JP2009288238A - Method of detecting fish egg protein with high sensitivity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of specifically detecting, with a high degree of accuracy and high sensitivity, fish egg protein of a particular type of fish egg such as salmon caviar contained in food. <P>SOLUTION: A method of specifically detecting, by means of a sandwich immunologic measurement method, fish egg protein of a particular type of fish egg such as salmon caviar contained in food includes the steps of: extracting fish egg protein; and using two types of antibody against the fish egg protein, the two types of antibody being the following combination (a) or (b): (a) a rabbit-derived antibody that serves as a primary antibody that turns into a solid phase and a rat-derived antibody that serves as a second antibody which binds to fish egg protein bonded to the primary antibody; or (b) a rat-derived antibody that serves as a primary antibody that turns into a solid phase and a rat-derived antibody that serves as a second antibody which binds to fish egg protein bonded to the primary antibody. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for measuring a specific protein such as an allergen protein in food. Specifically, the present invention relates to a method for measuring fish egg protein in food.

  In recent years, an increase in allergic diseases has become a social problem, and it has become clear that food-related allergies tend to increase not only in children but also in adults.

  Food allergy, which is one of allergic diseases, is a disease that causes allergic symptoms such as eczema, urticaria, diarrhea, discomfort in the oral cavity, and wheezing caused by food. Although it is predominantly seen in childhood, the number of patients is increasing in adults. Various causative foods have been reported, but some foods may cause severe anaphylactic shock, so caution is required. In patients with severe food allergies, anaphylactic shock can be caused even by licking, and it has been the case that such patients cannot eat conventional commercial foods unless they are informed.

  In other words, the conventional food labeling does not indicate raw materials that contain only trace amounts or processed food raw materials when processed foods are used as raw materials. I couldn't determine if it included food that I had to have. Thus, the range of food choices for patients has been narrowed, and this has had a significant impact on daily diet.

  In April 2001, the Ministry of Health, Labor and Welfare revised the Food Sanitation Law Enforcement Regulations in order to improve the situation and prevent the occurrence of consumer health hazards. As a result, the “labeling of foods containing allergens” system started, and after a one-year grace period, it was fully implemented in April 2002. Based on this system, the labeling of raw materials is obligatory for foods that may cause allergies (5 specified raw materials) or recommended (recommended 20 items according to specific raw materials).

  The salmon roe included in the recommended 20 items according to specific raw materials is mainly chum salmon eggs, but in recent years the number of fish allergy patients, mainly salmon roe, is increasing. In the 2005 nationwide monitoring survey for patients with immediate food allergies, fish eggs are the sixth cause of food (4% of total), peanuts (7th), fish (8th), It became clear that it was ranked higher than buckwheat (9th) and soybean (10th) (see Non-Patent Document 1). Further, as fish eggs other than salmon roe, it has been reported that tarako, which is an egg of the walleye pollack, tobiko, which is a flying fish, and the like cause food allergies (see Non-Patent Document 2).

  A fish egg consists of an egg membrane and egg yolk, and has no egg white. The egg yolk protein is derived from vitellogenin. Vitellogenin synthesized in the liver by estrogen is secreted into the blood and taken into the egg. The vitellogenin is cleaved and accumulated by a cathepsin D-like enzyme into a protein called β'-component which is an egg yolk protein peculiar to lipoviterin, phosvitin, and teleost. According to the examination using the serum of salmon allergic patients, it is considered that β'-component is a major allergen at least in salmon roe (see non-patent document 3).

  For proper labeling of foods that contain allergens, it is considered necessary to have a highly sensitive and specific method for detecting raw egg raw materials in various processed foods. There was no report on egg testing.

Takanari Imai et al., 57th Annual Meeting of Japanese Society of Allergology, Allergy, 56, 952, 2007 Watanabe Kazuhiko et al., Examination of usefulness of CAP-RAST for fish egg allergy, Allergy, 50, 309, 2001 Satoshi Nakamura, Yoji Iikura, latest food allergy, Nagai Shoten, Osaka, pp. 252-256, 2002

  It is an object of the present invention to provide a method capable of specifically and accurately detecting fish egg proteins of specific fish species including salmon roe contained in food.

  The present invention is an immunological method for specifically detecting fish protein of a specific fish species contained in foods with high accuracy and sensitivity, wherein antibodies are prepared by immunizing multiple types of animals. In addition to combining the obtained antibodies and adding a surfactant and a reducing agent to the buffer used to extract the sample, it is possible to construct a measurement system that is specific and sensitive to the specific fish species. Make it possible. Specifically, antibodies are prepared using, for example, rabbits and rats as immunized animals used for antibody production, and a sandwich ELISA system is constructed by combining rabbit antibodies and rat antibodies. Further, for example, sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) are added to the buffer solution used for sample extraction. By measuring the specimen thus obtained with the sandwich ELISA system, it was possible to improve specificity to a specific fish species and to detect fish egg protein with high sensitivity.

That is, the present invention is as follows.
[1] A method for specifically detecting a fish egg protein of a specific fish species contained in food by sandwich immunoassay, wherein the fish egg protein is extracted and two kinds of antibodies against the fish egg protein are extracted. Wherein the combination of the two types of antibodies is a combination of the following (a) or (b):
(a) The primary antibody to be immobilized is a rabbit-derived antibody, and the secondary antibody that binds to fish egg protein bound to the primary antibody is a rat-derived antibody;
(b) The primary antibody to be immobilized is a rat-derived antibody, and the secondary antibody that binds to fish egg protein bound to the primary antibody is a rat-derived antibody.
[2] The method of [1], which is an ELISA.
[3] The method according to [1] or [2], wherein the specific fish species is a salmonid.
[4] The method according to [1] or [2], wherein the specific fish species is a cod fish.

[5] The method according to any one of [1] to [4], wherein the fish egg protein is β′-component.
[6] The method according to any one of [1] to [5], wherein the secondary antibody is an enzyme-labeled antibody.
[7] Any one of [1] to [6], wherein a surfactant and a reducing agent are added to a buffer used for extraction of fish egg protein and / or a buffer used for antigen-antibody reaction. the method of.

[8] The surfactant is selected from the group consisting of sodium dodecyl sulfate, Tween 20 (Polyoxyethylene (20) sorbitan monolaurate), and Triton X-100 (Polyoxyethylene (10) octylphenyl ether), and the reducing agent is 2-mercaptoethanol, dithiotray. [7] The method according to [7], which is selected from the group consisting of Toll, sodium sulfite and Tris (2-carboxymethyl) phosphine.
[9] A fish egg protein comprising at least a carrier obtained by immobilizing a rabbit-derived or rat-derived anti-mushroom-derived β'-component antibody, a rat-derived anti-mushroom-derived β'-component antibody, and a surfactant and a reducing agent An ELISA kit for specifically detecting chum salmon fish egg protein contained in a food containing an extraction reagent.

[10] The ELISA kit according to [9], wherein the rat-derived anti-mushroom-derived β′-component antibody is an enzyme-labeled antibody.
[11] The surfactant is selected from the group consisting of sodium dodecyl sulfate, Tween 20 (Polyoxyethylene (20) sorbitan monolaurate) and Triton X-100 (Polyoxyethylene (10) octylphenyl ether), and the reducing agent is 2-mercaptoethanol, dithiotray [9] or [10] ELISA kit selected from the group consisting of Thor, sodium sulfite and Tris (2-carboxymethyl) phosphine.

  According to the method of the present invention, it is possible to specifically and accurately detect fish protein of specific fish species including salmon roe contained in food. In particular, an antibody against chum salmon β'-component (hereinafter, anti- (β'-component) antibody is referred to as a-β) is used as an antibody, and rat is used as a secondary antibody that binds to fish protein bound to the primary antibody. When the derived antibody is used, it is possible to specifically detect salmon roe contained in food.

It is a figure which shows the result of having prepared the extract from various fish eggs of chum salmon, walleye pollock, herring, Asaba flounder, Baba flounder, and caperin, and performing Western blot analysis using rabbit a-β. In addition to chum salmon, all fish species except herring were detected with components that showed immunological cross-reactivity with β'-component. It is a figure which shows the result of having constructed a sandwich ELISA system using unlabeled rabbit a-β as a primary antibody and using biotin-labeled rabbit a-β as a secondary antibody and measuring various fish egg extracts (YEx). . In this sandwich ELISA system, chum salmon YEx and walleye YEx showed significant reactivity, and the reactivity to the former was particularly high. On the other hand, Yex of Asaba flounder, Baba flounder and Caperin showed little reactivity. A sandwich ELISA system was constructed using unlabeled rat a-β as the primary antibody, unlabeled rabbit a-β as the secondary antibody, and HRP-labeled anti-rabbit IgG antibody as the tertiary antibody. It is a figure which shows the result of having measured the extract (YEx). In this sandwich ELISA system, among 6 types of YEx, it showed strong reactivity to chum salmon YEx, but almost no reactivity to other fish species was observed, but when the concentration of walleye pollock YEx reached 50 ng / ml or more. A clear reaction signal was observed. A sandwich ELISA system was constructed using unlabeled rabbit a-β as the primary antibody, unlabeled rat a-β as the secondary antibody, and HRP-labeled anti-rat IgG antibody as the tertiary antibody. It is a figure which shows the result of having measured the extract (YEx). In this sandwich ELISA system, chum salmon YEx alone showed strong reactivity in a concentration-dependent manner, indicating that the reactivity to other fish species including walleye pollock was extremely low. A sandwich ELISA system was constructed using unlabeled rabbit a-β as the primary antibody and HRP-Fab 'fragment type rat a-β as the secondary antibody, and the results of measuring various fish egg extracts (YEx) FIG. In this sandwich ELISA system, it was confirmed that the blank value was reduced while showing strong reactivity only with chum salmon YEx. We constructed a sandwich ELISA system using unlabeled rat a-β as the primary antibody and HRP-Fab 'fragment type rat a-β as the secondary antibody, and measured the results of various fish egg extracts (YEx). FIG. This sandwich ELISA system also showed high specificity for chum salmon YEx and reduced the blank value, but the sensitivity of this system was lower compared to the system using rabbit a-β as the primary antibody shown in FIG. . A sandwich ELISA system using rabbit a-β as the primary antibody and HRP-Fab 'fragment type rat a-β as the secondary antibody was constructed, and the fish egg extracts of six fish species (AEx) extracted with the reagent for extracting specific raw materials It is a figure which shows the result of having measured. The reaction signal of chum salmon AEx increased in a concentration-dependent manner, and a sufficiently strong signal was detected, but walleye pollack AEx was hardly detected in this concentration range. A sandwich ELISA system using rabbit a-β as the primary antibody and HRP-Fab ′ fragment type rat a-β as the secondary antibody was constructed, and AEx of various fish eggs extracted with the reagent for extracting specific raw materials was used. It is a figure which shows the result of having measured with the protein density | concentration which was only diluted 20 times according to a user's manual (namely, the sample which diluted the fish egg homogenate 400 times). In this case, no reaction signal was observed with AEx other than chum salmon. Six kinds of fish extracted from specific raw material extraction reagents by constructing sandwich ELISA system using unlabeled rat a-β as primary antibody and HRP-Fab 'fragment type rat a-β as secondary antibody It is a figure which shows the result of having measured an egg extract (AEx). Similar to the system using rabbit a-β as the primary antibody and the HRP-Fab ′ fragment type rat a-β as the secondary antibody described in FIG. It became clear that the sensitivity of was inferior to the system using rabbit a-β as the primary antibody. A sandwich ELISA system using rabbit a-β as the primary antibody and HRP-Fab 'fragment type rat a-β as the secondary antibody was constructed, and various extracts extracted with extraction reagents containing surfactant but not reducing agent The figure which shows the result of having measured AEx (-) of a fish egg with the high protein concentration (namely, the sample which diluted the fish egg homogenate 400 times) only diluted 20 times according to the instruction manual of the reagent for extraction of specific raw materials is there. In this case, all AEx (-) other than chum salmon also showed cross-reactivity.

Hereinafter, the present invention will be described in detail.
In the present invention, foods to be measured include foods containing animal-derived meat and processed foods containing plants. Examples of animals include animals belonging to mammals, birds, fish, crustaceans, mollusks and the like. In addition, foods containing milk and edible eggs are also targeted. Plants include rice grains, wheat such as wheat, miscellaneous grains such as buckwheat, beans such as soybeans, flours such as flour and soybean flour, starches, fruits, vegetables and the like.

  Processed foods include agricultural processed foods (Japanese standard product classification number 72, processed vegetable products, noodles / breads, processed cereal products, confectionery, etc.), livestock processed foods (73, meat) Products, dairy products, processed egg products, etc.), processed fishery products (74, processed seafood, processed seaweeds), other food products (75, seasonings and soups, cooked foods), beverages, ice and manufactured tobacco (No. 76, beverages not containing alcohol, etc.).

  The fish species to be examined include salmonid salmon (Ikura), codfish walleye (Tarako), herring family Herring (Kazunoko), flounder family Asaba-Kalei and Babakarei, cucumber family Kapelin and shishamo, flying fish The present invention measures the eggs of these fish species. In addition, the fish species here do not necessarily indicate taxonomic species (Species), and may be genera (Genus) or family (Family). That is, for example, in the case of salmonid fish, the whole species belonging to the salmonid family, the whole species belonging to the genus Salmonaceae (Ito, etc.), the whole species belonging to the genus Ivana (such as Iwana, Oshorokoma, Brook trout, etc.), Salmonidae salmo All species belonging to the genus (Atlantic salmon, brown trout, etc.) and all species belonging to the Salmonidae salmon genus (chum salmon (salmon), rainbow trout, cherry salmon, calaft trout, coho salmon, sockeye salmon, king salmon, etc.) are targeted. Similarly, the cod family includes the genus Komai, Walleye genus, Madara, Minamidara, Lanikeps, Arctogadus, Boreogadus, Gadiculus, Melanogrammus, Merlangius, Microgadus, Polllachius, Trisopterus, All species belonging to the above, and all species belonging to each of the above genera are targeted. Among these, chum salmon, rainbow trout, cherry salmon, calaft trout, coho salmon, sockeye salmon, and king salmon fish are preferred, and chum salmon fish are particularly preferred.

  It does not specifically limit as a fish egg protein used as a measuring object, All proteins contained in a fish egg can be used as a target. Examples thereof include proteins that can be allergens for humans. Examples of fish egg proteins that can be allergens for humans include β'-component derived from vitrogenin contained in the egg yolk of fish eggs, lipoviterin, phosvitin, and the like. Among these, β′-component is preferable.

Protein is extracted from the food to be measured and measured. Protein extraction is performed by homogenizing food using a blender, homogenizer, food cutter, etc., adding an extraction reagent, and allowing it to stand for several to tens of hours or shaking. The buffer used for the extraction reagent is not limited. For example, a Tris buffer or phosphate buffer may be used, and it may further contain serum albumin such as NaCl or bovine serum albumin (hereinafter referred to as BSA). . In the present invention, such extraction reagents include sodium dodecyl sulfate (SDS), Tween20 (registered trademark) (Polyoxyethylene (20) sorbitan monolaurate), Triton X-100 (registered trademark) (Polyoxyethylene (10) octylphenyl). Add surfactants such as ether) and reducing agents such as 2-mercaptoethanol (2-ME), dithiothreitol (DTT), sodium sulfite (Na ₂ SO ₃ ), Tris (2-carboxymethyl) phosphine Is preferred. By adding a surfactant and a reducing agent, fish egg protein can be measured in a fish species-specific manner. Specified raw material extraction reagent marketed as an allergen extraction reagent (Morinaga Bioscience Institute, FASPEK / Extraction Reagent For Specified Ingredient, Watanabe Y. et al., Journal of Immunological Methods, 300, 115-123, 2005. Composition) : Tris buffer containing BSA, SDS and 2-ME). The concentration of the surfactant contained in the extraction reagent is 0.2 to 2%, preferably 0.5 to 1%, and the concentration of the reducing agent is 1 to 10%, preferably 2 to 7%. As the surfactant, SDS is preferable, and as the reducing agent, 2-ME, sodium sulfite and the like are preferable. In protein extraction, 5 to 50 mL, preferably 10 to 30 mL, and more preferably 15 to 25 mL of extraction reagent may be added to 1 g of food. After extraction, the supernatant is collected by centrifugation and the resulting supernatant is used for measurement. At this time, the supernatant is preferably filtered using filter paper.

  The protein in the supernatant may be measured by an immunoassay using an antibody against the protein to be measured. For example, when the protein to be measured is β′-component, an anti- (β′-component) antibody may be used. In the present invention, in order to increase the specificity and sensitivity of a specific fish species to the fish egg protein, an immunized animal used for antibody production is selected, and in some cases, a plurality of immunized animals are used in combination. Examples of immunized animals used for antibody production include horses, monkeys, dogs, pigs, cows, goats, sheep, rabbits, rats, guinea pigs, hamsters, mice and other mammals, and birds such as pigeons and chickens. Not. Any method can be used for immunization, but it is preferable to inject the antigen mainly intravenously, subcutaneously or intraperitoneally. Further, the immunization interval is not particularly limited, and immunization is performed 2 to 10 times at intervals of several days to several weeks, preferably at intervals of 1 to 3 weeks. The antigen can be purified from fish eggs by a known method. For example, β′-component can be used after purification from chum salmon fish.

  Blood is collected from an immunized animal with an increased antibody titer, and the obtained serum is allowed to stand at room temperature or 4 ° C., and then centrifuged to obtain a serum containing polyclonal antibodies. The obtained serum containing the polyclonal antibody can be used as it is, but known methods such as ammonium sulfate salting out, ion exchange chromatography, affinity chromatography using a carrier on which protein A, protein G, etc. are immobilized, molecular sieve chromatography, etc. It is preferable to purify the antibody fraction by appropriately selecting these methods or by combining them.

  In the present invention, a sandwich immunoassay system is constructed using the thus prepared polyclonal antibody. Sandwich immunoassays include sandwich radioimmunoassay (RIA), sandwich enzyme immunoassay (EIA), sandwich fluorescence immunoassay (FIA), sandwich luminescence immunoassay (CLIA), sandwich All sandwich immunoassay methods such as luminescent enzyme immunoassay (CLEIA method) and immunochromatography based on the sandwich method can be mentioned. Among these, ELISA (Enzyme-Linked ImmunoSorbent Assay) which is an EIA method is preferable. The ELISA will be described below, but other sandwich immunoassays are the same in that the antigen to be measured is sandwiched between two types of antibodies, and those skilled in the art can construct a system as appropriate. An unlabeled polyclonal antibody is immobilized on a carrier such as a 96-well microtiter plate or polystyrene beads as a capture antibody (primary antibody). As the detection antibody (secondary antibody), an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), a fluorescent substance such as FITC, or a polyclonal antibody labeled with biotin or the like is used. In this case, an enzyme or the like can be labeled on the Fab ′ fragment obtained by digesting the antibody molecule with pepsin. Alternatively, after reacting with an unlabeled secondary antibody, an antibody that specifically binds to the secondary antibody labeled with an enzyme, a fluorescent substance or the like can be used as a tertiary antibody for detection. A polyclonal antibody prepared from various immunized animals as described above is used as a primary antibody or a secondary antibody, and a sandwich ELISA is performed according to a usual method to select a polyclonal antibody having high specificity for the protein to be measured. At this time, the immunized animal of the primary antibody and the secondary antibody may be the same or different. When a polyclonal antibody labeled with biotin is used, it may be affected by biotin present in fish eggs. In this case, an antibody not labeled with a secondary antibody is used instead of an antibody labeled with biotin as a secondary antibody, and a labeled antibody that binds to the secondary antibody as a tertiary antibody (for example, the secondary antibody is a rabbit antibody). In this case, an enzyme-labeled anti-rabbit IgG antibody) may be used, or an antibody against fish egg protein or a functional fragment such as Fab ′ thereof labeled with an enzyme may be used as a secondary antibody.

  Moreover, the surfactant used for the antigen-antibody reaction may contain a surfactant and a reducing agent derived from the extraction reagent. In this case, the concentration of the surfactant is 0.01 to 0.1%, preferably 0.025 to 0.05%, and the concentration of the reducing agent is 0.05 to 0.5%, preferably 0.1 to 0.35%. As the surfactant, SDS is preferable, and as the reducing agent, 2-ME, sodium sulfite and the like are preferable. That is, in the measurement method of the present invention, a surfactant and a reducing agent are added to a buffer solution used for extraction of fish egg protein and / or a buffer solution for performing an antigen-antibody reaction.

  In this way, antibodies are prepared by immunizing a plurality of types of animals, the obtained antibodies are combined, and a surfactant is added to a buffer used for sample extraction and / or a buffer used for antigen-antibody reaction. By adding a reducing agent, it is possible to specifically and accurately detect the egg protein of a specific fish species such as salmonaceae and cod family contained in the processed food.

  In one embodiment of the present invention, chum salmon fish egg protein in food is specifically measured. In the measurement, as a combination of a primary antibody and a secondary antibody immobilized on an ELISA titer plate, a combination of a rabbit anti-Chum salmon β′-component antibody and a rat anti-Chum salmon β′-component antibody or a rat anti-Chum salmon β′-component A combination of an antibody and a rat anti-chumy salmon β'-component antibody can be mentioned. In the former combination, an unlabeled antibody may be used as the secondary antibody, a labeled anti-rat IgG antibody may be used as the tertiary antibody, or any rat anti-chum salmon β′-component antibody labeled as the secondary antibody may be used. It can also be applied to combinations. In this case, for example, chum salmon fish eggs can be specifically detected, and no walleye pollack, herring, red flounder, baba flounder and caperin roe are detected.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Preparation of various antibodies (1) Preparation of rabbit and rat anti- (β′-component) antibodies β′-component was purified from chum salmon and immunized to rabbits by a conventional method. The obtained antiserum was absorbed by purified lipoviterin to increase the specificity for β′-component. This antiserum was applied to a HiTrap Protein A HP column (manufactured by GE Healthcare) to purify the IgG fraction, and rabbit anti- (β'-component) antibody (hereinafter, anti- (β'-component) antibody was -β).
Further, β′-component purified from chum salmon was immunized to rats by a conventional method, and the obtained antiserum was applied to a HiTrap Protein G HP column to purify the IgG fraction to obtain rat a-β.

(2) Preparation of labeled antibody Rabbit a-β was biotinylated with Sulfo-NHS-LC-Biotin (PIERCE). Unreacted biotin was removed using a desalting PD-10 column (GE Healthcare) to obtain biotin-labeled rabbit a-β.

Further, rat a-β was treated with pepsin to prepare an F (ab ′) ₂ fragment and labeled with horseradish peroxidase (HRP) by the maleimide method to obtain an HRP-Fab ′ fragment.

Example 2 Examination of Specificity by Western Blot Analysis Extracts were prepared from various fish eggs of chum salmon, walleye pollock, herring, Asaba flounder, Baba flounder and caperin, and subjected to SDS-polyacrylamide gel electrophoresis. The protein in the gel was transferred to a PVDF membrane, and Western blot analysis was performed using various antibodies.

  When rabbit a-β is used, all fish species, except chum salmon and herring, show β'-component (in Fig. 1, a band with a molecular weight of about 20kD or less. The molecular weight varies depending on the fish species) and shows immunocrossing properties. Components were detected. That is, it was suggested that this antibody may be reactive with fish protein of a wide range of fish species (FIG. 1).

Example 3 Preparation of Various Fish Egg Extracts (1) YEx Preparation 0.5 M NaCl, 20 mM Tris-HCl (pH 8.0) was added and homogenized to various fish eggs of chum salmon, walleye pollock, herring, Asaba flounder, Baba flounder and caperin. Thereafter, the extract obtained by removing insolubles by centrifugation (20,000 × g, 15 minutes) was designated as YEx. The protein concentration of the obtained YEx was measured with a Protein assay rapid kit (manufactured by Wako Pure Chemical Industries, Ltd.).

(2) Preparation of AEx Various fish eggs such as chum salmon, walleye pollock, herring, Asaba flounder, Baba flounder and caperin were homogenized and weighed 1 g, and a specific raw material extraction reagent containing a high concentration of surfactant and reducing agent (Mao Nagamori) 19 mL) was added and dispersed well, followed by extraction with shaking at room temperature for 12 hours. The obtained extract was centrifuged at 3,000 × g for 20 minutes, and the supernatant was filtered to obtain AEx. The protein concentration of the obtained AEx was measured by 2-D Quant Kit (manufactured by GE Healthcare).

Example 4 Sandwich ELISA with Rabbit a-β
Unlabeled rabbit a-β as a primary antibody was immobilized on a 96-well microtiter plate, blocked with a buffer containing BSA, and various fish egg extracts (YEx) were added. After incubating at 25 ° C. for 60 minutes, the wells were washed, and biotin-labeled rabbit a-β was added as a secondary antibody. After incubating at 25 ° C. for 60 minutes, the wells were washed, and Streptavidin-HRP conjugate (manufactured by Tagoimmunologicals) was added. After incubating at 25 ° C. for 60 minutes, the wells were washed, a coloring substrate (3, 3 ′, 5, 5′-tetramethylbenzidine, hereinafter referred to as TMB) was added and incubated for 20 minutes. The enzyme reaction was stopped by adding 1 mol / L sulfuric acid, and the absorbance at 450 nm was measured with a plate reader.

  In this sandwich ELISA system, chum salmon YEx and walleye YEx showed significant reactivity, and the reactivity to the former was particularly high (FIG. 2). On the other hand, Yex of Asaba flounder, Baba flounder and Caperin showed little reactivity. The western blot analysis of Example 2 found the presence of a protein band that reacts with rabbit a-β in all YEx except herring, so detection of fish egg protein by sandwich ELISA using rabbit a-β Therefore, it was considered that the difference in reactivity between fish species was larger than that in Western blot analysis using the same antibody.

  In addition, it has been reported that biotin is present in fish eggs, and it is known that biotin is often present in a state bound to a protein. Therefore, in order to examine the effect of fish egg-containing biotin in this sandwich ELISA system, YEx of chum salmon and walleye pollock was subjected to debiotin treatment and then subjected to measurement. As a result, it was confirmed that the reactivity of both chum salmon YEx and walleye YEx was significantly reduced (results omitted). Furthermore, it was also revealed that when both YEx were treated with debiotin, the protein recovery rate decreased to about 85%. These results suggest the presence of biotin bound to β'-component in YEx. That is, the reaction signal of chum salmon YEx and walleye YEx obtained in this sandwich ELISA system seems to be influenced by coexisting biotin.

Example 5 Sandwich ELISA with rat a-β and rabbit a-β
Aiming to improve fish species selectivity and eliminate the influence of biotin in fish eggs, primary antibody and secondary antibody are combined with rabbit and rat a-β, and HRP-labeled anti-rabbit (or rat) IgG antibody as the third antibody A sandwich system was constructed using

(1) When rat a-β is used as a primary antibody Unlabeled rat a-β as a primary antibody is immobilized on a 96-well microtiter plate, blocked with a buffer containing BSA, and various fish egg extracts ( YEx) was added. After incubating at 25 ° C. for 60 minutes, the wells were washed, and unlabeled rabbit a-β was added as a secondary antibody. After incubating at 25 ° C. for 60 minutes, the wells were washed, and an HRP-labeled anti-rabbit IgG antibody (manufactured by Bio-Rad) was added as a tertiary antibody. After incubating at 25 ° C. for 60 minutes, the wells were washed, and a chromogenic substrate (TMB) was added and incubated for 20 minutes. The enzyme reaction was stopped by adding 1 mol / L sulfuric acid, and the absorbance at 450 nm was measured with a plate reader.

  This sandwich ELISA system showed strong reactivity to chum salmon YEx among 6 types of YEx, and the reaction signal (absorbance) was also concentration-dependent (FIG. 3). On the other hand, there was almost no reactivity to other fish species, but a clear reaction signal was observed when the concentration of Alaska pollock YEx was 50 ng / ml or higher, and the intensity of 1/2 of chum salmon YEx was observed at 1,000 ng / ml. Reached.

(2) When rabbit a-β is used as a primary antibody Unlabeled rabbit a-β as a primary antibody is immobilized on a 96-well microtiter plate, blocked with a buffer containing BSA, and then extracted with various fish egg extracts ( YEx) was added. After incubating at 25 ° C. for 60 minutes, the wells were washed, and unlabeled rat a-β was added as a secondary antibody. After incubating at 25 ° C. for 60 minutes, the wells were washed, and an HRP-labeled anti-rat IgG antibody (manufactured by Calbiochem) was added as a tertiary antibody. After incubating at 25 ° C. for 60 minutes, the wells were washed, and a chromogenic substrate (TMB) was added and incubated for 20 minutes. The enzyme reaction was stopped by adding 1 mol / L sulfuric acid, and the absorbance at 450 nm was measured with a plate reader.

  In this sandwich ELISA system, only chum salmon YEx showed strong reactivity in a concentration-dependent manner, and it was revealed that reactivity to other fish species including walleye pollock was extremely low (FIG. 4).

  The above results show that a fish-specific fish egg detection method can be established by constructing a sandwich ELISA by combining two types of a-βs obtained from different organisms.

Example 6 Sandwich ELISA using HRP-Fab ′ fragment as secondary antibody
Example 5 shows that a fish-specific sandwich ELISA system can be established by using rabbit a-β as the primary antibody, rat a-β as the secondary antibody, and HRP-labeled anti-rat IgG antibody as the tertiary antibody. However, in order to put this system into practical use, it was necessary to suppress the complexity of the operation due to the use of three types of antibodies and the increase in blank value (related to improvement in detection accuracy). Therefore, in order to improve these problems, it was decided to construct an assay system using HRP-Fab ′ fragment type a-β as the secondary antibody.

(1) Reactivity with YEx Unlabeled rabbit a-β as a primary antibody was immobilized on a 96-well microtiter plate, blocked with a buffer containing BSA, and various fish egg extracts (YEx) were added. . After incubating at 25 ° C. for 60 minutes, the wells were washed, and HRP-Fab ′ fragment type rat a-β was added as a secondary antibody. After incubating at 25 ° C. for 60 minutes, the wells were washed, and a chromogenic substrate (TMB) was added and incubated for 20 minutes. The enzyme reaction was stopped by adding 1 mol / L sulfuric acid, and the absorbance at 450 nm was measured with a plate reader.

  In this sandwich ELISA system, it was confirmed that the blank value was reduced to about 1/3 while maintaining the characteristic of the system of Example 5 (2), which showed strong reactivity only to chum salmon YEx ( FIG. 5). In the walleye pollock YEx, a reaction signal appeared from about 100 ng / ml, and a clear reaction signal was obtained at 1000 ng / ml, but this was weak enough to be observed with 2 ng / ml chum salmon YEx. . That is, this system was determined to be a system that suppressed the improvement in operability, which is a drawback of the system of Example 5 (2), and the increase in blank value (related to the improvement in detection accuracy). That is, it was confirmed that the introduction of HRP-Fab ′ fragment type rat a-β is extremely effective for the construction of a chum salmon fish egg (Ikura) detection system that can obtain a simpler and more stable measurement value.

  Subsequently, various fish egg extracts (YEx) were measured using a system in which unlabeled rat a-β was immobilized as a primary antibody and HRP-Fab ′ fragment type rat a-β was used as a secondary antibody. This sandwich ELISA system also showed high specificity for chum salmon YEx and reduced blank values (FIG. 6). However, the sensitivity of this system is considerably lower than that of the system using rabbit a-β as the primary antibody, and the system using rabbit a-β as the primary antibody is more suitable for constructing a practical measurement system. It was thought that.

(2) Reactivity with AEx Rabbit a-β is the primary antibody of (1) above in the range of 1.6 to 1,000 ng / ml of 6 fish egg extracts (AEx) extracted with the reagent for extracting specific raw materials. Then, it was subjected to a system using HRP-Fab ′ fragment type rat a-β as a secondary antibody, and its effect and fish species selectivity were evaluated. As a result, the reaction signal of chum salmon AEx increased in a concentration-dependent manner, and a sufficiently strong signal was detected (FIG. 7). Furthermore, walleye pollack AEx was hardly detected in the above concentration range. Based on this result, each AEx extracted with the reagent for extracting specific raw materials remains at a high protein concentration (ie, a sample obtained by diluting the fish egg homogenate 400 times) according to the reagent's instructions. When subjected to the system (FIG. 8), no reaction signal was observed with AEx other than chum salmon.

  The above results indicate that a specific raw material extraction reagent containing a surfactant and a reducing agent can be used in this sandwich ELISA system, and that the use of such an extraction reagent makes it possible to select fish species for this system such as salmon roe ( It shows that it contributes to improvement of specificity.

  Furthermore, in the system using unlabeled rat a-β as a primary antibody and HRP-Fab ′ fragment type rat a-β as a secondary antibody, 6 fish species extracted with specific raw material extraction reagents Egg extract (AEx) was measured. As a result, similar to the system using rabbit a-β as the primary antibody and HRP-Fab ′ fragment type rat a-β as the secondary antibody, it showed extremely high specificity only for chum salmon AEx. However, as with the YEx measurement in (1) above, it was revealed that the sensitivity of this system was significantly inferior to the system using rabbit a-β as the primary antibody (FIG. 9).

  Patients with food allergies may present symptoms even with a very small amount of allergen intake, so the sensitivity of foods that cause allergy is required to be high enough to detect several mg / kg or less (Poms RE et al. , Food Addit Contam, 21, 1-31, 2004). When extracting protein in food according to the instruction manual for the reagent for extracting specific raw materials, extract by adding 19 mL of extraction reagent to 1 g of food (ie, 20-fold dilution), and the resulting extract Is further diluted 20 times and subjected to ELISA measurement, the sample at the time of measurement is diluted 400 times. In this case, in order to detect a protein contained in several mg / kg in the food to be examined, it is necessary to have a sensitivity capable of sufficiently measuring a protein diluted to several tens of ng / mL at the time of measurement. From this point of view, in the system using HRP-Fab 'fragment type rat a-β as the secondary antibody, the use of rabbit a-β as the primary antibody is the first to show practical high sensitivity. It is considered to be.

Comparative Example 1 Examination Using Extraction Reagent Containing Only Surfactant According to the investigation using AEx in Example 6 (2), the use of an extraction reagent containing a surfactant and a reducing agent is a fish species selectivity (specific However, it is widely known that the use of surfactants suppresses nonspecific reactions in immunoassays. Therefore, the fish species selectivity when using an extraction reagent containing only a surfactant and no reducing agent was examined.

  In this study, an extraction reagent (including a surfactant) obtained by removing a reducing agent component from a reagent for extracting a specific raw material (manufactured by Morinaga Bioscience Institute) was used. After homogenizing various salmon roe, walleye pollock, herring, sea bream flounder, caperin fish eggs, weigh 1 g, and after adding 19 mL of extraction reagent (without reducing agent) containing surfactant, well dispersed The mixture was extracted with shaking at room temperature for 12 hours. The obtained extract was centrifuged at 3,000 × g for 20 minutes and the supernatant was filtered. The filtrate obtained was designated as AEx (−). This ELISA system (primary sample) with 6 types of AEx (-) at a high protein concentration (ie, a sample diluted 400-fold with fish egg homogenate) that is 20-fold diluted according to the instructions for using the reagent for extracting specific raw materials When subjected to a system using rabbit a-β as an antibody and HRP-Fab ′ fragment type rat a-β as a secondary antibody) (FIG. 10), a strong reactivity was observed with chum salmon AEx (−). Furthermore, it was found that cross-reactivity was observed to some extent in other fish species.

  From the above results, the improvement of fish species selectivity (specificity) for the salmon roe of this system is not simply due to the non-specific reaction suppression effect of surfactants as conventionally known. It was thought to be based on a novel mechanism in which cross-reactivity was suppressed by combining reducing agents.

Claims (11)

A method for specifically detecting a fish egg protein of a specific fish species contained in food by sandwich immunoassay, wherein the fish egg protein is extracted and two kinds of antibodies against the fish egg protein are used, The combination of the two types of antibodies is the following combination (a) or (b):
(a) The primary antibody to be immobilized is a rabbit-derived antibody, and the secondary antibody that binds to fish egg protein bound to the primary antibody is a rat-derived antibody;
(b) The primary antibody to be immobilized is a rat-derived antibody, and the secondary antibody that binds to fish egg protein bound to the primary antibody is a rat-derived antibody.   The method of claim 1 which is an ELISA.   The method according to claim 1 or 2, wherein the specific fish species is a salmonid.   The method according to claim 1 or 2, wherein the specific fish species is a cod fish.   The method according to any one of claims 1 to 4, wherein the fish egg protein is β'-component.   The method according to any one of claims 1 to 5, wherein the secondary antibody is an enzyme-labeled antibody.   A surfactant and a reducing agent are added to a buffer solution used for extraction of fish egg protein and / or a buffer solution when performing an antigen-antibody reaction, according to any one of claims 1 to 6, Method.   The surfactant is selected from the group consisting of sodium dodecyl sulfate, Tween 20 (Polyoxyethylene (20) sorbitan monolaurate) and Triton X-100 (Polyoxyethylene (10) octylphenyl ether), and the reducing agent is 2-mercaptoethanol, dithiothreitol, sulfite. 8. The method of claim 7, wherein the method is selected from the group consisting of sodium and Tris (2-carboxymethyl) phosphine.   A fish egg protein extraction reagent comprising at least a carrier obtained by immobilizing a rabbit-derived or rat-derived anti-mushroom-derived β'-component antibody, a rat-derived anti-mushroom-derived β'-component antibody, and a surfactant and a reducing agent A kit for ELISA for specifically detecting chum salmon fish egg protein contained in foods containing.   10. The ELISA kit according to claim 9, wherein the rat-derived anti-Chum salmon-derived β′-component antibody is an enzyme-labeled antibody.   The surfactant is selected from the group consisting of sodium dodecyl sulfate, Tween 20 (Polyoxyethylene (20) sorbitan monolaurate) and Triton X-100 (Polyoxyethylene (10) octylphenyl ether), and the reducing agent is 2-mercaptoethanol, dithiothreitol, sulfite. The ELISA kit according to claim 9 or 10, wherein the ELISA kit is selected from the group consisting of sodium and Tris (2-carboxymethyl) phosphine.

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