JPH07122622B2 - Immunosensor - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunosensor. More specifically, the present invention relates to an immunosensor for an enzyme immunoassay, characterized in that a fibroin film entrapping and immobilizing an antibody is attached in close contact with an oxygen electrode via an oxygen permeable membrane.

[Conventional technology]

It is widely practiced to detect and quantify a specific antigen or antibody contained in a sample by utilizing the high specificity of the antigen-antibody reaction, and to make it useful for diagnosis or treatment of diseases and the like. Among them, the enzyme immunoassay (EIA) is simple,
From the viewpoint of safety, as a microanalysis method in clinical tests,
Its usefulness is increasing.

In EIA, generally, an antibody (or antigen) corresponding to the antigen (or antibody) to be measured is immobilized in advance on an appropriate insoluble carrier by the chemical binding method, adsorption method, entrapment method, etc. A solid phase method in which an antibody (or an antigen) is reacted with an antigen (or an antibody) to be measured is used.

By the way, recently, as an application of this solid-phase method, various immunosensors for EIA in which a membrane (film) on which an antibody is immobilized are attached to an oxygen electrode have been variously studied because of their sensitivity and ease of handling.

[Problems to be solved by the invention]

In the EIA, various studies were conducted on a new type of immunosensor that can be used for repeated measurements and has excellent reproducibility.

[Means for solving problems]

As a result of various investigations by the present inventors in view of the above points, an immunosensor for an enzyme immunoassay characterized in that a fibroin film entrapping and immobilizing an antibody is closely attached to an oxygen electrode through an oxygen permeable membrane. The present invention has been completed by discovering that the above-mentioned object is suitable for the above purpose.

The immunosensor of the present invention is obtained by closely mounting a fibroin film (antibody-immobilized membrane) on which antibodies are entrapped and immobilized on the outer side of an oxygen permeable membrane of a galvanic or polarographic oxygen electrode. (See FIG. 1).

The immunosensor of the present invention is incorporated into a reaction cell as shown in FIG. 1, and a flow system (see FIG. 2) in which an immune reaction solution, an enzyme substrate solution or a washing solution is sequentially introduced into the reaction cell, or respectively, It is used for measurement by successively immersing it in a container containing the solution.

The fibroin film in which the antibody used in the immunosensor of the present invention is entrapped and immobilized can be produced by the method described in JP-A-60-142259 or JP-A-60-155129.

A polyethylene film, a Teflon film, or the like is used as the oxygen permeable film of the oxygen electrode.

Antigens that can be measured by the immunosensor of the present invention include, for example, insulin and chorionic gonadotropin (hC
G), placental lactogen, polypeptide hormones such as luteinizing hormone, and serum proteins such as IgG, IgA, IgM, IgE, α-fetoprotein (AFP), carcinoembryonic antigen, and haptoglobin.

When measuring the above-mentioned antigens, a fibroin film on which the corresponding antibodies are entrapped and immobilized is used.

The measurement is preferably performed by the sandwich method.

As the labeling enzyme, oxidase such as glucose oxidase that directly consumes oxygen and catalase can be used, but among them, catalase, which is a stable enzyme with high enzymatic activity, is particularly preferable.

In order to use catalase as a labeling enzyme, it is necessary to combine catalase with an antibody in advance.

The conventional cross-linking method using glutaraldehyde is
The binding of catalase and antibody proceeds too much to precipitate a high molecular weight product, which lowers enzyme activity and antibody activity,
Or because it has drawbacks such as low yield and poor reproducibility,
It is preferable to use a labeled antibody for catalase produced as described below.

That is, the above-mentioned catalase-labeled antibody has the same general formula (I) as catalase. (In the formula, R ¹ represents a C ₁ -C ₃ alkyl group, and n is 2-4.
Represents the integer. ) Is reacted with a mercaptoimidate compound, (N is the same as above) is introduced into the amino group of catalase, and then the antibody of the general formula (II) (In the formula, R ² represents a divalent residue of an aliphatic hydrocarbon or an aromatic hydrocarbon.) A succinyl maleimide compound represented by (R ² is the same as above.) Is introduced into the amino group of the antibody, and the resulting thiolated catalase is reacted with a maleimidated antibody, (N and R ² are the same as above), and catalase is bound to an antibody.

The above manufacturing method can be performed as follows.

The thiolated catalase comprises a catalase and a mercaptoimidate compound (I) in an atmosphere of an inert gas such as nitrogen or argon, or in the presence of ethylenediaminetetraacetic acid or a salt thereof, a weakly alkaline buffer solution, for example, 0.05. It is obtained by reacting in M phosphate buffer (pH 8.0) at 0 ° C to 40 ° C for 0.5 to 20 hours.

The thiol group introduced into catalase is preferably 2 to 6 molecules per one molecule of catalase, and therefore the molar ratio of mercaptoimidate compound (I) to catalase is:
1:10 to 1: 500 is preferable, and the concentration of catalase is 0.01 to 1
mM is preferred.

As the mercaptoimidate compound (I), for example,
Methyl 4-mercaptobutyrimidate, methyl 3-mercaptopropioimidate and the like can be mentioned.

Addition amount of ethylenediaminetetraacetic acid or its salt is 10 ^-4 M ~ 1
0 ^-2 M is preferred. As the salt of ethylenediaminetetraacetic acid, its disodium salt is preferable.

Catalase may be derived from animals and plants, but is preferably derived from animals, especially the liver thereof.

The thiolated catalase thus obtained can be purified by dialysis, gel chromatography and ultrafiltration, with ultrafiltration being particularly preferred.

The thiolated catalase is preferably stored in an inert gas atmosphere or in a neutral buffer containing ethylenediaminetetraacetic acid or a salt thereof at 10 ⁻⁴ M to 10 ⁻² M.

Next, the maleimidated antibody is prepared by combining the antibody against the antigen to be measured and the succinylmaleimide compound (II) at 0 ° C. in a neutral buffer such as 0.05M phosphate buffer (pH 7.0).
Obtained by reacting at -40 ° C for 0.5-20 hours.

The number of maleimide groups introduced into an antibody is 5 per antibody molecule.
.About.20 molecules are preferable, so that the molar ratio of the succinyl maleimide compound (II) to the antibody is preferably 1: 5 to 1: 300, and the concentration of the antibody is preferably 0.005 to 0.1 mM.

As the succinyl maleimide compound (II), R ² in the general formula (II) is a divalent residue of an aliphatic hydrocarbon such as propylene or pentylene, or 1,3-phenylene, 1,4-
A compound that is a divalent residue of an aromatic hydrocarbon such as phenylene can be mentioned.

The maleimidated antibody thus obtained can be purified by dialysis and gel chromatography.

Finally, the catalase-labeled antibody is a thiolated catalase and a maleimidated antibody thus obtained, under an atmosphere of an inert gas such as nitrogen or argon, or in the presence of ethylenediaminetetraacetic acid or a salt thereof. Sex buffer,
For example, it can be produced by reacting in a 0.05 M phosphate buffer (pH 7.0) at 0 ° C to 40 ° C for 1 to 20 hours.

The molar ratio of maleimidated antibody to thiolated catalase is preferably 1: 1 to 6: 1.

Addition amount of ethylenediaminetetraacetic acid or its salt is 10 ^-4 M ~ 1
0 ^-2 M is preferred. As the salt of ethylenediaminetetraacetic acid, its disodium salt is preferable.

It is preferable to block unreacted maleimide group and thiol group after the reaction in order to improve the stability of the catalase-labeled antibody. To block the maleimide group,
A thiol compound such as cysteine or mercaptoethanol is used, and a maleimide compound such as N-ethylmaleimide or 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) is used to block a thiol group.

Further, the obtained catalase-labeled antibody can be purified by gel chromatography.

In order to repeatedly use the immunosensor of the present invention, the immobilized antibody membrane may be once immersed in a buffer solution having a pH of about 2 to 4 after the measurement.

The buffer solution having a pH of about 2 to 4, for example, has a concentration of 0.05 to 0, respectively.
A 2 M glycine-hydrochloric acid buffer solution or a tartaric acid-sodium hydroxide buffer solution is used.
By immersing the immobilized antibody membrane for 10 minutes, the bound antigen and labeled antibody can be easily dissociated from the antibody immobilized on the fibroin film without impairing its binding ability.

If the pH exceeds the above range, the bond is unlikely to be dissociated, and conversely, if the pH is lowered, the immobilized antibody is easily denatured, which is not preferable.

If the buffer solution contains 0.5 to 10% by weight of a neutral salt such as sodium chloride, potassium chloride or sodium sulfate, the dissociation operation can be performed more easily.

〔The invention's effect〕

As shown in the following test results, the immunosensor of the present invention can be repeatedly used for EIA and has excellent reproducibility, and for example, it can be applied to a continuous automatic measurement device to easily process a large number of samples. It can be carried out.

Test Example 1 Repeated measurement test of human AFP (preparation of standard curve of human AFP): Using the immunosensor prepared in Example 1, a flow-type measurement device as shown in FIG. 2 was assembled, and human AFP0,2.5, 5,20n
The standard curve of human AFP was prepared by repeating the measurement 4 times for each concentration of g / ml as follows.

The reaction cell (volume 0.2 ml) was filled with distilled water, and the standard human AFPs were each 0, 2.5, 5, 20 ng / ml, 10% by weight of horse serum, and the catalase-labeled monoclonal anti-human AF obtained in Production Example 1.
0.2 ml of a 0.1 wt% bovine serum albumin physiological saline solution containing 1 μg / ml of P antibody was introduced, left standing for 30 minutes, and then distilled water was flown for 1 minute at a flow rate of 20 ml / min to wash the reaction cell. Then, a 0.1 M phosphate buffer solution (pH 7.
0) 0.2 ml was introduced into the reaction cell, and the current value proportional to the oxygen concentration of the oxygen electrode was obtained. Subsequently, 0.1 M glycine-hydrochloric acid buffer solution (pH 2.5, containing 2% by weight of NaCl) was added at a flow rate of 20 ml / min for 30 minutes.
After flowing for 2 seconds, the bound antibody was dissociated by allowing to stand for 3.5 minutes to dissociate the bound antibody and further, distilled water was flowed for 1 minute at a flow rate of 20 ml / min to wash the reaction cell. Next, the standard AFP solution was again introduced into the reaction cell, and the same operation was repeated four times for each concentration to prepare a standard curve of human AFP (Fig. 3).
(The operation was carried out at 37 ° C. for the immune reaction and at room temperature for the others.) Test Example 2 hCG Repeated Measurement Test (Preparation of hCG Standard Curve): Second test using the immunosensor prepared in Example 2 Assemble a flow-type measurement device as shown in the figure, and replace h with human AFP.
HCG was prepared in the same manner as in Test Example 1 except that CG and the catalase-labeled monoclonal anti-hCG antibody obtained in Production Example 2 were used.
A standard curve of hCG (Fig. 4) was prepared by repeatedly measuring each concentration of 0, 25, 50, 100, 200 mIU / ml four times.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to Examples and Production Examples.

Production of Catalase-Labeled Monoclonal Anti-Human AFP Antibody: (1) Production of Thiolated Catalase: Catalase (derived from beef liver, Sigma)
Dissolved in 2.5 ml of acid buffer (pH8.0), and aerated with nitrogen (60 ml / m
in) for 10 minutes, then methyl 4-mercaptobutyl
Add 1 mg of imidate and let it react under nitrogen atmosphere at 4 ℃ for 2 hours.
I responded. After the reaction is complete, the diaflow membrane (A
Ultrafiltration under nitrogen atmosphere using
Add 3 ml of M phosphate buffer (pH 7.0) and perform ultrafiltration again.
It was. This operation was repeated 3 times to obtain unreacted methyl 4-me
After removing the rucaptobutyrimidate,
3 ml of the thallase solution was obtained.

The introduction amount of thiol group determined by the following method was 4 molecules per 1 molecule of catalase.

Quantification of thiol group: 1 ml (5 mg) of thiolated catalase plus 1 mg DTNB
After the reaction for a period of time, unreacted low molecular weight compounds such as DTNB were removed by ultrafiltration.

Add 2 ml of distilled water, reduce with dithiothreitol or 2-mercaptoethanol to release 5-mercapto-2-nitrobenzoic acid, add 0.05 ml of 50% trichloroacetic acid and leave still, then centrifuge (12000 rpm, The precipitate was removed (for 5 minutes). After adjusting to pH 8 with 1N NaOH, bring the total volume to 5 ml and
The amount of free 5-mercapto-2-nitrobenzoic acid was determined from the absorbance at 12 nm, and the amount of thiol group introduced into catalase was calculated.

(2) Production of Maleimidated Monoclonal Anti-Human AFP Antibody: Monoclonal anti-human AFP antibody (immunized animal; mouse, which has a different recognition site from the monoclonal antibody used in Example 1 (2)) is 0.05M. Phosphate buffer (pH
7.0) 5.0 ml, N- (γ-maleimidobutyroxy) succinimide (GMBS) 0.5 mg was dissolved in dioxane 0.5 ml and added. After reacting at 4 ° C for 2 hours, dialyzed twice with 1000 ml of 0.05M phosphate buffer (pH 7.0) (2 hours) twice to give 6 ml of maleimidated monoclonal anti-human AFP antibody solution.
Got

The introduced amount of maleimide group determined by the following method was 10 molecules per 1 molecule of antibody.

Quantification of maleimide group: 0.5 mg of maleimidated antibody in 0.05M phosphate buffer (pH 7.0)
After making the total volume to 1 ml, 13.4 mM cysteine 10μ and
Add 10μ of 0.1M EDTA solution and incubate at 37 ℃ for 30 minutes under nitrogen atmosphere. After adding 1 ml of 0.05 M phosphate buffer (pH 7.0), 0.2 ml of 0.67 mM DTNB solution was added, and unreacted cysteine was measured by measuring the absorbance of 5-mercapto-2-nitrobenzoic acid at 412 nm. It was quantified, the amount of cysteine consumed was determined, and the amount of the introduced maleimide group was determined from the amount of cysteine consumed per antibody molecule.

(3) Production of tacarase-labeled monoclonal anti-human AFP antibody: 2 ml (10 mg) of the thiolated tacarase solution and 3 ml (5 mg) of maleimidated monoclonal anti-human AFP antibody solution were mixed under a nitrogen atmosphere and reacted at 4 ° C. for 16 hours. Let To block unreacted maleimide group, 1 mg of cysteine was added and reacted at 4 ° C. for 30 minutes, followed by dialysis with 1000 ml of 0.05M phosphate buffer (pH 7.0) (2 hours). Further, 1 mg of N-ethylmaleimide was added to block unreacted thiol groups, and the mixture was reacted at 4 ° C. for 30 minutes, and then dialyzed with 1000 ml of 0.05M phosphate buffer (pH 7.0) (2 hours). .
Then, after removing the precipitate by centrifugation (12000 rpm, 5 minutes), high performance liquid column chromatography [column; Sephadex G-3000SW (manufactured by Toyo Soda Co., Ltd.), eluate;
0.05M phosphate buffer solution (pH 7.0)], collect the first fraction, and collect catalase-labeled monoclonal anti-human AFP antibody solution 12m
got l.

Production Example 2 Production of Catalase-Labeled Monoclonal Anti-hCG Antibody: Catalase-labeled monoclonal anti-hCG antibody was produced in the same manner as in Production Example 1 except that the monoclonal anti-hCG antibody (immunized animal; mouse) was used instead of the monoclonal anti-human AFP antibody of Production Example 1. 12 ml of antibody solution was obtained.

Example 1 Preparation of immunosensor for human AFP measurement: (1) Preparation of fibroin aqueous solution: 100 g of raw silk was immersed in 5,000 ml of 1.0 wt% Marcel soap aqueous solution and scoured at 80 ° C. for 3 hours. After washing with water, further dip it in 5000 ml of 0.5 wt% Marcel soap solution, and stir at 80 ℃
72 g of fibroin raw material was obtained by substantially refining sericin and the like for a long time. Dissolve 150 g of calcium chloride in a kneader containing 100 g of water and 80 g of ethyl alcohol, raise the temperature to 75 ° C., add 70 g of the above fibroin raw material, and stir 1 while stirring.
Dissolved for hours. Next, 180 g of warm water (75 ° C.) was added and diluted and mixed. After cooling the solution of fibroin, it was dialyzed and desalted against running water using a hollow fiber type dialyzer to obtain 1200 ml of a 5.7 wt% fibroin aqueous solution. The residual amount of calcium chloride was 0.08% by weight.

(2) Production of fibroin film immobilized with monoclonal anti-human AFP antibody: A monoclonal anti-human AFP antibody (immunized animal; mouse) was dissolved in physiological saline to prepare a 250 μ / ml antibody solution. Next, this solution was cast on a glass plate having four sides so that the amount of antibody was 10 μg / cm ^2, and dried at 15 ° C. for 3 hours. Glycerin was added to the above fibroin aqueous solution so as to be 30% by weight with respect to fibroin, and the solution was cast from above and dried at 20 ° C. for 10 hours to form a film, which was then cut into a circle having a diameter of 0.6 cm. A fibroin film having a thickness of 60 μm and having the described monoclonal anti-human AFP antibody immobilized thereon was obtained.

(3) Preparation of immunosensor for measuring human AFP: As shown in FIG. 1, the reaction cell contains the monoclonal anti-human AFP antibody-immobilized fibroin film, oxygen permeable membrane (polyethylene membrane), O -Ring and galvanic oxygen electrodes (AN type, Oriental Electric Co., Ltd.)
(Manufactured by Mitsui Chemical Co., Ltd.) were sequentially mounted to prepare an immunosensor for human AFP measurement.

Example 2 Preparation of immunosensor for hCG measurement: (1) Production of fibroin film on which polyclonal anti-hCG antibody was immobilized (see Japanese Patent Laid-Open No. 60-155129): Polyclonal anti-hCG antibody (immunized animal; goat) was added to a physiological saline solution. To prepare a 250 μg / ml antibody solution. Next, add 10 μl of this solution to a glass plate that is partitioned on all sides.
It was applied so as to be g / cm ² and dried at 20 ° C. for 3 hours. Then, the aqueous fibroin solution obtained in the same manner as in Example 1 (1) was concentrated to give a 15.7 wt% fibroin aqueous solution, and a solution in which glycerin was added so as to be 30 wt% with respect to fibroin was cast thereon. Then, it was dried at 20 ° C. for 8 hours to form a film, which was then cut into a circle having a diameter of 0.7 cm to obtain a 90 μm-thick polclonal anti-hCG antibody-immobilized fibroin film.

(2) Preparation of immunosensor for hCG measurement: The polyclonal anti-hCG antibody-immobilized fibroin film obtained in (1) was applied to a galvanic oxygen electrode (AN type, Oriental Electric Co., Ltd.) in the same manner as in Example 1 (3). ))
An immunosensor for hCG measurement was prepared by mounting it on the.

[Brief description of drawings]

FIG. 1 shows a schematic diagram of the immunosensor prepared in Examples 1 and 2. FIG. 2 shows a schematic diagram of the flow-type measuring device used in Test Examples 1 and 2. FIG. 3 shows the standard curve of human AFP of Test Example 1,
The figure shows the hCG standard curve of Test Example 2.

Continuation of the front page (56) References JP-A-60-142259 (JP, A)

Claims (1)

[Claims] 1. An immunosensor for an enzyme immunoassay comprising a fibroin film entrapping and immobilizing an antibody, which is closely attached to an oxygen electrode through an oxygen permeable membrane.