JP2020159830A - Antigen peptide detection method - Google Patents

Abstract

To provide a method for detecting an antigen peptide capable of detecting an antigen peptide with high sensitivity.SOLUTION: Provided is an antigen peptide detection method in which a step (I) of performing the antigen-antibody reaction using a detection sample and a step (II) of reacting a detection sample obtained through the step (I) with a solid phase carrier and evaluating an antigen amount in the detection sample for each peptide are at least performed using the solid phase carrier to which the peptide is immobilized. Also provided is a kit for detecting the antigen peptide, which includes: the solid phase carrier to which the peptide is immobilized; and antibody which is subjected to antigen-antibody reaction with the detection sample.SELECTED DRAWING: None

Description

The present technology relates to a method for detecting an antigen peptide. More specifically, the present invention relates to an antigen peptide detection method capable of detecting an antigen peptide with high sensitivity, and a kit.

In recent years, consumer awareness of food safety and security has increased significantly, and food allergies are one example. The Food Sanitation Law recommends labeling that it contains raw materials that are prone to cause food allergies from the viewpoint of preventing the occurrence of health hazards due to food allergies of consumers. In particular, the seven "specific raw materials" for shrimp, crab, wheat, buckwheat, eggs, milk and peanuts, which are highly necessary to be labeled in consideration of the number and severity of food allergies, include these specific raw materials. Manufacturers are obliged to indicate the fact.

Under these circumstances, there is a demand for a technique for quickly and easily detecting allergens from the viewpoint of food maintenance and prevention of sudden accidents. Furthermore, considering the safety of protecting consumers, high accuracy is required for its detection.

As a method for detecting an allergen, for example, in Patent Document 1, a specific raw material is obtained by extracting a component in a food using sulfite, which is commonly used as a food additive, as a reducing agent for extracting the component from the food. Is disclosed as a method of detecting.

Further, for example, Patent Document 2 describes a screening method for a diagnostic agent for an egg allergic disease, which is recognized by IgE of a patient having an IgE specific to the allergen of the allergic disease to be diagnosed and exhibiting allergic symptoms. The amino acid sequence of the epitope to be diagnosed is compared with the amino acid sequence of the epitope recognized by the IgE of a patient having an IgE specific to the allergen of the allergic disease to be diagnosed and showing no allergic symptoms, and the amino acid sequence is compared. A method is disclosed that comprises selecting a polypeptide comprising.

Japanese Unexamined Patent Publication No. 2013-33062 JP-A-2007-217303

As mentioned above, some methods are known to those skilled in the art as prior art, but in order to allow consumers to consume food with greater peace of mind and to allow producers to more accurately grasp the condition of the product. , Further development of technology is desired.

Therefore, the main object of this technique is to provide an antigen peptide detection method capable of detecting an antigen peptide with high sensitivity.

That is, in the present technology, first, an antigen-antibody reaction is carried out using a detection sample using a solid phase carrier on which a peptide is immobilized, and a detection sample that has undergone the step (I) is used as the solid phase carrier. Provided is a method for detecting an antigen peptide, wherein at least the step (II) of evaluating the amount of antigen in the detection sample for each peptide is carried out.
In the method according to the present technology, the solid phase carrier may be a peptide array.
In addition, in the method according to the present technology, the peptide may contain an amino acid sequence constituting a milk protein.

The present technology also provides a kit for detecting an antigenic peptide, which comprises a solid-phase carrier on which the peptide is immobilized and an antibody that reacts with the detection sample.
In the kit according to the present technology, the solid phase carrier may be a peptide array.
Further, in the kit according to the present technology, the peptide may contain an amino acid sequence constituting a milk protein.

According to this technique, antigen peptides can be detected with high sensitivity.
The effect of the present technology is not necessarily limited to the effects described herein, and may be any of the effects described in the present disclosure.

It is a drawing substitute graph which shows the result of Example 1. FIG. It is a drawing substitute graph which shows the result of Example 2. FIG.

Hereinafter, a suitable mode for carrying out the present technology will be described.
It should be noted that the embodiments described below show typical embodiments of the present technology, and the scope of the present technology is not narrowly interpreted by this.

<1. Antigen peptide detection method>
The method according to the present technology is characterized in that at least step (I) and step (II) are carried out using a solid-phase carrier on which a peptide is immobilized.

(1) Solid-phase carrier on which a peptide is immobilized In this technique, the peptide is not particularly limited, but from the viewpoint of detecting an antigen derived from milk protein, which is one of the typical allergens, the amino acid sequence constituting milk protein is used. It is preferable to include it. Further, it is more preferable that the length is appropriate (preferably 15 to 20 residues) based on the amino acid sequence constituting the milk protein.

Milk protein can be, for example, considered to be associated with allergic diseases. Specifically, for example, one or more selected from the group consisting of α-lactalbumin, β-lactoglobulin, α _S1 -casein, α _S2 -casein, β-casein, and κ-casein can be mentioned. Be done. It is preferably one or more selected from the group consisting of α _S1 -casein, α _S2 -casein, β-casein, κ-casein, and β-lactoglobulin, and more preferably α _S1 -casein. , And one or more selected from the group consisting of β-lactoglobulin.

The peptide typically consists of a specific amino acid sequence and may be modified by known techniques, such as amino acid substitutions, deletions or additions. It is also possible to impart solubility and antigen-antibody reactivity suitable for various uses. The peptide can be produced by a known peptide synthesis method, for example, a method using a fully automatic peptide synthesizer, gene recombination with yeast, Escherichia coli, mammalian cells, or the like.

The peptide may optionally be in the form of a salt, preferably in the form of a physiologically acceptable acid addition salt. Such salts include salts of inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartrate). , Citrate, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) salts and the like.

In the present technology, the peptides are particularly preferably peptides having the amino acid sequences of SEQ ID NOs: 1 to 50 and / or peptides of SEQ ID NOs: 51 to 88, which are shown in Examples described later.

In the present art, the peptide is immobilized on a suitable solid phase carrier. As used herein, "immobilization" is a concept that includes linking, adsorbing, encapsulating, embedding, or supporting a peptide on a solid phase carrier. The material and shape of the solid phase carrier are not particularly limited as long as it is a carrier insoluble in a solvent in the reaction system of the antigen-antibody reaction, and a known solid phase carrier can be used. As the shape of the solid phase carrier, an appropriate shape may be selected according to the purpose of use, and examples thereof include a test plate shape, a bead shape, a spherical shape, a disc shape, a tube shape, and a filter shape. It is preferably a flat plate such as a test plate, a disk, or a filter. Further, as the material thereof, for example, it can be usually used as a carrier for immunoassay. Specifically, for example, synthetic resins such as polypropylene, polystyrene, and polyacrylamide, or those in which a reactive functional group such as a sulfonic acid group or an amino group is introduced by a known method, glass, polysaccharides, silica gel, etc. Examples include porous ceramics and metal oxides. More specifically, the solid-phase carrier is, for example, a cellulose membrane, a glass plate, a microwell plate, or the like.

The method for immobilizing the peptide on the solid phase carrier is not particularly limited, and known methods such as a physical adsorption method, a covalent bond method, an ionic bond method, and a cross-linking method can be used.

In the present technology, the peptide-immobilized solid-phase carrier is preferably in the form of a peptide array. In the present specification, the “peptide array” refers to a plurality of peptides adhered to the surface of a solid-phase carrier. Further, in the present technology, the solid phase carrier is more preferably a peptide array containing the amino acid sequences constituting the milk protein, and further preferably a peptide array covering the amino acid sequences constituting the milk protein, which will be described later. It is particularly preferable to use a peptide array consisting of peptides having the amino acid sequences of SEQ ID NOs: 1 to 50 or a peptide array consisting of the peptides of SEQ ID NOs: 51 to 88 shown in the examples.

If necessary, the solid phase carrier on which the peptide is immobilized may be treated to prevent non-specific adsorption on the structure. For this treatment, it is preferable to coat with a blocking agent so as not to lose the activity of the carried peptide. The blocking agent is not particularly limited, and examples thereof include serum proteins such as collagen, gelatin, skim milk, casein, and BSA. In addition, any compound that does not react with a protein and contains a hydrophobic portion and a hydrophilic portion can be used.

(2) Step (I)
Step (I) is a step of carrying out an antigen-antibody reaction using the detection sample.

The detection sample is not particularly limited, and may be a substance containing an antigen to be detected, a substance suspected of containing an antigen to be detected, or the like. Specifically, for example, meat, seafood, eggs, milk, grains, beans, potatoes, vegetables, wild vegetables, seaweed, seeds and seeds, fruits, herbs, and their processed foods, cosmetics, pharmaceuticals, for example, water supply. Examples include substances existing in the environment such as water, lake water, seawater, and sludge, and biological substances such as blood and biopsy.

Further, the detection sample may be derived from the above-mentioned substance and preliminarily pulverized, homogenized, separated, extracted, diluted or the like so as to be suitable for application to the method according to the present technology. ..

For example, when preparing a detection sample derived from food, the following operation is performed. The desired food is ground, homogenized and the extract is added. Then, after stirring with a vortex mixer or the like, the mixture is centrifuged. Then, the filtrate is collected by filtration. The collected filtrate is diluted with a diluent, if necessary. The solution thus obtained can be used as a detection sample. The extract used here may contain a reducing agent such as Na ₂ SO ₃ , a surfactant such as SDS, Tween 20, NP-40, and Triton X-100.

As used herein, the term "antigen" refers to any component capable of immunologically specific binding to an antibody. Further, in the present specification, the “antigen peptide” refers to a peptide capable of immunologically specific binding to an antibody. In the method according to the present technology, an antigen peptide, which is one of the typical allergens, is particularly targeted for detection. The antibody may be an antibody capable of recognizing and binding to a specific substance as an antigen, and examples thereof include IgG, IgM, IgA, IgD, and IgE, and fragments thereof. In addition, the antibody is produced by using a natural antibody such as a polyclonal antibody or a monoclonal antibody, a chimeric antibody that can be produced by using gene recombination technology, a humanized antibody or a single-stranded antibody, a human antibody-producing transgenic animal, or the like. It may be a human antibody or the like to be obtained.

The antibody used in step (I) is referred to herein as the "primary antibody". In step (I), an antigen-antibody reaction is carried out by contacting the detected sample with serum or the like containing an antibody and imparting conditions for causing an antigen-antibody reaction to these. The conditions for causing the antigen-antibody reaction are not particularly limited, and for example, the antigen-antibody reaction can be caused by adjusting the pH with an appropriate buffer solution and causing the reaction. In the step (I), the primary antibody may be reacted with the detection sample to promptly move to the step (II), or the detection sample may be allowed to wait for an appropriate time before moving to the step (II).

(3) Step (II)
In step (II), the detection sample that has undergone the step (I) is reacted with the solid-phase carrier, and the amount of antigen in the detection sample is evaluated for each peptide.

The detection sample that has undergone the step (I) is, that is, a detection sample that has undergone an antigen-antibody reaction using a primary antibody, and contains a complex composed of an antigen and an antibody, and a free antibody. In step (II), the free antibody is reacted with the peptide on the solid-phase carrier by reacting the detection sample with the solid-phase carrier on which the peptide is immobilized.

In step (II), the specific binding between the free antibody on the solid-phase carrier and the peptide that has reacted with the free antibody is then detected. The antigen-antibody reaction on the solid phase carrier can be detected by using, for example, a labeling substance usually used in an immunoassay. Examples of the labeling substance include fluorescent substances, luminescent substances, dyes, enzymes, coenzymes, radioisotopes and the like. Further, the labeling substance may be used by directly binding to the primary antibody or the secondary antibody, or may be indirectly used by using an antibody that recognizes the labeling substance, an avidin-biotin system, or the like. That is, in the present technique, a method of quantifying the primary antibody using the secondary antibody can be used, or a method of directly quantifying the labeled primary antibody (quantification method without using the secondary antibody) can be used. it can.

The method for evaluating the amount of antigen in the detection sample for each peptide is not particularly limited. For example, when the solid-phase carrier is a peptide array, strength information based on the labeling substance is provided for each peptide sequence constituting the peptide array. By obtaining it, the amount of the antigen is quantified. The intensity information can be acquired as the magnitude of a specific signal by using a detection device or the like according to the type of signal based on the labeling substance. The intensity information is acquired as, for example, the emission intensity, fluorescence intensity, etc. detected based on the labeling substance. Then, from this intensity information, information on the amount of antigen contained in the detection sample can be obtained. In the present specification, the “detection sample” in the “amount of antigen in the detection sample” refers to the detection sample before the step (I).

More specifically, the difference between the luminescence intensity measured using the detection sample and the luminescence intensity measured using the positive control is obtained, and the larger the difference, the higher the residual antigenicity. Can be evaluated as.

That is, since the positive control does not contain an antigen peptide, the peptide immobilized with the primary antibody on the solid-phase carrier reacts with the antigen-antibody, and the value of the luminescence intensity becomes the largest. On the other hand, when a detection sample is used, since the detection sample has undergone step (I), the more the detection sample contains the antigen peptide, the more the antigen peptide reacts with the primary antibody. Antigen-antibody reaction occurs. Therefore, in step (II), the more the antigen peptide is contained, the less the reaction with the immobilized peptide is performed, or the antigen-antibody reaction with the immobilized peptide is competitively inhibited, and the luminescence intensity is increased. The value becomes smaller. Therefore, when the emission intensity of the detection sample is subtracted from the emission intensity of the positive control and the difference is large, it means that the detection sample contains a large amount of antigen peptide. In addition, in this technology, since it is possible to evaluate each peptide using a solid-phase carrier on which a plurality of peptides are immobilized, which peptide in the detection sample is contained in a large amount as an antigen is quantitatively instantaneous. Can be grasped.

As described above, in the method according to the present technology, instead of the antigen-antibody reaction targeting the entire protein, the protein is divided into peptide units and the antigen-antibody reaction targeting each peptide is performed. Antigen detection sensitivity is dramatically improved as compared with the prior art. In addition, by performing a competitive immunoassay on a solid-phase carrier on which a peptide is immobilized, the antigen peptide present in the detection sample can be detected with high sensitivity and quantitatively for each sequence.

<2. Kit ＞
The present technology also provides a kit for detecting an antigen-peptide, which comprises a solid-phase carrier on which the peptide is immobilized and an antibody that reacts with the detection sample and an antigen-antibody. By performing the method according to the present technology using the kit according to the present technology, as described above, antigen can be detected quantitatively with high sensitivity for each peptide.

Various embodiments already described in the method according to the present technology can be applied to the kit according to the present technology.

Hereinafter, the present technology will be described based on examples.
It should be noted that the examples described below show an example of typical examples of the present technology, and the scope of the present technology is not narrowly interpreted by this.

<Peptide array>
The peptide arrays used in Examples 1 and 2 described later are prepared for two types of milk proteins, β-lactoglobulin and α _S1 -casein, respectively, which are shown in Table 1 below, SEQ ID NOs: 1 to 50 and SEQ ID NO: 51, respectively. The peptides described in ~ 89 were chemically synthesized, and a peptide array was prepared using a peptide solid phase synthesizer (manufactured by INTAVIS). A cellulose membrane was used as the solid phase carrier.

In Table 1 above, SEQ ID NOs: 1 to 50 are sequences in which the amino acid sequence of β-lactoglobulin is shifted by 3 residues by 16 residues from the N-terminal. In addition, SEQ ID NOs: 51 to 88 are sequences in which the amino acid sequence of α _S1 -casein is shifted by 5 residues by 16 residues from the N-terminal. Moreover, SEQ ID NO: 89 is a negative sequence having weak binding to an antibody.

<Example 1>
In Example 1, a solid-phase carrier (peptide array) on which the peptides of SEQ ID NOs: 1 to 50 shown in Table 1 above were immobilized was used for β-lactoglobulin.

[blocking]
The peptide array was blocked with a blocking agent at 37 ° C. for 1 hour with stirring.

[Detection sample pretreatment]
Four types of each detection sample (Conventional Infant Formula, Partially Hydrolyzed Formula, Extensively Hydrolyzed Formula 1, and Extensively Hydrolyzed Formula 2) were selected in 0.1% Tween / so that the protein content was in the range of 0.01 to 2%. It was dissolved in PBS, centrifuged at 3000 rpm at room temperature for 15 minutes, and the supernatant was filtered through a 45 μm filter.

[Step (I)]
Each detection sample (positive control is 0.1% Tween / PBS) was mixed 1: 1 with a 20,000-500,000-fold diluted primary antibody and pre-incubated at 37 ° C. for 1 hour. As the primary antibody, "anti-β-lactoglobulin, Rabbit-IgG antibody" was used.

[Step (II)]
The peptide array after blocking was washed and reacted with each detection sample that had undergone step (I) at 37 ° C. for 2 hours with stirring.

The peptide array after the reaction was washed and reacted with a 10,000-fold diluted secondary antibody at 37 ° C. for 1 hour with stirring. As the secondary antibody, "anti-Rabbit, HRP-labeled, Goat-IgG antibody" was used.

The peptide array after the secondary antibody reaction was washed, a luminescent substrate for detection was added, and the luminescence intensity was detected and quantified. As the scanner, a ChemiDoc imaging system manufactured by BioRad was used. For the numerical analysis, the Image Lab software attached to the same system was used.

The results of the antigenicity evaluation using the anti-β-lactoglobulin antibody of Example 1 are shown in FIG. In FIG. 1, the vertical axis shows the antigenicity score [-] for β-lactoglobulin, and the horizontal axis shows the peptide SPOT No. The antigenicity score [-] was calculated according to the following formula [Equation 1].

(However, the emission relative value is a value obtained by standardizing the average value of all SPOTs of the emission amount in the positive control as "1" and converting the emission amount of each SPOT as a relative value with respect to the standard value.)

In addition, a part of the data shown in FIG. 1 is excerpted, and the numerical values are shown in Table 2 below. The "β-LG antigenicity" shown in Table 2 below indicates the average value of the antigenicity scores of all SPOTs, and the inhibition rate [%] was calculated according to the following formula [Equation 2].

<Example 2>
In Example 2, for α _S1 -casein, a solid phase carrier (peptide array) in which the peptides of SEQ ID NOs: 51 to 89 shown in Table 1 above were immobilized was used, and as detection samples, Conventional Infant Formula, Partially Hydrolyzed Formula, The experiment was carried out in the same manner as in Example 1 except that three types of Extensively Hydrolyzed Formula 2) were used and "anti-casein, Rabbit-IgG antibody" was used as the primary antibody.

The results of the antigenicity evaluation by the anti-casein antibody of Example 2 are shown in FIG. In FIG. 2, the vertical axis shows the antigenicity score [-] for casein, and the horizontal axis shows the peptide SPOT No. The antigenicity score [-] on the vertical axis was calculated according to the above formula [Equation 1].

In addition, a part of the data shown in FIG. 2 is excerpted, and the numerical values are shown in Table 3 below. The “α _S1- CN antigenicity” shown in Table 3 below indicates the average value of the antigenicity scores of all SPOTs, and the inhibition rate [%] was calculated according to the above formula [Equation 2]. ..

<Comparative example 1>
Β-lacto for each detection sample (Conventional Infant Formula, Partially Hydrolyzed Formula, Extensively Hydrolyzed Formula 1, and Extensively Hydrolyzed Formula 2) using a general plate-type inhibition assay method (Inhibition ELISA method) The amount of residual antigen of globulin was measured.

[coating]
The antigen (β-lactoglobulin) was diluted with a solidified buffer (0.1 M sodium carbonate buffer) at 0.1 mg / ml, 100 μl was dispensed into a 96-well plate, and the mixture was allowed to stand at 37 ° C. for 2 hours to allow the antigen to stand. Fixed to the plate.

[Sample preparation]
(I) The inhibitor sample (milk powder) was dissolved in a washing buffer (0.05% Tween / PBS) so that the protein content was 1%, centrifuged at 3000 rpm for 15 minutes, and the supernatant was filtered through a 45 μm filter. ..
(Ii) The antibody (anti-β-lactoglobulin, Rabbit-IgG antibody) was diluted 10,000-fold with washing buffer.

[Primary antibody reaction (inhibition reaction)]
The immobilized plate is washed, the prepared sample (positive control is 0.05% Tween / PBS) and the antibody are mixed 1: 1 and 100 μl each is dispensed into the plate and allowed to stand at 37 ° C. for 1 hour. Reacted with.

[Secondary antibody reaction (anti-Rabbit, HRP labeling, Goat-IgG antibody)]
The plate after the primary antibody reaction was washed and reacted with the secondary antibody diluted 40,000 times at 37 ° C. for 45 minutes.

[Detection (luminescence reaction by enzyme)]
The plate after the secondary antibody reaction was washed, the luminescent substrate for detection was dispensed into the plate, and the reaction was carried out for 30 minutes under shading, and then the reaction was stopped with a reaction terminator solution (3MH ₂ SO ₄ ). The absorbance of each well (main wavelength: 492 nm, sub-wavelength: 630 nm) was measured with a plate reader.

The results of Comparative Example 2 are shown in Table 4 below. The inhibition rate [%] was calculated according to the following formula [Equation 3].

<Conclusion>
The antigenicity quantification value of Extensively Hydrolyzed Formula 2 was below the detection limit as shown in Table 4 in Comparative Example 1, whereas it could be quantified in peptide SPOT No. 27 as shown in Table 2 in Example 1. It was. Therefore, it was found that Example 1 was superior in detection sensitivity. Further, in Example 1, unlike Comparative Example 1, the antigenicity can be quantified for each peptide sequence, which makes it possible to evaluate the difference for each peptide sequence. Furthermore, from the results of Example 2, it was confirmed that the antigenicity evaluation for α _S1 -casein is also possible by this technique.

Claims (6)

Using a solid-phase carrier on which peptides are immobilized,
A step (I) of performing an antigen-antibody reaction using a detection sample, and a step (II) of reacting the detection sample that has undergone the step (I) with the solid-phase carrier and evaluating the amount of antigen in the detection sample for each peptide. ,
At least a method for detecting an antigen peptide. The method of claim 1, wherein the solid phase carrier is a peptide array. The method of claim 1 or 2, wherein the peptide comprises an amino acid sequence that constitutes a milk protein. A kit for detecting antigenic peptides
With a solid-phase carrier on which peptides are immobilized,
Antibodies that react with the detection sample
Including, kit. The kit according to claim 4, wherein the solid phase carrier is a peptide array. The kit according to claim 4 or 5, wherein the peptide contains an amino acid sequence constituting a milk protein.