JPH08145997A - Composition for method for measuring enzyme immunity and kit for measuring enzyme immunity - Google Patents

Abstract

PURPOSE: To improve reproducibility by incorporating tetraaokylbenzene or its salt and oxidizing substance, and incorporating, by a specific ratio, dialkylsulfoxyde and a compound selected from heterocyclic compound containing nitrogen. CONSTITUTION: A composition contains tetraaklylbenzene or its salt and oxidizing substance, and contains dialkysulfoxide and one type of compound selected from heterocyclic compound containing nitrogen at 30-60% (V/V). One example of dialkylsulfoxido or a compound (compound 1 group) belonging to the heterocyclic compound containing nitrogen is piperidine. The compound selected from the compound group 1 is contained in the composition for measurement in the ratio of 30-60% (V/V), thus drastically improving the linearity of the calibration curve in the system of an enzyme immunity measuring method using antibody or antigen which is labeled by peroxidase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for measuring peroxidase-like substances and oxidative substances, which is used in the fields of enzyme immunoassay, biochemical assay and the like, and a kit for measuring those substances. Regarding More specifically, it relates to the use of this composition or kit for medical diagnosis or biological evaluation.

[0002]

2. Description of the Related Art Many benzidine compounds used for measuring peroxidase-like substances or oxidative substances have low mutagenicity or have no mutagenicity,
Moreover, it is known for its good sensitivity. Therefore, many studies and improvements have been made on benzidine compounds, especially tetraalkylbenzidine. Conventionally, problems that have been mentioned as problems when using tetraalkylbenzidine include low solubility of tetraalkylbenzidine, storage stability during long-term storage, and how easily a color developing solution is prepared at the time of use. Or, etc.
These problems are solved by the methods disclosed in JP-A-62-134100, JP-A-63-199270, JP-A-1-104196, JP-B-6-38757, JP-A-6-165696 and the like. Has been resolved.

[0003]

However, as a result of examining these known methods, the inventor discovered a surprising fact that has not been a problem at present. The problem is a phenomenon that the calibration curve is not linear in the currently known method using tetraalkylbenzidine, especially in a measurement system using a peroxidase-labeled antibody such as EIA and ELISA. This phenomenon is not observed in the system using free peroxidase, but only in the reaction system using the peroxidase-labeled antibody. Further, even in a measurement system using a peroxidase-labeled antibody, the calibration curve becomes almost linear when a non-benzidine-based coloring agent such as o-phenylenediamine is used. From this, this problem is peculiar to the benzidine-based color-developing agent and has been observed only in the reaction system using the peroxidase-labeled antibody.

According to the examination of the publicly known method shown above, even when an organic solvent such as dimethylformamide (DMF) or methanol is used, or when a method of dissolving in a highly acidic solution is used, It became clear that the phenomenon that the calibration curve was not linear was observed reproducibly.

It is clear that this phenomenon reduces the reproducibility of the results and the accuracy of the measurement, and also affects the reliability of the data obtained. Particularly, since the field to be used is a medical field, a large error entails a risk that it may lead to misdiagnosis. Nevertheless, this phenomenon has not been reported so far, no problem has been raised, etc.
It is believed that it did not attract attention. Naturally, none of the previously reported methods have solved this problem.

The object of the present invention is to make the preparation easy and the color development sensitivity,
It is intended to provide a composition for an enzyme immunoassay having excellent linearity of a calibration curve, and to provide a highly reproducible measurement system by using this composition. Furthermore, the present invention aims to improve reproducibility when a tetraalkylbenzidine or a salt thereof is used in a solid-phase enzyme immunoassay using an antibody or antigen molecule bound with peroxidase.

[0007]

The above objects of the present invention can be achieved by the following constitutions.

1) 30 to 60% (v / v) of at least one compound containing tetraalkylbenzidine or a salt thereof and an oxidizing substance and selected from dialkyl sulfoxide and a nitrogen-containing hetero 6-membered ring compound. ) The composition for enzyme immunoassay, characterized in that the composition is contained.

2) Containing tetraalkylbenzidine or a salt thereof and an oxidizing substance, and containing at least two compounds represented by the following general formula [I] in a proportion of 30 to 60% (v / v). A composition for enzyme immunoassay characterized by comprising:

[0010]

Embedded image

In the formula, R represents a substituted or unsubstituted alkyl group.

3) At least one compound containing tetraalkylbenzidine or a salt thereof and an oxidizing substance and selected from a dialkyl sulfoxide and a nitrogen-containing heterocyclic compound and represented by the above general formula [I]. 30-60% (v /
A composition for enzyme immunoassay, characterized in that it is contained in the ratio of v).

4) 30 to 60% (v / v) of at least one compound containing tetraalkylbenzidine or a salt thereof and selected from dialkyl sulfoxide and nitrogen-containing heterocyclic compound A kit for enzyme immunoassay, characterized in that it is contained in a ratio.

5) At least two compounds containing tetraalkylbenzidine or a salt thereof and selected from the compounds represented by the above general formula [I] are added to the reaction solution in an amount of 30
An enzyme immunoassay kit, characterized in that the kit is contained at a ratio of -60% (v / v).

6) It is selected from at least one compound containing tetraalkylbenzidine or a salt thereof and selected from a dialkyl sulfoxide and a nitrogen-containing heterocyclic compound, and a compound represented by the above general formula [I]. 30-60% (v / v) of at least one compound in the reaction mixture
A kit for enzyme immunoassay, characterized in that it is contained at a ratio of.

7) The tetraalkylbenzidine is 3,3 ',
The composition chromogen for enzyme immunoassay or the kit for enzyme immunoassay according to 1, 2, 3, 4, 5 or 6, which is 5,5′-tetramethylbenzidine.

In the present invention, a dialkyl sulfoxide or a nitrogen-containing heterocyclic compound (hereinafter referred to as compound group 1)
The compounds belonging to, for example, dimethylsulfoxide, piperidine, N-methylpiperidine, 2-methylpiperidine, 1-methyl-4-piperidinone, 2- (2-pyridyl) ethanol, 2-pyridylmethanol, 3-pyridyl Methanol, morpholine, N-formylmorpholine and the like can be mentioned.

The inventor has found that, by containing a compound selected from the compound group 1 at a high concentration in the composition for assay, the linearity of the calibration curve is remarkably improved. . In the already known methods, it was common knowledge that the measuring composition contains 5 to 30% of the additive and the concentration of the additive is kept as low as possible. However, as a result of the inventor's study, it was found that the linearity of the calibration curve tends to be improved by containing 30% or more of the compound selected from the compound group 1, and in particular, it is contained at a high concentration of 35% or more. It has been found that extremely good linearity can be obtained. In addition, if the mixing ratio is increased above 60%, a decrease in sensitivity is observed.
It is effective to use in the range of up to 60%. Further, it is possible to select a plurality of compounds selected from the compound group 1. In this case, it is also possible to use two or three types in combination as long as the total amount of the compounds in the composition for measurement is 35 to 60%. Then, by using this color-developing solution, the linearity of the calibration curve in the enzyme immunoassay system using the antibody or antigen labeled with peroxidase was significantly improved, and the problem was solved.

The compound represented by the general formula [I] of the present invention is, for example, N-methyl-2-pyrrolidinone, N-ethyl-2-pyrrolidinone, N-propyl-2-pyrrolidinone or N-butyl-2-
Pyrrolidinone, N-octyl-2-pyrrolidinone, N-dodecyl-2-pyrrolidinone, N-hydroxymethyl-2-pyrrolidinone, N- (2-hydroxyethyl) -2-pyrrolidinone, N- (3-hydroxypropyl) -2 -Pyrrolidinone, N- (5-hydroxypentyl) -2-pyrrolidinone, N- (8-hydroxyoctyl)
-2-pyrrolidinone, 2-pyrrolidinone-1-acetic acid, N- (2-carboxyethyl) -2-pyrrolidinone, N- (3-carboxypropyl) -2-pyrrolidinone and the like can be mentioned.

The enzyme immunoassay method using an antibody or antigen molecule bound to peroxidase in the present invention refers to the "enzyme immunoassay method" (edited by Eiji Ishikawa et al.), Which was first published in 1982 by the Medical Shoin. The enzyme immunoassay method is as described on pages 30-49 and pages 8-19 of "Enzyme Immunoassay" (P.TIJSSEN, Eiji Ishikawa) published in 1989 by Tokyo Kagaku Dojin. Then, any method can be applied as long as it is a method of measuring the amount of peroxidase activity using a peroxidase-labeled antibody or antigen and a color-forming liquid composed of an oxidizing substance and a color-developing agent.

In the tetraalkylbenzidine used in the present invention, examples of the substituted alkyl group include a methyl group, an ethyl group and a propyl group, and a methyl group is preferable.
A particularly preferred tetraalkylbenzidine is 3,3 ′, 5,
It is 5'-tetramethylbenzidine.

In the assay composition or assay kit of the present invention, the concentration of tetraalkylbenzidine or a salt thereof is 0.01 to 5.0 mg / ml, preferably 0.05 to 2.0 mg / ml.

The composition for assay or the assay kit of the present invention preferably contains a buffering agent.
An organic acid such as citric acid, acetic acid or oxalic acid and an organic acid salt or an inorganic acid salt such as sodium salt or potassium salt such as sodium citrate or sodium phosphate can be selected. A more preferable buffering agent is citric acid.

As components for assisting color development, chelating agents (JP-A-63-199270), nonionic surfactants (JP-A-62-294099), tripolyphosphoric acid (JP-A-1-243)
No. 998) can be added to the reaction system to stabilize the chromogen.

Examples of the peroxidase-like substance according to the present invention include horseradish peroxidase, cytochrome c peroxidase, lactoperoxidase, myeloperoxidase, glutathione peroxidase, and metals and metal compounds such as iron, gold and silver, hemoglobin and the like. However, horseradish peroxidase is preferable.

Further, the oxidizing substance described in the present invention may be any substance as long as it can oxidize tetraalkylbenzidine in the presence of peroxidase. Specifically, hydrogen peroxide, alkyl hydroxyperoxide, p-nitroperoxybenzoic acid,
Cumene hydroxyperoxide, urea peroxide and the like can be mentioned, but hydrogen peroxide is particularly preferable. The concentration of the oxidizing substance is preferably 0.001 to 0.1%. If it is 0.001% or less, the sensitivity is deteriorated, and if it is 0.1% or more, the enzyme activity is inhibited and a good judgment cannot be performed, which is not preferable in terms of reproducibility. More preferably, it is 0.005 to 0.02%. As long as the conditions described above are satisfied, excellent results can be obtained in terms of sensitivity as compared with reagents that have been conventionally used for similar purposes.

When the present invention is applicable to an enzyme immunoassay using an antibody or an antigen molecule to which a peroxidase is bound, the obtained peroxidase activity is used as a standard substance according to the method usually used in EIA, ELISA and the like. The concentration (or amount, unit) of the substance to be measured in the sample can be determined by comparing the thus obtained peroxidase activity with a calibration curve.

[0028]

The present invention will be described in detail below with reference to examples, but the invention is not limited thereto.

Example 1 Preparation of Coloring Solution A composition for assay method for use in the enzyme immunoassay method was prepared. 3,3 ′, 5,5′-Tetraalkylbenzidine was dissolved in 2-methylpiperidine (MePi), and 0.1M citric acid-sodium citrate buffer solution (pH) was added to this solution at the ratio shown in Table 1.
3.5) was added and stirred. This was used as a color forming liquid. Similarly, Me
Dimethyl sulfoxide (DMSO), N-methyl instead of Pi
-2-Pyrrolidinone (NMP) and dimethyl sulfoxide 1: 1
The mixture was dissolved in a mixture (NMP + DMSO) and a 1: 1 mixture of 2-methylpiperidine and morpholine (MePi + Mo) to prepare a color developing solution.

[0030]

[Table 1]

As a comparative example, as shown in Table 2, 3,3 ′, 5,
Dissolve 5 mg of 5'-tetramethylbenzidine in methanol, ethanol or dimethylformamide (DMF), and then add 0.1M citric acid-sodium citrate buffer (pH 3.5).
A coloring solution mixed with was also prepared.

Example 2 Determination of the amount of galactosyl transferase 3,3 ', containing the compound of the present invention prepared in Example 1
An enzyme immunoassay system was used to examine the color development solution of 5,5'-tetramethylbenzidine and the color development solution containing 3,3 ', 5,5'-tetramethylbenzidine prepared by the conventional method. went.

Anticancer-related galactosyltransferase (hereinafter abbreviated as GAT) described in Japanese Patent Laid-Open No. 3-259093
Monoclonal antibody MAb8513 containing 1M sodium chloride
Dilute to 10 μg / ml in 0.1 M carbonate buffer (pH 9.15) and add polystyrene beads (Sekisui Chemical Co., Ltd.) to this solution at 4 ° C.
・ Immersion was carried out for 24 hours to immobilize the antibody.

Next, a 0.02 M phosphate buffer solution (150 mM NaCl-containing pH 7.3, hereinafter referred to as PBS) containing 1% bovine serum albumin (hereinafter abbreviated as BSA) was prepared as a blocking solution, and this solution was prepared. Transfer the beads after antibody immobilization, 37 ℃,
After immersion for 10 hours and standing, coating (blocking) of antibody non-binding sites on the bead surface was performed. Through these series of operations, the antibody-immobilized beads used below were obtained.

Cancer patient pool serum containing GAT was used as a sample. 50 μl of this sample was added with 0.02M phosphate buffer (pH
200 μl of 6.5, 150 mM NaCl and 0.01% Tween 20) was added, and the antibody-immobilized beads prepared by the method described above were immersed therein to carry out the primary reaction (45 ° C., 2 hours). After the reaction, washing operation was performed with PBS. HRP-labeled anti-G labeled with horseradish peroxidase (hereinafter referred to as HRP)
AT antibody MAb8628 (described in JP-A-3-259093) was appropriately diluted in a PBS solution containing 1% BSA, and 250 μl of this was added to carry out a secondary reaction. After that, a washing operation with PBS was similarly performed.

Further, 300 μl of each color-developing solution prepared in Example 1 was added, and a color-developing reaction was carried out at room temperature for 30 minutes. Further, the same reaction was performed for the color forming liquid prepared as a comparative example and the color forming liquid containing no organic solvent. After the reaction, add 1 ml of 1N H ₂ SO ₄ to each to stop the reaction.
Absorbance at 50 nm was measured. The results were compared by the correlation coefficient of the calibration curve. The results are shown in FIG. 1 and Table 2.

[0037]

[Table 2]

As a result, the color developing solution of 3,3 ', 5,5'-tetramethylbenzidine of the present invention was compared with the conventional color developing solution using 3,3', 5,5'-tetramethylbenzidine. As a result, excellent linearity was obtained. In particular, it was found that the mixing ratio of the compound of the present invention was good in the range of 30 to 60%. Also, as for the mixing ratio when a plurality of compounds are mixed, good results are obtained when the ratio of the total amount of the compounds is in the range of 30 to 60%.

Example 3 <Production of color-developing solution kit> A color-developing solution kit to be included in the enzyme immunoassay kit was produced.

(1) Preparation of color-developing agent 3.95 g of citric acid monohydrate is weighed, and about 20 ml of distilled water is added and dissolved. To this solution of citric acid monohydrate, 3,3 ', 5,
Add 50 mg of 5'-tetramethylbenzidine and stir to dissolve. After dissolution, add distilled water to adjust the total volume to 25 ml. The 3,3 ', 5,5'-tetramethylbenzidine solution thus obtained is dispensed into brown vials in an amount of 1 ml and freeze-dried.
This was used as a color former.

(2) Preparation of substrate solution 9.18 g of trisodium citrate (dihydrate) is weighed and dissolved in about 250 ml of distilled water. Dimethylsulfoxide and N-methyl-2-pyrrolidinone were added to this solution, respectively.
After adding 00 ml and further adding hydrogen peroxide solution to a final concentration of 0.02% by weight and stirring well, distilled water is added to bring the total amount to 500 ml. 20 ml of this solution in a brown vial
Dispense each one and save. This is the substrate solution.

(3) Preparation of color developing solution The color developing agent prepared in (1) and the substrate solution prepared in (2) were mixed to prepare a color developing solution. At this time, tetramethylbenzidine was completely dissolved and no precipitation was observed.

[0043]

Industrial Applicability According to the present invention, the linearity of the calibration curve is dramatically improved in the enzyme immunoassay system in which the enzyme-labeled antibody is immobilized on the solid phase, and the reproducibility and reliability of the results are dramatically improved. . In particular, in a solid-phase enzyme immunoassay using a peroxidase-bound antibody or antigen molecule, a remarkable effect was obtained when tetraalkylbenzidine or a salt thereof was used.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between the antigen concentration and the absorbance when the enzyme immunoassay composition of the present invention and a conventional enzyme immunoassay composition are used.

[Explanation of symbols]

1 Calibration curve using a coloring solution containing a 1: 1 mixture of dimethyl sulfoxide and N-methyl-2-pyrrolidinone at a mixing ratio of 40% (invention) 2 Coloring containing a mixture ratio of 40% dimethylformamide Calibration curve using liquid (comparative example)

Claims (7)

[Claims] 1. At least one compound containing tetraalkylbenzidine or a salt thereof and an oxidizing substance and selected from dialkyl sulfoxide and a nitrogen-containing hetero 6-membered ring compound is 30 to 60% (v / v). ) The composition for enzyme immunoassay, characterized in that the composition is contained. 2. A tetraalkylbenzidine or a salt thereof and an oxidizing substance, and at least 2 kinds of compounds represented by the following general formula [I] in a proportion of 30 to 60% (v / v). A composition for enzyme immunoassay characterized by comprising: Embedded image In the formula, R represents a substituted or unsubstituted alkyl group. 3. At least one selected from a dialkyl sulfoxide and a nitrogen-containing heterocyclic compound, containing tetraalkylbenzidine or a salt thereof and an oxidizing substance.
Composition and at least one compound selected from the compounds represented by the above general formula [I] in an amount of 30 to 60% (v / v). . 4. A reaction liquid containing at least one compound selected from the group consisting of dialkyl sulfoxide and nitrogen-containing heterocyclic compound containing tetraalkylbenzidine or a salt thereof in an amount of 30 to 60% (v / v). A kit for enzyme immunoassay, characterized in that it is contained in a ratio. 5. A reaction liquid containing at least two compounds containing tetraalkylbenzidine or a salt thereof and selected from the compounds represented by the above general formula [I] in an amount of 30 to 60.
% (V / v) is contained in the enzyme immunoassay kit. 6. A compound containing tetraalkylbenzidine or a salt thereof and selected from at least one compound selected from dialkyl sulfoxide and a nitrogen-containing heterocyclic compound and a compound represented by the above general formula [I]. A kit for enzyme immunoassay characterized in that at least one compound is contained in the reaction solution at a rate of 30 to 60% (v / v). 7. The tetraalkylbenzidine is 3,3 ′, 5,
The composition chromogen for enzyme immunoassay according to claim 1, 2, 3, 4, 5 or 6, which is 5'-tetramethylbenzidine, or the kit for enzyme immunoassay.