JP2002040027A - Immunological measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method of pathogenic bacteria or pathogenic viruses in a fishery product, poisons produced therefrom, or antibodies against them, characterized by executing a reaction between the pathogenic bacteria, the pathogenic viruses, the poisons produced therefrom, or the antibodies against them and a test body in the presence of a milk protein, in the immunological measuring method of the pathogenic bacteria or the pathogenic viruses existing in body fluid of the fishery product, the poisons produced therefrom, or the antibodies against them. SOLUTION: The pathogenic bacteria or the pathogenic viruses existing in the body fluid of the fishery product, the poisons produced therefrom, or the antibodies against them can be measured accurately by this immunological measuring method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for accurately measuring pathogenic bacteria, pathogenic viruses, toxins produced therefrom, or toxins produced therefrom, or antibodies thereto, in a body fluid of fish and shellfish.

[0002]

BACKGROUND OF THE INVENTION Infectious diseases caused by the pinated rod DNA virus (PRDV) are:
Since the spread of prawns and seedlings imported from China in 1993 in Japan, they have seriously affected shrimp aquaculture and seedling industries. This infection has a very high mortality rate and has been shown to cause rapid mass death in a short period of time (Nakano et al., 1994).

[0003] As a method for detecting the virus, dark field microscopy, electron microscopic observation by negative staining, PCR, in-situ hybridization and Western blot have been reported to date. In the method, virus particles in shrimp epithelial cells (gastric epithelial cells) and body fluids (hemolymph) are examined by dark-field microscopy or PCR.

[0004] However, the above method has a problem that technical skill is required because special equipment and biochemical experimental techniques are used. In addition, since the facilities that can perform the inspection are limited, a quick and simple inspection method that can be used even in a farming field or the like has been demanded.

On the other hand, the present inventors have proposed PRDV
To develop an antibody with high specificity for PRDV, and to propose a method to detect PRDV quickly and easily without limiting the working environment by using immunological agglutination particles to which the antibody is adsorbed or bound. (Japanese Patent Laid-Open No. 11-2791)
No. 98). By using this method, it was clarified that PRDV in gastric epithelial cells, which is one of the sites of infection, can be accurately detected.

However, when hemolymph is used as a sample without dilution, PRDV cannot be specifically detected because some factors in the hemolymph and the above-mentioned agglutination particles aggregate non-specifically. Was. Therefore, it is necessary to dilute the sample to such an extent that aggregation does not occur non-specifically (10000 times or more). Therefore, the virus concentration is also reduced by dilution, so that the number of detectable samples is limited or accurate. Problems such as inability to make a judgment have occurred.

[0007] Accordingly, an object of the present invention is to provide a fish and shellfish pathogenic bacterium and pathogen typified by PRDV accurately without non-specific aggregation even when a fish lymphatic fluid is used as a specimen. An object of the present invention is to provide an immunological assay capable of detecting a sexual virus, a toxin produced by the virus, and an antibody thereof.

[0008]

Means for Solving the Problems Accordingly, the present inventors have conducted intensive studies for the purpose of specifically detecting viruses and the like present in body fluids of fish and shellfish without being affected by nonspecific agglutination. As a result, the non-specific reaction factors in the body fluid of the prawn were about 670 kDa and about 142 kD.
a, at least three types of proteins of about 30 kDa, and milk proteins do not react with these factors in the body fluids of fish and shellfish among various components. The present inventors have found that the immunological measurement of viruses and bacteria can be accurately performed because a reactive reaction can be prevented, and have completed the present invention. That is, the present invention provides an immunoassay for a pathogenic bacterium, a pathogenic virus, a toxin produced by them, or an antibody against them, which is present in a body fluid of a fish or shellfish. A method for assaying pathogenic bacteria and pathogenic viruses of fish and shellfish, toxins produced by them, and antibodies to them, characterized in that the reaction between a produced toxin or an antibody against them and a sample is performed in the presence of milk protein. Is what you do.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The assay method of the present invention relates to the determination of pathogenic bacteria, pathogenic viruses, toxins produced by them (hereinafter collectively referred to as antigens), or antibodies against them, in the body fluid of fish and shellfish. Any method may be used as long as it is an immunological measurement method for reacting the sample with a sample. For example, the sample may be fish or shellfish blood, lymph, tissue, or the like, or may be used after homogenization. More specifically, an immunoassay for detecting a pathogenic bacterium, a pathogenic virus, or a toxin produced by the specimen using an antibody specific thereto, or an infection with a pathogenic bacterium or a pathogenic virus. And immunoassays for detecting an antibody whose production has been promoted in a body fluid by using an antigen of the antibody (pathogenic bacteria, pathogenic viruses, toxins produced by the antibody).

The immunoassay includes, for example, immunoagglutination such as latex agglutination and reverse passive latex (red blood cell) agglutination (microtiter), immunochromatography, fluorescent antibody, enzyme antibody, and radioimmunoassay. But
From the viewpoint of simplicity and rapidity, an immunoagglutination method and an immunochromatography method are preferred. Among the immunoagglutination methods, a microtiter method, which is an indirect agglutination reaction using red blood cells or latex particles bound to an antigen or an antibody, is particularly preferable.

The milk protein used in the present invention includes milk protein itself such as milk casein and whey protein, and a mixed protein obtained by mixing these. The milk protein is 0.1 to 2.0 (w / v)% by dry weight, especially 0.5 to 0.5% by dry weight in the blocking solution in view of the effect of preventing nonspecific reaction.
It is preferable to add so that it may be 1.0 (w / v)%.

These milk proteins need only be present in the reaction site between the antibody or antigen and the sample, and the means for adding them is not particularly limited, and any means such as addition to a buffer may be used. . Above all, using immobilized particles in which an antigen or antibody specific to the antibody or antigen in the sample is carried on an immobilized carrier,
It is preferable to use this when reacting with a sample from the viewpoint of quickness and simplicity, and it is particularly preferable that the immobilized carrier is used after blocking with a milk protein. Any of latex particles, colloidal gold particles, erythrocytes and the like may be used as the immobilization carrier used at this time, but the use of latex particles is preferred from the viewpoint of the stability of the reagent after antigen or antibody sensitization.

[0013] Pathogenic bacteria or pathogenic viruses present in the body fluids of fish and shellfish include baculovirus midgut gland necrosis, PRDV infection (PAV) (also called WSV infection (white spot syndrome)), yellow Head disease, causative virus such as viral infectious disease such as Taura's syndrome and bacterial infectious disease such as vibrio disease caused by Vibrio spp., PRDV is particularly preferable in terms of measurement accuracy and the like. preferable. Antibodies against these pathogenic bacteria, pathogenic viruses, and toxins produced by immunizing animals such as rabbits, mice, rats, chickens, sheep, and horses using these infectious agents as antigens are antiserum (polyclonal antibodies) You just need to collect Also,
Monoclonal antibodies may be used. As the anti-PRDV antibody, for example, a polyclonal antibody described in JP-A-11-279198 is preferable because the antigen can be easily purified. In addition, when an antibody against a pathogenic bacterium or the like present in a specimen is detected by an antigen, the pathogenic bacterium or the pathogenic virus may be used as it is, but it is preferable to extract and use an antigen recognition site. preferable.

The fish and shellfishes to which the present invention can be applied are not particularly limited, and freshwater fish such as eel, carp, rainbow trout, ayu, tilapia, and crucian carp are used. Crustaceans such as kurosoi, tiger puffer, amberjack, tuna, etc. include crab shrimp, black tiger, prawn, shrimp, white shrimp, lobster, crayfish and the like. Among them, crustaceans are preferable because they have a high effect of suppressing nonspecific reactions due to milk proteins, and shrimp are particularly preferable.

The specimen may be any body tissue or body fluid of a fish or shellfish in which a pathogenic bacterium, a pathogenic virus, a toxin produced by them, or an antibody against them is present. Hemolymph is preferred.

Hereinafter, the case of the immunoagglutination method will be described in more detail by way of example. Examples of the insoluble carrier used for the immunological agglutination particles include red blood cells, inorganic compound particles such as colloidal gold particles, and organic polymer particles such as latex particles. Was preferred. As the latex particles to be used, high specific gravity latex particles having a specific gravity of 1.0 g / mL or more, preferably about 1.5 mg / mL are used, and the average particle diameter is further preferably 0.1 to 2.0 μm. Preferably, latex particles of about 1.0 μm are used as suitable ones, and further, latex particles colored are used as suitable ones. Such latex particles include, for example, Bacto latex 0.81 (manufactured by Difco,
(Average particle size 0.81 μm, specific gravity 1.0 g / mL), H090
1, H0902, H2002 (manufactured by JSR, average particle size and specific gravity are 0.94 μm · 1.5 g / mL, 0.9
8 μm · 1.5 g / mL, 2.22 μm · 1.5 g / mL, including colored latex) and poly beads # 1570
6, $ 15709 (manufactured by Polyscience) and the like, but are not limited thereto, and any type of product having the same effect as these can be used.

There are two methods for carrying these antibodies on carrier particles to form immunological agglutination particles. There are a physical adsorption method and a chemical bonding method. Both methods are preferably used for the immunological agglutination reaction of the present invention. Can be. When latex particles are used as the agglutination reaction particles, the antibody can be sufficiently sensitized by a physical adsorption method.

When sensitizing the above latex particles,
It is preferably performed in a buffer. That is, a suspension of latex particles and an antibody solution diluted to an appropriate concentration are mixed, left to stand for a while, and then washed to produce sensitized latex particles. As the buffer used for dilution, a buffer having an ionic strength and pH that does not inhibit the reaction of the supported antibody with the antigen, which is the substance to be measured, is generally selected. For example, TBS, PBS, GBS, phosphate buffer and borate buffer can be used. Among them, phosphate buffer,
The borate buffer and GBS are preferably used because of the cohesiveness of the sensitized latex particles and the difficulty of self-aggregation. In general, the pH is selected in the range of 5 to 10, particularly preferably in the range of pH 6 to 9.

The concentration of latex particles at the time of sensitization is 0.01
~ 0.5 (w / v)% is suitable, especially 0.05 ~
Good results are obtained at about 0.25 (w / v)%. In addition, the antibody protein concentration of the antibody solution when sensitizing the antibody is appropriately 1.0 to 1000 μg, otherwise it is difficult to determine positive / negative due to the decrease in sensitivity or self-aggregation of the sensitized latex particles. Become.

Further, the temperature during the sensitization reaction is from 0 to
The reaction is carried out within the range of 60 ° C., but when the reaction is carried out at room temperature or at a temperature slightly higher than room temperature (℃ 40 ° C.), latex particles having good sensitivity can be obtained.

After sensitizing the latex particles with the antibody, the exposed portions of the particles that are not bound to the antibody are blocked with milk protein.

Also, utilizing immunological agglutination particles,
When detecting and quantifying pathogenic bacteria, pathogenic viruses, toxins produced by them, or antibodies against them, pathogens of fish and shellfish, surfactants such as Triton X100 and Tween 20, and water-soluble polymers such as polyethylene glycol It is known that the addition of additives enhances the sensitivity and suppresses nonspecific antigen-antibody reactions. In the present invention, these additives can be added as needed.

In order to detect and quantify a test substance using the above latex particles, a microtiter method is preferable. The microtiter method refers to a liquid reagent containing immunological agglutination particles in which a substance (such as an antibody) having the property of specifically binding to a test substance is bound to a high-density labeling substance (such as latex particles). This is a method in which a target substance is detected or quantified by mixing a sample containing a test substance and observing the aggregated sedimentation image of a complex formed as a result of the mixing with the naked eye. More specifically, a test sample serially diluted with a buffer solution is added to a U-shaped 96-well microtiter plate, an equal amount of reagent is dispensed into each well, and the mixture is allowed to stand at room temperature for 4 to 17 hours. The aggregated image after being placed is observed and determined using the naked eye or a loupe of about 10 times.

As the immunological agglutination particles used in the microtiter method, the latex particles exemplified in the above sensitization are preferably used. That is, those having a specific gravity of about 1.5 g / mL and a particle size of about 1.0 μm promptly sediment the aggregated latex particles,
It is suitable for easy and accurate judgment even with the naked eye.

In the present invention, in addition to this, a method of mixing the latex particles according to the present invention and a test sample on a slide glass and judging the success or failure of an aggregation image by an optical microscope (microscope latex method), Needless to say, it can be used for various qualitative / quantitative methods using immunological agglutination particles, such as immunochromatography.

[0026]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 Search for a blocking agent that does not bind to the purification of non-specific agglutinating factors contained in body fluids of fish and shellfish (1) Preparation of blood plasma solution As an anticoagulant for hemolymph, an ASP buffer solution (27 mM citric acid) was used. Acid Na, 336 mM NaCl, 115 mM glucose,
9.0 mM EDTA, 5.0 mM PMSF pH 7.0) was used. After mixing an equal amount of the hemolymph fluid collected from the prawn and the above anticoagulant, a fluid from which blood cells and the like were removed by centrifugation at 2000 G for 10 minutes was used as a blood plasma fluid.
Blood plasma solution is prepared for each 50 male and female,
Stored at −30 ° C. until use. For partial purification of the non-specific agglutinating factor using cation exchange chromatography, a mixed solution of blood plasma solutions for 5 males and 5 males was used.

(2) Preparation of Latex Reagent for Nonspecific Aggregation Judgment BSA (fraction V: Iwai Chemical Co., Ltd.) was
It was diluted to 1.0% (w / v) using M glycine buffered saline (GBS, pH 8.2). 0.25% (w / w) of 10 mM GBS (pH 7.5)
v) latex particles (HSR manufactured by JSR)
0902Y, particle size 0.98μm, specific gravity 1.5g / mL) 1
After adding the mixture and mixing well, the mixture was allowed to stand at 37 ° C. for 1.0 hour to adsorb BSA to the latex particles. Thereafter, unadsorbed BSA is removed by centrifugal washing, and then 10 mM GBS (pH 7.0) so that the latex particle concentration becomes 0.25%.
5, 1.0% BSA, 0.08% sodium azide) to give a latex reagent for nonspecific aggregation determination.

(3) Internal Purification of Nonspecific Aggregation Factor in Blood Plasma by Cation Exchange Chromatography 1.0 mL of this blood plasma was used for Macro-Prep high S (Bio-R
AD), and then citric acid solution (27 mM citric acid N
a pH 7.0) and a gradient of NaCl with a citric acid solution (pH 7.0) containing 1.0 M NaCl. The fractionation conditions were 1.0 mL / min. It was 3.0 mL / tube and 22 fractions were collected. Each fraction 2
5 μL and 25 μL of a latex reagent for non-specific agglutination determination,
96-well UV plate (800901; Sanko Junyaku Co., Ltd.)
The mixture was added to the middle well, and the plate was carefully stirred with a vortex mixer, and then allowed to stand at room temperature for about 4 hours. Table 1 shows the aggregation results after the standing reaction. The non-specific agglutination reaction is the fraction No. Nos. 3 to 5 (peak 1) and fraction Nos. The peaks were observed at 12 to 22 (peak 2). As a result of comparison between the two using the aggregation strength per unit protein as an index, it was considered that the substance contained in peak 2 was strongly acting as an aggregation factor for BSA.

[0030]

[Table 1]

(2) Purification of Nonspecific Aggregation Factor by Gel Filtration Chromatography Among the fractions obtained by cation exchange chromatography, fraction No. 1
Regarding No. 4, the aggregation factor was further purified by gel filtration chromatography. Column is Hiprep16 / 60 Sephacr
y "S300 (Pharmacia) and AS buffer (2
7 mM Na-citrate, 336 mM NaCl, 11
After equilibration with 5 mM glucose, 9.0 mM EDTA, 500 μL of the fraction (8% as protein content)
5 μg). The fractionation conditions were 0.5 mL / min. 1.5 m
L / tube and 112 fractions were collected. 25 μL of each fraction and 25 μL of a nonspecific agglutination determination latex reagent were added to a well of a 96-well UV plate (800901; Sanko Junyaku Co., Ltd.) and mixed, and the plate was carefully stirred with a vortex mixer. Let stand for hours. Aggregation was observed in three peaks. The molecular weight estimated from the elution pattern of the standard substance is
From the largest one, they are about 670 kDa, about 142 kDa and about 30 kDa, and it has been clarified that three or more proteins having different properties are involved in this non-specific aggregation.

Example 2 Search for a blocking agent that does not bind to an agglutinating factor contained in the body fluid of fish and shellfish (1) Preparation of latex particles having a blocking agent adsorbed Various blocking agents (Table 2) were added to a 10 mM glycine buffered saline solution. Using (GBS) (pH 7.5), the mixture was diluted to an appropriate concentration. One volume of this diluent contains 10 mM GBS
(PH 7.5) and diluted to 0.25% (w / v) with polystyrene high specific gravity latex solution (manufactured by JSR,
H0902Y, particle size 0.98 μm, specific gravity 1.5 g / mL)
One volume was added, mixed well, and allowed to stand at 37 ° C. for 1.0 hour to adsorb the blocking agent to the latex particles. Then, after the unadsorbed blocking agent is removed by centrifugal washing, the latex particle concentration is adjusted so that the latex particle concentration becomes 0.25%.
1M glycine buffered saline (blocking agent at each concentration,
It was suspended in 0.08% NaN ₃ (pH 7.5) to give a screening reagent.

(2) Reaction of Latex Reagent with Aggregation Factor Mixture An agglutination factor mixture solution (25 μL) obtained by mixing three equal fractions containing the agglutination factor obtained in Example 1 was combined with only various blocking agents. Add 25 μL of latex particles to 9
The solution was added to a well of a 6-well UV plate (800901; Sanko Junyaku Co., Ltd.), and the plate was carefully stirred with a vortex mixer and allowed to stand at room temperature for about 4 hours. Table 2 shows the results after the standing reaction. Latex particles combined with casein, block ace and skim milk powder hardly aggregated with the blood plasma solution. Since other blocking agents coagulated similarly to BSA, it was found that they were not suitable as blocking agents at the time of preparing the latex reagent. In addition, a latex reagent blocking hemocyanin derived from horseshoe shrimp could not be used because self-aggregation occurred in a buffer solution.

[0034]

[Table 2]

Example 3 Examination of Dilution Ratio of Hemolymph Fluid (1) Preparation of Blood Plasma Solution As a living prawn, a cultured prawn with an average weight of 10 g was used. 0.5 mL of ASP buffer (27 mM Na citrate, 33
6 mM, NaCl, 115 mM glucose, 9.0 mM E
500 μL of hemolymph was collected from the first abdominal node of the prawn using a 1.0 mL syringe containing DTA, 5.0 mM PMSF pH = 7.0) and rinsed in advance. And
A liquid from which contaminants such as blood cells were removed by centrifugation at 2000 G for 10 minutes was used as a blood plasma liquid. The blood plasma solution was prepared for each 50 males and females and stored at -30 ° C until use.

(2) Preparation of Latex Particles Adsorbing Milk Protein Block Ace shown in Example 1 was diluted with purified water to an appropriate concentration. One volume of this diluent was added to a high-density polystyrene latex solution (manufactured by JSR, particle size: 0,1).
One volume of 98 μm, specific gravity 1.5 g / mL) was added, mixed well, and allowed to stand at 37 ° C. for 1.0 hour to adsorb the blocking agent to the latex particles. Thereafter, the unadsorbed blocking agent was removed by centrifugal washing, and then prepared using Block Ace (containing 0.08% NaN ₃ ) diluted 40 times so that the latex particle concentration became 0.25%.

(3) Reaction of Latex Reagent with Shrimp Blood Plasma Solution 25 μL of the latex reagent prepared in (2) and 25 μL of each of 2-, 4- and 40-fold diluted blood plasma solutions were added to 96
The solution was added to a well of a well UV plate (800901; Sanko Junyaku Co., Ltd.), and the plate was carefully stirred with a vortex mixer, and then allowed to stand at room temperature for about 4 hours.
After the reaction, those in which agglutination was observed in the wells were judged to have nonspecific agglutination.

The results are shown in Table 3. Regarding the blood plasma solution diluted twice with the anticoagulant, non-specific aggregation was observed in 44% of the individuals. However, it was found that by diluting the blood plasma solution 4-fold with the anticoagulant, the incidence of non-specific aggregation was reduced to 3.0% or less. In this experiment, no difference in aggregation was observed between males and females.
In addition, the BSA-blocked latex reagent produced non-specific aggregation with the blood plasma solution at any dilution ratio tested this time. Therefore, if the blood plasma solution is diluted to 4 times, the PR can be obtained without being affected by contaminants.
It has been found that DV can be detected.

[0039]

[Table 3]

Example 4 Blood collection by the microtiter method
(1) Anti-PRDV polyclonal antibody sensitized high density rat
Preparation of anti-PRDV polyclonal antibody protein was added to 10 mM glycine
To 50 μg / mL using a buffered saline solution (pH 8.2).
Dilution. Latex soluble in 1 volume of this antibody protein
Liquid H0902Y (manufactured by JSR, particle size 0.98 μm, specific gravity
1.5 g / mL), add 1 volume, mix well, then
Gently shake for 1.0 hour to absorb the antibody into the latex particles.
I wore it. After adsorption, add Block Ace diluted 40 times
After stopping the reaction by adding an excess amount, remove the supernatant by centrifugation.
Excess antibody was removed by removal. Latex particles
Block dilution to a concentration of 0.25%
Solution (0.8% NaN _Three, 0.016% TRITON X1
00 containing) was used as a PRDV detection reagent. What
The lower detection limit of the reagent is the protein content of the purified PRDV.
Was about 300 ng. Also anti-PRDV polyclonal
Latec sensitized with normal rabbit IgG instead of antibody
Particles were prepared and used as control reagents.

(2) PRD from mock-infected hemolymph
V detection test As the living body of the prawn, a cultured prawn with an average body weight of 10 g purchased on the market was used. 0.3 mL of ASP buffer (27 mM Na citrate, 33
6 mM NaCl, 115 mM glucose, 9.0 mM E
DTA, 5.0 mM PMSF pH = 7.0), and 300 μL of hemolymph was collected from the first abdominal node of the prawn using a 1.0 mL syringe which had been rinsed beforehand. And
A liquid from which contaminants such as blood cells were removed by centrifugation at 2000 G for 10 minutes was used as a blood plasma liquid. 200 μL of an ASP buffer solution containing 3.0 μg of purified PRDV was mixed with 200 μL of the blood plasma solution to prepare a pseudo-infected blood plasma solution (containing 1.5 μg of purified PRDV) diluted 4 times.

Each of 25 μL of the PRDV detection reagent and the control reagent prepared in (1) was mixed with the mock-infected blood plasma solution (4
Twenty-five μL of a 96-well UV plate (800 times dilution)
901; Sanko Junyaku Co., Ltd.), and carefully vortex the plate with a vortex mixer.
It was left at room temperature for about 4 hours. After the reaction, those in which agglutination was observed in the wells were judged to have nonspecific agglutination.
Table 4 shows the results. The control reagent using Block Ace as the blocking agent did not cause nonspecific agglutination with the mock-infected blood plasma solution. Therefore, the agglutination observed in the wells containing the detection reagent was PRDV
And a specific agglutination reaction between the antibody and the antibody, and it was possible to determine that the PRDV was positive. On the other hand, in the case of the latex reagent prepared using BSA as a blocking agent, strong aggregation occurred even in the wells to which the control reagent was added, and there was no difference in the aggregation strength between the detection reagent and the control reagent. could not.

[0043]

[Table 4]

[0044]

According to the present invention, pathogenic bacteria and pathogenic viruses present in the body fluid of fish and shellfish can be accurately measured by immunoassay.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Kazuaki Hamasa, 1-1-1 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Shuta Yamamoto 1-1-1, Higashi-Shimbashi, Minato-ku, Tokyo No. 19 Yakult Honsha (72) Inventor Tomiko Nagai 1-19-1 Higashi-Shimbashi, Minato-ku, Tokyo (72) Inventor Akio Kanemitsu 3-4 Marunouchi, Chiyoda-ku, Tokyo No.1 New International Building 8F806-808-ku Miyako Chemical Co., Ltd. (72) Inventor Toshiro Kono 3-4-1 Marunouchi Chiyoda-ku, Tokyo New International Building 8F806-808-ku Miyako Chemical Co., Ltd.

Claims (4)

[Claims] 1. A pathogenic bacterium present in a body fluid of a fish or shellfish,
In immunoassays for pathogenic viruses, toxins produced by them or antibodies to them, the pathogenic bacteria,
A pathogenic virus, a pathogenic bacterium of a fish or shellfish, characterized in that a reaction of a specimen with a toxin produced by them or an antibody against them in the presence of a milk protein, a pathogenic virus produced by them, or a toxin produced by them. Method for measuring antibodies. 2. An immunoagglutination method comprising blocking an immobilized carrier on which a pathogenic bacterium, a pathogenic virus or an antibody against them is immobilized with a milk protein, and reacting the blocked immobilized carrier with a sample. 1. The measuring method according to 1. 3. A pathogenic bacterium present in a body fluid of a fish or shellfish,
Pathogenic viruses, toxins produced by them, or antibodies against them are used for the pined rod DNA virus (PRD).
The method according to claim 1 or 2, which is V). 4. The method according to any one of claims 1 to 3, wherein the fish and shellfish is at least one member selected from the group consisting of a prawn, a black tiger, a prawn, a shrimp, a white shrimp, a lobster and a crayfish.