, and the time elapsed since ingesting the food that caused FPIES. This seems to be related to the variation in . Therefore, it is better to make a judgment by combining the serum IL-2 concentration and serum IL-10 concentration, rather than making a judgment based only on the serum IL-2 concentration or serum IL-10 concentration. It can be said to be highly specific and lead to accurate diagnosis. From the results in Tables 1 to 3, for example, when the concentration of IL-2 protein in serum is 4 pg/ml or more, or the concentration of IL-10 protein in serum is 7 pg/ml or more, it is preferable that, for example, IL-2 protein in serum Neonatal-infant gastrointestinal allergy can be determined when the concentration of 2 proteins is 4 pg/ml or higher and the serum IL-10 protein concentration is 7 pg/ml or higher.

[Example 2] Serum IL-2 and serum IL-10 concentrations over time in FPIES patients IL-2 in serum over time in one of the FPIES patients (No. 5) in Example 1 and serum IL-10 concentration was measured. IL-2 concentration in serum and serum after 0, 0.5, 1, 1.5, 2, and 48 hours when milk was ingested 6 times every 15 minutes, and the first milk intake was defined as 0 hours. The concentration of IL-10 in the sample was measured in the same manner as in Example 1. The results are shown in Figure 5.

As is clear from FIG. 5, both serum IL-2 and serum IL-10 were detected 2 hours after the first milk intake. On the other hand, neither IL-2 in the serum nor IL-10 in the serum was detected after 48 hours. Therefore, it has become clear that it is necessary to measure serum IL-2 and serum IL-10 concentrations at least 48 hours after ingesting the food that caused FPIES.

It can be used for quick and simple diagnosis of neonatal-infant gastrointestinal allergy (FPIES).

Claims (3)

Neonatal-Infant Food Protein-Induced Gastroenteritis (FPIES) , characterized in that it comprises the step of measuring interleukin-2 (IL-2) and interleukin-10 (IL-10) protein levels in serum collected from a subject. , a method for collecting data for the diagnosis of food protein-induced proctal colitis (FPIAP), or food protein-induced gastroenteropathy (FPE) , where the IL-2 protein level in serum is 4 pg/ml or higher. The method further comprises the step of obtaining an indicator that the subject has developed FPIES, FPIAP, or FPE . Method according to claim 1, characterized in that FPIES, FPIAP or FPE is FPIES. Neonatal-Infant Food Protein-Induced Gastroenteritis (FPIES), comprising the step of measuring interleukin-2 (IL-2) and interleukin-10 (IL-10) protein levels in serum collected from a subject. A method for collecting data for the diagnosis of a subject who has a serum IL-2 protein level of 4 pg/ml or more and a serum IL-10 protein level of 7 pg/ml or more. The above-mentioned method includes the step of obtaining an indicator that the above-mentioned FPIES has developed.