JP4299332B2 - Enzyme immunoassay method and developing composition used therefor - Google Patents

Description

  The present invention relates to an enzyme immunoassay method and a composition for development used therefor. More specifically, the present invention enables highly sensitive detection in a short time in an enzyme immunoassay method using a benzidine-based derivative that detects a peroxide-degrading substance such as peroxidase, an oxidizing substance, etc. as a chromogenic reagent. It is related to the technology.

  In recent years, an immunochromatographic method that can easily detect various substances in a sample using an immunological reaction such as an antibody-antigen reaction has attracted attention in a wide range of fields including clinical diagnosis.

  This method is performed as follows, for example.

  First, a test substance in a sample, for example, a first immune substance that specifically reacts with an antigen, for example, a monoclonal antibody is immobilized on a stationary phase comprising a water-absorbing substrate. After the test substance is bound to the fixing part, a labeled second immunity substance capable of binding to the test substance, for example, a labeled antibody, is developed to form a complex. Subsequently, the test substance in the sample is qualitatively or quantitatively measured by detecting the labeled second immunity substance bound at the fixing part.

  By applying such immunochromatography method, pregnancy test kits, hepatitis B diagnostic kits, etc. have been developed and marketed by various companies, but these are all not using enzymes as labels. Absent.

  The immunoassay method using an enzyme as a label is highly sensitive by amplifying the signal by the reaction of the enzyme labeled with the immunity substance component and its chromogenic substrate component, as represented by ELISA (Enzyme Linked Immuno Sorbent Assay). Measurement is possible.

  For this reason, in the immunochromatography method as described above, development of methods using enzymes such as peroxidase and alkaline phosphatase as a label is underway.

  For example, Patent Document 1 and Patent Document 2 disclose a method in which alkaline phosphatase is used as a label and color is developed with a chromogenic substrate, a fluorescent substrate, a luminescent substrate, or the like. Patent Document 3 discloses a method in which peroxidase is used as a label and a benzidine derivative is used as a chromogenic substrate.

  However, it has been pointed out that there is a problem in the storage stability of such an enzyme substrate in a developing solution, and that the decomposition occurs due to the storage over a long period of time and the color development sensitivity decreases. In particular, some benzidine derivatives have the advantage of being highly toxic and have high sensitivity, but they have a problem in storage stability because they are only slightly soluble in water and precipitate at low temperatures.

  Therefore, conventionally, a benzidine derivative is conventionally dissolved in an aqueous solution containing an organic solvent such as methanol and 1-methyl-2-pyrrolidone at a high concentration by dissolving the benzidine color former in a high concentration with a buffer solution before use. Diluted and used. However, conventional benzidine-based indicators containing a large amount of organic solvent are relatively stable when the organic solvent before the preparation of the indicator is at a high concentration, but unstable after being diluted to the use concentration and used as an indicator. However, there is a problem that the degree of color development is lowered, and since it cannot be stored at a use concentration for a long time, it is necessary to prepare it immediately before use. These are significant limitations in developing products such as kit-made diagnostic sets using the immunochromatography method described above.

In order to improve instability in the solution, it has also been proposed to dry-fix the enzyme substrate on the chromatographic piece. However, the enzyme substrate dried and fixed needs to be completely redissolved by the developing solution that develops in the chromatographic piece. However, if the dissolution is insufficient, the enzyme reaction does not occur sufficiently. As a result, the color developability deteriorates, and if not sufficiently dissolved, there is a problem that the chromatographic piece made of the porous substrate may be clogged to cause development failure. .
JP 2000-329767 A JP 2000-193665 A JP 2001-13144 A

  Accordingly, it is an object of the present invention to provide an enzyme immunoassay method that solves the above-described problems in the prior art and a composition for development used therefor. The present invention also provides an enzyme immunoassay method in which a benzidine derivative is used as a chromogenic reagent, and a test substance is detected using a second immunity substance labeled with an enzyme using the benzidine derivative as a substrate. It is an object of the present invention to provide an enzyme immunoassay method capable of highly sensitive detection in time and a developing composition used therefor.

  As a result of intensive studies to solve the above problems, the present inventors have used a benzidine derivative as a color-developing reagent and a second immunity substance labeled with an enzyme using the benzidine derivative as a substrate. Surprisingly, in the enzyme immunoassay method used to detect the test substance, the presence of carboxymethylcellulose in the reaction system can amplify the detection sensitivity (chromogenicity) without extending the detection time. This is the headline and the present invention.

  That is, the present invention that solves the above-described problems uses enzyme immunization that uses a benzidine derivative as a color-developing reagent and detects a test substance using a second immunity substance that is labeled with an enzyme that uses the benzidine derivative as a substrate. In the assay method, the enzyme immunoassay method is characterized in that carboxymethylcellulose is contained in the mobile phase.

  The present invention also shows the enzyme immunoassay method described above, wherein the benzidine derivative is 3,3 ′, 5,5′-tetramethylbenzidine.

The present invention further immobilizes a first immunity substance that specifically reacts with a test substance in a sample on the downstream side of a stationary phase comprising a water-absorbing substrate,
On the upstream side of the stationary phase, an enzyme-labeled second immunological substance that can bind to the test substance is brought into contact with the test substance in the sample to form a reaction system,
Furthermore, from the upstream side, the reaction system is developed downstream by flowing down a mobile phase containing a benzidine derivative and carboxymethyl cellulose,
The test substance that has reacted with the enzyme-labeled second immunity substance is bound to the immobilized first immunity substance, and color is developed by the reaction between the benzidine derivative and the enzyme labeled with the second immunity substance. The enzyme immunoassay method for detecting a test substance in a sample is shown.

The present invention also immobilizes a first immunity substance that specifically reacts with a test substance in a sample on the downstream side of a stationary phase comprising a water-absorbing substrate,
On the upstream side of the stationary phase, a test substance in the sample is mixed with an enzyme-labeled second immunity substance that can compete with the test substance to form a mixed system,
Furthermore, from the upstream side, a mobile phase containing a benzidine derivative and carboxymethyl cellulose is flowed down, and the mixed system is developed downstream,
An enzyme-labeled second immunity substance and a test substance are competitively bound to the immobilized first immunity substance, and a reaction between the benzidine derivative and the enzyme labeled with the second immunity substance is performed. The above enzyme immunoassay method for developing a color and detecting a test substance in a sample according to the degree or presence thereof is shown.

  The present invention also shows the enzyme immunoassay described above, wherein the enzyme using a benzidine derivative as a substrate is peroxidase.

  In the present invention, the oxidase is immobilized at a position upstream from the position where the first immunity substance of the stationary phase is immobilized, while the substrate substance of the oxidase is added to the developing solution, The enzyme immunoassay method described above, which generates active oxygen when a developing solution comes into contact with an immobilized oxidase, is shown.

  Furthermore, the present invention that solves the above-described problems uses an enzyme immunity that uses a benzidine derivative as a color-developing reagent and detects a test substance using a second immunity substance labeled with an enzyme using the benzidine derivative as a substrate. A developing solution composition used in an assay method, wherein the developing solution composition contains carboxymethyl cellulose.

  The present invention also shows a developing solution composition in which the benzidine derivative is 3,3 ′, 5,5′-tetramethylbenzidine.

  The present invention further shows the above-described developing solution composition in which the carboxymethyl cellulose is blended in the developing solution at a ratio of 0.003% by mass or more.

  The present invention further shows the above developing solution composition, wherein the carboxymethylcellulose has an average degree of substitution (number of carboxytyl groups per glucose unit) of 0.6 or more.

  According to the present invention, an enzyme immunoassay method using a benzidine derivative as a chromogenic reagent enables highly sensitive detection in a short time, and greatly contributes to the detection and diagnosis of various pathogenic substances. can do.

  Hereinafter, the present invention will be described in detail based on specific embodiments.

  The present invention relates to an enzyme immunoassay method that uses a benzidine derivative as a color-developing reagent and detects a test substance using a second immunity substance labeled with an enzyme using the benzidine derivative as a substrate. This is an enzyme immunoassay method characterized by containing carboxymethylcellulose.

  As a measurement principle of the enzyme immunoassay method according to the present invention, either a sandwich method or a competitive method can be used. Furthermore, when the labeled test substance (for example, antigen) is bound to the immobilized immune substance (for example, antibody), the test substance is not directly bound, but the test substance. In advance, an immune substance (for example, a primary antibody) is reacted in advance, and an immune substance (for example, a secondary antibody) that specifically binds to the immune substance is immobilized, and a test substance is indirectly bound to the test substance. It is also possible to use a double or multiple sandwich method or a double or multiple competition method for bonding.

  The enzyme immunoassay method according to the present invention includes, for example, the following steps, but is not limited thereto.

  First, in an embodiment applying the sandwich method as an enzyme immunoassay method according to the present invention, (a) a specific reaction with a test substance in a sample downstream of a stationary phase comprising a water-absorbing substrate. And (b) contacting an enzyme-labeled second immunity substance that can bind to the test substance in the sample upstream of the stationary phase. To form a reaction system, and (c) from the upstream side, a mobile phase containing a benzidine derivative and carboxymethylcellulose is flowed down to develop the reaction system downstream, and (d) the enzyme is labeled A test substance that has reacted with a second immunity substance is bound to the immobilized first immunity substance, and a color is developed by a reaction between a benzidine derivative and an enzyme labeled with the second immunity substance. Detect a test substance.

  Further, in one embodiment in which a competitive method is applied as the enzyme immunoassay method according to the present invention, (a) a specific reaction with a test substance in a sample downstream of a stationary phase comprising a water-absorbing substrate. (B) an enzyme-labeled second immunity substance capable of competing with the test substance in the sample upstream of the stationary phase. (C) Furthermore, from the upstream side, a mobile phase containing a benzidine derivative and carboxymethylcellulose is flowed down, and the mixed system is developed downstream, and (d) the enzyme is labeled. The second immunity substance and the test substance are competitively bound to the immobilized first immunity substance, and color is developed by the reaction between the benzidine derivative and the enzyme labeled with the second immunity substance, Specimen in sample depending on degree or presence To detect.

  The test substance that can be detected by the enzyme immunoassay method according to the present invention is not particularly limited as long as it can be detected by the sandwich method or the competitive method as described above. For example, fibrin degradation products in detecting thrombosis, acute leukemia, α2-plasmin inhibitor / plasmin complex (PIC), vitamin K deficient protein-II (PIVKAII) in detecting severe liver disease, etc. It is also a marker for diseases associated with vitamin K deficiency and liver cancer); pathogenic bacteria such as Escherichia coli O-157, methicillin-resistant Staphylococcus aureus, Salmonella, Vibrio parahaemolyticus, Helicobacter pylori and Chlamydia trichomatis, and components thereof Toxins produced by bacteria such as verotoxin and streptolysin O; for example, proteins such as human transferrin, human albumin, human immunoglobulin, microglobulin and C-reactive protein; HBs, HBc, HBe antigens and antibodies of hepatitis B virus; , Hepatitis C virus antigen And viral antigens and antibodies such as human immunodeficiency virus antigens and antibodies, rotavirus antigens and antibodies, and adenovirus antigens and antibodies; and other microorganisms such as yeast and mold or antibodies thereto.

  The first immunological substance that can specifically react with the test substance is a substance that can specifically bind to the test substance to be detected and can be selected according to the target test substance. is there. Specific examples include antibodies or antigens (wherein antigens include proteins, peptides, haptens, etc.), depending on the analyte to be detected, known ones used in the sandwich method or the like are appropriately used. select. For example, when the test substance is an antigen, a corresponding antibody can be used as an immunity body. In this case, a polyclonal antibody and a monoclonal antibody can be used as the second immunity substance used as a component of the first immunity substance to be immobilized and the enzyme-labeled specific conjugate described later. When is a monoclonal antibody, the other immune body preferably recognizes an antigenic determinant different from that of the monoclonal antibody. When the test substance is an antibody, the corresponding antigen can be used as the immunity substance. In this case, anti-antibodies (anti-immunoglobulin antibodies) against the corresponding antigen and antibody as the test substance may be used as the first immunity substance and the second immunity substance, respectively.

  The benzidine derivative used as a color-developing reagent in the present invention is not particularly limited, and examples thereof include benzidine, 3,3 ′, 5,5′-tetra (alkyl) benzidine, 3,3 ′, 5. , 5′-tetra (alkyl) benzidine dihydrochloride, 3,3′-diaminobenzidine, 3,3′-diaminobenzidine tetrahydrochloride, and the like, and mixtures thereof. The alkyl substituents of the 3,3 ′, 5,5′-tetra (alkyl) benzidine to 3,3 ′, 5,5′-tetra (alkyl) benzidine dihydrochloride include methyl, ethyl, and n-propyl. And isopropyl, and various alkyl groups having 1 to 6 carbon atoms such as butyl, pentyl and hexyl.

  Of these, 3,3 ', 5,5'-tetraalkylbenzidine is preferable, and 3,3', 5,5'-tetramethylbenzidine is particularly desirable. Among these benzidine derivatives, those in the salt form can be added as they are to the aqueous medium, and those not in the salt form can be dissolved in a small amount of an organic solvent and added to the aqueous medium.

  On the other hand, as the enzyme used as a labeling enzyme in the present invention, the enzyme using the benzidine derivative as a substrate is, for example, derived from a plant extracted from horseradish, soybeans, rice straw, etc., or derived from animals extracted from milk, leukocytes, etc. Among them, various peroxidases or catalases are preferable. Among them, peroxidase is preferable, and horseradish peroxidase is particularly preferable.

  As carboxymethylcellulose, carboxymethylcellulose or water-soluble carboxymethylcellulose salts such as carboxymethylcellulose sodium salt can be used. The carboxymethyl cellulose is not particularly limited, but the average degree of substitution (number of carboxymethyl groups per glucose unit) is 0.6 or more, more preferably 1.3 or more, and still more preferably 2.3. The above is desirable. In particular, when the average substitution degree is 2.3 or more, a more effective sensitizing effect can be expected.

The molecular weight (weight average molecular weight Mw) of carboxymethyl cellulose is not particularly limited, but is, for example, 10,000 to 2,000,000, more preferably 50,000 to 1,000,000, and still more preferably about 90000 to 700,000.
Even when the average degree of substitution of carboxymethyl cellulose is relatively low (for example, 0.6 to 0.9), an effective sensitizing effect can be expected by increasing the molecular weight.

  In the present invention, in the developing solution which is a mobile phase containing a benzidine derivative and carboxymethyl cellulose, the content of the carboxymethyl cellulose is, for example, 0.003% by mass or more, more preferably 0.010% by mass or more. is there. This is because if the content of carboxymethyl cellulose is less than 0.003% by mass, an effective sensitizing effect may not be expected. In addition, even if the content of carboxymethyl cellulose is 0.010% by mass or more, the sensitization effect cannot be expected to be improved as much as 0.010% by mass. Since the detection time may be delayed, the upper limit concentration is about 1.000 mass%, more preferably about 0.100 mass%.

  Note that the composition of the developing solution includes an appropriate water-soluble organic solvent for dissolving the benzidine derivative in addition to water. The water-soluble organic solvent is not particularly limited as long as it can dissolve the benzidine derivative and does not inhibit the enzyme reaction and cause corrosion of the water-absorbing substrate or the like at the concentration contained. Specifically, for example, dioxane, acetone, dimethylformamide (DMF), N, N-dimethylacetamide (NMP), N-methylpyrrolidone, dimethyl sulfoxide (DMSO), 2-methoxyethanol, 2-ethoxyethanol and the like can be mentioned. DMSO or DMF is preferred from the viewpoint of being difficult to inhibit enzyme activity. Although not particularly limited, when DMSO is used, its content is about 0.1 to 10% by mass. Moreover, when using DMF, although it does not necessarily limit, the content is about 0.1-10 mass%.

  In addition, the developing solution may contain an appropriate buffer component for adjusting to an appropriate pH range that does not inhibit the reaction of a labeling enzyme such as peroxidase, for example, pH 4 to 8, more preferably pH 5 to 7. Examples of such buffer components include, but are not limited to, citrate buffer, phosphate buffer, borate buffer, Tris-HCl buffer, Bis-Tris-HCl buffer, and imidazole buffer. The concentration of the buffer component can be appropriately set and is not particularly limited, but is preferably about 5 mM to 0.2 M.

  Furthermore, hydrogen peroxide can be added to the developing solution. However, since hydrogen peroxide has poor storage stability, an embodiment in which the developing solution is stored in advance in a test instrument such as a diagnostic kit. Is not very suitable. In this case, the first immunity substance is immobilized in the middle of the flow path of the immobilization layer, preferably downstream of the position where the enzyme-labeled second immunity substance capable of binding to the test substance is located. An oxidase enzyme is arranged at a position upstream from the position, and the substrate substance in the developing solution is contained, and the substrate substance is developed and contacted with the oxidase enzyme to react with it, so that hydrogen peroxide (active oxygen) is added to the reaction system. ) Is preferably provided.

  As the oxidase enzyme used in such a form, for example, glucose oxidase, cholesterol oxidase and the like which can be appropriately selected from various known ones that do not inhibit the reaction in the immune reaction system can be used. Cholesterol or the like can be used as a substrate substance.

  Further, in the developing solution, surfactants such as nonionic surfactants and cationic surfactants, and amine polyacetates or salts thereof for increasing storage stability can be added as necessary. Examples of amine polyacetate include N- (2-hydroxylethyl) ethylenediaminetriacetic acid, ethylenediaminetetraacetic acid, cyclohexylenediaminetetraacetic acid, nitrilotriacetic acid, iminodiacetic acid, α-ethylenediaminediacetic acid dipropionic acid, and β-ethylenediamine. And dipropionic acid diacetate, hydroxylethylaminodiacetic acid, and mixtures thereof. Moreover, sodium, potassium, etc. are mentioned as these salts.

  In addition, the water-absorbing substrate constituting the stationary phase is not particularly limited as long as it is a porous material or fiber material exhibiting capillary action, and for example, filter paper containing nitrocellulose, cellulose, glass fiber, or the like can be used.

Hereinafter, the present invention will be described more specifically based on examples. The following examples are provided only for the purpose of facilitating the understanding of the present invention, and are not intended to limit the present invention.
Example 1
Detection was carried out using two kinds of detection antibodies (1 mg / ml) shown in Table 1 and respective detection objects. The assay was started by dropping 20 μl of a plasma sample containing each detection target onto the conjugate pad and immediately flowing the developing solution from the upstream side to the stationary phase. The obtained results are shown in Table 1 and FIG.

  As the developing solution, 100 mM Bis-Tris HCl pH 6.0 containing 1% glucose, 0.01% carboxymethylcellulose and 1 mM 3,3 ′, 5,5′-tetramethylbenzidine was used, and the conjugate composition was 4 50 mM PBS, pH 7.0 containing an antibody for detection of μl / ml peroxidase was used, and 1 mg / ml anti-mouse IgG antibody was used as a control antibody.

Example 2
Next, the influence of the difference in the degree of substitution of carboxymethylcellulose (CMC) on the detection sensitivity was examined.

Example 3
Next, the effect of differences in CMC concentration on detection sensitivity was examined.
The CMC substitution degree was> 2.3 (molecular weight was 200,000). Under the following detection conditions, CMC concentrations were prepared in six ways: 0, 0.001, 0.003, 0.010, 0.032, and 0.100%, and the differences were observed.
(A) Detection condition Detection antibody: Anti-FDP D dimmer monoclonal antibody Detection time: 5 minutes Detected: FDP D dimmer, 20 μl (500 ng / ml)
Other detection conditions (detection solution composition, conjugate composition, and composition on membrane) were the same as in Example 1.
(B) Detection method The same method as in Example 1 was used.
The obtained results are shown in FIG .
As shown in FIG. 3 , no detection line was seen when CMC was not added. Moreover, although the detection line can be confirmed when the concentration of CMC is 0.003% or more, a clearer line can be obtained at 0.01%. In addition, even when the CMC concentration was increased, the sensitization effect was not substantially improved.
Example 4
Next, the influence of the difference in molecular weight of carboxymethyl cellulose (CMC) on the detection sensitivity was examined.
The degree of CMC substitution was 0.7 to 0.9, and the CMC concentration was 0.01%.
Under the following detection conditions, CMC molecular weights of 90,000, 250,000 and 700,000 were prepared, and the differences were observed.
(A) Detection condition Detection antibody: Anti-FDP D dimmer monoclonal antibody Detection time: 5 minutes Detected: FDP D dimmer, 20 μl (500 ng / ml)
Other detection conditions (detection solution composition, conjugate composition, and composition on membrane) were the same as in Example 1.
(B) Detection method The same method as in Example 1 was used.

  The obtained results are shown in FIG. As shown in FIG. 4, even when the degree of substitution of CMC was low (CMC substitution degree: 0.7 to 0.9), it was detectable if the molecular weight was large. In addition, although the molecular weight of CMC tends to require detection time, it was able to be detected within 5 minutes even when the molecular weight was 700,000 (however, the degree of substitution of CMC was 0.7 to 0.9).

It is a drawing substitute photograph which shows the detection result in an Example. It is a drawing substitute photograph which shows the detection result in another Example. It is a drawing substitute photograph which shows the detection result in another Example. It is a drawing substitute photograph which shows the detection result in another Example.

Claims (10)

(A) a first immunity substance that specifically reacts with a test substance in a sample immobilized on a stationary phase, and (b) a benzidine derivative as a coloring reagent, and the benzidine derivative as a substrate. a second immune-based material that will be labeled by an enzyme, the enzyme immunoassay method for detecting an analyte using the benzidine derivative, a second immune system to be labeled the analyte, and by the enzymes in the mobile phase used for moving a substance to the immobilized first immunogenic substance side, an enzyme immunoassay method which is characterized in that is carboxymethylcellulose.   The enzyme immunoassay method according to claim 1, wherein the benzidine derivative is 3,3 ', 5,5'-tetramethylbenzidine. Immobilizing a first immunity substance that specifically reacts with a test substance in a sample on the downstream side of a stationary phase comprising a water-absorbing substrate,
On the upstream side of the stationary phase, an enzyme-labeled second immunological substance that can bind to the test substance is brought into contact with the test substance in the sample to form a reaction system,
Furthermore, from the upstream side, the reaction system is developed downstream by flowing down a mobile phase containing a benzidine derivative and carboxymethyl cellulose,
The test substance that has reacted with the enzyme-labeled second immunity substance is bound to the immobilized first immunity substance, and color is developed by the reaction between the benzidine derivative and the enzyme labeled with the second immunity substance. The enzyme immunoassay method according to claim 1, wherein a test substance in the sample is detected. Immobilizing a first immunity substance that specifically reacts with a test substance in a sample on the downstream side of a stationary phase comprising a water-absorbing substrate,
On the upstream side of the stationary phase, a test substance in the sample is mixed with an enzyme-labeled second immunity substance that can compete with the test substance to form a mixed system,
Furthermore, from the upstream side, a mobile phase containing a benzidine derivative and carboxymethyl cellulose is flowed down, and the mixed system is developed downstream,
An enzyme-labeled second immunity substance and a test substance are competitively bound to the immobilized first immunity substance, and a reaction between the benzidine derivative and an enzyme labeled with the second immunity substance is performed. The enzyme immunoassay method according to claim 1, wherein the test substance in the sample is detected based on the degree or presence of color development.   The enzyme immunoassay method according to any one of claims 1 to 4, wherein the labeling enzyme using a benzidine derivative as a substrate is peroxidase. Upstream position than the first immunogenic substance of the stationary phase was immobilized position, leave immobilized oxidase, whereas, during said movement phase possible to add the substrate material of this oxidase, mobile phase immobilized The enzyme immunoassay method according to any one of claims 3 to 5, wherein active oxygen is generated by contact with the oxidized oxidase. (A) a first immunity substance that specifically reacts with a test substance in a sample immobilized on a stationary phase, and (b) a benzidine derivative as a coloring reagent, and the benzidine derivative as a substrate. a second immune-based material that will be labeled by an enzyme, the enzyme immunoassay method for detecting an analyte using the benzidine derivative, a second immune system to be labeled the by analyte and the enzyme A developing solution composition used for transferring a substance to the immobilized first immunizing substance side, which contains carboxymethylcellulose.   The developing solution composition according to claim 7, wherein the benzidine derivative is 3,3 ', 5,5'-tetramethylbenzidine.   The developing solution composition according to claim 7 or 8, wherein the carboxymethyl cellulose is blended in the developing solution at a ratio of 0.003% by mass or more.   The developing solution composition according to any one of claims 7 to 9, wherein the carboxymethyl cellulose has an average degree of substitution (the number of carboxytyl groups per glucose unit) of 0.6 or more.