JP2001272406A - Container for high-sensitivity analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immune analysis container that is highly sensitive and has less repetition determination error, and further can eliminate a blocking operation. SOLUTION: In the high-sensitivity immune analysis container where a hydrophilic macromolecule having a functional group is grafted to a base part that comes into contact with at least the reagent solution of a container, or the functional group and the hydrophilic macromolecule is grafted to the base part that comes into contact with at least the reagent solution of the container, and the contact angle with water of the base part in contact with at least the reagent solution is equal to or less than 1 degree, the functional group can be combined with at least one of peptide, protein, and oxygen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunoassay for detecting an antibody or an antigen utilizing an antigen-antibody reaction, which is used in a solid phase method in which a molecule such as an antibody or an antigen is immobilized on a container for measurement. It relates to the material and surface treatment of the container.

[0002]

2. Description of the Related Art In a conventional immunoassay using a solid phase method, a molecule such as an antibody or an antigen is immobilized on a plastic substrate such as polystyrene by using nonspecific adsorption, and the immobilized molecule is immobilized on the immobilized molecule. A method of capturing target molecules in a sample solution (analyte) using an immune reaction, and finally capturing and quantifying molecules labeled with radioisotopes, enzymes, fluorescent substances, etc. Generally done. However, in such a method using non-specific adsorption, molecules to be added after immobilization are also non-specifically adsorbed on the substrate, so that only the amount of molecules originally captured by the specific antigen-antibody reaction is used. Should be taken as a signal,
The amount of contaminants remaining on the substrate due to non-specific adsorption comes out as noise.

[0003] Therefore, it is generally necessary to perform an operation for preventing adsorption of foreign substances such as blocking. The blocking is an operation of contacting a protein that is easily adsorbed and does not affect the system to the surface after immobilization, thereby masking a portion that is not immobilized. However, even if the surface of the base material is completely covered with the blocking protein, the protein generally loses its higher-order structure due to adsorption (denaturation) and induces secondary adsorption. In many cases, nonspecific adsorption occurs, and a sufficient effect cannot be obtained. Furthermore, since the blocking protein is a biological substance, there is a problem in quality stability, and reproducibility cannot be obtained in many cases on different measurement dates. Artificial blocking agents using hydrophilic polymers such as polyvinylpyrrolidone are also commercially available, but these hydrophilic polymers have weak interaction with the hydrophobic container base material, and the non-specific adsorption causes the base material to become non-specific. It is very difficult to introduce it on the surface, and it is difficult to obtain a sufficient effect because it is easily peeled off by washing.

[0004] Next, considering the step of immobilizing a molecule such as an antibody or an antigen, the immobilization utilizing the non-specific adsorption of the present state requires the immobilization of an immobilized molecule having a concentration higher than the saturation adsorption concentration. Generally, washing is performed after contacting the solution.However, when multi-layer adsorption is present on immobilized molecules, molecules to be reacted later, such as analytes and labeled antibodies, react with the multi-layer part with relatively weak adsorption power. Then, it is washed away in the subsequent washing, and a signal corresponding to the amount of the target molecule in the sample cannot be obtained, or the variation becomes large. In other words, at present, in the immunoassay using the solid-phase method, most of the causes of noise and variations are caused by non-specific adsorption to the container substrate, and non-specific adsorption should be excluded. Regardless, the contradiction that non-specific adsorption must be used for immobilization on the container base material has not been solved.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made as a result of intensive studies in order to solve such conventional problems, and it is possible to stably immobilize a solid phase and eliminate the need for a blocking step. It is intended to provide a container for measurement.

[0006]

That is, a first aspect of the present invention is characterized in that a hydrophilic polymer having a functional group is grafted on at least a substrate portion of a container which comes into contact with a reagent solution. And a functional group and a hydrophilic polymer are grafted on at least a substrate portion of the container in contact with the reagent solution. Container.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The feature of the present invention is that a non-specific adsorption is reduced by introducing a hydrophilic polymer and a functional group on the surface of a container substrate, and only a target molecule is surely covalently bonded. This is the point that the solid phase can be formed. The hydrophilic polymer is polymethacrylic acid, methacrylic acid-alkyl methacrylate copolymer, polyhydroxyalkyl methacrylate, hydroxyalkyl methacrylate-alkyl methacrylate copolymer, polyoxyalkylene group-containing methacrylate polymer or a copolymer containing the same, ethylene Vinyl alcohol copolymers, phospholipid / polymer complexes, polyethylene oxide, polyvinylpyrrolidone, and the like, and polymers having at least one of these molecules in the side chain are preferable. The functional group is not particularly limited as long as it can bind to a molecule to be solid-phased, and examples thereof include an amino group, a carboxyl group, and a mercapto group.

As a method for grafting a hydrophilic polymer having a functional group, a polymerization starting point is formed on the surface of a molded container substrate, and a monomer having a functional group and a hydrophilic group is consequently added thereto. Polymerization, a method of growing, or a method of forming a functional group on the surface of a molded container base material, and bonding a hydrophilic polymer having a functional group to the functional group, or a method of adding a functional group to the raw material of the container base material A method of adding a graft copolymer of a hydrophilic polymer having the compound and molding the mixture can be considered. Among the above methods, the method which can be introduced most easily is the method described below. In general, a carboxyl group is generated by oxygen plasma treatment on a container base material formed of polystyrene or an amino group is generated by ammonia plasma. The present invention can be achieved by coupling a bifunctional hydrophilic polymer having a carboxyl group or an amino group on one surface with carbodiimide or the like.

In the present invention, it is sufficient that the functional group and the hydrophilic polymer coexist on the surface, and the functional group and the hydrophilic polymer are not used as described above. May be introduced into each surface, and even in that case, the above-mentioned methods (1) to (4) can be basically used. An example of a schematic diagram of the functional group and the hydrophilic polymer introduced on the surface as described above is shown in the drawing. Note that the present invention is not limited to these.

The reason for introducing a hydrophilic polymer in order to reduce non-specific adsorption to a container substrate is that non-specific adsorption in an aqueous solvent largely depends on hydrophobic bonds, and the surface hydrophilicity is wide. This is because non-specific adsorption of the kinds of molecules can be reduced. If non-specific adsorption can be eliminated at all, the noise is theoretically zero and the most ideal.However, in practice there is no problem if the noise is sufficiently smaller than the signal to be obtained. There is no need to make it zero.

That is, the introduction of the hydrophilic polymer depends on the amount of the molecule to be immobilized, the number of functional groups, the reaction efficiency between the functional group and the molecule to be immobilized, and the amount of the target substance to be measured in the sample. The amount may be changed. Generally, the contact angle with water on the surface is 1
When a hydrophilic polymer is introduced so as to have a degree of lower than the degree, sufficient nonspecific adsorption controllability for a wide range of molecules can be obtained. In addition, the density of the functional group introduced into the container substrate depends on the reactivity between the functional group of the container substrate and the immobilized molecule.
It is preferably at least 10 pmol / cm ² .

In the procedure for actually performing an immunoassay using the present invention, first, an arbitrary solid-phased molecule is bonded to a functional group on the surface of a container substrate. The method of bonding varies depending on the type of the functional group and the like, but when the amino group of the immobilized molecule is reacted with the carboxyl group on the surface of the container base material, a condensing agent such as carbodiimide is used. When reacting the amino group of the solid-phased molecule with the amino group of the material, glutaraldehyde may be used. Next, the unreacted solid-phased molecules are washed, and if necessary, the unreacted functional groups are inactivated.
Thereafter, the normal immunoassay step may be continued, but the blocking step is not required. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

[0013]

EXAMPLES (Example) Polystyrene 96-well multiwell plate (MS-8496F, manufactured by Sumitomo Bakelite)
Was irradiated with γ-rays (50 kGy) to form hydroxyl groups on the substrate surface. A 1.5% methanol solution of polyethylene glycol (manufactured by Wako Pure Chemical Industries, average molecular weight: 400) was dispensed into the above plate at 200 μl / well, and after 10 minutes, aspirated and discarded. Next, γ- (2-aminoethyl) aminopropyltrimethoxysilane (SH-60 manufactured by Toray Dow Silicone)
The 3% methanol solution of 20) was dispensed at 200 μl / well, stirred for 60 seconds with a plate mixer, and allowed to stand at room temperature for 4 hours. After standing, the well solution was discarded, and the plate was washed with a 5% aqueous ethanol solution and dried with a dryer at 50 ° C. for 3 hours. A plate having a polyethylene glycol chain and an amino group introduced into the surface was prepared through the above steps, and the plate was used as an example.

Comparative Example 1 A 96-well multi-well plate made of polystyrene having an amino group on its surface (MS-8696F manufactured by Sumitomo Bakelite) was used as Comparative Example 1. (Comparative Example 2) Polystyrene was added to a 96-well multiwell plate made of polystyrene (MS-8496F, manufactured by Sumitomo Bakelite).
2-hydroxyethyl methacrylate (Sigma P-
3932) in 200 μl / well
The plate was dispensed in 1 l and air-dried to make the surface hydrophilic. Comparative Example 3 An untreated polystyrene 96-well multiwell plate (Sumitomo Bakelite MS-8496F) was used as Comparative Example 3.

Using the plates of the above Examples and Comparative Examples 1 and 2, the surface contact angle was measured, the amount of adsorbed protein was compared, and the amount of immobilized protein after the treatment with the coupling agent was compared. 1. Measurement of surface contact angle The contact angle was measured using a contact angle meter (CAS-150, manufactured by Kyowa Interface Science). The contact measurement was performed by cutting out the bottom of the well of each plate. Table 1 shows the measurement results. 2. Comparison of Protein Adsorption Amount of protein adsorption was determined by directly contacting the enzyme-labeled antibody solution with each plate, and measuring the amount remaining after washing by the amount of color developed in the substrate solution.

Alkaline phosphatase-labeled human IgG
(Sigma A3150) phosphate buffer (pH 7.
4) Add 200 μl of solution (0.2 μg / ml) to each plate
1 / well and allowed to stand at 37 ° C. for 1 hour for adsorption. After standing, the solution in the well is discarded, and 0.05% Tween is added.
The plate was washed three times with a phosphate buffer containing n20. After washing
Add 10 phenolphthalein monophosphate substrate solutions to each well.
After dispensing at 0 μl / well and reacting at room temperature for 30 minutes, 50 μl of disodium hydrogen phosphate + EDTA aqueous solution was added.
The reaction was stopped by adding / well. The absorbance at 550 nm was measured with a plate reader (MTP-32 manufactured by Corona). Table 1 shows the measurement results.

3. Comparison of the amount of immobilized protein with and without the coupling agent treatment Each plate was previously prepared using glutaraldehyde (Wako ab
t. 2% phosphate buffer solution (for 20% electron microscope)
After dispensing at 00 μl / well, allowing to stand at room temperature for 20 minutes, washing with pure water, It was measured by the measurement method used for comparison of the protein adsorption amount. Table 1 shows the measurement results.

[0018]

[Table 1]

In the example and the comparative example 2 in which the contact angle was 0 degree, the amount of adsorbed protein was low, and in the example, it was recognized that the amount of solid phase increased by performing the coupling agent treatment. That is, in the examples, it was shown that the physical adsorption of the protein was suppressed by the hydrophilicity of the surface, and the protein was immobilized by covalent bond by the functional group on the surface. Next, measurement sensitivity was compared by an actual immunoassay (sandwich method) using Example and Comparative Example 3. In the examples, 200 μl of a 2% phosphate buffer solution of glutaraldehyde (manufactured by Wako, abt. 20% electron microscope) was previously added to each plate.
The mixture was dispensed at 1 / well, left at room temperature for 20 minutes, and then washed with pure water.

Subsequently, a phosphate buffer (pH 7.4) solution (10 μg / ml) of an anti-human CRP antibody (manufactured by Towns) was added to each well of each plate of the Examples and Comparative Examples.
The solution was dispensed at 00 μl / well and allowed to stand at 37 ° C. for 2 hours for adsorption. After standing, the solution in the well is discarded and 0.05% Tw
The plate was washed three times with a phosphate buffer containing een20. After washing, for a comparative example, 200 μl / well of a 3% BSA (International's 31473) phosphate buffer solution (blocking solution) was dispensed into each well and allowed to stand at room temperature for 4 hours. After standing, discard the solution in the well, and add 0.05%
The plate was washed three times with a phosphate buffer containing Tween 20.

Next, a CRP standard serum (Towns) phosphate buffer solution (0.001, 0.01, 0. 1) was added to each well.
1, 1, 10, 100 μg / ml) were dispensed at 100 μl / well in two rows (16 wells) of each concentration plate,
It was left at room temperature for 30 minutes. After standing, discard the solution in the well,
The plate was washed three times with a phosphate buffer containing 0.05% Tween20. After washing, each well contains POD-labeled anti-human CRP.
2.0 μg / m phosphate buffer solution of antibody (Towns)
1 was dispensed and allowed to stand at room temperature for 30 minutes. After standing, the solution in the well was discarded, and the well was washed three times with a phosphate buffer containing 0.05% Tween20. After washing, the color was developed using a POD color development kit (ML-1120T manufactured by Sumitomo Bakelite), and the absorbance at 450 nm was measured with a plate reader. Table 2 shows the results.

[0022]

[Table 2]

In the comparative example, the specimen (CR
Although no increase in signal was observed in accordance with the concentration of (P standard serum), in Examples, an increase in signal in accordance with the concentration of the sample was observed in the entire range from low to high concentrations. In the comparative example, a decrease in the signal was confirmed at the highest concentration, which is considered to be due to the inhibition of the reaction of the labeled antibody due to the adsorption of the contaminating protein of the sample. That is, in the comparative example, the noise (background) due to the nonspecific adsorption of the labeled antibody is high in the low concentration region, and the signal corresponding to the concentration of the sample is obtained at the high concentration due to the reaction inhibition by the adsorption of the contaminating protein. In contrast, in the examples, non-specific adsorption was excluded, and it was shown that an accurate signal depending only on the antigen-antibody reaction was obtained from a low concentration to a high concentration region.

[0024]

The container for high-sensitivity immunoassay of the present invention eliminates nonspecific adsorption from the immunoassay measurement system by using a hydrophilic polymer and a functional group grafted on the surface, and removes immobilized molecules. By solid-phase immobilization by covalent bonding, a blocking operation is omitted, and high-sensitivity measurement according to the analyte concentration can be performed from a low concentration to a high concentration region.

[Brief description of the drawings]

FIG. 1 is a graft of a polymer chain having a functional group at a terminal and a hydrophilic group at a side chain.

FIG. 2 is a graft of a polymer chain having a hydrophilic group at a terminal and a functional group at a side chain.

FIG. 3 shows a graft of a hydrophilic polymer having a terminal functional group.

FIG. 4 is a graft of a polymer chain having a hydrophilic group and a functional group in a side chain.

FIG. 5 is a graft of a polymer chain having a hydrophilic group and a functional group in a side chain.

FIG. 6 shows a graft of a hydrophilic polymer and a functional group.

[Explanation of symbols]

 1. Functional group 2. 2. hydrophilic groups or hydrophilic polymers; Polymer chains

Claims (4)

[Claims] 1. A highly sensitive immunoassay container characterized in that a hydrophilic polymer having a functional group is grafted on at least a substrate portion of the container that comes into contact with a reagent solution. 2. A highly sensitive immunoassay container characterized in that a functional group and a hydrophilic polymer are grafted on at least a substrate portion of the container that comes into contact with the reagent solution. 3. The high-sensitivity immunoassay container according to claim 1, wherein the contact angle with water of at least the substrate portion in contact with the reagent solution is 1 degree or less. 4. The highly sensitive immunoassay container according to claim 1, wherein the functional group can bind to at least one of a peptide, protein, nucleic acid, and enzyme.