JP2003156433A - Surface plasmon resonance method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface plasmon resonance method that can restrain a non-singular adsorption with respect to a sensor chip. SOLUTION: This surface plasmon resonance method is characterized in that a surfactant is added into a mobile layer. The non-singular adsorption to the sensor chip for the surface plasmon resonance method can be remarkably suppressed thereby. Measuring sensitivity can be also enhanced greatly by applying the method of the present invention to measurement of antigen-antibody reaction, and by carrying an immunoreactive substance in the mobile layer on a polymer particle.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a surface plasmon resonance method.

[0002]

2. Description of the Related Art The surface plasmon resonance (SPR) method is used when the refractive index of a medium such as a solution in contact with the lower surface of a sensor chip (a glass substrate on which metal such as gold is deposited) changes. This is a method of measuring the change in the refractive index of the medium by utilizing the change in the angle of the reflected light of reduced intensity among the reflected light of the light incident at various angles on the upper surface of the metal thin film through the prism. Kenichi Kasai, Protein Nucleic Acid Enzyme, Vol.37 No.15 (1992), pp.2977-2984). The intensity of the reflected light that is reflected at a specific angle is reduced because resonance occurs when the evanescent wave generated at the interface between the glass and the metal thin film and the wave number of a surface wave called surface plasmon generated on the metal thin film surface match, It occurs because part of the energy of light is used to excite surface plasmons and the amount of light returning as reflected light is reduced. When one of the reactants involved in the chemical reaction is bound to the lower surface of the sensor chip, the reaction of the reactant with the other reacts to change the refractive index of the medium in contact with the lower surface of the sensor chip. The angle of reflected light with reduced intensity (this angle is called the "resonance angle") changes momentarily as the chemical reaction progresses.
Therefore, the progress of the chemical reaction can be observed in real time by measuring the change in the resonance angle in real time. Therefore, SPR is used for studies of chemical reaction kinetics, and is also used for analysis of antigen-antibody reaction in the field of biochemistry. Particularly, in the analysis of the antigen-antibody reaction, unlike the usual immunoassay, there is no need to use a label at all, which is convenient, and there is an advantage that the label does not interfere with the antigen-antibody reaction at all.

As described above, one of the reactants is bonded onto the metal film of the SPR sensor chip, but it is often difficult to bond the reactant directly onto the metal film. Usually, a structure for binding a reactant is provided on the metal film. As one of such structures, a so-called self-assembled monolayer (Self Assembled Monolayer,
SAM) is known to be used. A thiol such as an alkyl thiol is known as a molecule constituting the self-assembled monolayer. When the gold film is immersed in the thiol solution, the thiol group is adsorbed and bonded to gold, and a thiol monomolecular film is spontaneously formed on the gold film. Such a spontaneously formed monolayer is called a self-assembled monolayer (SAM). Conventionally, as a molecule constituting SAM, alkyl thiol or alkyl thiol, such as carboxyalkyl thiol or aminoalkyl thiol, which facilitates protein binding by substituting the opposite end of the thiol with a carboxyl group or amino group, etc. It has been known. Dextran and the like are also used.

[0004]

When the surface plasmon resonance method is performed using a conventional sensor chip, a substance irrelevant to the reaction to be measured is nonspecifically adsorbed on the sensor chip, which results in accurate measurement. There is a problem of being hindered.

Therefore, an object of the present invention is to provide a surface plasmon resonance method capable of suppressing non-specific adsorption on a sensor chip.

[0006]

As a result of earnest research, the inventors of the present invention have found that non-specific adsorption to a sensor chip can be significantly suppressed by adding a surfactant to the moving layer. The present invention has been completed.

That is, the present invention provides a surface plasmon resonance method characterized by adding a surfactant to the moving layer in the surface plasmon resonance method.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the SPR method itself is a well-known method. That is, one of the reactants involved in the reaction is bound to the surface of the gold film of the sensor chip, and the solution containing the other reactant is moved as a moving layer while being in contact with the surface of the gold film, and the binding of the reactants is detected by the sensor. The measurement is performed by measuring the reflected light from the substrate side of the chip. Since a device for performing the SPR method and a sensor chip are also commercially available, the method of the present invention can be easily performed using such a commercially available device.

The method of the present invention is characterized by adding a surfactant to the moving layer. The surfactant to be added is not limited at all, and may be any surfactant having a hydrophobic portion and a hydrophilic portion, such as a nonionic surfactant, a cationic surfactant and an anion. It may be either a surfactant or an amphoteric surfactant. As a preferred surfactant, a nonionic surfactant,
In particular, but not limited to, Tween (registered trademark) series surfactants having a polyoxyethylene moiety.

The concentration of the surfactant added to the moving bed is
The concentration range is not particularly limited, and an appropriate concentration range can be easily set by routine experiments according to the properties of the surfactant, the reaction conditions to be measured, and the like. Normal,
The final concentration of the surfactant in the moving layer is appropriately 0.01 to 0.1%, but is not limited to this range.

The method of the present invention can be carried out in exactly the same manner as the conventional SPR method except that a surfactant is added to the moving layer, and the reaction to be measured is not limited at all. In addition, the SAM of the sensor chip is not limited to the alkylthiol-based molecule used in the following examples, but may be a dextran-based molecule that is widely used, or a dialkylsulfide-based or dialkyldisulfide-based molecule. It may be.

As will be concretely shown in the following examples, by adding a surfactant to the transfer layer, non-specific adsorption can be remarkably suppressed, and accurate reaction can be measured.

Further, as described above, the SPR method is widely used for measuring the antigen-antibody reaction. In this case, one reaction substance of the antigen-antibody reaction (ie, antibody or antigen-binding fragment thereof (Fab fragment, F (ab ') ₂ fragment, etc.) or antigen) is bound to the surface plasmon resonance sensor chip, and the other Reactants (ie, antigens or antibodies or antigen-binding fragments thereof) are included in the mobile phase. As described above, in the method of the present invention, since the surfactant is present in the transfer layer, nonspecific adsorption is remarkably suppressed, but further, the reaction substance (that is, the antigen or the antibody or By supporting the antigen-binding fragment) on the hydrophobic polymer particles, the measurement sensitivity of the antigen-antibody reaction can be significantly improved, which is preferable.

As preferred examples of the hydrophobic polymer constituting the hydrophobic polymer particles, a polymer having an aromatic compound (polystyrene, polyimide), a polymer having a cyclo ring compound (polypiperazine, polyvinyl dioxane), a polymer having a carbonyl group ( Examples thereof include, but are not limited to, polyacrylamide and polycarbonate).

The diameter of the hydrophobic polymer particles is not particularly limited, but is usually about 50 nm to 1 μm, preferably about 100 nm to 700 nm.

The hydrophobic polymer particles can be easily produced by a well-known ordinary method, and since there are some commercially available latex particles and the like, commercially available particles can be used.

The binding of the antigen or the antibody or the antigen-binding fragment thereof (hereinafter, these may be referred to as "immunoreactive substances" for convenience) onto the hydrophobic polymer particles is carried out, for example, in a solution of the antigen or the like. This can be easily carried out based on a well-known method in which polymer particles are added to physically adsorb an antigen or the like, or to form a covalent bond using a linker. In addition, since a variety of hydrophobic polymer particles to which an immunoreactive substance is bound are commercially available as reagents for the immunoagglutination method, a commercially available immunoagglutination method may be used depending on the type of antigen-antibody reaction to be measured. It is also possible to use a reagent for use.

The amount of the hydrophobic polymer particles supporting the immunoreactive substance to be subjected to the reaction depends on the amount of the immunoreactive substance to be subjected to the antigen-antibody reaction and the amount of the antigen supported on each hydrophobic polymer particle. Can be calculated based on
The appropriate amount can be easily set by routine experimentation.

When the SPR method of the present invention is used for the measurement of the antigen-antibody reaction, the method in which the immunoreactive substance in the moving layer is carried on the hydrophobic polymer particles is subjected to the reaction,
Except for adding a surfactant in the moving layer and subjecting the immunoreactive substance in the moving layer to the reaction in the state of being loaded on the hydrophobic polymer particles, the antigen-antibody reaction using the conventional SPR method The measurement can be performed in exactly the same manner.

In this case, not only the non-specific adsorption is remarkably suppressed by the surfactant but also the measurement sensitivity is remarkably improved.

[0021]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Comparative Examples 1 to 5 As thiol molecules forming SAM on the sensor chip, thiols (A) to (D) having the following structures were used.

Thiol (A) HS- (CH ₂ ) ₁₁ -OH (Comparative Example 1) Thiol (B) HS- (CH ₂ ) ₁₁ -COOH (Comparative Example 2) Thiol (C) HS- (CH ₂ ) ₁₁ -NH ₂ (Comparative example 3) thiol _{(D) HS- (CH 2)} 11 -CH 3 ( Comparative example 4)

A 2.5 mM ethanol solution of each thiol was prepared, and a commercially available SPR sensor chip (glass plate having a gold film thickness of 50 nm and size 9 mm x 9 mm) having a gold film formed on a glass substrate was placed in each solution at room temperature. It was immersed under for 24 hours to form SAM. Further, a commercially available sensor chip in which the gold film was exposed was used as it was without forming SAM (Comparative Example 5).

Using each of the above sensor chips, a commercially available S
The SPR method was performed using a PR device (Bia Core 2000 type manufactured by Pharmacia). The mobile layer is PBS (10mM phospate, 15
The measurement was carried out under the conditions of 0 mM NaCl) pH 7.4, flow rate 10 μl / min, temperature 25 ° C., sample introduction time 60 seconds. Serum protein (total protein: 500ppm),
Human serum albumin (HSA: 300 ppm) or human immunoglobulin G (hIgG: 90 ppm) was used. The values (resonance units) of each response measured 100 seconds after the sample was introduced are shown in Table 1 below.

Examples 1 to 5 Exactly the same operation as in Comparative Examples 1 to 5 was performed except that the surfactant Tween 20 (registered trademark) was added to the moving layer at a final concentration of 0.05%. Examples using thiols (A) to (D) are Examples 1 to 4, and example using a gold film is Example 5. 10 measured from the time of sample introduction for each sample
The value of each response (resonance unit) after 0 seconds is shown in Table 1 below.

[0027]

[Table 1]

The comparative examples and examples described in each row of Table 1 are different only in whether or not a surfactant is added to the moving layer, and the other conditions are measured under exactly the same conditions. As is clear from Table 1, the addition of a surfactant to the moving bed significantly reduces nonspecific adsorption.

Examples 6 and 7 Measurement of antigen-antibody reaction by SPR method (1) Preparation of sensor chip As thiol molecules, 10 mol% of the above thiol (B) and 90 mol% of thiol (E) having the following structure Thiol solution containing
A sensor chip was prepared in the same manner as Comparative Examples 1 to 5 except that it was used for producing SAM. Thiol _{(E) HS- (CH 2)} 11 - (OC 2 H 5) 3 -OH

An anti-BSA (rabbit IgG) antibody was bound to the carboxyl group of the SAM thiol molecule of the sensor chip prepared as described above. This operation was specifically performed as follows. 0.4M EDC / 0.1M NHS (EDC: N-Ethyl-N '-(3
-Dimethylaminopropyl) carbodiimide hydrochloride, NHS: N-hydroxysuccinimide) was used to activate the carboxyl group. EDC / NHS is ultra pure water (Mili Q
Water). The activation time was 7 minutes at a flow rate of 10 μl / min. The activated carboxyl group was coupled using the amino group of the protein. Antibody concentration is 10
It was dissolved in 10 mM acetate buffer pH 4.5 at 0 μg / ml.
The immobilization time was 7 minutes. In addition, the unreacted activated carboxyl group was inactivated with 1M ethanolamine pH 9.0.

(2) Preparation of Antigen-Binding Polystyrene Particles Based on a styrene monomer, polystyrene particles having a diameter of about 500 nm (3.64
w / v%) was synthesized. The antigen BSA was bound to this. This operation was specifically performed as follows. Polystyrene particles were prepared by dissolving BSA in 10 mM HEPES (pH 7.4) and then at 37 ° C for 2
A time incubation was performed. The amount of BSA bound was calculated by the 280 nm absorbance of the residue (derived from tyrosine and tryptophan).

(3) SPR method Using the sensor chip prepared in (1), the antigen-antibody reaction was measured by the SPR method. The moving layer is 10 mM HEPES (pH
7.4), flow rate 10 μl / min, measurement temperature 25 ° C, sample volume 10
μl (in 10 mM HEPES), the dissociation solution was 10 mM (glycine-H
Cl pH 2.0) and various concentrations of antigen B as a sample
SA was used (Example 6). As the antigen, various concentrations of the antigen prepared in (2) and supported on polystyrene particles were used (Example 7). The response 10 seconds after the introduction of the sample is shown in FIG.

As shown in FIG. 1, the measurement sensitivity of Example 7 in which the antigen was supported on the polystyrene particles and the reaction was higher than that in Example 6 in which the antigen was subjected to the reaction in the free state. It can be seen that is greatly improved.

[0034]

According to the method of the present invention, non-specific adsorption to the sensor chip for surface plasmon resonance method can be significantly suppressed. Further, by applying the method of the present invention to the measurement of the antigen-antibody reaction, and carrying the immunoreactive substance in the moving layer on the hydrophobic polymer particles, the measurement sensitivity can be greatly improved.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of SPR when an antigen is provided in a moving phase in a free state or in a state of being supported on polystyrene particles.

Claims (7)

[Claims] 1. The surface plasmon resonance method, wherein a surfactant is added to the moving layer in the surface plasmon resonance method. 2. The concentration of the surfactant in the moving bed is 0.01 to 0.
The surface plasmon resonance method according to claim 1, which is 1%. 3. The surface plasmon resonance method according to claim 1, wherein the surfactant is a nonionic surfactant. 4. The reaction to be measured by the surface plasmon resonance method is an antigen-antibody reaction, one reaction substance of the antigen-antibody reaction is bound to a sensor chip for surface plasmon resonance method, and the other reaction substance is hydrophobic. The surface plasmon resonance method according to claim 1, wherein the surface plasmon resonance method is present in the moving layer in a state of being supported on the polymer particles. 5. The surface plasmon resonance method according to claim 4, wherein the hydrophobic polymer is polystyrene. 6. The diameter of the hydrophobic polymer particles is 100 nm.
The surface plasmon resonance method according to claim 4, wherein the surface plasmon resonance method is from 1 to 1 μm. 7. The antibody or antigen-binding fragment thereof is bound to the sensor chip, and the hydrophobic polymer particles are
7. The surface plasmon resonance method according to claim 4, wherein an antigen that reacts with the antibody or an antigen-binding fragment thereof in an antigen-antibody reaction is bound.