JPS6215194B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a simple clinical chemistry test device in which physiologically active substances such as enzymes, antigens, and antibodies are fixed to the inner wall of a polystyrene test tube 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, but
Even if these are subjected to surface treatment with nitric acid, hydrogen fluoride, etc. to expand the reaction surface area of physiologically active substances, the immobilized physiological activity per unit area is too low for industrial use. No satisfactory results are obtained. That is, the immobilization rate of physiologically active substances on the inner walls of silicon 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, it is also possible to efficiently immobilize physiologically active substances such as enzymes by amination, amidation, alkylation, etc. However, although nylon tubes have the potential to be put to practical use, they have not been put to practical use due to their strength and other factors, so it is currently impossible to apply them to immobilize physiologically active substances on nylon test tubes. 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. (C.Living et al. Journal of Immunology 109 p. 834 (1972) C.Living etal, J.
Immunology 109 , 834, '72 and E. Engbor et al., Journal of Immunology 109.
Vol. 129 (1972) E. Engvall etal, J.
Immunology 109 , 129, '72) The principle of this method is to fill a test tube with an alkaline solution containing a physiologically active substance and leave it at 37°C overnight to allow the biologically active substance to adsorb to the inner wall of the test tube. Because the 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 any method other than measuring supercollection. .

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, they can be used as simple tests for diagnostic enzymatic assays, radioimmunoassays, enzyme immunoassays, etc. This means that utensils can be manufactured.

Conventionally, as a method for immobilizing proteins such as enzymes on polystyrene resin (powder form), aminopolystyrene was prepared from chloromethylstyrene [M.
J.H. Horn and R.A. Rosen, Huebsletter vol. 36, p. 285 (1973), MJHorn & R.
A. Laursen, FEBS letters, 36 , 285 ('73)]
However, a method is known in which proteins such as enzymes are immobilized thereon.

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 present inventors 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, γ was placed in a polystyrene test tube.
- A few drops of a stock solution of an organic silane such as aminopropyltriethoxysilane are applied to evenly coat the inner wall of the test tube, and the test tube is left in the air for about an hour, and then washed 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. A 0.2 to 1% aqueous glutaraldehyde solution is added as a crosslinking agent to the polystyrene test tube obtained in this way, and the mixture is reacted at 4°C for 1 to 2 hours. In this case, since it is generally thought that polystyrene and organic silane do not react directly, it is not clear whether a direct covalent bond is formed between polystyrene and organic silane as a result of the above reaction.

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 is completed, the sample is thoroughly washed with the buffer used in the reaction and 0.5M NaCl, and then thoroughly washed with 0.2M ethanolamine buffer (PH8.0), and unreacted groups are blocked with amino groups. Note that the concentration range of the physiologically active substance solution is wide, and sufficient immobilization is possible even at about 0.01 mg/ml.

However, in polystyrene test tubes treated with γ-mercaptopropyltrimethoxysilane as the organic silane, 1.5mM of 2,2-dipyridyl sulfide (2,2 −
After treatment with 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
-aminoethyl-γ-aminopropyltrimethoxysilane, N-β-aminoethyl-α-methyl-
γ-Aminopropyldimethoxymethylsilane, N
-bis-β-hydroxyethyl-γ-aminopropyltriethoxysilane and the like.

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

In the physiologically active substance immobilized product obtained by this immobilization method, the physiologically active substance is immobilized on the inner wall of a polystyrene test tube via an organic silane and a crosslinking agent. This immobilized product becomes 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. That is, blood sugar, urea nitrogen, uric acid, creatinine, ammonia, cholesterol, neutral lipids, phospholipids, GOT, GPT, coagulation fibrinolysis test, complement fixation reaction, etc.

In addition, examples of application to radioimmunoassay and enzyme immunoassay include IgG, IgA, IgM,
IgD, IgE, α-fetoprotein, Hb antigen,
Examples include quantitative measurements of CRP, insulin, various steroid hormones, thyroid hormones, and various peptide hormones, as well as tests for drug toxicity.

As examples of enzymes, antigens, and antibodies as physiologically active substances in the present invention, almost all enzymes used in the above clinical measurement items are immobilized on polystyrene test tubes, but the following are particularly exemplified. .

That is, the enzymes include glucose oxidase, peroxidase, catalase, hexokinase, G-6-P dehydrogenase, etc. for glucose determination, urease for urea nitrogen determination, uricase for uric acid determination, glutamate dehydrogenase for ammonia determination, Creatinine amide hydrolase for creatinine determination, creatinine deiminase, cholesterol ester hydrolase for cholesterol determination, cholesterol oxidase, lipase for triglyceride determination, glycerol kinase, pyruvate kinase, lactate dehydrogenase, phospholipase C for phospholipid determination , alkaline phosphatase, ATPase
etc. However, it is not limited to the above enzymes,
Any diagnostic enzyme can be applied.

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, a simple clinical chemistry test device in which physiologically active substances such as enzymes, antigens, and antibodies are immobilized on a polystyrene test tube via an organic silane and a crosslinking agent can be used for diagnostic enzymatic measurement methods, radioimmunoassays, etc. The characteristics when used in clinical chemistry simple test methods such as enzyme immunoassay and enzyme immunoassay are (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, in the enzymatic measurement method, measurements can be repeated in one test tube for 1 to 2 months.) (5) It can be easily immobilized in a test tube and used for measurements as is, making it economical. can give.

Next, examples of the present invention will be described.

Example 1 Polystyrene test tube (10 mm x 50 mm or 15 mm x
100 mm), drop several drops of γ-aminopropyltriethoxysilane stock solution, coat the inner wall of the test tube evenly to a specified height (10 to 50 mm), leave it in the air for about 1 hour, and after washing with water, coat it with 1% glutaraldehyde. Add 5ml of (PH7.4) and react for 1 hour under ice cooling. After washing, glucose oxidase (15EV/
ml) - Add 0.5 ml of peroxidase (50V/ml) (both types manufactured by Sigma) (PH7.4) and mix at room temperature.
Rotate the test tube for ~2 hours to uniformly immobilize both enzymes on the inner wall of the test tube. After immobilization, wash with 0.5M NaCl to remove unimmobilized enzyme, and then wash thoroughly with 0.2M ethanolamine (PH8.0) (using a thermomixer) to block unreacted groups with amino groups.
Among these immobilized enzymes, the immobilization rate of glucose oxidase is 22%, and the immobilization rate of peroxidase is 25%.
%. (As protein amount) Reference Example 1 A coloring solution consisting of 0.6mM 4-aminoantipyrine, 10mM phenol, and 50mM potassium phosphate (PH7.4) was placed in a polystyrene test tube coated with the immobilized enzyme obtained according to Example 1. Add 1.0ml and 10-20μ of serum (human) to start the reaction at room temperature, and after 10 minutes, remove the reaction solution from the test tube.
Measure with a colorimeter (500 nm). Note that the amount of glucose in the 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 supplied serum obtained by the reaction is determined by making it correspond 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 color develops that is visible to the naked eye within a few seconds after adding the sample, so it is sufficient to measure if the sample is left for 30 seconds to 1 minute, but when measuring at the end point, it is recommended to leave it for about 10 minutes. Good. Furthermore, a feature of this operation is that no reaction termination operation is required.

Reference Example 2 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 Reference Example 1 (4-aminoantipyrine-phenol-
Add Triton After a few minutes have elapsed, the reaction solution is taken out from the test tube, the OD at 500 nm is measured using a colorimeter, and the amount of free cholesterol is measured using a cholesterol calibration curve determined in advance. When measuring the total cholesterol amount, cholesterol ester hydrolase is immobilized in another polystyrene test tube (10 x 50 mm), and this test tube contains 20μ of serum and 0.1% Triton X-100.
Add 0.5 ml of 0.1M phosphate buffer (PH7.0) to previously hydrolyze cholesterol ester (at 37°C).
(react for 20 minutes), 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 total cholesterol amount from the cholesterol calibration curve.

Reference Example 3 According to Example 1, anti-human was added to a polystyrene test tube.
IgG antiserum was immobilized with peroxidase immobilized IgG (S. Avrameas & B. Guilbert.
Biochimie, 54, 837, ('72)) and
Sample containing IgG (serum diluted 10,000 times) 10-50μ
was added together with phosphate buffer (PH7.4) to competitively bind the antibody, and the supernatant was discarded and thoroughly washed.
After adding hydrogen peroxide as a substrate and reacting with the reaction solution (4-aminoantipyrine phenol buffer) used in Reference Example 1, perform color comparison (500 nm), and compare the obtained OD value with the standard curve determined in advance. ,
Determine the amount of IgG in the sample.

Claims (1)

[Claims] 1. A simple clinical chemistry test device in which physiologically active substances such as enzymes, antigens, and antibodies are chemically bonded to the inner wall of a polystyrene test tube via an organic silane and a crosslinking agent.