JPS5921596B2 - Physiologically active substance immobilization method and clinical chemistry simple test device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing physiologically active substances such as enzymes, antigens, antibodies, etc. onto the inner wall of polystyrene test tubes using an organic silane and a crosslinking agent.

Conventionally, methods have been used in which physiologically active substances are immobilized on porous glass using organic silane and a crosslinking agent, or inorganic or organic carriers containing silicon (glass, silica, silicon, etc.) are used instead of porous glass. Methods of immobilizing physiologically active substances have also been attempted; however, even if surface treatments such as nitric acid or hydrogen fluoride are applied to expand the reaction surface area of physiologically active substances, the amount of immobilized physiological substances per unit area is small. Since the activity is too low, satisfactory results cannot be obtained for industrial use.

That is, the immobilization rate of physiologically active substances on the inner walls of silicone tubes, glass test tubes, etc. is only 10% or less compared to that on the inner walls of polystyrene test tubes of the present invention.

Since nylon tubes have peptide bonds in their structure, physiologically active substances such as enzymes can be efficiently immobilized by amination, amidation, alkylation, etc.

However, although nylon tubes have been put into practical use, they have not been put to practical use due to their strength and other factors, so the application of immobilizing physiologically active substances to nylon test tubes is currently only possible.

On the other hand, in an attempt to simplify radioimmunoassay and enzyme immunoassay, attempts have been made to adsorb physiologically active substances directly onto the inner walls of polypropylene and polystyrene test tubes for use in tests.

(Shi Lieving et al. Journal of Immunology 10
Volume 9, page 834 (1972) C, Living eta
l, J.

Immunology 109.834, '72 and E. Engval et al., Journal op Immunology - vol. 109, p. 129 (1972) E. Engval
letal, J. Immunology 10
9.129, '72) This method is based on the principle of filling a test tube with an alkaline solution containing a physiologically active substance and storing the volume at 37°G overnight so that the physiologically active substance is adsorbed to the inner wall of the test tube. Because the adsorbed amount is extremely small and it is relatively easy to separate from the inner wall, repeated measurements in the same test tube are impossible, and it cannot be applied to anything other than ultra-trace measurement methods. .

Enzymes, antigens, and antibodies are generally expensive, and it is a serious problem from an economic standpoint that they are thrown away when used for simple daily measurement tests.

Therefore, the present inventor attempted to immobilize physiologically active substances such as enzymes, antigens, and antibodies on the inner wall of polystyrene test tubes.

In other words, if physiologically active substances such as enzymes, antigens, and antibodies can be directly immobilized on polystyrene test tubes by some kind of treatment, simple testing methods such as diagnostic enzymatic assays, radioimmunoassays, and enzyme immunoassays can be used. It will be established.

Conventionally, as a method for immobilizing proteins such as enzymes on polystyrene resin (powder), aminopolystyrene was prepared from chloromethylstyrene (M. J. Horn and R. N. Rosen, Feb. 36, p. 285 (1973). ,M.

J, Horn & R, A, LaursenlF
EBSletters, 36.285 ('73))
L. A method of immobilizing proteins such as enzymes on this is known.

However, even when trying to apply this method to immobilization in polystyrene test tubes, phenomena such as swelling and dissolution were observed due to the solvents used, such as chloromethyl ether and dioxane, as polystyrene test tubes contain additives in addition to polymers. , physiologically active substances are not immobilized.

As a result of extensive research, the inventors of the present invention succeeded in immobilizing a physiologically active substance on the inner wall of a polystyrene test tube by interposing an organic silane and using a crosslinking agent.

In detail, first, a few drops of a stock solution of organic silane such as γ-aminopropyltriethoxysilane was added to a polystyrene test tube, the inner wall of the test tube was evenly coated, and the tube was left in the air for about an hour. Wash with deionized water.

As a result of this operation, the polystyrene test tube becomes cloudy like ground glass, but the surface is relatively smooth and there is little swelling.

The thus obtained polystyrene test tube was filled with 0 as a crosslinking agent.
．． Add 2-1% glutaraldehyde aqueous solution and incubate at 4°C.
Allow to react for ~2 hours.

In this case, it is generally thought that polystyrene and organic silane do not react directly, so as a result of the above reaction,
It is not clear whether polystyrene and organosilane form a direct covalent bond.

After washing with water, a solution of physiologically active substances such as enzymes, antigens, and antibodies is added and allowed to react at room temperature for 2 hours or overnight at 4° C. while gently rotating the test tube.

After the reaction, the buffer used in the reaction and 0.5 MNa
After thorough washing with Cl, the solution is thoroughly washed with 0.2M ethanolamine buffer (pH 8,0), and unreacted groups are blocked with amine groups.

The concentration range of the physiologically active substance solution is wide < 0.01 m
Sufficient immobilization is possible even with a volume of about 97 ml.

However, in polystyrene test tubes treated with γ-mercaptopropyltrimethoxysilane as the organic silane, 1.5 mM 2,2-dipyridyl sulfide (2
After treatment with 2-dipyridylsulfide and washing, a physiologically active substance solution must be added for immobilization.

In addition to γ-aminopropyltriethoxysilane and γ-mercaptopropyltrimethoxysilane, the organic silanes used for this immobilization include N-β-7minoether γ-
Examples include aminopropyltrimethoxysilane, N-β-aminoether-α-methyl-γ-aminopropyldimethoxymethylsilane, and N-bis-β-hydroxyethyl-γ-aminopropyltriethoxysilane.

In addition to the above-mentioned glutaraldehyde and 2,2-dipyridyl sulfide, hydroxyadipaldehyde, which is commonly used as a crosslinking agent for enzymes, and various isocyanate derivatives can be used as the crosslinking agent.

In the physiologically active substance immobilized product obtained by the immobilization method of the present invention, the physiologically active substance is immobilized on the inner wall of a polystyrene test tube via an organic silane and a crosslinking agent on the upper layer thereof.

This immobilized product provides an excellent simple clinical chemistry test device.

When the simple clinical chemistry test device of the present invention is applied to a diagnostic enzymatic measurement method, the following test items can be mentioned.

Namely, blood sugar, urea nitrogen, uric acid, creatinine, ammonia, cholesterol, neutral lipids, phospholipids, GOT, GP
T, coagulation fibrinolysis test, complement fixation reaction, etc.

Further, examples of application to radioimmunoassay and enzyme immunoassay are IgG, IgA, and IgM.

IgD, IgEl α-fetoprotein, Hb antigen,
CRP, ear/nilin, various steroid hormones,
Examples include quantitative measurements of thyroid hormones, various peptide hormones, etc., and tests for drug toxicity.

As examples of enzymes, antigens, and antibodies as physiologically active substances in the present invention, almost all the enzymes used in the clinical measurement items described above are immobilized in polystyrene test tubes in the present invention. It is a kimono.

That is, the enzymes include glucose budoxidase, peroxidase, catalase, hexokinase, G-6-P dehydrogenase, etc. for glucose determination, urease for urea nitrogen determination, uricase for uric acid determination, and glutamate dehydrogenase for ammonia determination. , createnine amide hydrolase for creatinine determination, createnine deiminase, cholesterol ester hydrolase for cholesterol determination, cholesterol oxidase, lipase for triglyceride determination, glycerol kinase, pyruvate kinase, lactate dehydrogenase, phospholipid determination phospholipase C1 alkaline phosphatase, ATPase, etc.

However, the present immobilization method is not limited to the above-mentioned enzymes, and can be applied to any diagnostic enzyme.

As antigens and antibodies, all antigens such as various immunoglobulins, hormones, and proteins, and antibodies against these antigens can be used.

As described above, in the present invention, physiologically active substances such as enzymes, antigens, and antibodies are immobilized in polystyrene test tubes via organic cysine and a crosslinking agent, and the immobilized physiologically active substances can be used in diagnostic enzymatic measurement methods, radiotherapy, etc. Characteristics of use in clinical chemistry simple test methods such as immunoassays and enzyme immunoassays include (1) good immobilization efficiency, (2) good quantitative performance, and (3) easy measurement.

(Measurements can be easily made anywhere by adding a buffer solution or reaction coloring solution.

) (4) Repeated long-term measurements are possible with a single test tube.

(For example, with enzymatic measurement, 1% can be measured repeatedly in one test tube for 2 months.

(5) It can be easily immobilized in a test tube and used for measurement as is, so it is economical.

Next, examples of the present invention will be described.

Example 1 Polystyrene test tube (10 mm x 50 mm or 15 m
A few drops of the stock solution of γ-aminopropyltriethoxysilane are dropped onto a 100 m
The inner wall of the test tube was evenly coated until the test tube was coated until 100 m), left in the air for about 1 hour, washed with water, 5 Tnl of 1% glutaraldehyde (pH 7,4) was added, and reacted for 1 hour under water cooling. After washing, glucose oxidase (15EV/7711) was added.
) - Add 5 ml of peroxidase (50 V/ml) (both Sigma type ■) (pH 7,4) and rotate the test tube at room temperature for 1 to 2 hours to uniformly immobilize both enzymes on the inner wall of the test tube. .

After immobilization, the enzyme was washed with 0.5M NaCl to remove unimmobilized enzyme, and then washed with 0.2M ethanolamine (pH 8,
Wash thoroughly with 0) and use a thermomixer) and block with unreacted amine groups.

Among the immobilized enzymes, the immobilization rate of glucose oxidase was 22%, and the immobilization rate of peroxidase was 25%.

(As protein amount) Example 2 0.6mM 4-aminoantipyrine, 10mM phenol, 50mM potassium phosphate (pH
7, 4) and 10 to 20 μl of serum (human) were added to start the reaction at room temperature. After 10 minutes, the reaction solution was taken out from the test tube and measured with a colorimeter (50 μl).
0 nm).

Note that the amount of glucose in serum is determined in advance by determining a standard calibration curve of an aqueous glucose solution, and on the other hand, the measured value of the sample serum obtained by reaction is determined by corresponding to the calibration curve.

This tester can be used continuously for at least one month even if left at room temperature and repeated addition of color fixing solution and washing.

In addition, during measurement, a visible color develops to the naked eye within a few seconds after adding the sample, so it is sufficient to measure by leaving it for 30 seconds to 1 minute, but when measuring at the end point, approximately 1
It is better to leave it for 0 minutes.

Furthermore, a feature of this operation is that no reaction termination operation is required.

Example 3 A mixture of cholesterol oxidase and peroxidase was immobilized on the inner wall of a polystyrene test tube according to Example 1, and the reaction solution used in Example 2 (4-aminoantipyrine-phenol-potassium phosphate (pH 7, Add TRITON Take out the reaction solution from the tube and measure it with O at 500 nm using a colorimeter.
D is measured, and the amount of free cholesterol is determined from a cholesterol standard curve determined in advance.

In addition, when measuring the total cholesterol amount, cholesterol ester hydrolase is immobilized in another polystyrene test tube (10 x 50 mm), and 20 μl of serum and 0.1M containing 0.1% Triton X-1oo are added to this test tube.
Process 0.5 ml of phosphate buffer C pH 7.0, take it in advance to hydrolyze cholesterol ester (2 ml at 37°C).
0 minute reaction), transfer this to a test tube for measuring free cholesterol, add the above coloring reaction solution, perform colorimetric measurement in the same manner as above, and determine the amount of cholesterol.

Example 4 Anti-human IgG antiserum was immobilized on a polystyrene test tube according to Example 1, and peroxidase was immobilized on IgG (S, Avrameas &B).

Gui lbert,,Biochimie
, 54.837, (prepared by the method of “72)) and IgG
(serum diluted 10,000 times) 10-50μl
was added together with a phosphate buffer (pH 7,4) to competitively bind the antibody, the supernatant was discarded, the mixture was washed thoroughly, hydrogen peroxide was added as a substrate, and the reaction solution used in Example 2 (4 to aminoantipyrine) was added. (phenol buffer) to increase the reaction size colorimetrically to 50
0 nm), and the obtained OD value is correlated with a standard curve determined in advance to determine the amount of IgG in the sample.

Claims (1)

[Claims] 1. A method for immobilizing a physiologically active substance, which comprises treating the inner wall of a polystyrene test tube with an organic silane, then treating it with a crosslinking agent, and immobilizing a physiologically active substance such as an enzyme, an antigen, or an antibody on the surface thereof.