JP2006220518A - Detection method of allergy-inducing substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and rapidly detecting an allergy-inducing substance. <P>SOLUTION: The detection method of the allergy-inducing substance includes a process (a) of bringing a substance to be inspected into contact with serum protein and a process (b) of confirming whether serum protein changed in its tertiary structure by the reaction of the substance to be inspected with serum protein is present by means of electrophoresis. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a simple method for detecting an allergen.

  Currently, it is said that about 55,000 kinds of chemical substances used in the Japanese industry alone are counted, and more than 500 kinds of chemical substances are added to labor every year as demand diversifies. It has been introduced on site. These chemical substances are naturally considered to contain chemical substances having a high allergenic potential, and it is extremely important to detect such allergenic substances from a large number of chemical substances.

  Whether or not a certain compound has an allergenic potential has been confirmed by experiments using animals. For example, as a type I allergenicity test, there are a systemic active anaphylaxis test (ASA test) using guinea pigs and rats, a local passive anaphylaxis test (PCA test), and the like. The ASA test is a test in which a test substance is repeatedly administered at regular intervals to check for the induction of anaphylactic shock symptoms. The PCA test is a test substance after intradermal administration of test substance-sensitized serum to normal animals. Is measured from the magnitude of inflammation caused by the anaphylactic reaction at the site of intradermal injection. In addition, as a type IV allergy inducing ability test, there is a method of measuring the inducing ability of delayed type hypersensitivity from the magnitude of inflammation of the skin site where the test substance is administered to guinea pigs or mice and the substance is re-administered or applied. There are Draize test, Buehler test, maximization test, etc. depending on the administration method of substances. In addition, a local lymph node assay (LLNA), which measures lymphocyte proliferative responses in the auricular lymph nodes that have been re-exposed to the substance instead of inflammation, is used as an alternative test method. Furthermore, as an application of the LLNA method, there is a method for analyzing a cytokine profile produced by lymph node cells.

  However, all of these methods require a relatively long test period with many experimental animals, and have major problems from the viewpoint of cost and time. There is also a problem that it is not always appropriate as a means for predicting allergy due to a substance. Therefore, development of a method for detecting allergens in a short time and simply has been desired.

On the other hand, regarding the binding property between serum proteins and chemical substances, Non-Patent Document 1 (Gauggel DL et al., J. Appl. Toxicol., 13, 307, 1993) states that albumin bound to a chemical substance (hapten) A method for detecting changes by high performance liquid chromatography (HPLC) is described. The above document has a short description of SDS-polyacrylamide gel electrophoresis and isoelectric focusing of hapten-protein conjugates, but no experimental data is shown. According to the studies by the present inventors, in contrast to the description in Non-Patent Document 1, in serum SDS-polyacrylamide gel electrophoresis, serum albumin reacted with allergens often gives only one band, In isoelectric focusing, albumin itself has already been confirmed to have multiple bands. Therefore, the credibility of this part of Non-Patent Document 1 is poor. Non-Patent Document 1 does not suggest or teach that allergens can be detected by SDS-polyacrylamide gel electrophoresis and / or isoelectric focusing.
J. Appl. Toxicol., 13, 307, 1993

  An object of the present invention is to provide a method for easily and rapidly detecting an allergen.

  As a result of diligent research to solve the above-mentioned problems, the inventor reacts with serum proteins such as serum albumin to change the tertiary structure of the allergen, and the serum has the tertiary structure changed. It was found that the protein can be separated from unreacted serum protein by electrophoresis. Further, in the system in which the test substance and the serum protein coexist, the presence of the serum protein whose tertiary structure has changed due to the reaction between the test substance and the serum protein is proved by electrophoresis as described above. It was found that the test substance can be determined to be an allergen. The present invention has been completed based on the above findings.

That is, according to the present invention, there is provided a method for detecting an allergen, which comprises the following steps:
(a) contacting the test substance with serum protein; and
(b) A method is provided which comprises a step of confirming by electrophoresis whether or not there is a serum protein whose tertiary structure has been changed by the reaction between a test substance and the serum protein.
According to a preferred embodiment of the present invention, the above method wherein the serum protein is albumin; the above method wherein electrophoresis is performed by SDS-polyacrylamide gel electrophoresis and / or isoelectric focusing; and the electrophoresis is performed using SDS-poly There is provided the above method performed by a combination of acrylamide gel electrophoresis and / or isoelectric focusing.

  Another aspect of the present invention is a method for diagnosing allergic diseases, in which serum proteins contained in blood collected from mammals including humans are subjected to electrophoresis to react the allergens with the serum proteins. The present invention provides a method comprising the step of confirming whether or not there is a serum protein having a changed tertiary structure.

  According to the method of the present invention, it is possible to quickly and easily confirm whether or not a test substance is an allergy-inducing substance without conducting a long-term complicated allergy-inducing test using animals.

The method of the present invention is a method for detecting an allergenic substance, wherein (a) a test substance is brought into contact with a serum protein; and (b) a tertiary structure is changed by a reaction between the test substance and the serum protein. It includes a step of confirming by electrophoresis whether or not serum protein is present.
The type of the test substance used in the step (a) is not particularly limited. For example, any low molecular organic compounds, high molecular organic compounds, proteins, sugar compounds, lipids, nucleic acids, etc. may be used as the detection target. Among these, a low molecular organic compound can be mentioned as a preferable test substance. The chemical structure of the low molecular weight organic compound is not particularly limited, and may have one or two or more arbitrary functional groups, and an arbitrary hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom). May be included. Including any structure consisting of a linear or branched, saturated, partially saturated, or aromatic ring structure (the ring structure may be monocyclic or polycyclic), or a combination thereof May be.

  The type of serum protein used in the step (a) is not particularly limited, and examples thereof include albumin and globin. Of these, albumin is preferable. As the albumin, in addition to those separated and prepared from human serum, those in any form or origin such as a commercial product provided as dry albumin may be used.

  The step of bringing the serum protein into contact with the test substance is performed, for example, by adding the test substance to an aqueous medium containing the serum protein. However, this operation is not particularly limited, and the serum protein and the test substance are contacted in the aqueous medium. Any operation may be selected as long as it is in contact with the test substance. As the aqueous medium, water, a buffer solution such as a phosphate buffer, physiological saline, or the like can be used. If necessary, a small amount of a water-miscible organic solvent such as glycerin, ethylene glycol, or dimethyl sulfoxide can be added. Good. The temperature and time for performing the above contact are not particularly limited, but in general, in a temperature range in which serum proteins do not denature (for example, about 0 to 40 ° C., preferably about 20 to 37 ° C.), several hours to several days The treatment is preferably performed for about 24 to 48 hours. The contact is preferably performed in the vicinity of neutrality equivalent to the physiological environment, for example, in the range of about pH 7.0 to 7.5. Although this contacting step may be performed in vitro as described above, it can also be performed in vivo by a method in which a test substance is administered to a mammal including a human and blood is collected. It is also possible to collect blood of mammals including humans that may have been exposed to the test substance, and replace with step (a).

  In the above step (a), when the test substance is an allergy-inducing substance, the test substance reacts with the serum protein, and as a result, a serum protein having a changed tertiary structure is generated. A serum protein having a changed tertiary structure is a protein formed by a polypeptide in which a reactive functional group of an allergen induces a reaction with an amino acid residue of the serum protein to form a covalent bond, and the allergen inducer is covalently bound. When the tertiary structure of the serum protein is different from the tertiary structure of the serum protein, or when the tertiary structure of the serum protein changes due to the physicochemical interaction between the allergen and the serum protein, Alternatively, it means a state in which some change has occurred in the three-dimensional structure of the serum protein, including the case where the serum protein associates into a macromolecule by reaction. In this specification, “reaction” must be interpreted in the broadest sense including a chemical reaction in which a covalent bond is formed, and should not be interpreted in a limited manner in any sense. In many cases, a chemical reaction with an allergen causes a covalent bond to be formed, resulting in a change in the tertiary structure of the serum protein.

  In the method of the present invention, serum proteins whose tertiary structure has been changed by reaction with an allergen can be analyzed by electrophoresis. Serum proteins whose tertiary structure has changed due to the reaction show different migration behavior from the serum protein before the reaction, and give a band at a different position from the serum protein before the reaction, so it is different from the serum protein before the reaction by electrophoresis By confirming the presence of the band, it can be proved that a serum protein whose tertiary structure is changed by the reaction is produced. The type and conditions of electrophoresis are not particularly limited, and any one may be used as long as it can separate a serum protein whose tertiary structure has been changed by a reaction with an allergen, and a serum protein before the reaction, For example, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) are suitable. By combining and analyzing these two electrophoresis methods, analysis with extremely high accuracy becomes possible.

  Without being bound by any particular theory, in order for foreign chemicals to be immunogenic, they bind to the serum protein and change the tertiary structure of the serum protein, and the protein after the reaction becomes a non-self protein. Need to be recognized. The present inventor found for the first time that a serum protein in which the tertiary structure has changed to such a degree that it can be recognized as a non-self protein can be separated from the pre-reaction serum protein by electrophoresis. It has been found that it can be easily and quickly determined by (a) a two-component contact process in vitro and (b) a subsequent electrophoresis process. Since the method of the present invention can screen allergic sensitization of a test substance by electrophoresis without using an immune reaction, the test period is significantly shortened compared to conventional methods, and animals are not used. And it is useful from both aspects of expenses.

  In addition, by applying the method of the present invention, it is possible to diagnose an allergic disease by examining the tertiary structure change of the serum protein of a patient. More specifically, in the above diagnosis, the serum protein contained in blood collected from mammals including humans is subjected to electrophoresis, and the tertiary structure is changed by the reaction between the allergen and the serum protein. This can be done by confirming whether serum protein is present. In order to perform such a diagnostic method, it is also possible to produce and use an automated diagnostic apparatus.

Example 1
Glutaraldehyde and toluene-2,4-diisocyanate are known to be allergens. On the other hand, 2,5-hexanedione is known to bind to proteins but is not allergenic. Human serum albumin and the above three chemicals (glutaraldehyde, toluene 2,4-diisocyanate (TDI), and 2,5-hexanedione) in phosphate buffer (pH 7.5) containing 50% dimethyl sulfoxide The sample was contacted for 24-48 hours, and the obtained sample was analyzed by SDS-PAGE and IEF. In addition, FIG. 1 shows the results of SDS-PAGE in which phthalic acid, phthalic anhydride, bisphenol A (BAP), bisphenol A diglycidyl ether (BADGE), and diaminodiphenyl ether were similarly treated. The results of IEF are shown in FIG.

  Albumin has a molecular weight of 66,000 and is stained as a single band (Lane 1) as shown in FIG. The sample treated with 2,5-hexanedione shows almost no change in the 66,000 band (Lane 2). It can be seen that the sample treated with glutaraldehyde and toluene-2,4-diisocyanate has a different migration pattern from untreated albumin (Lane 1) (Lane 3 and 10). From these results, allergens glutaraldehyde and toluene-2,4-diisocyanate react with albumin to change the tertiary structure of albumin, and 2,5-hexane, which is not allergenic. It can be seen that dione does not substantially change the tertiary structure of albumin even when it reacts with albumin. In addition, no change was observed in the electrophoretic pattern of the samples treated with phthalic acid, bisphenol A, and diaminodiphenyl ether, which are non-allergenic. On the other hand, in the sample treated with allergen-inducing substances phthalic anhydride, bisphenol A diglycidyl ether, and a mixture of bisphenol A diglycidyl ether and diaminodiphenyl ether, the appearance position of the band is different from untreated albumin (Lane 1). It was observed that the tertiary structure of was changed.

  In isoelectric focusing (FIG. 2), albumin gives several bands in the vicinity of isoelectric point 4.7 to 5.2 (Lane 1). Almost no change in the isoelectric point of albumin was observed in the sample treated with 2,5-hexanedione (lane 2). On the other hand, in the sample treated with glutaraldehyde and toluene-2,4-diisocyanate, it can be seen that a protein having an isoelectric point different from that of untreated albumin (Lane 1) was produced (Lane 3 and Lane 10). ). From these results, allergens glutaraldehyde and toluene-2,4-diisocyanate react with albumin to change the isoelectric point of albumin, and are not allergenic 2,5- It can be seen that hexanedione does not substantially change the isoelectric point of albumin even when it reacts with albumin. In addition, none of the samples treated with phthalic acid, bisphenol A, and diaminodiphenyl ether, which are non-allergenic, substantially changed the isoelectric point of albumin. On the other hand, samples treated with allergen-inducing substances phthalic anhydride, bisphenol A diglycidyl ether, and a mixture of bisphenol A diglycidyl ether and diaminodiphenyl ether produce proteins with various isoelectric points due to the tertiary structure change of albumin. It had been.

2 is a photograph showing the result of analyzing a sample by SDS-polyacrylamide gel electrophoresis after contacting a test substance and serum protein (human albumin) by the method of the present invention. Lane 1: Human albumin (HSA) 10 mg / ml, Lane 2: HSA 10 mg / ml + 2,5-hexanedione (10 mM), Lane 3: HSA 10 mg / ml + glutaraldehyde (10 mM), Lane 4: HSA 10 mg / ml + phthalic acid (10 mM), Lane 5: HSA 10 mg / ml + phthalic anhydride (10 mM), Lane 6: HSA 10 mg / ml + bisphenol A (10 mM), Lane 7: HSA 10 mg / ml + Bisphenol A diglycidyl ether (10 mM), Lane 8: HSA 10 mg / ml + diaminodiphenyl ether (10 mM), Lane 9: HSA 10 mg / ml + bisphenol A diglycidyl ether + diaminodiphenyl ether (each 10 mM), Lane 10: HSA 10 mg / ml + toluene 2,4-diisocyanate (10 mM), M: molecular weight marker (Nakarai) It is the photograph which showed the result of having analyzed the sample by isoelectric focusing after making the test substance and serum protein (human albumin) contact by the method of this invention. Lane 1: Human albumin (HSA) 10 mg / ml, Lane 2: HSA 10 mg / ml + 2,5-hexanedione (10 mM), Lane 3: HSA 10 mg / ml + glutaraldehyde (10 mM), Lane 4: HSA 10 mg / ml + phthalic acid (10 mM), Lane 5: HSA 10 mg / ml + phthalic anhydride (10 mM), Lane 6: HSA 10 mg / ml + bisphenol A (10 mM), Lane 7: HSA 10 mg / ml + Bisphenol A diglycidyl ether (10 mM), Lane 8: HSA 10 mg / ml + diaminodiphenyl ether (10 mM), Lane 9: HSA 10 mg / ml + bisphenol A diglycidyl ether + diaminodiphenyl ether (each 10 mM), Lane 10: HSA 10 mg / ml + toluene 2,4-diisocyanate (10 mM), M: IEF standard sample (Bio-Rad, 161-0310)

Claims (3)

A method for detecting an allergen, comprising the following steps:
(a) contacting the test substance with serum protein; and
(b) A method comprising a step of confirming by electrophoresis whether or not there is a serum protein having a tertiary structure changed by a reaction between a test substance and the serum protein. The method according to claim 1, wherein the serum protein is albumin. The method according to claim 1 or 2, wherein the electrophoresis is performed by SDS-polyacrylamide gel electrophoresis and / or isoelectric focusing.