JPH02297063A - Manufacture of food allergen and method and kit for measuring food allergy - Google Patents

Abstract

PURPOSE: To enable the producing of food allergens and the measuring of food allergies by bringing proteins or peptides in food into reaction with lipid peroxides or its clearage-product mixture. CONSTITUTION: A complex of lipid peroxide clearage products and proteins or peptides in food are taken as an allergen. And it becomes possible to correctly diagnose food allergies by the use of these allergens. In addition, this complex presents higher allergenicity than a food protein does and the positive rate of diagnosis is improved. Therefore allergy diagnosing methods and diagnostic medicine are improved. Furthermore, this complex can be obtained by bringing lipid peroxides that lipids are oxidized, lipid peroxide clearage products such as malondialdehyde or these mixtures into reaction with proteins or peptides in food.

Description

Detailed Description of the Invention "Industrial Application Field" The present invention relates to a method for producing a food allergen, a method for measuring a specific antibody in a biological fluid sample using the allergen, and a kit for measuring the same. .

"Conventional techniques and issues" Food allergies include milk, eggs, soybeans, wheat, rice, etc.
It is caused by an extremely wide variety of food groups, and its clinical symptoms range from anaphylactic shock to eczema, measles, bronchial asthma, diarrhea, vomiting, abdominal pain, and purpura-like symptoms.

Diagnostic methods for such allergies include in vitro methods, such as measuring specific 1gE or IgE4 antibodies in the blood against food allergens, and routine clinical practice, such as skin tests in which food allergens are injected subcutaneously and erythema observed. It is held at the venue. Among these, measuring specific antibodies in blood is a good method since it is less burdensome to patients, but it cannot be said that the measured values of antibodies necessarily reflect clinical symptoms well. This varies from the presence of specific antibodies to the onset of clinical symptoms (sometimes due to the presence of fleeting stages).
Compared to pollen, etc.), there are many cases where the specific antibody titer and clinical symptoms do not match. One of the reasons for this is that there are problems with allergens in the specific antibody measurement system.

"Means for Solving the Problems" The inventors of the present application have conducted extensive research to solve the above-mentioned conventional problems, and have come to the following findings.

That is, in food, allergenic proteins or peptides coexist with lipids, and it is thought that there is an interaction between them. Particularly when lipids contain unsaturated fatty acids, the lipids undergo autooxidation to produce peroxidized lipids, which then produce various decomposition products such as malondialdehyde, alkenals, keto acids, alkanals, and diketones. These lipid peroxide decomposition products are known to generate various complexes by interacting with the amide 7 group in proteins or peptides (S, Matsushita A.
gric, Biol, Chem, 42781. (
197g)).

In the present invention, an attempt was made to use a complex (hereinafter referred to as a complex) of the above lipid peroxide decomposition product and a protein or peptide in food as an allergen.

These complexes are not included in conventional allergens, and specific antibodies against them have so far been overlooked. Therefore, the use of such allergens has made it possible to measure antibody titers that have been overlooked so far, making it possible to more accurately diagnose food allergies. Alternatively, these complexes showed higher allergenicity than food proteins, and improved diagnostic positivity rates were also observed. Based on these facts, it was considered that the use of such a complex would make it possible to significantly improve conventional allergy cattle diagnostic methods or diagnostic agents.

Such complexes are produced by reacting lipid peroxides or lipid peroxide breakdown products such as malondialdehyde, alkenals, keto acids, alkanals, diketones, etc. or mixtures thereof with proteins or peptides in foods. However, it can also be obtained by preserving foods containing lipids under conditions that facilitate peroxidation reactions. Lipid peroxidation normally occurs at 3
It can be sufficiently carried out by leaving it at 0°C to 50°C for one day or more, but blowing in air or oxygen, heating it to a higher temperature, and adding enzymes such as Lipo-Cow Shigenase or catalysts such as heavy metals are recommended. It can also be promoted by adding acid or alkali.

In the present invention, soybean globulin, egg albumin, egg white conalbumin, and milk β-lactoglobulin are used as protein peptides in foods that can be allergens, but they are not limited to these proteins. It is also applicable to other proteins in other food products (eg, rice, wheat, other grains, fish, vegetables, fruits, etc.). Complex formation occurs at pH 2~
13, preferably in a suitable buffer at pH 4-10.
This can be achieved by keeping the temperature between 4°C and 50°C (preferably between 4°C and 50°C).

Methods for measuring antibodies such as specific IgE and fg04 in biological fluids using the thus obtained allergen as an antigen include enzyme immunoassay (EIA), radioimmunoassay (RIA), Fluorescence immunoassay (FIA),
Chemiluminescent immunoassays and the like are available.

The present inventors used these complexes to measure specific IgE antibodies in the blood of various allergic patients by ELISA, which is a modified enzyme immunoassay (EIA) method. As a result, we were able to obtain measurement conditions that are significantly more sensitive than conventional measurements using food allergens themselves.

"Example" Hereinafter, the present invention will be specifically explained with reference to Examples.

(Example 1) This example is an example of producing a food allergen using a lipid peroxide or a mixture of lipid peroxide decomposition products and a food protein.

Soybean oil, rapeseed oil, and peanut oil at 40°C1 in the atmosphere.
By incubating for 3 days, each lipid peroxide and a mixture of lipid peroxide decomposition products are obtained. Each of these 0.5-
was added to 10-phosphate buffer (pH 7,0) containing 0.5 g of food protein, mixed, and then shaken at 40°C for 1 to 5 days. Afterwards, it was defatted with ethanol and the protein was freeze-dried to make it into an allergen.

The peroxide value (POV) of each oil when using soybean 2S globulin is shown in FIG. PO-V is the thiocyanate method [Hlitsuda et al., Nutrition and Foods.

19210 (1966)].

(Example 2) This example is an example of producing a food allergen using malondialdehyde (MDA), which is one of the lipid peroxide decomposition products, and food protein.

Mix 0.5 m (l) of malondialdehyde and 0.5 cm (l) of protein solution and incubate at 37°C for 1 day. After dialyzing this against an appropriate buffer solution, freeze-dry.
It was considered an allergen.

(Example 3) This example is an experiment using the food allergen of Example 1 or 2.
d immun.

This is an example of 5orbent assay).

2-10 μg/1 in a 96-well microtiter plate
Add s (l) of food allergen solution to adsorb the allergen.
: Add 50 μQ of a serum sample diluted to 200 to allow the allergen to react with the specific 1gE antibody in the serum. After removing the serum sample, a peroxidase-labeled anti-human IgE antibody was reacted with 0-phenylene-diamin.
The absorbance at 492 nm is measured.

FIG. 2 shows an example in which a soybean 2S globulin complex reacted with peroxidized soybean oil was used as an allergen. The ELISA values of 3 normal subjects and 3 soybean allergic patients increased as the antigen concentration increased.

On the other hand, when soybean 2S globulin itself was used as the allergen, no difference was observed between normal subjects and patients.

(Example 4) This example was carried out according to the above-mentioned Example 3,
FIG. 3 shows an example in which a complex of ovalbumin, conalbumin, β-lactoglobulin, and 2S-globulin reacted with peroxidized soybean oil was used as an allergen.

EL for any allergen as the allergen concentration increases
An increase in ISA value was observed.

On the other hand, when these proteins themselves were used as allergens, no differences were observed between normal subjects and patients.

(Example 5) This example was also carried out according to the above-mentioned Example 3,
FIG. 4 shows an example in which complexes of soybean 7S globulin, ovalbumin, conalbumin, and β-lactoglobulin reacted with malondialdehyde and intact proteins were used as allergens.

Table 1 shows the average and standard deviation of the ELISA values of the 6 allergic patients.

ELISA values of proteins
1 incubated with MDA (Abet) (
Example 6) This example is a 1nh test for the ELISA method of Example 3.
This is an example of the ibition test.

A 100 μσ allergen solution was added to 50 μQ of diluted patient serum in the ELISA method of Example 3, and the rest of the measurement was performed using the method of Example 3.

In FIG. 4, as the ELISA immobilized allergen,
An example was shown in which a complex obtained by reacting linoleic acid peroxide and ovalbumin was used, and the above complex and ovalbumin were used as a 1nhibitor.

Further, in FIG. 5, a complex obtained by reacting malondialdehyde and soybean 7S globulin is used as an ELISA immobilized allergen, and the above complex and soybean 7S globulin are used as an inhibitor. An example is shown below.

In both examples, 1 nhibitor was observed due to the complex of lipid peroxide degradation product and protein, and E
LISA was shown to measure specific antibodies.

(Example 7) This example shows an example of analysis of the amino acid composition of 78 globulin and 7S globulin of malondialdehyde.

The protein or complex is dissolved in 6 N HCQ, degassed and sealed in a glass tube. This is 110°C1
The mixture was hydrolyzed for 24 hours and subjected to an amino acid analyzer. The results are shown in Table 2.

The number of lysine residues in the complex was reduced compared to 7S globulin, indicating that the ε-amine group of lysine reacted with the lipid peroxide decomposition product.

(Blank below) (Table 2) ^m1no acid composition or
soY, -globulin reacted t
o MDA (mo1%) "Effects of the Invention" As explained above, according to the present invention, it is possible to obtain measurement conditions that are significantly more sensitive than conventional measurements using food allergens themselves.

[Brief explanation of drawings]

Figure 1 shows the change in peroxidation of fats and oils over time, Figure 2 and Figure 3 show the following:
ELISA values for various allergens of allergic patients and normal subjects are plotted against allergen concentration (Example 3.4), Figures 4 and 5 show the EL
This represents a 1 nbibition test of the ISA method (Example 5.6).

Claims (6)

[Claims] (1) A method for producing a food allergen, which comprises reacting a protein or peptide in food with a lipid peroxide or a mixture of its decomposition products. (2) The method for producing a food allergen according to claim 1, wherein the lipid peroxide decomposition product is at least one of malondialdehyde, alkenal, keto acid, alkanal, and diketone. (3) A method for measuring food allergy in which a specific antibody in a biological fluid against a food allergen is immunologically measured, wherein the food allergen is produced by the method according to any one of claims 1 to 2. A food allergy measurement method characterized by using a food allergen. (4) The immunoassay method is radioimmunoassay (RIA),
Enzyme immunoassay (EIA), fluorescence immunoassay (FIA)
4. The method for measuring food allergy according to claim 3, which is a chemiluminescence immunoassay. (5) A food allergy measurement kit for immunologically measuring a specific antibody in a biological fluid against a food allergen, wherein the food allergen is the method according to any one of claims 1 to 2. A food allergy measurement kit characterized by using a manufactured food allergen. (6) The immunoassay method is radioimmunoassay (RIA),
Enzyme immunoassay (EIA), fluorescence immunoassay (FIA)
The method for measuring food allergy according to claim 5, which is a chemiluminescence immunoassay.