JPH0315987B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a plastic molded article for immunoassays, on which an antigen or antibody having a primary aminosilane is immobilized and used as a carrier. Immunological assays that apply antigen-antibody reactions are used as methods for detecting or quantifying microorganisms such as viruses, mormons, enzymes, certain drugs, and antibodies against these antigenic substances. In addition to macroscopically detecting the precipitation phenomenon caused by the antigen-antibody reaction and optically quantifying turbidity, this immunoassay method involves binding a labeling substance to the antigen or antibody. There is a method of detecting or quantifying a reaction phenomenon using a labeling substance. Animal red blood cells, latex microspheres, as labeling substances
Hemagglutination reactions, latex agglutination reactions, enzyme immunoassays (EIA), fluorescence immunoassays (FIA), or radioimmunoassays (RIA), etc. using enzymes, fluorescent substances, or radioactive substances are widely applied. . Among these, in recent years, in order to increase detection sensitivity and simplify measurement operations, complexes (B) have been developed in which antigens or antibodies are immobilized on the surface of glass, silicone rubber, or plastic called carriers, and antigen-antibody reactions occur.
Separation of unreacted antigen or antibody (F) (B/
A solid-phase method that allows separation of F separation by washing with water without centrifugation is attracting attention. The most important thing in this solid-phase method is that in order to improve measurement accuracy, detection sensitivity, and detection range, it is desirable to immobilize antibodies or antigens uniformly and in large quantities on each carrier, and the selection of carrier materials and immobilization Various improvements have been made to the conditions. The main conventional immobilization methods utilize the phenomenon of nonspecific physical adsorption of antigens or antibodies onto the surface of the carrier. It has the disadvantage that it is almost impossible to immobilize low molecular weight antigens (eg haptens) and large antigens such as viruses and bacteria on the carrier surface. On the other hand, as a conventional technique for enhancing the immobilization of antigens or antibodies on carriers, ARNeurath et al. (J.
Virological Methods (1981) introduced a method of immobilizing antibodies by nitrating polystyrene microplates with fuming nitric acid, chemically reducing them to introduce amino groups, and chemically covalently bonding the antibodies via glutaraldehyde. is suggesting. However, this method has drawbacks such as discoloration and deterioration of the plate, dangerous reaction reagents, and problems with waste liquid disposal, making it extremely difficult to produce in large quantities. Another known method is to treat a glass carrier with an aminosilane coupling agent; Not only does glass have the disadvantage that it is not suitable for pipes, etc., but it also has an excessive density of hydroxyl groups that react with the aminosilane coupling agent, so the presence of the carefully added amino groups in close proximity causes glutaraldehyde A chemical covalent bond occurs between aminosilanes, which has the disadvantage that the efficiency of binding and immobilizing antibodies, etc. is extremely low. As a result of various studies to overcome the drawbacks of the above-mentioned carriers for immunological assays, the present invention can be applied to plastic molded products of all shapes, and can introduce primary amino groups easily, safely, and under mild conditions. Furthermore, we have discovered a chemical treatment method that can control the amount of introduced antibodies, making it possible to immobilize large amounts of antibodies or antigens extremely efficiently.Antigens or antibodies are immobilized on a surface and used as a carrier for immunoassays. This led to the invention of a method for manufacturing molded products. That is, the surface of the plastic carrier is oxidized in advance by physicochemical means to introduce hydroxyl groups, and then a primary aminosilane coupling agent and the hydroxyl groups on the carrier surface are subjected to a condensation reaction, thereby chemically forming the surface of the carrier. It has been found that it is possible to impart a covalently bonded primary amino group. The shape of the plastic carrier of the present invention is as follows:
Microplates having one or more wells, test tubes, spectroscopic cells, or spherical and cylindrical tubes called balls, beads, latex, etc., made of plastic and suitable for immunoassays. If so, it is not limited to the above example. Materials for the plastic molded product of the present invention include polystyrene, polyethylene, polypropylene, polycarbonate, polyvinyl chloride, polyester, polymethyl methacrylate, polyvinyl acetate, vinyl chloride, vinyl acetate copolymer, and ethylene-vinyl acetate copolymer. Coalescence, styrene/methyl methacrylate copolymer, acrylonitrile/styrene copolymer, acrylonitrile/butadiene/styrene copolymer,
6 nylon, 6,6 nylon, polymethylpentene, silicone, Teflon, etc. Among these, polystyrene, styrene copolymers, and styrene copolymers, which have excellent transparency, are inexpensive, easy to mold, and can easily incorporate primary aminosilane. Polyvinyl chloride is preferred. Examples of oxidation treatment methods for introducing hydroxyl groups into the plastic carrier of the present invention include physicochemical oxidation treatment methods such as ultraviolet irradiation, electron beam irradiation, γ-ray irradiation, corona discharge, and low-frequency or high-frequency low-temperature plasma discharge treatment. However, among these, low-frequency or high-frequency low-temperature plasma discharge treatment is preferable because it is simple, is not limited by shape, has no problem in disposing of the oxidation reaction reagent, and is easy to adjust the density of introduction of hydroxyl groups.
When performing low-temperature plasma discharge treatment, a plastic carrier is set in the plasma generator and the temperature is set at 10Torr.
Oxidation treatment is performed by exposing to low temperature plasma while passing single or mixed gas such as oxygen gas, argon gas, carbon dioxide gas or nitrogen gas under the following reduced pressure, but preferably in the presence of oxygen gas or carbon dioxide gas.
A high frequency power of 1 Torr or less, 13.56 MHz, 50 W or more and 5 KW or less is applied for 10 seconds or more, more preferably 150 W or more for 2 minutes or more. The primary aminosilane coupling agent used in the present invention has the general formula NH ₂ -R-SiXnY _3-o (wherein, X and Y are an alkoxy group, a chloro group,
Hydrolyzable group such as acetoxy group, alkylamino group, propenoxy group, n is 2 or 3, R is [-
(CH ₂ ) _x NH〕− _n (CH ₂ ) _y − or −CONH〔−
(CH ₂ ) _x NH〕− _n (CH ₂ ) _y −, x and y are each 0
or an integer of 20 or less, m is 0 or an integer of 10 or less), such as γ-aminopropyltrimethoxysilane, N-β(aminoethyl)γ-aminopropyltrimethoxysilane, mino)propyltrimethoxysilane, γ
Preferred examples include ureidopropyltrimethoxysilane, but in place of trimethoxysilane, which is a hydrolyzable group, triethoxysilane, methyldimethoxysilane, ethyldiethoxysilane, trichlor group, triacetoxy group, etc. Derivatives of are also effective. As a method for introducing a primary aminosilane onto the carrier surface through a condensation reaction between the hydroxyl group introduced into the plastic carrier according to the present invention and the primary aminosilane coupling agent, for example, the above-mentioned aminosilane coupling agent may be mixed with methanol, ethanol, acetone, Dilute water, etc., alone or in a mixed solvent to a ratio of 0.1 to 98% by weight, for 5 minutes to 72 hours, preferably in a solvent with a water:methanol ratio of 0 to 80:100 to 20.
It may be diluted to a proportion of 50% by weight, immersed or poured for 1 to 48 hours to react, and then washed with distilled water or a buffer solution, and the reaction temperature is 4°C to 65°C, preferably 24°C to 60°C. Good. Next, examples of the present invention will be described. Example 1 A polystyrene 96 flat-bottom well microplate was heated to 0.5 Torr under reduced pressure while oxygen gas was vented.
After generating 13.56MHz, 150W high-frequency low-temperature plasma and performing oxidation treatment for 3 minutes, γ-aminopropyltrimethoxysilane (manufactured by Toray Silicone) 10
% methanol solution and left for 16 hours for aminosilane treatment. Then wash with distilled water and dry. Next, a 0.05M phosphate buffer solution (PH8.0) of 4% glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well of the aminosilane-treated microplate.
Dispense 200μ each and wash with water. Mouse IgG labeled with POD (peroxidase) (manufactured by Miles,
POD-IgG) 0.3mg was diluted to 1/100,000, and 200μ was dispensed into each well of the microplate.
Leave at 4°C for 16 hours to immobilize POD/IgG on the inner surface of the well by covalent bonding. For comparison, 200μ of the above POD/IgG was used without glutaraldehyde treatment.
It is dispensed in portions and fixed by simple physical adsorption. Next, unimmobilized POD/IgG was removed from each well of the microplate with immobilized POD/IgG by suction, washed three times with 0.1M phosphate buffer (PH7.0), and then the substrate solution (H Add 200μ of ₂ O ₂ , phenol, 4-aminoantipyrine) to allow an enzymatic color reaction between POD and the substrate. After reacting at 37℃ for 40 minutes and adding 50μ of 0.5% sodium nitride aqueous solution to stop the enzymatic coloring reaction, the absorbance was measured at a wavelength of 500nm and the POD/IgG immobilized in the wells of the aminosilane-treated microplate was measured. I asked for the degree. The absorbance measurement results are shown in Table 1.

[Table] Microplates treated with aminosilane are
Compared to the untreated plate, the physical adsorption amount of POD/IgG (corresponding to the untreated plate with glutaraldehyde) increased to approximately 2 times, and the amount of chemically covalently bound POD/IgG treated with glutaraldehyde increased to approximately 3 times A double increase in the amount was observed. Example 2 A polystyrene 96 flat bottom microplate is treated with aminosilane in the same manner as in Example 1. Next, after glutaraldehyde treatment in the same manner as in Example 1, 0.4 mg/ml (80 U/ml) of POD (manufactured by Sigma) itself was immobilized in the wells by covalent bonding instead of POD/IgG. Example 1 for comparison
Similarly, the POD itself is immobilized on the inner surface of the well by physical adsorption. After fixation, unfixed
POD was removed by suction, washed in the same manner as in Example 1,
Furthermore, the degree of immobilization of POD in the wells of the aminosilane-treated microplate was determined by performing an enzymatic coloring reaction with the substrate. The absorbance measurement results are shown in Table 2.

[Table] As in Example 1, the aminosilane-treated microplate had about 4 times the physical adsorption amount of POD compared to the untreated plate, and the glutaraldehyde-treated chemically covalently bonded microplate had about 10 times more POD than the untreated plate. A double increase in the amount was observed. Comparing Examples 1 and 2, the aminosilane-treated microplate shows higher molecular weight proteins (POD has a molecular weight of approximately 30,000) than high molecular weight proteins (POD/IgG has a molecular weight of approximately 180,000). It was observed that the amount of immobilization due to physical and chemical covalent bonds was increased, and it was found that this method is particularly effective for immobilizing low molecular weight antigen proteins. Example 3 A polyvinyl chloride resin microplate (diameter 8 mm, depth 6 mm - 10 wells) was subjected to oxidation treatment by exposing it to high-frequency low-temperature plasma while passing argon gas and oxygen gas in accordance with Example 1. ,
Aminosilane treatment is carried out in the same manner as in Example 1. After treating the wells of a microplate treated with aminosilane with glutaraldehyde in the same manner as in Example 2, POD is immobilized by chemical covalent bonding.
For comparison, POD was immobilized by physical adsorption in the same manner as in Example 2. After immobilization, POD was subjected to an enzymatic coloring reaction with the substrate in the same manner as in Example 2, and the degree of immobilization of POD in the wells of the aminosilane-treated microplate was determined. Table 3 shows the absorbance measurement results.

[Table] In the polyvinyl chloride resin microplate treated with aminosilane in the same manner as in Example 2, it was observed that the amount of POD adsorbed and fixed by physical and chemical covalent bonds was increased compared to the untreated product. In particular, in the oxidation treatment, oxygen gas low-temperature plasma treatment is more effective than argon gas low-temperature plasma treatment because it increases the amount of protein immobilized. As is clear from these results, by oxidizing a plastic molded article by physicochemical means according to the present invention and then treating it with aminosilane, the amount of immobilized antigenic substances such as antibodies or small molecules can be increased. A molded article can be obtained by immobilizing the antibody on the surface and using it as a carrier.

Claims (1)

[Claims] 1. After the surface of the plastic product is oxidized in advance by physicochemical means, the general formula NH ₂ -R-Si-XnY ₃ -n (wherein, X and Y are an alkoxy group or a chloro group) , an acetoxy group, an alkylamino group, a hydrolyzable group such as a propenoxy group, n is 2 or 3, R is [-(CH ₂ ) _x -NH]- _n (CH ₂ ) _y - or -CONH[-(CH ₂ ) _x −NH〕− _n (CH ₂ ) _y , x, y are each 0 or an integer of 20 or less, m is 0
or an integer less than or equal to 10. ) is reacted with a primary aminosilane coupling agent represented by the formula to form a primary aminosilane on the surface of the formed product, and an antigen or antibody is immobilized on the surface and used as a carrier for immunological measurement. Method of manufacturing plastic molded products.