JP2000304749A - Specific binding immunoanalytical container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a container for immunoassay, which can perform the immunoassay and whose sensitivity, reproducibility and reliability are superior. SOLUTION: This specific binding immuoanalytical container uses a base material which is molded out of a material whose adsorbability is low with reference to molecules used for analysis, and/or the container uses the base material whose surface is covered with the material. A functional group which can be bonded to the molecules which are generated on the surface and which are changed into a solid phase is changed into a solid phase by using the functional group, Since a molecular chain which connects the surface of the low- adsorbability base material to the functional group is not hydrolyzed, the adsorption of the low-adsorbability material is at 1×10-1 pmol/cm2 or lower.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay for detecting an antibody or an antigen using an antigen-antibody reaction.
Alternatively, the present invention relates to a material and surface treatment of a container used in a solid phase method for performing measurement by immobilizing a molecule such as an antibody on 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. Generally, the target molecule in the sample solution is captured using an immune reaction, and finally the molecule labeled with a radioisotope, enzyme, fluorescent substance, etc. is specifically captured and quantified. Is being done. However, in such a method using non-specific adsorption, molecules after immobilization are also adsorbed to the substrate, so only the amount of molecules captured by the specific antigen-antibody reaction should be taken as a signal. However, the amount of impurities 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, the effect of blocking changes due to slight fluctuations in the pH of the protein solution, temperature, and humidity, and the background value varies with each measurement, resulting in inconsistent results. As a result, the noise cannot be completely suppressed, and further reproducible data cannot be obtained. Another problem of the conventional solid-phase immobilization by non-specific adsorption is the reduction of signals and the occurrence of variations due to the desorption of solid-phase molecules.

[0004] In particular, when used for a clinical test, a large number of solid-phased molecules are prepared and stored at once, instead of starting from solid phase during testing. In general, at the time of inspection, it is common to take out the substrate that has already been immobilized and use it.However, it can be said that the molecules immobilized by non-specific adsorption are stably retained on the carrier. However, depending on the storage conditions, they are detached from the carrier and often vary with time, and the reproducibility and reliability of the resulting data poses a problem.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made as a result of intensive studies to solve the above-mentioned conventional problems. A high signal / noise ratio is obtained and the immobilization of an immobilized antibody is prevented. It is an object of the present invention to provide an immunoassay container capable of performing highly accurate and reliable measurement without occurrence of variation due to separation.

[0006]

That is, the present invention provides a method of molding a material having low adsorption to molecules used for analysis,
The present invention relates to a specific binding immunoassay container characterized in that the surface of the substrate covered with the surface has a functional group capable of binding to a molecule to be solid-phased.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The substrate for immunoassay according to the present invention is formed of a material having low adsorption to molecules used for analysis, and / or a solid phase is applied to the surface of the substrate which is covered. It is characterized by the fact that a functional group capable of binding to the molecule to be formed is generated, and after the solid-phased molecule is first bound to the functional group, the molecule becomes non-specifically larger in amount on the substrate surface. Without adsorption, only the target molecule is captured on the substrate surface by a specific immunoreaction, and as a result, a highly sensitive and stable result can be obtained.

Therefore, in the present invention, a step called conventional blocking is not required. Here, the surface having the best sensitivity and stability has no non-specific adsorption of molecules other than the target, but there is no problem if a measurable signal / noise ratio can be obtained. For that purpose, the adsorption of the molecule that causes noise is 1/5 to 1 of the signal molecule.
/ 10 or less. Generally, when performing an immunoassay by a solid phase immobilization method, the immobilized amount of the solid phase molecule is 5 × 10
^Since it is about ⁻³ nmol / cm ² , there is no problem if the nonspecific adsorption of the molecule is 1 × 10 ⁻¹ pmol / cm ² or less.

As a material used to obtain a surface having low adsorption to molecules used for analysis, a hydrophilic polymer such as polyhydroxyethyl methacrylate (PHE) is used.
MA), a polymer such as propionate ester polymer or polytetrafluoroethylene (PTFE). When a material which is apt to cause non-specific adsorption such as polystyrene is used, carboxyl groups and / or Alternatively, when a hydroxyl group is introduced, or when polymethyl methacrylate is used, a surface having low adsorptivity can be obtained by hydrophilization by surface modification such as introduction of a carboxyl group by partial surface hydrolysis with an alkali. Further, a material having low adsorption may be coated on the surface of the substrate. The functional group capable of binding to the solid phase molecule on the substrate surface is not particularly limited. However, when the functional group on the substrate surface is an amino group, the functional group having an amino group in the molecule is used. An immobilized molecule can be immobilized via glutaraldehyde, and an immobilized molecule having a carboxyl group in the molecule can be immobilized using carbodiimide.

When the functional group on the surface of the substrate is a carboxyl group, any solid-phased molecule having an amino group in the molecule can be solid-phased using carbodiimide. As a method for imparting a functional group to the surface of the base material, a general method of imparting a functional group by plasma exposure can be used. For example, an amino group can be provided by ammonia plasma or a mixed plasma of nitrogen and oxygen, and a carboxyl group can be introduced by plasma of an inert gas such as argon or helium or oxygen plasma. Hereinafter, the present invention will be described more specifically with reference to examples.

[0011]

(Example 1) Commercially available 96-hole E made of polystyrene
Plate for LISA (MS-84, manufactured by Sumitomo Bakelite)
96F) After coating the surface with polyhydroxyethyl methacrylate, apply 15
Example 1 was obtained by subjecting the substrate to a hydroxyl group and an amino group on the surface of the substrate. (Example 2) A 96-well microplate was molded with polymethyl methacrylate, and after molding, 2N sodium hydroxide was dispensed into the wells, and treated at 50 ° C for 10 hours to partially hydrolyze the surface. Example 2 in which a carboxyl group was generated on the surface by plasma treatment was used as Example 2.

Comparative Example 1 A commercially available polystyrene 96-well ELISA plate (MS-8 manufactured by Sumitomo Bakelite)
496F) was used as Comparative Example 1. (Comparison of non-specific adsorptivity) To compare non-specific adsorptivity, an enzyme-labeled anti-human IgG antibody (Cosmo Bio
100 ng / ml, 1 μg / ml, 10 μg / m
In a concentration series of 100 μg / ml, each concentration was dispensed in 24 wells and adsorbed for 3 hours. In addition, about the plate of Example 1 and Example 2, after deactivating amino group or carboxyl group beforehand, it was used for examination. The labeling enzyme is
Using peroxidase, determine the amount of adsorption from the absorbance,
The density was calculated from the contact area between the well and the solution. The results are as shown in FIG. 1, and it was confirmed that the non-specific adsorptivity of both the plates of Example 1 and Example 2 was lower than that of the comparative example.

[0013]

FIG.

(Comparison of immunoassay sensitivity) As a comparison of immunoassay sensitivity, an anti-rat albumin antibody as an immobilized antibody,
Immunoassay was performed using rat albumin as a measurement antigen and peroxidase-labeled anti-rat albumin antibody as a labeled antibody. The measurement method for each plate is as follows.

Example 3 Each well of a plate was adjusted to pH
200 μL of glutaraldehyde (20% manufactured by Wako Pure Chemical Industries, Ltd. for electron microscope) diluted to 2% with a phosphate buffer of 7.4
Per well and allowed to stand at 37 ° C. for 2 hours. Next, after washing three times with 300 μL / well of a washing solution (phosphate buffer solution of pH 7.4 containing 0.05% Tween 20), p
An anti-rat albumin antibody (manufactured by Cosmo Bio) diluted to 5 μg / ml with a phosphate buffer of H7.4 was added at 200 μL /
The mixture was dispensed into wells and allowed to stand at 37 ° C. for 2 hours. Next, after washing three times with 300 μL / well of a washing solution, rat albumin (manufactured by Cosmo Bio) was added at 1 μg / ml and 0.5 μg / ml.
The solution was dispensed at a concentration of 24 μg / ml, 0.25 μg / ml, and 0.125 μg / ml at 100 μL / well for each 24 wells, and allowed to stand at room temperature for 1 hour. Next, 300 μL /
After washing three times with the wells, a peroxidase-labeled anti-rat albumin antibody (manufactured by Cosmo Bio) diluted to 2 μg / ml with a phosphate buffer at pH 7.4 was dispensed at 200 μL / well and allowed to stand at room temperature for 1 hour. . Next, the cleaning liquid 30
After washing three times with 0 μL / well, color development was performed using a color development kit (color development kit T for peroxidase, manufactured by Sumitomo Bakelite), and the absorbance at a wavelength of 450 nm was measured with a plate reader.

Example 4 Each well of a plate was adjusted to pH
A water-soluble carbodiimide (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to 10% with a phosphate buffer solution of 5.8 was dispensed at 200 μL / well and allowed to stand at 37 ° C. for 1 hour. Thereafter, the procedure from antibody dispensing to color development was performed under the same conditions as in Example 3 above. (Comparative Example 2) 5 μg / ml with a phosphate buffer of pH 7.4
Anti-rat albumin antibody (Cosmo Bio) diluted in water
Was dispensed at 200 μL / well and allowed to stand at 4 ° C. for 20 hours. Next, after washing three times with 300 μL / well of a washing solution, 200 μL / well of a blocking agent (skim milk was adjusted to 5% with a phosphate buffer of pH 7.4) was dispensed,
It was left at room temperature for 2 hours. Thereafter, the procedure from albumin dispensing to color development was performed under the same conditions as in Example 3. The results are as shown in FIG. 2. In both Examples 3 and 4, linearity was obtained in the absorbance value with respect to the albumin concentration as compared with Comparative Example 2, and the coefficient of variation (CV%) of the data of 24 wells at each concentration was low and stable. Was.

[0017]

FIG. 2

[0018]

The substrate for immunoassay according to the present invention is formed of a material having low adsorption to molecules used for analysis, and / or a solid phase is applied to the surface of the substrate covered with the surface. By having a functional group capable of binding to a molecule, a highly sensitive and stable immunoassay can be performed without leaving a molecule other than the target in the measurement system and affecting the result. Further, since the immobilized molecule is immobilized by covalent bonding, it is stable even after immobilization, and excellent reproducibility can be obtained even when a substrate in which the molecule is immobilized and stored is used.

Claims (3)

[Claims] 1. A substrate surface molded and / or covered with a material having low adsorptivity to molecules used for analysis has a functional group capable of binding to a molecule to be solid-phased. A specific binding immunoassay container characterized by having: 2. The specific binding immunoassay container according to claim 1, wherein the molecular chain connecting the functional group and the surface of the substrate having low adsorption does not undergo hydrolysis. 3. Adsorption of a material having low adsorbability is 1 × 10 ⁻¹
3. The specific binding immunoassay container according to claim 1, which has a pmol / cm ² or less.