JPH05322891A - Method for preventing nonspecific adsorption for immunological measurement - Google Patents

Abstract

PURPOSE:To reduce nonspecific adsorption and perform highly reliable and highly sensitive immunological measurement even in a low-concentration range by using a kind of protein in which hydrophobic and hydrophilic areas exist in a separated state as an effective component. CONSTITUTION:At the time of performing immunological measurement, a kind of protein in which hydrophobic and hydrophilic areas exist in a separated state is used as an effective component. The protein used as an effective component has a three-dimensional structure. The principal component of the protein is beta-casein, kappacasein, or their mixture. The beta-casein and kappa-casein can be taken as an example of the protein in which hydrophobic and hydrophilic areas are separated like the amino acid sequence. About a quarter part of beta-casein on its N-terminus side is hydrophilic and the rest is hydrophobic. On the contrary, about a half part of kappa-casein on its C-terminus side is hydrophilic and the remaining half is hydrophobic.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for preventing nonspecific adsorption in an immunoassay utilizing an antigen-antibody reaction.

[0002]

2. Description of the Related Art Substances contained in blood, urine, etc.,
Alternatively, immunoassays are used to measure trace substances contained in the environment such as wastewater. The immunoassay method is based on the specific strong binding property of the antigen-antibody reaction, and it is possible to specifically and sensitively measure the target substance even from a sample in which various substances are latent.

In order to increase the reliability of the measurement and perform the measurement with high sensitivity, a specific signal from the antigen-antibody reaction caused by the substance to be measured is distinguished from other non-specific background signals. Must. It is desirable to minimize nonspecific background signals without reducing specific signals.

The solid phase method is often used in immunoassay.
In the solid phase method, an antibody is used when the substance to be measured is an antigen, an antigen is used when the substance to be measured is an antibody, or one of the complex pairs, for example, either the avidin-biotin complex pair.
(See Japanese Patent Application No. 2-335863) or protein A of immunoglobulin-protein A complex pair is immobilized on a solid phase. In the following description, for the sake of brevity, the case where the measurement target substance is an antigen will be described as an example.
The present invention is not limited to this.

After immobilizing the antibody, a nonspecific adsorption preventing operation called procking is performed in order to reduce the background signal due to nonspecific adsorption. As a blocking agent, bovine serum albumin (for example, Ishikawa et al., Enzyme Immunoassay 3rd Edition, Gakushoin, 1987), casein (for example, JP-A 1-217266), gelatin, etc. are often used and are mainly physical. Non-specific adsorption is reduced by binding to the solid phase by adsorption. Even with these blockings, when microplates or beads were used as a solid phase and measurement was performed with ordinary measurement sensitivity, there was a blocking effect in practice and immunoassay was possible.

However, in the conventional blocking method, the blocking effect is weak and satisfactory results are obtained when highly sensitive measurement is performed, or when a porous carrier having a large surface area is used as the solid phase, or when a label that easily adsorbs is used. Was not obtained. In addition, a more effective non-specific adsorption prevention method was needed to establish a measurement method that can be performed by anyone without requiring short-time measurement or special technology.

[0007]

[Means for Solving the Problems] It is considered that the main binding force for adsorbing a blocking agent on a solid phase is hydrophobic binding. In order to reduce non-specific adsorption, it is effective to make the surface of the solid phase hydrophilic. Therefore, the blocking agent is required to have both hydrophobicity and hydrophilicity. However, in the conventional blocking method, a protein such as bovine serum albumin or gelatin in which the hydrophobic region and the hydrophilic region are not clearly separated in terms of amino acid sequence order or three-dimensional structure is used as it is, or αs-, Since the whole casein including β- and κ-casein or the mixture of the partial decomposition products of gelatin was used as it was, the pocking effect was poor.

Therefore, the blocking method using a protein in which the hydrophobic region and the hydrophilic region are clearly separated in the order of amino acid sequence, the three-dimensional structure, or both is extremely effective in reducing nonspecific adsorption. As a result of intensive studies, the present invention has been completed.

That is, the present invention provides immunoassay
Provided is a method for preventing non-specific adsorption, which comprises using a protein existing in a state where a hydrophobic region and a hydrophilic region are separated as an active ingredient.

Examples in which the hydrophobic region and the hydrophilic region are separated in order of amino acid sequence include β-casein and κ-casein. About 1/4 of the N-terminal side of β-casein is hydrophilic, and the remaining part is hydrophobic. κ-Casein, on the other hand, is approximately C-terminal half hydrophilic and the other half hydrophobic. On the other hand, αs-casein does not have such a sequence. (Structure of casein micelles by Yoshikawa and Chiba, “Physical properties of food”, 6th 153
~ 173).

[0011] β-casein or κ-casein or a mixture of β-casein and κ-casein in any proportion is suitable as a blocking agent, αs-casein is not suitable as a blocking agent such as the present invention. is there. However, in practical use, αs-casein may be contained to the extent that the blocking effect is not impaired. For example
αs- casein was dialyzed bivalent because the property of precipitating a metal ion Ca ²⁺ was added to the casein solution, the supernatant or the supernatant was removed by precipitation most of αs- casein Ca ^2+, etc. The solution is a practically good blocking solution.

The hydrophobic region and the hydrophilic region may or may not be separated in the order of amino acid sequence or in a three-dimensional structure. For proteins whose three-dimensional structure has not been elucidated, commonly used methods such as the method of Chow and Fassman [J. Mol. Biol. 115 , 135-175 (197)
It suffices if they are separated on the structure estimated by 7).

The steric separation may be in a single molecule or in the state where several subunits are associated with each other. For example, the glucose transporter is an example of the former [Scientific American
an, January 1992), Pauline is an example of the latter (E
XPERIENTIA, 1990 46 (2) No. 16
7-173).

The hydrophobic region referred to in the present invention is a region containing a large number of hydrophobic amino acid residues, and specifically includes phenylalanine, tryptophan, isoleucine, leucine, proline, methionine, valine, alanine and the like. .. The hydrophilic region is a region containing many hydrophilic amino acid residues, and specifically, lysine, glutamine,
Includes aspartic acid, glutamic acid, threonine, asparagine, arginine, serine and the like.

[0015]

Industrial Applicability According to the present invention, non-specific adsorption can be reduced in immunoassay. The present invention can be applied to various immunoassay methods. First, it reduces non-specific background signals without reducing the size of the specific signal from the measurement target, enabling reliable measurement even in the low concentration range, enabling highly sensitive measurement. become. In addition, a nonspecific background signal can be reduced to allow the use of a solid phase having a large surface area, and the measurement range can be expanded. Furthermore, the use of a solid phase having a large surface area with porosity shortens the time required for the diffusion of the reactants, and enables short time measurement. It is possible to easily utilize fine particles, fluorescent or chemiluminescent labeling substances that are easily adsorbed.

Particularly, in a heterogeneous immunoassay system,
The measurement procedure can be largely omitted. Until now, the heterogeneous immune system required large amounts of liquid for cleaning. However, by using the present invention, it is possible to reduce the amount of liquid required in a heterogeneous immunoassay system. This is effective in applying the dry analysis, which is currently generalized in the biochemical test field, to the field of immunoassay.

[0017]

EXAMPLES Examples will be shown below to more specifically describe the present invention, but the present invention is not limited to these examples.

Example 1 Preparation of Casein Fractionation 1. Bovine milk casein (manufactured by Nacalai Tesque) is dissolved in PIPES buffer (0.1 M, pH 6.4) at 10 mg / ml. 2. CaCl ₂ was dissolved in 1. to 0.3 mol / l, and α
Precipitate s-casein. 3.2. Centrifuge and use the supernatant as the casein fraction.

Preparation of various blocking agents and blocking 1. β-casein, κ-casein, and the casein fraction were prepared using PIPES buffer (0.1M, pH6.
Using 4), make the protein concentration 1 mg / ml. These are used as blocking agents by the respective caseins. 2. Nitrocellulose filter for each blocking agent
(Pore size 5 μm, manufactured by Toyo Roshi Kaisha, Ltd.) is immersed at room temperature for 2 hours. 3. Blow dry at 40 ° C for 30 minutes and punch out into a circle with a diameter of 6 mm to obtain a measurement sample.

1. Anti-human C-reactive protein (CRP
Abbreviated as rabbit antibody and blue fine particles coated with bovine serum albumin (diameter 0.2 μm, Bangs Laboratories
Prepare a 3% suspension of Incorporated (Bangs Laboratories, Inc. USA). 2. As a washing solution, 10 mM phosphate buffer pH
7.4 (sodium chloride 150 mM, Tween 20 0.1
%, Bovine serum albumin 0.1%).
3. 4 μl of the above suspension was spotted on each blocked nitrocellulose filter and allowed to stand at 37 ° C. for 2 minutes. It is washed twice with 30 μl of the washing solution of 4.2. Each nitrocellulose filter is measured from the reflectance (R) at 640 nm. These measurements indicate the amount of non-specific adsorption.

As a control, the same experiment was carried out using αs-casein, whole casein, or a commercially available blocking agent prepared from milk protein (Block Ace, Snow Brand Milk Products Co., Ltd.). The results are shown in Table 1.

When the supernatant obtained by adding Ca ²⁺ to the β-casein, κ-casein, and total casein solutions was used, it was more nonspecific than when αs-casein, whole casein or block ace was used. Adsorption was significantly reduced.

[0023]

[Table 1] Blocking agent Log (1 / R) β-casein 0.071 κ-casein 0.078 Supernatant obtained by adding Ca ²⁺ to a total casein solution 0.101 αs-casein 0.482 total casein 0.199 Block Ace 0.202

Example 2 Measurement of CRP Using the casein fraction prepared in Example 1 for a nitrocellulose filter having an anti-CRP goat antibody adsorbed thereon, the "preparation of various blocking agents and blocking" of Example 1 was performed. Blocking was carried out in the same manner as 1 and 2 and drying was performed. This was uniformly impregnated with the blue fine particle suspension used in Example 1, dried and then punched into a circle having a diameter of 6 mm to obtain a sample.

4 μl of a solution having a known concentration of CRP was spotted on the sample filter and reacted at 37 ° C. for 2 minutes, and then 30
The plate was washed twice with the same washing solution as the μl of the 10 mM phosphate buffer used in Example 1. After washing, the amount of blue fine particles in the nitrocellulose filter was measured from the reflectance (R) at 640 nm and expressed as Log (1 / R). The results are shown in Figure 1.
In the figure, □ indicates Log (1) of reflectance (R) at each CRP concentration.
/ R). Clearly, a quick and easy measurement of CRP was possible.

[Brief description of drawings]

FIG. 1 is a graph showing the results of Example 2.

Claims (4)

[Claims] 1. A method for preventing non-specific adsorption, which comprises using a protein that exists in a state where a hydrophobic region and a hydrophilic region are separated in an immunoassay as an active ingredient. 2. The method for preventing non-specific adsorption according to claim 1, wherein a protein in which the separation of the hydrophobic region and the hydrophilic region is recognized in at least the amino acid sequence is used. 3. The method for preventing non-specific adsorption according to claim 1, wherein a protein in which a hydrophobic region and a hydrophilic region are separated in a three-dimensional structure is used. 4. The method for preventing non-specific adsorption according to claim 1, wherein the main component of the protein is β-casein or κ-casein or a mixture of β-casein and κ-casein.