JPH0740957B2 - Chromogen aqueous solution for peroxidase activity measurement - Google Patents

Description

TECHNICAL FIELD The present invention relates to an aqueous chromogenic solution for measuring peroxidase enzyme activity. More specifically, in an enzyme immunoassay method using a peroxidase-labeled antibody or an antigen, a stable and reproducible 3,3 ′, 5,5′-tetraalkylbenzidine or a salt thereof for measuring the enzyme activity is used. The present invention relates to a chromogen aqueous solution and a method for measuring the peroxidase activity using a substrate solution comprising the chromogen aqueous solution and an aqueous peroxide solution.

b. Conventional Technology Recently, an enzyme immunoassay in which an antigen or an antibody is labeled with an enzyme has been widely used as a method for measuring a component in a body fluid, for example, an antigen or an antibody in blood or urine. As a typical method, there is a sandwich method. In this method, (1) First, the first method for the substance (antigen) to be measured
The antibody is immobilized on a carrier such as beads, and an antigen is added to the carrier to bind the antigen to the immobilized antibody. (2) Next, a second antibody labeled with an enzyme is added to form a solid-phased antibody-antigen-labeled antibody complex. (3) Then, the enzyme-labeled antibody that has not bound to the antigen is washed and removed, and then the amount of antigen is determined by measuring the enzyme activity bound to the solid phase. That is, the enzyme activity is determined by the amount of the enzyme-labeled antibody bound to the solid phase, and the amount of the enzyme-labeled antibody is determined by the amount of the antigen bound to the immobilized antibody, so that the enzyme activity represents the amount of the antigen. Here, as the enzyme, peroxidase is widely used because of its relatively small molecular weight, rapid reaction, and expected highly sensitive measurement in a short time.

The peroxidase activity is measured by various reaction methods. The most commonly used method is to colorimetrically quantify a dye by oxidizing a chromogen in the presence of peroxide. There is. Various chromogens are used, but tetraalkylbenzidine, among which very sensitive sensitivity is obtained, and is non-carcinogenic 3,
3 ', 5,5'-Tetramethylbenzidine (hereinafter referred to as TMB) was developed (Tetrahedron Vol. 30, p. 3299, 1974), and because of its usefulness, a test strip (JP-A-52-
21892) and the sandwich method (JP-A-62-28666).

However, for example, TMB has a very poor solubility in water, and thus, conventionally, a method of dissolving TMB in an organic solvent such as methanol (US Pat. No. 4,503,143) is used.
Alternatively, a method of dissolving in an aqueous medium containing N-methylpyrrolidone (JP-A-62-500882) or a method of dissolving in 70% isopropyl alcohol and then adding a buffer solution to obtain a solution having a pH of about 8 ( JP-A-62-28666) has been devised and used.

However, the method of dissolving TMB in methanol is not only harmful to the human body but also a flammable dangerous substance, and is significantly limited in commercial production.
Further, the solubility is not always sufficient, and therefore, when the amount required for the enzyme reaction is dissolved and stored at 4 ° C., crystals are precipitated or methanol is liable to volatilize. Furthermore, when used as a substrate solution for an enzymatic reaction, an aqueous medium containing hydrogen peroxide and a methanol solution of TMB are mixed and subjected to the reaction, but sometimes turbidity occurs during this mixing, or In some cases, an oxidant may be precipitated after the enzymatic reaction, which is not yet satisfactory in terms of accuracy and reproducibility in measuring peroxidase activity.

In addition, the method of dissolving in an aqueous medium containing N-methylpyrrolidone is not preferable in terms of handling an organic solvent, and is not necessarily sufficient in terms of stability and reproducibility of a substrate solution composed of TMB and hydrogen peroxide.

The method of adjusting the pH to about 8 by adding a buffer solution after dissolving it in 70% isopropyl alcohol also has drawbacks such as complicated preparation operations in addition to the same problems.

In order to solve the above-mentioned problems of the prior art, a method using an aqueous solution containing TMB, a peroxide and a buffer substance adjusted to a pH range of 2.5 to 3.9 has recently been disclosed (Japanese Patent Laid-open Publication) 62-134100).

However, according to the present inventors, this method is still unsatisfactory in terms of sensitivity in measuring peroxidase activity.

c. Means for Solving the Problems In view of such circumstances, the present inventors have made it possible to easily prepare without using an organic solvent without impairing the properties of tetraalkylbenzidine such as TMB, and to preserve the storage stability. As a result of earnest research to develop a chromogenic aqueous solution and an accurate and reproducible substrate solution for the enzyme immunoassay of tetraalkylbenzidine or a salt thereof, which has excellent properties and sensitivity, tetraalkylbenzidine in a specific concentration and pH range is obtained. Or an aqueous solution of its salt, and by mixing such an aqueous solution with a peroxide, a peroxidase activity can be obtained by using a substrate solution containing tetraalkylbenzidine at a specific concentration and having a limited pH range. It was confirmed that the above object was achieved by measurement, and the present invention was achieved.

That is, the first invention of the present invention comprises 0.01 to 0.5% by weight of tetraalkylbenzidine or a salt thereof and has a pH of 1.0.
It is an aqueous chromogen solution for enzyme immunoassay of ~ 2.5.

With tetraalkylbenzidine of such a chromogen aqueous solution,
It contains 1 to 3 carbon atoms in the alkyl moiety, and among these, 3,3 ', 5,5-tetramethylbenzidine is preferable. In addition, examples of the salt of tetraalkylbenzidine include inorganic acid salts such as hydrochlorides and sulfates, but in view of solubility and commercial availability, 3,3 ′, 5,
The dihydrochloride salt of 5'-tetramethylbenzidine is preferred.

In the case of tetraalkylbenzidine, the chromogen aqueous solution of the present invention can be obtained by dissolving it in an acidic aqueous solution and then adjusting the pH. In the case of the salt, it can be obtained by a method such as simply dissolving in water and then adjusting the pH.

In the chromogen aqueous solution of the present invention, the concentration of tetraalkylbenzidine or its salt is 0.01 to 0.5% by weight.
If it is 0.5% by weight or more, there is a problem in solubility, and if it is 0.01% by weight or less, it is insufficient to develop a color with high sensitivity. It is preferably 0.02 to 0.2% by weight.

The pH of the chromogen aqueous solution is 1.0 to 2.5, and the pH is 2.5.
When it is larger, the solubility is insufficient, and the stability of the aqueous chromogen solution is insufficient, so that the base of the aqueous solution is easily colored during storage. On the other hand, when the pH is lower than 1.0, the pH control becomes insufficient, and when the substrate solution is prepared by mixing with the aqueous peroxide solution, the pH tends to fluctuate, which is not preferable in terms of reproducibility. The pH is preferably 1.5 to 2.5, and the best result is obtained in this range. Further, when the chromogen aqueous solution is prepared, the chromogen, water and acid are sufficient, but salts, stabilizers and the like may be added to the chromogen as necessary.

A second invention of the present invention is a method using the chromogenic aqueous solution of the first invention, which is an enzyme immunoassay method using a peroxidase-labeled antibody or an antigen, wherein (1) 0.01 to 0.5% by weight of tetraalkyl A chromogen aqueous solution containing benzidine or a salt thereof and having a pH of 1.0 to 2.5 and (2) an aqueous solution containing a peroxide are mixed to form 0.003 to 0.15% by weight of a tetraalkylbenzidine or a salt thereof. And 1 to 20 mM peroxide,
And create a substrate solution whose pH is greater than 3.9 and less than or equal to 4.5,
An enzyme immunoassay method characterized by measuring peroxidase activity using the substrate solution.

The amount of tetraalkylbenzidine or its salt and peroxide contained in the substrate solution of the present invention largely depends on the property and concentration of peroxidase to be measured, but preferably tetraalkylbenzidine or its salt is 0.01 to 0.06.
% By weight, peroxide in the range of 2 mM to 6 mM.

And the important thing is to keep the pH of the substrate solution above 3.9 and below 4.5. The pH is preferably 4.0 to 4.2. If the pH is too low, the enzyme activity will be inhibited and the color development will be insufficient, thus decreasing the sensitivity. For example, color development at pH 4.0 is 100
Then, it decreases to about 63 at pH 3.5 and about 13 at pH 3.0. On the other hand, if the pH is too high, precipitation of chromogen will be observed, which is also inconvenient, depending on the concentration.

By controlling the pH, chromogen concentration, and peroxide concentration of the substrate solution in this way, peroxidase activity can be measured with sufficiently high sensitivity and reproducibility without impairing the properties of tetraalkylbenzidine and the like. be able to.

The peroxide of the present invention is capable of oxidizing a chromogen in the presence of peroxidase, and examples thereof include hydrogen peroxide, methylhydroperoxide, urea peroxide, and the like. Is preferred. The aqueous peroxide solution of the present invention is preferably an aqueous buffer solution in order to carry out an enzymatic reaction at a stable pH. Such a buffer can be easily prepared from, for example, disodium hydrogen phosphate and citric acid. Further, the concentration of the peroxide in the buffer solution may be adjusted so that the concentration of the peroxide when mixed with the chromogen aqueous solution of the present invention is 1 to 20 mM.

Therefore, the mixing ratio and mixing method of the aqueous chromogen solution and the aqueous peroxide solution of the present invention are not particularly limited.

In addition, a third invention of the present invention, in an enzyme immunoassay kit using a peroxidase-labeled antibody or an antigen, comprises (1) 0.01 to 0.5% by weight of tetraalkylbenzidine or a salt thereof as a substrate solution And an aqueous solution containing a chromogen aqueous solution having a pH of 1.0 to 2.5, and (2) an aqueous solution containing 1.25 mM to 40 mM peroxide.

The chromogen aqueous solution and the peroxide aqueous solution described here can be supplied in the form of measurement kits, which are stored in separate containers, and are mixed at a ratio of, for example, 2: 8 to 8: 2 during measurement. By doing so, the substrate solution of the second invention described above is formed.

The enzyme immunoassay method using a peroxidase-labeled antibody or antigen as referred to in the present invention means an enzyme immunoassay method as described in “Enzyme Immunoassay” edited by Eiji Ishikawa et al. However, 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 substrate solution consisting of a peroxide and a chromogen. Examples of the peroxidase include those extracted from horseradish, milk, yeast, white blood cells, red blood cells and the like, and horseradish peroxidase is particularly preferable.

In addition, the substance to be measured by the immunoassay is not particularly limited, and examples thereof include human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH). , Hormones such as prolactin, or their antibodies; α ₂ -plasmin inhibitor (α ₂ PI), α ₂ -plasmin inhibitor-plasmin complex, protein C, protein S, plasminogen, antithrombin III, antithrombin
Coagulation-related factors such as III-thrombin complex, tissue plasminogen activator, plasminogen activator inhibitor and these complexes or their antibodies; α-fetoprotein, haptoglobulin, HBs antigen, lung surfactant protein, especially apoprotein ( LSP), immune complex, TNF, interleukin I, interleukin II
Such proteins or their antibodies; herpes simplex virus,
Examples thereof include viruses such as cytomegalovirus or antibodies thereof. The antibody may be either a polyclonal antibody or a monoclonal antibody.

Examples of the peroxidase-labeled antibody of the present invention include a polyclonal antibody or a monoclonal antibody against such a substance to be measured. Among them, anti-protein C antibody, anti-protein S antibody, anti-α ₂ -plasmin inhibitor antibody, anti-LSP antibody, anti-HCG. Antibodies and anti-IgA-α chain antibody-F (ab ') ₂ fragments are preferred. Of these, these monoclonal antibodies are particularly preferable.

In the present invention, examples of the anti-protein C monoclonal antibody include those disclosed in JP-A-61-134399 and JP-A-61-134399.
No. 283868, and examples of the anti-α ₂ PI monoclonal antibody include, for example, JP-A-60-222426 and JP-A-61-91.
Those obtained by the method described in detail in Japanese Patent Publication No. 200 can be used.

Further, as the anti-human LSP-monoclonal antibody, for example, those obtained by the method described in JP-A-61-277699 and JP-A-61-274678, particularly mouse anti-human antibody
PC6 and PE10 which are LSP-monoclonal antibodies can be used.

e. Effect Thus, according to the present invention, it is possible to supply an aqueous chromogen solution which can be easily and safely prepared without impairing the properties of tetraalkylbenzidine and the like and which is also excellent in storage stability and sensitivity. It was Further, by using a controlled substrate solution consisting of the chromogen aqueous solution and the peroxide aqueous solution, it became possible to measure the peroxidase activity accurately, with good reproducibility and with high sensitivity.

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

In the following, "%" means "% by weight".

Reference Example 1 Production and Purification of Anti-Human Protein S (PS) Monoclonal Antibody Purified human PS was immunized four times at 14-day intervals with two female Balb / C mice (4 weeks old). The first immunization was 50 μg human PS dissolved in phosphate buffered saline (PBS).
An equal volume of Freund's complete adjuvant.
und's adjuvant), the emulsion was administered intraperitoneally (0.5mg / head), and the second and third times were the same.
50 μg of human PS was mixed with Freund's incomplete adjuvant and intraperitoneally administered. The final immunization is 30 μg human PS in PBS
As a solution, it was additionally administered through the tail vein of the mouse. Spleen cells of immunized mice were used for cell fusion 3 days after the final immunization.

Spleen cells from immunized mice and myeloma cells (P3U1) from syngeneic mice were mixed at a ratio of about 2: 1 to about 15: 1, and 50% polyethylene glycol 1540 (Wako Pure Chemical Industries, Ltd.) was used as a fusion accelerator. As a result, cell fusion was performed according to the method of Kohler and Milstein. The cells after fusion were suspended in 10% FCS-RPMI-1640 medium at a cell concentration of 1 × 10 ⁶ cells / ml, and 100 μl was dispensed to 96 wells microplate (Coster) per well.

The fused cells were cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C), the medium was exchanged with a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), and the cells were grown in HAT medium to produce spleen. A hybridoma consisting of cells and myeloma cells was screened.

The antibody in the culture supernatant of the hybridoma is ELISA using a microtiter plate coated with the antigen human PS.
It was detected by the method. As the second antibody, a rabbit anti-mouse IgG antibody labeled with alkaline phosphatase was used, and the binding property to the antigen PS was examined. Colonies were formed in 487 wells out of a total of 494 wells in which the fused cells were seeded, and 94 wells of antibody production showing binding to the antigen PS were 94 wells.

Cloning by the limiting dilution method was repeated twice for 4 of these antibody production positive wells to obtain 6 clones. 90% of the obtained clones
Fetal calf serum (FCS) was suspended in 10% DMSO and stored in liquid nitrogen. The monoclonal antibody produced by each clone was propagated in the abdominal cavity of Balb / C mice and purified from the ascites using a Protein A-Sepharose 4B column.

The above is summarized in Table 1.

Reference Example 2 Properties of Purified Monoclonal Antibody IgG of each clone purified from mouse ascites was tested for class and human PS binding properties.

The class of mouse monoclonal antibody was determined by the Ouchterlony method using anti-mouse antiserum of each class specificity.

The results are shown in Table 2 below.

The binding to human PS can be detected with goat anti-mouse IgG labeled with alkaline fasfactorase by reacting human PS immobilized on a microtiter plate with a monoclonal antibody diluted to an appropriate concentration. It was evaluated by

As a result, the binding strength of the 6 kinds of monoclonal antibodies to human PS was 2B9F12 to 2B9C10>3C3G8>3C4G4> 2B9G.
3 >> 2 E12C7 was found.

C4bp-of these 6 kinds of monoclonal antibodies in human plasma
When the binding property with the PS complex was examined, the binding strength was found to be 2E12C7 >> 2B9F10 to 2B9C12>3C3G8>3C4G4> 2B9G3.
It turned out to be

From the above results, 2B9F12 and 2B9C10 were obtained as monoclonal antibodies that do not recognize the complex of C4bp and PS and specifically recognize free human PS.

Example 1 <Stability of Chromogen Aqueous Solution> (1) Preparation of Chromogen Aqueous Solution 3,3 ′, 5,5′-Tetramethylbenzidine dihydrochloride (hereinafter T)
MB.2HCl) (0.25 g) was dissolved in distilled water (500 ml) with stirring to obtain a 0.05% aqueous solution. Similarly, a 0.2% aqueous solution was obtained using 1 g of TMB · 2HCl. These are HCl aqueous solution or NaOH
Aqueous solutions were prepared to prepare chromogen aqueous solutions (No. 1 to 1) having different pHs as shown in Table 3.

(2) Acceleration test of chromogen aqueous solution Put the test tube containing chromogen aqueous solution prepared in (1) at 50 ℃
It was put in a thermostat and the acceleration test was carried out. The change with time in color of the aqueous solution was measured at a wavelength of 650 nm, and the results are shown in Table 3.

From Table 3, it can be seen that the chromogen aqueous solution is stable in the range of pH 1.0 to 2.5 of the present invention.

On the other hand, those with a pH of 3.5 or higher did not dissolve, and when the pH was higher than 2.5, the solubility was insufficient and coloring was observed in the chromogen aqueous solution during storage. When the pH was lower than 1.0, it was stable in terms of coloration, but it was difficult to control the re-pH when adjusting the substrate solution.

Example 2 <Stability of Substrate Solution> (1) Preparation of Substrate Solution 1) Preparation of 3,3 ′, 5,5′-tetramethylbenzidine Aqueous Solution pH 2.0 containing 0.25% and 0.05% TMB · 2HCl Two types of chromogen aqueous solutions were prepared.

2) Preparation of hydrogen peroxide aqueous solution 5. Add hydrogen peroxide to each of 0.1M disodium hydrogen phosphate-citrate buffer solution adjusted to pH 4.10, 4.30, 4.50, 4.80.
It was made to contain 0 mM, and four types of hydrogen peroxide aqueous solutions were prepared.

3) Preparation of substrate solution The chromogen aqueous solution prepared in 1) and the hydrogen peroxide aqueous solution prepared in 2) were mixed in the combination shown in Table 4 at a volume ratio of 3: 7, and the substrate solution (No. 2 --- a, 2 --- b) 8
The type was adjusted.

Among the combinations shown in Table 4, those using the H ₂ O ₂ aqueous solution having a pH of 4.80 (No. 2--a and -b) had a final pH of 4.65 and TMB was precipitated.

On the other hand, the final pH of the one using the H ₂ O ₂ aqueous solutions of pH 4.5, pH 4.3 and pH 4.1 (2- to -a and -b) is within the range of the present invention, and is 4.35, respectively. 4.18 and 4.02, and precipitation of TMB was not observed.

(2) Stability of substrate solution Among the substrate solutions prepared in (1), No.2-b (0.01
5% TMB · 2HCl, 3.5 mM / H ₂ O ₂ , pH 4.02) was allowed to stand at room temperature, and the color change of this solution was measured with time at 650 nm. The results are shown in Table 5.

As is clear from Table 5, the substrate solution of the present invention showed almost no problematic change until after 2 hours.

Example 3 <Measurement of Protein C> (1) Preparation of Substrate Solution A substrate solution (pH 4.02) containing 0.015% TMB · 2HCl and 3.5 mM hydrogen peroxide was prepared in the same manner as in Example 2.

(2) Measurement of protein C 0.01 M PBS (pH 7.
4) was added to prepare an antibody solution having a protein concentration of 10 μg / ml, polystyrene beads were added to this, and the mixture was immersed overnight.
The beads were separated, washed with PBS, and then immersed in 0.5% bovine serum albumin-containing PBS (BSA-PBS) for one day to obtain anti-protein C.
Antibody-immobilized polystyrene beads were obtained. Protein C 100ng
/ mL, 50ng / ml, 12.5ng / ml, 0ng / ml in 0.5% BSA-10mM C
200 μl of aCl ₂ -0.05M Tris / HCl solution (pH 7.4) was placed in each test tube, and 1 each of the antibody-fixing balls described above and 0.5 of a labeled anti-protein C monoclonal antibody labeled with horseradish peroxidase according to a conventional method were used. % BSA-PBS solution 2
00 μm was added, and the mixture was reacted at 37 ° C. for 30 minutes.

Then, after washing the beads with a physiological saline solution containing 10 mM CaCl ₂ , 400 μl of the substrate solution prepared in (1) above was added to
The color was developed by reacting at 7 ° C for 15 minutes.

Further, 1.0 ml of a 2% acetic acid aqueous solution containing 0.1% NaF was added thereto to stop the reaction, and then the absorbance was measured at 650 nm.

In addition, the solution after the color development is stopped is left at room temperature, and 650n
The absorbance at m was measured, and the results are shown in Table 6.

(3) Effect of pH on TMB color development in protein C measurement In the above (2), protein C 100 ng / ml and 12.5 nm / ml
In the case of, instead of the substrate solution prepared in (1),
Using a substrate solution obtained by mixing a chromogen aqueous solution containing 0.05% TMB · 2HCl and a 5.0 mM hydrogen peroxide solution, this pH
Changed to 3.0-6.0, the effect of pH during TMB color development is 650 nm
It was measured at. The results are shown in Fig. 1. From Fig. 1, considering that the value of protein C in adult humans is 3 to 4 ng / ml, the sensitivity is remarkably good around pH 4.0, while the sensitivity is not so good below pH 3.9. It is clear.

Comparative Example 1 Instead of the substrate solution of Example 3, a methanol solution containing 0.1% TMB and a 5.0 mM aqueous hydrogen peroxide solution were mixed in a volume ratio of 3: 7.
The same operation as in Example 3 was carried out except that the substrate solution obtained by mixing the above was used, and the results are shown in Table 6.

This substrate solution contained 0.03% TMB and 3.5 mM H ₂ O ₂ , p
It was H4.49.

As is clear from Table 6, in the case of the present invention in which the substrate solution is an aqueous solution (Example 3), the TMB content is 1/2 as compared with the case of the methanol solution (Comparative Example 1). Nevertheless, it shows excellent sensitivity.

Example 4 <HCG measurement kit> An HCG measurement kit comprising the following constituent reagents was prepared.

(1) 0.06% TMB · 2HCl aqueous solution (pH 2.0) (2) 5 mM H ₂ O ₂ -containing 0.1 M disodium hydrogen phosphate-citrate buffer (pH 4.1) (3) Anti-HCG monoclonal antibody-immobilized beads (4) 0.5% BSA-0.4% sucrose-0.01M PBS solution (pH containing peroxidase-labeled anti-HCG monoclonal antibody)
7.4) (5) 0.1% NaF These reagents were stored in a refrigerator at 2 to 8 ° C. Urine containing 100 mIU / ml of HCG was added to 0.75 ml of the labeled antibody of (4), the beads of (3) were added thereto, and the mixture was reacted at 25 ° C for 30 minutes. Then, wash the beads with physiological saline, and put them in the substrate solution prepared by mixing 0.3 ml of the solution of (1) and 0.7 ml of the solution of (2).
The reaction was performed at 15 ° C for 15 minutes. The adjusted substrate solution had a pH of 4.0. Then, 1 ml of (5) was added to stop the color reaction, and the absorbance was measured at 650 nm. The absorbance was 0.610.

On the other hand, when the substrate solution prepared from the solution (1) and the solution (2) left for 3 days in an incubator at 50 ° C. was measured in the same manner, the absorbance was 0.580 and the decrease rate was 5%, which is good. It has good stability.

Example 5 <Measurement of α ₂ PI-plasmin complex> (1) Preparation of substrate solution of chromogenic aqueous solution of 0.045% TMB · 2HCl and 5.0 mM hydrogen peroxide
0.1M phosphate-citrate buffer is mixed 1: 1 to prepare 0.1M phosphate-citrate buffer (pH 4.0) containing 0.023% TMB · 2HCl and 2.5 mM hydrogen peroxide as a substrate solution. did.

(2) Measurement of α ₂ PI-plasmin complex Rabbit anti-human plasminogen antibody in 0.01MPBS solution (pH
7.4) was added to prepare an antibody solution having a protein concentration of 20 μg / ml, polystyrene beads (diameter 6 mm) were added thereto, and the mixture was immersed overnight. Next, after separating the beads and washing with PBS, 0.5
It was immersed in% BSA-PBS for 24 hours to obtain anti-human plasminogen antibody-immobilized polystyrene beads.

α ₂ PI-plasmin complex 200ng / ml, 100ng / ml, 50ng / ml, 0
200 μm of 0.5% BSA-PBS containing ng / ml was placed in each test tube, and 0.25% skim milk containing 1 each of the above-mentioned antibody-immobilized beads and an enzyme-labeled anti-α ₂ PI monoclonal antibody labeled with horseradish peroxidase according to a conventional method was contained. PBS200
μ was added and reacted at 37 ° C. for 60 minutes.

After completion of the reaction, the reaction solution is separated, the beads are washed with physiological saline, and then the substrate solution 400 μm prepared in (1) above is used.
Was added and reacted at 37 ° C. for 30 minutes to develop color.

Further, 1.0 ml of a 2% acetic acid aqueous solution containing 0.1% NaF was added thereto to stop the reaction, and then the absorbance at a wavelength of 650 nm was measured using a spectrophotometer. The results are shown in Table 7.

From Table 7, it can be seen that the substrate solution (pH 4.0) of the present invention enables sensitive measurement of α ₂ PI-plasmin complex.

Example 6 <Measurement of lung surfactant apoprotein (LSP)> (1) Investigation of substrate solution In the same manner as in Example 2, 0.1M phosphoric acid-citrate containing 0.015% TMB · 2HCl and 3.5 mM hydrogen peroxide. Acid buffer (pH 4.
0) was prepared as a substrate solution.

(2) Measurement of LSP PBS was added to mouse anti-human LSP-monoclonal antibody (PC6) to prepare an antibody solution having a protein concentration of 10 μg / ml, and polystyrene beads (diameter 6 mm) were added thereto and immersed overnight, The beads were separated, washed with PBS, and then immersed in 0.5% BSA-PBS for a whole day and night to obtain anti-LSP-monoclonal antibody-immobilized polytirelin beads. Next, human LSP 300 ng / ml, 150 ng / ml, 75 ng / m separated and purified from lung lavage fluid of patients with alveolar proteinosis
l, 0ng / ml in 0.5% BSA, 3% Triton X-100 and 0.6%
200 μl of PBS containing sodium lauryl sulfate (SDS) was placed in each test tube, and 0.25 containing each of the above-mentioned antibody-immobilized beads and enzyme-labeled mouse anti-human LSP-monoclonal antibody (PE10) labeled with horseradish peroxidase according to a conventional method. Add PBS containing 200% skim milk at 45 ℃
Allowed to react for 30 minutes.

After the reaction was completed, the reaction solution was decomposed, the beads were washed with physiological saline, 400 μl of the substrate solution prepared in (1) above was added, and the mixture was reacted at 45 ° C. for 15 minutes to develop color.

Further, 1.0 ml of a 2% acetic acid aqueous solution containing 0.1% NaF was added to stop the reaction, and a wavelength of 650 n was measured using a spectrophotometer.
The absorbance at m was measured, and the results are shown in Table 8.

From Table 8, it can be seen that in the case of the measurement of human lung surfactant apoprotein, the substrate solution of the present invention can measure it with high sensitivity.

Example 7 <Measurement of IgA type immune complex> Rabbit anti-human C3 antibody was treated with plasmin to remove the CH3 region to prepare an anti-human C3 antibody-Facb fragment, and PB
S was added to prepare an antibody solution with a protein concentration of 10 μg / ml,
After adding polystyrene beads (diameter 6 mm) to this and immersing it overnight, the beads were separated and washed with PBS, then 0.5% BSA-P
It was immersed in BS for 24 hours to obtain anti-human C3 antibody-Facb fragment-immobilized polystyrene beads.

Next, human IgA and human C3 were covalently bound by a binding agent I
The IgA-C3 complex (see Japanese Patent Application Laid-Open No. 60-192263) was used as a standard substance for the measurement of IgA-type immune complex.
Complex 400ng / ml, 200ng / ml, 100ng / ml, 50ng / ml, 25ng / ml, 1
PBS 400 containing 0.5% BSA containing 2.5 ng / ml and 0 ng / ml and 400 μm PBS containing 0.25% skimmed milk were placed in test tubes, and 1 each of the above-mentioned antibody-immobilized beads was added thereto and reacted at 37 ° C. for 60 minutes.

After the reaction was completed, the reaction solution was separated, the beads were washed with physiological saline, and then the enzyme-labeled goat anti-human IgA-α chain antibody-F labeled with horseradish peroxidase according to a conventional method was used.
400 μl PBS containing 0.5% BSA and 0.25% skim milk containing the (ab ′) ₂ fragment was added and reacted at 37 ° C. for 60 minutes.

Next, after separating the reaction solution and washing the beads with a physiological saline solution, a substrate solution 400 obtained by adjusting in Example 6 (1) was obtained.
μ was added and reacted at 37 ° C. for 30 minutes to develop color.

Furthermore, 1.0 ml of a 2% acetic acid aqueous solution containing 0.1% Naf was added to stop the reaction, and then a wavelength of 650 n was measured using a spectrophotometer.
The absorbance at m was measured, and the results are shown in Table 9.

From Table 9, it can be seen that the IgA type immune complex can be measured with high sensitivity by the substrate solution of the present invention.

Example 8 <Measurement of human protein S> Polystyrene beads (diameter 6 mm) were used as goat anti-human PS.
An antibody (polyclonal antibody: American Diagnostica) at a concentration of 20 μg / ml in a 0.01 M PBS solution of pH 7.4 at a temperature of 4 ° C. was allowed to stand for 1 day and then washed with PBS.
The antibody-immobilized beads were obtained by carrying out post-coating treatment by leaving it in a 0.5% BSA aqueous solution at a temperature of 4 ° C. for one day.

Monoclonal antibody (2B9F12) that specifically recognizes free human protein S was added to 1.0 ml of 1.0 mg / ml PBS solution.
10 mg / ml dimethylformamide solution of N- (m-maleimidobenzoic acid) -N-succinimide ester (MBS)
After adding 50μ and reacting for 30 minutes at a temperature of 25 ° C, using a column packed with Sephadex G-25, gel filtration with 0.1M phosphate buffer (pH 6.0) was performed, and a maleimidated monoclonal antibody was used. And unreacted MBS were separated.

Meanwhile, horseradish peroxidase (HRP) 1.0 mg / ml
N-succinimidyl-3- (2-
After adding 10 mg / ml ethanol solution of pyridylthio) propionate (SPDP) and reacting at 25 ° C for 30 minutes, use a column packed with Sephadex G-25 and gel with 0.01M acetate buffer (pH 4.5). The fractions containing pyridyl disulfide HRP were collected and concentrated in a collodion bag under ice cooling to about 10 times. Then to this 0.85
1 ml of 0.1 M acetate buffer (pH 4.5) containing 0.1% NaCl and 0.1 M dithiothreitol was added and stirred at 25 ° C for 30 minutes.
After reducing the pyridyl disulfide group introduced into the HRP molecule, the fraction containing thiolated HRP was obtained by gel filtration with 0.1 M phosphate buffer (pH 6.0) using a Sephadex G-25 column. It was

Next, the obtained maleimidated monoclonal antibody and thiolated HRP were mixed, concentrated to a protein concentration of 4 mg / ml under ice-cooling using a collodion bag, allowed to stand at 4 ° C. for one day, and then Ultrogel AcA44 ( An HRP-labeled monoclonal antibody was obtained by gel filtration with PBS using a column packed with LKB, France.

Goat anti-human protein S antibody-immobilized beads 1 each
Individual and purified human PS at concentrations of 0, 50, 100, 200, 400 ng / ml at a concentration of 0.1% BSA and 2% skimmed milk in PBS (pH 7.4) 200 μ, and HRP-labeled monoclonal antibody at 0.1% BSA and 2 PBS solution containing% skim milk (p
H7.4) 200μ and added to each test tube (n = 2) 37 ℃
Was incubated for 1 hour.

Next, after removing the solution in the test tube by suction, it was washed twice with PBS, and then 400 μl of buffer solution (pH 4.0) containing 0.02% TMB · 2HCl and 1.5 mM hydrogen peroxide was added to each test tube. In addition,
After incubating at 37 ° C. for 30 minutes, 1 ml of an aqueous solution containing 0.1% NaF and 2% acetic acid as a reaction terminator was added to each test tube to stop the enzymatic reaction.

Next, this solution was measured for absorption intensity at a wavelength of 650 nm using a spectrophotometer, and by plotting this with human PS concentration, a calibration curve for human PS concentration measurement having concentration dependence was obtained. (See Figure 2).

To measure the concentration of human PS in plasma samples, normal mixed human plasma was dissolved in PBS containing 0.1% BSA and 2% skim milk (pH 7.
Add 200μ of the solution diluted 50 times in 4) to a test tube along with antibody-immobilized beads and 200μ of HRP-labeled monoclonal antibody solution, conduct immunoreaction and color reaction in the same manner as above, and then measure the absorbance with a spectrophotometer. Was measured. This value was converted to plasma concentration using the calibration curve in Fig. 1 for human PS
As a result of obtaining the concentration, the plasma concentration was 10.4 μg / ml.

[Brief description of drawings]

Fig. 1 shows the relationship between the absorbance and the pH of the substrate solution. In the figure, ○-○ is the protein C concentration of 5 µg / ml, □-□ is the protein C concentration of 1.25 µg / ml, and △-△ is the protein. The C concentration is 0 μg / ml. This protein C concentration is converted into plasma concentration assuming a system in which a plasma sample is diluted 100 times and measured. FIG. 2 shows a calibration curve for human / PS measurement when the substrate solution of the present invention is used.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hideaki Suzuki 4-3-2 Asahigaoka, Hino City, Tokyo Inside Teijin Biomedical Research Institute (56) Reference JP-A-62-134100 (JP, A)

Claims (3)

[Claims] 1. An enzyme immunoassay method using a peroxidase-labeled antibody or an antigen, which comprises (1) an aqueous chromogen solution containing 0.01 to 0.5% by weight of tetraalkylbenzidine or a salt thereof and having a pH of 1.0 to 2.5. (2) mixed with an aqueous solution containing a peroxide to contain 0.003 to 0.15% by weight of tetraalkylbenzidine or a salt thereof and 1 to 20 mM of a peroxide,
And create a substrate solution whose pH is greater than 3.9 and less than or equal to 4.5,
An enzyme immunoassay method characterized by measuring peroxidase activity using the substrate solution 2. A tetraalkylbenzidine is 3,3 ′, 5,5 ′.
-The enzyme immunoassay method according to claim 1, which is tetramethylbenzidine. 3. A peroxidase-labeled antibody is an anti-protein C antibody, an anti-protein S antibody, an anti-α ₂ -plasmin inhibitor antibody, an anti-pulmonary surfactant apoprotein antibody, an anti-IgA-α chain antibody-F (ab ') ₂ fragment. And the enzyme immunoassay method according to claim 1 or 2, wherein the antibody is any one antibody selected from the group consisting of anti-human chorionic gonadotropin antibody.