JP2002202312A - Diagnostic kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a proper method for preparing a diagnostic kit constituted of an immunological agglutination reaction reagent reduced in non-specific agglutination reaction and capable of detecting a very small amount of a substance to be measured. SOLUTION: The diagnostic kit is constituted of the immunological agglutination reaction reagent, which is prepared by supporting an antigen or antibody, preferably, antigen protein originating from a C-type hepatitis virus gene and a blocking agent, preferably, casein on an insoluble carrier, and a reagent dispersion containing the same substance as the blocking agent supported on the immunological agglutination reaction reagent and a buffer solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological agglutination reagent utilizing an antigen-antibody reaction used for diagnosis of disease, and more particularly to a diagnostic kit comprising an immunological agglutination reagent for diagnosing hepatitis C. .

[0002]

2. Description of the Related Art Immunological measurement methods utilizing a specific reaction between an antigen and an antibody are widely used in the field of clinical examinations because of their high specificity and high measurement sensitivity.

[0003] Immunological measurement methods used in clinical tests include radioimmunoassay using a radioactive substance, enzyme immunoassay using an enzyme-labeled antibody, and immunoturbidimetry using a change in turbidity due to an antigen-antibody reaction. And a latex agglutination method using latex particles, and a microtiter method in which an antigen or antibody is adsorbed to erythrocytes or the like to form an image of tube bottom aggregation on a microtiter plate by an antigen-antibody reaction. Of these, immunological agglutination reagents using carriers such as the latex agglutination method and the microtiter method are widely used in clinical tests because they can easily, quickly and accurately determine. The immunological agglutination reagent includes an antigen or antibody immobilized on carrier particles (hereinafter, the antigen or antibody immobilized on carrier particles is also referred to as a sensitizing substance) and an antibody or analyte to be measured contained in a subject. A diagnostic agent that diagnoses a patient's disease by reacting with an antigen (hereinafter, the antigen or antibody to be detected is also referred to as a measurement target substance) as an antigen-antibody reaction.

[0004] As with other immunodiagnostic agents, it is essential for an immunological agglutination reagent to perform an accurate early diagnosis. That is, 1) there is no agglutination reaction not based on the antigen-antibody reaction (hereinafter, also referred to as non-specific agglutination reaction), 2)
It is essential to satisfy the two points of being capable of detecting a trace amount of the target substance in the subject (hereinafter, also referred to as high sensitivity).

The causes of non-specific agglutination in the immunological agglutination reagent are considered as follows. That is, one of the causes is that the contaminant component in the specimen forms nonspecific binding with the carrier particle itself without depending on the antigen-antibody reaction. In such a non-specific agglutination reaction, after the sensitizer is carried on the carrier particles, the gaps on the carrier particles are carried with an immunologically inactive blocking agent (hereinafter, such an operation is referred to as blocking. ) Can be reduced. Another possible cause of the non-specific agglutination reaction is that contaminants other than the target substance cause non-specific binding to the sensitizer in the specimen. Therefore, a non-specific agglutination reaction can be reduced by adding a substance that absorbs and removes the contaminant (hereinafter, also referred to as an absorbent) to a dispersion medium used as a diluent for the specimen. It can be said that the examination of the blocking agent and the absorbent added to the dispersion medium is a factor in preventing the nonspecific agglutination of the immunological agglutination reagent.

As a known method for preparing an immunological agglutination reagent capable of avoiding non-specific agglutination, carrier particles carrying a sensitizer (hereinafter referred to as carrier particles carrying a sensitizer) ) For bovine serum albumin (hereinafter, referred to as
It is considered effective to use normal rabbit serum as an absorbent as a dispersion medium, and it is actually used, but it is still insufficient in avoiding non-specific agglutination. .

On the other hand, early detection of disease is important for immunological agglutination reagents, and for that purpose, it is essential to be able to detect a trace amount of a target substance, that is, to increase sensitivity. For example, in the case of a disease such as a viral infection, the amount of the measurement target substance is small in the early stage of infection, and it is important that the sensitivity be high in order to make a diagnosis in the early stage of infection. The only method for increasing the sensitivity of an immunological agglutination reagent is to carry as many sensitizers as possible on carrier particles. However, when the sensitizer is excessively loaded on the carrier particles, the sensitivity is improved. On the other hand, the sensitizers mutually bind to each other and a region of the sensitizer that is effective for an antigen-antibody reaction with the measurement target substance ( Hereinafter, this phenomenon will be referred to as an active site), and the measurement target substance cannot be measured. Therefore, there is an upper limit to the amount of a sensitizer that can be supported on an insoluble carrier for preparing an immunological agglutination reagent, and it is not possible to increase the sensitivity by supporting a certain amount or more of a sensitizer. However, a specific method for increasing the sensitivity has not yet been found.

[0008]

With the above background,
An object of the present invention is to develop a method suitable for preparing a diagnostic kit comprising an immunological agglutination reagent having a small amount of non-specific agglutination and capable of detecting a trace amount of an object to be measured. .

[0009]

Means for Solving the Problems The present inventors have made intensive studies to solve the above technical problems. As a result, the same substance as the blocking agent was added to the reagent dispersion, and when an antigen protein derived from the hepatitis C virus gene was used as the antigen, the above procedure was performed using casein as the blocking agent. In other words, 1) prevention of non-specific agglutination, 2) detection of trace amounts of target substances to be measured 2
The present inventors have found that the kit is a diagnostic kit using an immunological agglutination reagent satisfying the above requirements, and have completed the present invention.

That is, the present invention provides an immunological agglutination reagent comprising an insoluble carrier carrying an antigen or an antibody and a blocking agent, the same substance as the blocking agent carried by the immunological agglutination reagent, and a buffer. And a reagent dispersion comprising the liquid.

Another invention is directed to an immunological agglutination reagent for diagnosing hepatitis C comprising an insoluble carrier carrying an antigen protein derived from the hepatitis C virus gene and casein, and a reagent dispersion comprising casein and a buffer. And a liquid for diagnosis of hepatitis C.

Hereinafter, the present invention will be described in detail.

As the insoluble carrier particles in the present invention, known carriers which can be used as diagnostic reagents for immunological agglutination reaction can be used without limitation. For example, high specific gravity composite particles obtained by coating an inorganic compound serving as a core with a dye (Japanese Patent Application Laid-Open No.
2-115366, hereinafter also abbreviated as HDP),
Sheep erythrocytes, polystyrene particles, gelatin particles and the like. Among them, HDP and erythrocytes are preferably used because they have a large adsorption amount of antigen or antibody. Particularly, HDP, which is an artificial carrier, can artificially add a surface functional group and change the surface state according to the type of antigen or antibody. It can be more preferably used.

The particle size of the insoluble carrier is also not particularly limited from the known range that can be used as a diagnostic reagent for immunological agglutination reaction, but the sensitizing substance is increased by increasing the specific surface area per carrier particle weight. In general, particles having a particle diameter of 0.01 μm to 3 μm are preferably used because the adsorption amount of the particles can be improved.

The specific gravity of the insoluble carrier may be 1.0 or more. In particular, the larger the specific gravity of the carrier particles for microtiter reagents utilizing the sedimentation reaction, the faster the sedimentation speed and the shorter the judgment time. The above are preferably used.

The antigen used in the present invention is not particularly limited as long as it has an antigenic activity with respect to an antibody in a subject and is bound by an antigen-antibody reaction. For example, a polypeptide,
Polysaccharides, lipids and the like. Among them, a polypeptide is suitably used as the antigen. The chain length of the polypeptide may be any length as long as the antibody can be recognized by the antigen-antibody reaction, and may be at least 3 amino acid residues which are generally considered to be able to recognize the antibody as an antigen site by the antigen-antibody reaction. Further, since the polypeptide is carried on insoluble carrier particles, 3000 amino acid residues or less are preferably used in consideration of adsorption efficiency.
When the hepatitis C virus gene-derived antigen protein is used as the antigen, the effect of the present invention is remarkably exhibited.

The antigen protein derived from the hepatitis C virus gene (hereinafter abbreviated as HCV antigen) as used in the present invention refers to a protein encoded by the hepatitis C virus gene. The hepatitis C virus (hereinafter abbreviated as HCV) gene is an RNA of about 10 kb (about 10,000 nucleotides) having a nucleotide sequence described in JP-A-4-144686 and JP-A-4-179482.
HCV is an RNA virus, but HCV-derived RNA
CDNA produced by reverse transcriptase also corresponds to the hepatitis C virus gene.

The hepatitis C virus gene is non-A
It can be obtained from a cDNA library prepared by separating viral genes from the serum of non-B hepatitis patients. For example, hepatitis C virus is first separated from patient serum by ultracentrifugation, then gene RNA is prepared from the virus, cDNA is synthesized from the RNA using reverse transcriptase, and then the cDNA fragment is A cDNA library is prepared by inserting into a plasmid vector or phage vector. Next, the cDNA library is subjected to immunoscreening using serum (a serum containing an anti-HCV antibody) of a non-A non-B hepatitis patient after blood transfusion to obtain a target gene. Also known HC
A DNA library may be synthesized based on the base sequence of the V gene, and a cDNA library may be screened by DNA / DNA hybridization. Another alternative is Proceedings of the Japan Academy, V
ol. 65, Ser. B, No. 9, pp. 219-223 (1989), that is, RT- which combines reverse transcriptase and PCR.
A method in which a gene is amplified in a target region by a PCR method and the amplified gene fragment is cloned is also effective.

The HCV antigen can be prepared by a known gene expression system, that is, Escherichia coli host vector system, Bacillus subtilis host vector system, yeast host vector system, insect cell or insect host vector system. The expression can be carried out by using an animal cell host / vector system or the like. Among them, Escherichia coli can be suitably used. To express the antigen protein using Escherichia coli, first, a HCV gene is inserted into a vector that can be expressed in Escherichia coli to prepare a recombinant vector. The vector is not particularly limited, and any vector can be used as long as it is a vector usually used as a vector for Escherichia coli. Particularly, a vector in which gene expression occurs at a high frequency is suitably used. For example, a series of pUC vectors (Takara Shuzo Co., Ltd. products), a series of pTV vectors (Takara Shuzo)
Products), a series of pTZ vectors (Toyobo Co., Ltd. products), a series of pET (Methods in enzymology, Vol. 185)
Etc.) can be used. Also, a series of pUEX
Vector (Amersham Japan Co., Ltd.), a series of p
If an EX vector (a product of Boehringer Mannheim Yamanouchi Co., Ltd.) is used, the antigen protein can be expressed as a fusion polypeptide with β-galactosidase. A vector that can be expressed in E. coli usually has a promoter for gene expression that works in E. coli and an operator that controls it. A recombinant vector is prepared by inserting an HCV gene using an appropriate restriction enzyme site downstream of the promoter of such a vector. Among the recombinant vectors thus produced, a recombinant vector produced using a pUEX vector is particularly preferred because the polypeptide obtained by gene expression has a high yield and is easy to purify. Selected. Escherichia coli is transformed with the recombinant vector, and the transformed Escherichia coli is cultured to express the inserted gene, thereby producing an antigen protein.

When gene expression is performed using a recombinant vector, a plurality of amino acids having a random sequence may be added to the N-terminal or C-terminal of the polypeptide. However, since the plurality of amino acids added to the N-terminal or C-terminal are random amino acids, they are irrelevant to the antigen-antibody reaction and do not affect the measurement using the antigen-antibody reaction.

The HCV antigen is obtained by culturing the cells obtained by culturing the above-described transformed Escherichia coli by sonication or the like, and isolated from the crushed cells by a known method. The HC
The V antigen can be purified by any known method, such as salting out, adsorption of an ion exchange resin, gel filtration, and the like. Preferably, a combination of the above methods is effective. The purified HCV antigen may be dispersed in any solution, but preferably contains 0.87% aqueous sodium chloride (hereinafter abbreviated as physiological saline) or 0.87% sodium chloride, Desirably, it is dispersed in a 20 mM phosphate buffer, pH 7.2 (hereinafter abbreviated as PBS). Since the reagent sensitivity depends on the number of epitopes of the HCV antigen carried per unit surface area of the insoluble carrier, the purification purity is increased as the purification purity is increased in order to increase the number of epitopes of the HCV antigen carried per unit surface area of the insoluble carrier. desirable.

The HCV antigen is a polypeptide having immunological reactivity to an anti-hepatitis C virus antibody (hereinafter, also abbreviated as anti-HCV antibody). That is, it has an epitope site for the antibody in the subject, and has the property of specifically binding to the antibody in the patient's serum and plasma by an antigen-antibody reaction.

The length of the polypeptide chain of the HCV antigen may be any length as long as the anti-HCV antibody can be recognized by the antigen-antibody reaction. It is sufficient if it is at least the group. Furthermore, since the HCV antigen is carried on insoluble carrier particles, 3000 amino acid residues or less are preferably used in consideration of the adsorption efficiency to the carrier particles.

The antibody used in the present invention is an immunoglobulin capable of binding to an antigen in a subject by an antigen-antibody reaction. As the antibody, a monoclonal antibody having one antigen recognition site of an antigen molecule or a polyclonal antibody obtained from antiserum can be suitably used. Since various types of antigen recognition sites can be detected, a mixture of several types of monoclonal antibodies or a polyclonal antibody is more preferably used.

The blocking agent used in the present invention refers to an immunologically inactive substance used for covering the gap between the sensitizing substances on the sensitizing particles. The purpose of covering the sensitizing particles with the blocking agent is two points: 1) avoiding the influence of contaminants, and 2) preventing mutual adsorption of the sensitizing substances. As the blocking agent, an immunologically inactive substance is adopted, for example,
Examples include proteins such as BSA and casein, amino acids such as glycine, serine, and alanine, and surfactants that are amphiphilic substances.

When an antigen protein derived from the hepatitis C virus gene is used as the sensitizer, casein is extremely effective as the blocking agent. When casein is used as a blocking agent, non-specific binding between sensitizers can be prevented, and more sensitizer can be supported on the insoluble carrier. Therefore, in addition to enabling higher sensitivity, non-specific agglutination due to the influence of contaminants in the specimen can be prevented as compared with the case where another blocking agent is used. Other than casein as a blocking agent, for example, BS
When A is used, the HCV antigens of the insoluble carrier cause mutual adsorption, and a nonspecific agglutination reaction occurs in both a healthy subject sample and a hepatitis C patient sample. Further, the use of normal rabbit serum as a blocking agent is not suitable because it is susceptible to contaminants in the subject and causes nonspecific increase in sensitivity in the measurement of a healthy subject.

The casein in the present invention can be used without any particular limitation as long as it is known. As the casein, an acidic casein which is a main component of milk protein and is prepared by adding an acid to milk to obtain a pH of 4.6 and performing isoelectric point precipitation is preferably used. The acidic casein can be generally separated electrophoretically into three components, α, β, and γ, but may be any of α, β, and γ, or may be used as a mixture of two or more types. In addition, physical decomposition such as heating and pressurizing,
Those whose molecular weight has been reduced by chemical decomposition with an alkali or the like or enzymatic decomposition with a proteolytic enzyme can also be used.

The term "support" as used in the present invention means that an insoluble carrier is allowed to adsorb antigens and antibodies as sensitizing substances, a blocking agent for blocking, etc. (hereinafter, the substance carried on the insoluble carrier is also referred to as an adsorbed substance). is there.

The order of loading the sensitizing substance and the blocking agent on the insoluble carrier particles is not particularly limited, and any one of the following methods is employed.

1) a method of loading a sensitizer on an insoluble carrier and then loading a blocking agent, 2) a method of loading a blocking agent on an insoluble carrier and loading a sensitizer,
3) A method in which a sensitizer and a blocking agent are simultaneously supported on an insoluble carrier. In any method, the effect of the present invention is sufficiently exhibited. However, in order to preferentially carry the sensitizing substance, the method of 1) carrying the sensitizing substance on the insoluble carrier and then carrying the blocking agent is preferably used. Can be

The supporting step of the above 1) is summarized as follows. An insoluble carrier is dispersed and mixed in a buffer solution in which a sensitizing substance is dissolved, and the sensitizing substance is supported on the insoluble carrier. And then carrying the blocking agent in the same manner.

As a method for supporting the sensitizing substance on the insoluble carrier, known methods such as a physical adsorption method such as a hydrophobic adsorption method and a chemical adsorption method such as a chromium chloride method are employed. After being supported on a carrier, the sensitizing substance needs to be supported under as mild conditions as possible in order to bind to the measurement target substance in the specimen by an antigen-antibody reaction, and therefore the hydrophobic adsorption method is particularly preferably used. Can be

The loading by the hydrophobic adsorption method is performed in a state where the insoluble carrier and the adsorbed substance are dispersed in a buffer having a buffering action. The buffer can be used without limitation as long as it has a buffering action, such as a phosphate buffer, a glycine buffer, a Tris buffer, an acetate buffer and the like. The pH of the buffer solution is selected from the optimum range depending on the adsorbing substance to be carried, and is generally adopted.
0 is desirable. The time for supporting the adsorbed substance on the insoluble carrier may be a time sufficient for the adsorbed substance to be uniformly supported on the surface of the insoluble carrier.
In the case of performing the hydrophobic adsorption method of P, 30 minutes or more is sufficient. Further, the temperature is in the range where the adsorbed substance to be supported is not thermally denatured, that is, 1 ° C. or more and 80 ° C. or less.

For the subsequent loading of the blocking agent on the insoluble carrier, the same method as the above-mentioned loading of the sensitizer can be adopted.

For example, in an immunological agglutination reagent for diagnosing hepatitis C, an insoluble carrier carrying an HCV antigen (hereinafter also referred to as an HCV antigen-sensitized carrier) is subjected to centrifugal washing to remove excess HCV antigen. The HCV antigen-sensitized carrier was treated with casein as a blocking agent in an amount of 0.1 to 5 (w / vol).
% Of the sensitized particles in a solution dissolved in a buffer solution having a buffering action at pH 6.0 to pH 8.0 near neutrality so as to have a concentration of 0.0001 to 30 (w / w)%. To carry the blocking agent.

The immunological agglutination reagent of the present invention refers to an insoluble substance for detecting an antigen or antibody present in a subject (serum, plasma, urine, stool, etc.) as described above by immunological agglutination. This is a diagnostic reagent in which a sensitizer and an immunologically inactive blocking agent are carried on a carrier.

As the immunological agglutination reagent, the agglutination method usually used for diagnosis is applied without any limitation. For example,
These include the qualitative diagnosis plate method, the semi-quantitative diagnosis microtiter method, and the quantitative diagnosis latex agglutination method, particle counting method, and the like. Among them, the effect of the present invention is particularly remarkable when applied to a microtiter method.

The above-mentioned immunological agglutination reagent is used by suspending it in a reagent dispersion. When it is to be stored for a long time, it is preferable to freeze-dry it. The freeze-drying method is not limited and may be performed by a usual method. For example, a method and conditions adopted for a freeze-drying method of sensitized red blood cells are used. Preferably, a method is employed in which the immunological agglutination reagent is suspended in an aqueous solvent in a vial or the like, immersed in liquid nitrogen or the like, rapidly preliminarily frozen, and then freeze-dried in a vacuum.

The conditions for the vacuum freeze-drying method are not particularly limited, but the vial containing the suspension of the sensitizing particles is rapidly preliminarily frozen, and then freeze-dried previously cooled to -40 to -60 ° C. A preferred method is to place it in a chamber of the machine and gradually raise the temperature over 24 to 72 hours and freeze-dry. At this time, the pressure in the chamber is 50 to 200 μmH.
g, the final drying temperature is suitably from 20 to 50 ° C. Then, a lyophilized immunological agglutination reagent (hereinafter, also referred to as a lyophilized reagent) is sealed in a vacuum state or filled with an inactivating gas and sealed.

The lyophilized reagent is suspended and dispersed in a reagent dispersion comprising a buffer containing the same substance as the blocking agent carried by the immunological agglutination reagent, and then subjected to measurement. Thus, by adding the same substance as the reagent blocking agent to the reagent dispersion, the blocking agent carried on the insoluble carrier can be prevented from being detached.
If a substance different from the blocking agent carried on the insoluble carrier is added to the particle solution, the blocking agent carried on the insoluble carrier may be eliminated by an exchange reaction. Such elimination of the blocking agent from the insoluble carrier causes nonspecific binding between the contaminant in the specimen and the surface of the insoluble carrier, causing a new nonspecific agglutination reaction.

The lyophilized reagent suspended and dispersed in the reagent dispersion liquid has the same performance as the immunological agglutination reagent before lyophilization. In addition, even when freeze-drying is not performed, the immunological agglutination reagent is suspended and dispersed in the reagent dispersion and used for measurement.

As the buffer used for the reagent dispersion, a known buffer is not particularly limited and used. For example, phosphate buffer,
Tris buffer, borate buffer, carbonate buffer, glycine buffer and the like. The pH of the buffer is not particularly limited,
PH 7 to pH 10 in a neutral to weakly alkaline range is preferably used so that the insoluble carrier itself does not cause aggregation. Therefore, a phosphate buffer, a Tris buffer, or the like having a buffering action from neutral to weakly alkaline is preferably used. The concentration of the buffer may be any concentration as long as it has a buffering action, and 5 mM to 500 mM is preferably used.

Further, a salt may be present in the reagent dispersion. The type and concentration of salt are not particularly limited,
Under conditions under which the antigen-antibody reaction preferably proceeds, for example, sodium chloride, magnesium chloride, calcium chloride, etc., a concentration of about 1 mM to 1 M is suitably used. Further, in order to prevent spoilage, about 0.1 (w / vol)% sodium azide or the like may be added.

Further, a polysaccharide or the like may be added to the reagent dispersion for stabilizing the tube bottom aggregation image.

The prepared reagent dispersion can be stored for about two years by refrigeration at 15 ° C. or lower.

In a typical method for preparing the reagent dispersion, the same substance as the blocking agent used for the preparation of the immunological agglutination reagent is used in a buffer at 0.0001 to 30 (w / vol).
%, And after dissolving, if necessary, acid,
Alternatively, the pH of the buffer is finely adjusted by adding an alkali to obtain a reagent dispersion. Further, if necessary, sodium chloride, magnesium chloride, potassium chloride and the like are added and dissolved.

In order to actually detect the substance to be measured in the specimen using the immunological agglutination reagent of the present invention, in addition to the particle solution for dispersing the immunological agglutination reagent, the analyte Requires a sample diluent that dilutes to a suitable concentration within the measurement range. The sample diluent is composed of a buffer containing an absorbent.

The sample diluent contains an absorbent and a buffer as main components, and contains a substance carried on an insoluble carrier as a blocking agent for the immunological agglutination reagent as an absorbent. It is preferred to use the same.

The blocking agent carried by the immunological agglutination reagent is generally used to prevent mutual adsorption between sensitizers and to prevent the contaminants in the specimen from adsorbing to the surface of the insoluble carrier. If the blocking agent carried on the carrier and the contaminant in the specimen cause non-specific adsorption, it causes a new non-specific agglutination reaction.

Therefore, by adding the same substance as the blocking agent to the sample diluent as an absorbent, foreign substances in the sample which cause non-specific adsorption with the blocking agent carried on the insoluble carrier are adsorbed and removed in advance. When a substance different from the blocking agent carried by the immunological agglutination reagent is added to the sample diluent, non-specific adsorption may occur with the blocking agent carried by the immunological agglutination reagent contained in the sample. The resulting contaminants are not absorbed and removed, and tend to cause nonspecific agglutination.

In the above sample diluent, the type of buffer solution,
The salt content, storage method, adjustment method, and the like may be performed in the same manner as in the case of the reagent dispersion.

When a diagnostic kit comprising the above immunological agglutination reagent and a reagent dispersion is used as a microtiter reagent, the reaction is usually performed on a microtiter plate. When performing a qualitative test, a test sample is diluted with a sample diluent to a specified dilution ratio, and then the above-mentioned immunological agglutination reagent suspended in a reagent dispersion is dropped to perform measurement.

[0053]

The sensitizing substance and the blocking agent are carried on the insoluble carrier to prevent the foreign substances in the sample from adsorbing to the surface of the insoluble carrier. Then, by adding the same substance as the blocking agent also in the dispersion liquid, it is possible to prevent the desorption of the blocking agent carried on the insoluble carrier, and in the subject due to the desorption of the blocking agent of the insoluble carrier. A new non-specific agglutination reaction due to non-specific binding between the contaminant and the surface of the insoluble carrier can also be prevented.

On the other hand, by supporting the sensitizing substance and the blocking agent on the insoluble carrier, an excessive sensitizing substance can be supported on the insoluble carrier without causing mutual bonding between the sensitizing substances. Therefore, interaction between the sensitizers can be prevented, and high sensitivity can be achieved by efficiently exposing more active sites on the surface of the carrier particles.

Further, when the same substance as the blocking agent is added to the sample diluent, contaminant substances in the sample which cause non-specific adsorption with the blocking agent carried on the insoluble carrier are removed by adsorption, and the insoluble carrier is removed. When the blocking agent carried on the sample and the contaminant in the specimen cause nonspecific adsorption, a new nonspecific agglutination reaction can be prevented. Therefore, only a true immunological agglutination reaction between the sensitizing substance and the measurement target substance occurs, and eventually, the sensitivity of the immunological agglutination reagent can be increased.

[0056]

The sensitizing substance and the blocking agent are carried on the insoluble carrier, and the same substance as the blocking agent is used in the reagent dispersion, so that contaminants in the sample due to the elimination of the blocking agent from the insoluble carrier can be reduced. Non-specific agglutination due to non-specific binding on the surface of the insoluble carrier can also be prevented, and non-specific agglutination can be avoided, and the detection of a trace amount of the target substance in the specimen, that is, high sensitivity can be achieved. .

In particular, when an antigen protein derived from the hepatitis C virus gene is used as an antigen, when casein is used as a blocking agent, non-specific agglutination can be avoided and trace amounts of the target substance in the sample can be reduced. It has become possible to prepare a detectable immunological agglutination reagent. Furthermore, when the above-mentioned casein was contained in the reagent dispersion, nonspecific agglutination was avoided, and the detection of a trace amount of the target substance in the sample and the improvement in sensitivity were further improved.

[0058]

The present invention will be described below in detail with reference to examples. However, these examples do not limit the technical scope of the present invention. Reference Example Preparation of Antigen Protein Derived from Hepatitis C Virus Gene As an antigen protein derived from the hepatitis C virus gene, C was used.
Four types of ore antigen, NS-3 antigen, NS-4 antigen and NS-5 antigen were used. Four kinds of antigen proteins were prepared as follows. The technique of the genetic manipulation experiment is described in Sambrook et al. [Sambrook, J., Fritsch, EF, Maniat.
is, T., Molecular Cloning, 2nd ed., Cold Spring Har
bor Laboratory Press, New York (1989)]. The restriction enzyme used was Takara Shuzo Co., Ltd. product. (Preparation of Core antigen) pHC obtained by inserting a Core region gene of HCV (a nucleotide sequence encoding the amino acid sequence from the 1st to 168th from the N-terminus) into pUEX2 (Amersham)
Recombinant Escherichia coli HB101 [pHCX01] having X01 (Japanese Patent Application No. 4-1988)
06, No. 13056) is converted to LB + Amp medium [Bacto
Tryptone 1.0%, Yeast extract 0.5%, NaCl 0.5%, ampicillin (Amp) 50 μg / ml] at 30 ° C. overnight, add glycerin to a final concentration of 15%, and add −80 ° C.
And stored frozen.

By culturing recombinant Escherichia coli HB101 [pHCX01] and performing gene expression, a Core antigen is produced as a fusion polypeptide with β-galactosidase. 1 ml of cryopreserved cells of recombinant E. coli HB101 [pHCX01]
One liter of LB + Amp medium was inoculated and cultured at 30 ° C. overnight. Subsequently, this culture was added to 20 liters of LB + Amp.
The medium was inoculated and cultured at 30 ° C. until the OD 540 reached 1.5. The culture temperature was increased to 42 ° C., and the culture was continued for 3 hours. After the culture, the cells were collected by centrifugation to obtain 57 g of wet cells. The cells were placed in 2 liters of TNE buffer (50 mM Tris) containing 0.6 M urea.
-Suspended in HCl (pH 8.3), 100 mM NaCl, 1 mM EDTA) and disrupted by sonication. 10,000 g of this crushed cell, 20
Insoluble granules containing the Core antigen were collected in the precipitated fraction by centrifugation for minutes. This precipitate is again used for 2 liters.
The insoluble granules were suspended in a TNE buffer containing 0.6 M urea, washed, and the precipitate was collected by centrifugation. Further, the precipitate is suspended in 2 liters of TNE buffer containing 3M urea, and the mixture is stirred at room temperature for 30 minutes to sufficiently wash the insoluble granules. did. 200 ml of 8 was added to the precipitate of the insoluble granules.
The precipitate was solubilized by adding TNE buffer containing M urea. this
The supernatant was collected by centrifugation at 16,000 g for 20 minutes and dialyzed against TNE buffer. After dialysis, the supernatant was separated by centrifugation at 16,000 g for 20 minutes to obtain a Core antigen. 980 mg of Core antigen was obtained from a 20 liter culture. (Preparation of NS-3 antigen) Recombinant Escherichia coli E. coli having a plasmid pHCV07 containing an NS-3 region gene (a nucleotide sequence encoding the amino acid sequence at positions 1323 to 1533 from the N-terminus).
coli HCV7 (Japanese Unexamined Patent Publication (Kokai) No. 4-179482, No. 11831 of Microbial Research Laboratories)
From pHCV07, digested with EcoRI and StuI,
A 338 bp fragment on the 5 'side was obtained. This 338 bp fragment is
After partial digestion with nfI, DNA polymerase I Klenow fragm
The ends were blunt-ended with ent to obtain a 263 bp fragment. PHCV
7 was digested with StuI, treated with CIP, and then digested with PstI, cDN
A 3'-side 400 bp fragment of A was obtained. On the other hand, pUEX1 was replaced with SmaI and PstI
And digested with CIP. A ligation reaction of this pUEX1 with a 263 bp fragment on the 5 ′ side of the cDNA and a 400 bp fragment on the 3 ′ side of the cDNA was performed to obtain a recombinant vector pCI07. This recombinant vector pCI07 contains a nucleotide sequence encoding the amino acid sequence at positions 1323 to 1533, counted from the N-terminus of HCV. Next, host E. coli HB101 was transformed with the recombinant vector pCI07, and the recombinant E. coli HB101 [pCI0
7]. Recombinant E. coli HB101 [pCI07] was converted to LB + Am
The cells were cultured overnight in a p-medium at 30 ° C, glycerin was added to a final concentration of 15%, and the cells were frozen and stored at -80 ° C.

The NS-3 antigen is produced as a fusion polypeptide with β-galactosidase by culturing recombinant Escherichia coli HB101 [pCI07] and performing gene expression. Co
Recombinant Escherichia coli HB101 [pCI
07], disruption of the cells, and separation and purification of the fusion polypeptide. Culture in 20 liters of LB + Amp medium,
1,000 mg of NS-3 antigen was obtained. (Preparation of NS-4 Antigen) Recombinant E. coli E. coli harboring a plasmid pHCV10 containing an NS-4 region gene (a nucleotide sequence encoding the amino acid sequence from the 1605th to 1798th from the N-terminus).
coli HCV10 (Japanese Unexamined Patent Publication No. 4-179482;
From pHCV10, digested with AvaII, blunt-ended with DNA polymerase I Klenow fragment,
The 583 bp fragment was isolated by digestion with BamHI. On the other hand, Sma with pUEX3
Digestion with I, CIP treatment, and digestion with BamHI. Thereafter, electrophoresis was performed to separate the target fragment. These were ligated to produce a recombinant vector pCI10. This recombinant vector pCI10 contains a nucleotide sequence encoding the amino acid sequence at positions 1605 to 1798, counted from the N-terminus of HCV. Next, the host E. coli HB101 was transformed with the recombinant vector pCI10, and the recombinant E. coli HB101 [pCI1
0]. Recombinant E. coli HB101 [pCI10] was converted to LB + Am
The cells were cultured overnight in a p-medium at 30 ° C, glycerin was added to a final concentration of 15%, and the cells were frozen and stored at -80 ° C.

The NS-4 antigen is produced as a fusion polypeptide with β-galactosidase by culturing recombinant Escherichia coli HB101 [pCI10] and performing gene expression. Co
Recombinant Escherichia coli HB101 [pCI
10], cell disruption, and separation and purification of the fusion polypeptide. Culture in 20 liters of LB + Amp medium,
720 mg of the NS-4 antigen was obtained. (Preparation of NS-5 antigen) Recombinant E. coli E. coli harboring plasmid pHCV14 containing the NS-5 region gene (base sequence encoding the amino acid sequence at positions 2111 to 2270 from the N-terminus).
coli HCV14 (Japanese Patent Application No. 4-179482;
From pHCV14, digested with PstI and XbaI, and then blunting
The end was blunt-ended with the kit, and a fragment containing 484 bp was isolated. On the other hand, pUEX2 was digested with SmaI and treated with CIP. afterwards,
The target fragment was separated by electrophoresis. These were ligated to produce a recombinant vector pCI14. In this recombinant vector pCI14, 2 counted from the N-terminus of HCV,
Includes the base sequence encoding the amino acid sequence from position 111 to position 2270. Next, the host Escherichia coli HB101 was transformed with the recombinant vector pCI14, and the recombinant Escherichia coli HB101 [pCI14] was transformed.
I got Recombinant Escherichia coli HB101 [pCI14] was cultured overnight at 30 ° C. in LB + Amp medium, glycerin was added to a final concentration of 15%, and frozen at −80 ° C.

By culturing recombinant Escherichia coli HB101 [pCI14] and performing gene expression, the NS-5 antigen is produced as a fusion polypeptide with β-galactosidase. Co
Recombinant Escherichia coli HB101 [pCI
[14], the cells were disrupted, and the fusion polypeptide was separated and purified. Culture in 20 liters of LB + Amp medium,
750 mg of NS-5 antigen was obtained. Comparative Example 1 Preparation of immunological agglutination reagent for diagnosis of hepatitis C using casein as a blocking agent (Sensitization) 1.8 μm diameter HDP (product of Tokuyama Soda Co., Ltd.)
Was suspended in PBS to a concentration of 5 (w / w)%, and HDP was suspended.
The suspension was used. The four kinds of HCV antigen proteins prepared in the above Reference Examples were mixed at 50, 100, and 200 μg / ml, respectively, to obtain a mixed antigen solution. The HDP suspension (1 ml) and the mixed antigen solution (1 ml) were mixed in a test tube, and allowed to stand at room temperature for 1 hour to be hydrophobically supported on the HDP surface (hereinafter, this adsorption operation is also referred to as sensitization).

(Washing operation) Thereafter, in order to remove excess HCV antigen protein, 2,500 rpm was added to the mixed solution.
m, and centrifuged for 5 minutes, and the centrifuged supernatant was removed. For washing, the centrifuged precipitate was added with 2 ml of PBS, suspended, centrifuged at 2,500 rpm for 5 minutes, and the supernatant was removed. The HDP to which the HCV antigen protein was adsorbed was used as HCV antigen-sensitized particles.

(Blocking operation) To the HCV antigen-sensitized particles (50 mg), 5% of 1% casein was added, and
Blocking was performed at ℃ for 3 hours. After blocking, the suspension was centrifuged at 2,500 rpm for 5 minutes,
The centrifuged supernatant was removed. The blocking operation is completed by the above operation, and an immunological agglutination reagent for hepatitis C diagnosis (hereinafter referred to as C
Hepatitis hepatitis diagnostic reagent) was prepared. This hepatitis C diagnostic reagent was added to 3 (vol / vol)% normal rabbit serum-containing P
0.5 (w / vo) in BS (hereinafter abbreviated as solution A)
l) The resulting suspension was subjected to the following measurement operation.

(Measurement operation) On the other hand, the sample
The solution was serially diluted by a factor of two to two. Next, 25 μl each of the diluted solution of the sample was placed in a 96-well microtiter plate.
It dripped from a hole to 18 holes. Subsequently, the above-mentioned suspension of the diagnostic reagent for hepatitis C was dropped in 25 μl of each well. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed.

(Results) The performance of the hepatitis C diagnostic reagent prepared as described above was evaluated using five samples of healthy subjects and five samples of hepatitis C patients. The results are shown in Table 1. Positive images were detected only up to 8-fold dilution in all healthy subjects, whereas in hepatitis C patients, positive images could be detected even at 32,768-fold dilutions or more at a sensitizing concentration of 200 μg / ml. Was.

[0067]

[Table 1]

Example 1 A kit for diagnosing hepatitis C comprising a reagent for diagnosing hepatitis C using casein as a blocking agent and a reagent dispersion to which casein was added (sensitization) Same as Comparative Example 1.

(Washing Operation) The same as in Comparative Example 1.

(Blocking operation) To the HCV antigen-sensitized particles, 5 ml of 1% casein was added, and blocking was performed at 37 ° C for 3 hours. After blocking, the suspension was centrifuged at 2,500 rpm for 5 minutes, and the supernatant was removed. The blocking operation was completed by the above operation, and a hepatitis C diagnostic reagent was prepared. 0.5% in PBS containing 1 (vol / vol)% casein (hereinafter abbreviated as solution B)
(W / vol)% and suspended for the following measurement operation.

(Measurement operation) On the other hand, the sample
The solution was serially diluted by a factor of two to two. Next, 25 μl each of the diluted solution of the sample was placed in a 96-well microtiter plate.
It dripped from a hole to 18 holes. Then, the suspension of the above-mentioned hepatitis C diagnostic reagent was dropped in 25 μl of each well. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed. Reagent sensitivity is indicated by titer.

(Results) The performance of the hepatitis C diagnostic reagent prepared as described above was evaluated using five samples of healthy subjects and five samples of hepatitis C patients. The results are shown in Table 2. A positive image was detected only up to 4-fold dilution in healthy subjects, whereas a sensitizing concentration of 200 in hepatitis C patients was not detected.
Positive images could be detected even at 32,768-fold dilution or more at μg / ml. When the healthy subject sample was compared with Comparative Example 1, it was found that the nonspecific agglutination reaction could be further reduced, confirming the effect of casein as an additional component to the reagent dispersion.

[0073]

[Table 2]

Comparative Example 2 Hepatitis C diagnostic reagent without blocking by casein (sensitization) The same procedure as in Comparative Example 1 was carried out.

(Cleaning operation) The same operation as in Comparative Example 1 was performed.

(Blocking Operation) The blocking operation performed in Comparative Example 1 was omitted in Comparative Example 2. The hepatitis C diagnostic reagent prepared by the sensitization and washing operations was suspended in solution A containing 3% normal rabbit serum to a concentration of 0.5 (w / vol)% and subjected to the following measurement operation.

(Measurement operation) The sample used for the test was
It was diluted by a factor of 1. Next, 25 μl of the diluted solution of the sample was dropped on each of the 96-well microtiter plates from 1 to 18 wells. Then, the suspension of the above-mentioned hepatitis C diagnostic reagent was dropped in 25 μl of each well. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed. Reagent sensitivity is indicated by titer.

(Results) The performance of the hepatitis C diagnostic reagent prepared as described above was evaluated in the same manner as in Comparative Example 1, using five samples of healthy subjects and five samples of hepatitis C patients. The results are shown in Table 3. When casein blocking was performed, non-specific agglutination was caused by 200 μg / ml sensitization in which non-specific agglutination did not occur in both healthy subjects and hepatitis C patients. This proved that casein blocking could sensitize more antigens, and that the sensitivity of the immunological agglutination reagent could be increased. In addition, blocking of a non-specific sensitivity can be prevented in a healthy subject sample by performing blocking.

[0079]

[Table 3]

Comparative Example 3 A kit for diagnosing hepatitis C comprising a reagent for diagnosing hepatitis C without blocking by casein and a reagent dispersion to which casein was added (sensitization) The same procedure as in Comparative Example 2 was carried out.

(Washing Operation) The same operation as in Comparative Example 2 was performed.

(Blocking Operation) In Comparative Example 3, the blocking operation performed in Comparative Example 1 was omitted. The hepatitis C diagnostic reagent prepared by sensitization and washing operations was suspended in solution B containing 1% casein to a concentration of 0.5 (w / vol)% and subjected to the following measurement.

(Measurement procedure) A sample to be used for the test was serially diluted by a factor of 2 with a solution B containing 1% casein. next,
A diluted solution of the sample was dropped on a 96-well microtiter plate in an amount of 25 μl each from 1 to 18 wells. Then, the suspension of the above-mentioned hepatitis C diagnostic reagent was dropped in 25 μl of each well.
After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed. Reagent sensitivity is indicated by titer.

(Results) The performance of the hepatitis C diagnostic reagent prepared as described above was evaluated in the same manner as in Comparative Example 1 using five samples of healthy subjects and five samples of hepatitis C patients. The results are shown in Table 4. When casein blocking was performed, non-specific agglutination was caused by sensitization at 200 μg / ml in which non-specific agglutination did not occur in both healthy subjects and hepatitis C samples. This proved that casein as an additional component of the reagent dispersion exhibited its effect only when used with casein as a blocking agent.

[0085]

[Table 4]

Comparative Examples 4 to 6 C using a blocking agent other than casein (BSA, normal rabbit serum, glycine)
Hepatitis C diagnostic kit comprising a hepatitis C diagnostic reagent and a reagent dispersion to which casein is added (sensitization) Same as Comparative Example 1. However, sensitizing HC
The V antigen concentration was only 200 μg / ml.

(Cleaning operation) The same as in Comparative Example 1.

(Blocking operation) To the sensitized particles for hepatitis C diagnosis (50 mg), 5 ml each of 1% BSA, 1% normal rabbit serum or 1% glycine was added.
Time blocking was performed. After blocking, the suspension was centrifuged at 2,500 rpm for 5 minutes, and the supernatant was removed. The above operation completes the blocking operation,
Three hepatitis C diagnostic reagents were prepared. Each hepatitis C diagnostic reagent was made up of PBS containing 1 (vol / vol)% casein.
Was suspended in a solution of 0.5% (w / vol)% to prepare respective hepatitis C diagnostic reagents and subjected to the following measurement operation.

(Measurement operation) On the other hand, the sample
The solution was serially diluted by a factor of two to two. Next, 25 μl each of the diluted solution of the sample was placed in a 96-well microtiter plate.
It dripped from a hole to 18 holes. Then, 25 μl of each of the three suspensions of the above hepatitis C diagnostic reagent was dropped into each well. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed. Reagent sensitivity is indicated by titer.

(Results) The performance of the hepatitis C diagnostic reagent prepared as described above was evaluated using five samples of healthy subjects and five samples of hepatitis C patients. Table 5 shows the results. When BSA or glycine was used as a blocking agent, non-specific agglutination was exhibited in all cases, and a normal rabbit serum showed non-specific increase in sensitivity of a healthy subject. Therefore,
The effectiveness of casein as a blocking agent for hepatitis C diagnostic reagent was confirmed.

[0091]

[Table 5]

Example 2 Hepatitis B diagnosis kit comprising an immunological agglutination reagent for diagnosis of hepatitis B using normal rabbit serum as a blocking agent and a reagent dispersion containing normal rabbit serum (sensitization) Diameter 1.8 μm HDP (product of Tokuyama Soda Co., Ltd.)
Was suspended in PBS to a concentration of 5 (w / w)%, and HDP was suspended.
The suspension was used. A monoclonal antibody against hepatitis B virus surface antigen was suspended in PBS at 250 μg / ml to prepare an antibody solution. 1 ml of the above HDP suspension and 1 m of the antibody solution
were mixed in a test tube, and allowed to stand at 37 ° C. for 1 hour.
Sensitized on the surface.

(Washing operation) Thereafter, in order to remove excess monoclonal antibody against the hepatitis B virus surface antigen, the mixture was centrifuged at 2,500 rpm for 5 minutes, and the centrifuged supernatant was removed. To the centrifuged precipitate, 2 ml of PBS was added for washing, and after suspension, 2,500 rpm
After centrifugation for 5 minutes, the supernatant was removed. The above-mentioned HCV antigen protein was adsorbed to obtain HBV antibody-sensitized particles.

(Blocking operation) 5% of 10% normal rabbit serum was added to the HBV antibody-sensitized particles, and blocking was performed at 37 ° C for 1 hour. After blocking, the suspension was centrifuged at 2,500 rpm for 5 minutes, and the supernatant was removed. The immunological agglutination reagent for hepatitis B diagnosis (hereinafter referred to as B
Abbreviated as hepatitis hepatitis diagnostic reagent). The hepatitis B diagnostic reagent was suspended in a PBS solution containing 3 (vol / vol)% normal rabbit serum to a concentration of 0.5 (w / vol)% and subjected to the following measurement operation.

(Measurement operation) On the other hand, the sample
The solution was serially diluted by a factor of two to two. Next, 25 μl each of the diluted solution of the sample was placed in a 96-well microtiter plate.
It dripped from a hole to 18 holes. Next, the above-mentioned suspension of hepatitis B diagnostic reagent was dropped in 25 μl of each well. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed.

(Results) The performance of the hepatitis B diagnostic reagent prepared as described above was evaluated using one healthy subject sample and one hepatitis B patient sample. The results are shown in Table 6. A positive image was detected only up to 4-fold dilution in a healthy subject sample, whereas a positive image was detected up to 128-fold dilution in a hepatitis B patient sample. Comparative Example 7 Preparation of Hepatitis B Diagnostic Reagent without Blocking with Normal Rabbit Serum (Sensitization) The procedure was the same as in Example 2.

(Washing operation) The same operation as in Example 2 was performed.

(Blocking Operation) The blocking operation performed in Example 2 was omitted in Comparative Example 7. The hepatitis B diagnostic reagent prepared by the sensitization and washing operations was suspended as it was in the solution A containing hepatitis B diagnostic reagent 3 (vol / vol)% normal rabbit serum at 0.5 (w / vol)%. It became cloudy and was subjected to the following measurement.

(Measurement procedure) The performance of the hepatitis B diagnostic reagent prepared in the same manner as in Example 2 was evaluated.

(Results) The performance of the hepatitis B diagnostic reagent prepared as described above was evaluated using one healthy subject sample and one hepatitis B patient sample in the same manner as in Example 2. The results are shown in Table 6. When normal rabbit serum blocking was performed, non-specific agglutination occurred at 250 μg / ml sensitization in which non-specific agglutination did not occur in both healthy subjects and hepatitis B samples. Thus, by performing normal rabbit serum blocking, many antibodies can be sensitized without causing nonspecific agglutination, and the sensitivity of the immunological agglutination reagent can be increased. In addition, blocking of a non-specific sensitivity can be prevented in a healthy subject sample by performing blocking. Comparative Example 8 A kit for diagnosing hepatitis B comprising a reagent for diagnosing hepatitis B using normal rabbit serum as a blocking agent and a reagent dispersion to which casein was added (sensitization) The same as in Example 2.

(Washing operation) The same as in the second embodiment.

(Blocking Operation) As in Example 2, B
Hepatitis hepatitis diagnostic reagent was added with 5 ml of 10% normal rabbit serum, and blocking was performed at 37 ° C. for 3 hours. After blocking, the suspension was centrifuged at 2,500 rpm for 5 minutes, and the supernatant was removed. The above procedure was used to complete the blocking operation, and was used as a hepatitis B diagnostic reagent. The above-mentioned hepatitis B diagnostic reagent was prepared using PBS containing 1 (vol / vol)% casein.
Was suspended in a liquid having a concentration of 0.5 (w / vol)% and subjected to the following measurement.

(Measurement operation) On the other hand, the sample
The solution was serially diluted by a factor of two to two. Next, 25 μl each of the diluted solution of the sample was placed in a 96-well microtiter plate.
It dripped from a hole to 18 holes. Next, the above-mentioned suspension of hepatitis B diagnostic reagent was dropped in 25 μl of each well. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed.

(Results) The performance of the hepatitis B diagnostic reagent prepared as described above was evaluated using one sample from a healthy subject and one sample from a hepatitis B patient in the same manner as in Example 2. The results are shown in Table 6. The sensitivity increased non-specifically in the healthy subjects. It turns out that it is effective to use the same protein component added to the reagent dispersion as the blocking agent.
Comparative Example 9 A kit for diagnosing hepatitis B comprising a reagent for diagnosing hepatitis B using casein as a blocking agent and a reagent dispersion to which normal rabbit serum was added (sensitization) The same procedure as in Example 2 was carried out.

(Cleaning operation) The same operation as in Example 2 was performed.

(Blocking operation) 5% of 1% casein was added to the hepatitis B diagnostic reagent, and blocking was performed at 37 ° C for 1 hour. After blocking, add 2,5
Centrifugation was performed at 00 rpm for 5 minutes, and the centrifuged supernatant was removed. The blocking operation was completed by the above operation, and a hepatitis B diagnostic reagent was prepared. 3 (vol)
/ Vol)% solution in PBS containing normal rabbit serum.
It was suspended to 5 (w / vol)% and subjected to the following measurement.

(Measurement operation) On the other hand, the sample
The solution was serially diluted by a factor of two to two. Next, 25 μl each of the diluted solution of the sample was placed in a 96-well microtiter plate.
It dripped from a hole to 18 holes. Next, the above-mentioned suspension of hepatitis B diagnostic reagent was dropped in 25 μl of each well. After dropping, the mixture was shaken with a plate mixer and allowed to stand for 30 minutes, and then an image of tube bottom aggregation was observed.

(Results) The performance of the hepatitis B diagnostic reagent prepared as described above was evaluated using one healthy subject sample and one hepatitis B patient sample. The results are shown in Table 6. A positive image was detected up to 8-fold dilution in a healthy subject sample, whereas a positive image was detected up to 128-fold dilution in a hepatitis B patient sample. As compared with Example 2, the sensitivity of the healthy subject was nonspecifically increased. Therefore, it can be seen that non-specific agglutination is suppressed by adding the same substance as the blocking agent to the reagent dispersion.

[0109]

[Table 6]

[0110]

Claims (2)

[Claims] 1. An immunological agglutination reagent comprising an insoluble carrier carrying an antigen or an antibody and a blocking agent, the same substance as the blocking agent carried by the immunological agglutination reagent, and a buffer. A diagnostic kit comprising a reagent dispersion. 2. An immunological agglutination reagent for hepatitis C diagnosis comprising a hepatitis C virus gene-derived antigen protein and casein supported on an insoluble carrier, and a reagent dispersion comprising casein and a buffer. A diagnostic kit for hepatitis C constituted.