JP2002131319A - Method for measuring protein for inflammation marker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring protein for inflammation marker capable of measuring an inflammation marker with high sensitivity by using a small amount of specimen sample of a low concentration, to provide a method for measuring protein for inflammation marker capable of reducing the measur ing cost by simply performing high-sensitivity measurement even if the amount of a specimen sample is small and the concentration thereof is low, and to provide a method for measuring protein for inflammation marker capable of performing the measurement a plurality of times by using the same sensor chip to reduce the measurement cost. SOLUTION: A sample liquid to be tested containing C-reactive protein is sent to an anti-C-reactive protein antibody fixed to a metallic thin film formed on the surface of a transparent substrate to specifically bond the protein to the protein antibody. Light is emitted from one side of a prism fast stuck to the substrate toward a boundary between the substrate and the thin film. A surface plasmon resonance angle is detected by reflected light from the boundary, and the protein in the sample liquid is measured based on displacement of the resonance angle caused by the specific bond between the protein antibody and the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring a protein for an inflammatory marker using surface plasmon resonance (hereinafter, referred to as SPR).

[0002]

In the clinical diagnosis of infectious diseases and inflammation tests, C-reactive protein (hereinafter referred to as CRP) as a protein for an inflammatory marker in serum or plasma is quantified and its increase or decrease ( Concentration), the presence or absence of inflammation in the body, the size of the inflamed site and the degree of inflammation are measured, and the course and prognosis of inflammation and infectious diseases are determined.

[0003] As a typical example of this inflammation marker measuring method, there are: Fluorescence ELISA,. A method in which a colloidal gold-labeled anti-CRP antibody is reacted with CRP or the like adsorbed on nitrocellulose, and measurement is performed. There is known a method in which CRP or the like is specifically bound to latex beads on which an anti-CRP antibody is immobilized, and measurement is performed by turbidimetry.

However, all of the methods have a CRP measurement limit of 0.1 mg / dl and a measurement range of 0.1 to 200 mg / dL.
Because of this, CRP requires a high concentration and a large amount of a sample to quantify CRP, and the measurement target is limited to adults.
For this reason, for example, in newborns and children, the CRP concentration exceeds the detection limit of the device, and it has been difficult to detect the CRP concentration.

[0005] This phenomenon can be solved by using a large measuring device. However, since the device itself is large and expensive, there is a problem that the measurement cannot be performed easily and the measuring cost increases.

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned conventional drawbacks, and an object of the present invention is to measure an inflammatory marker with high sensitivity using a small amount of a sample sample at a low concentration. It is an object of the present invention to provide a method for measuring a protein for an inflammatory marker which can be performed.

Another object of the present invention is to provide a method for measuring an inflammatory marker protein, which can easily perform high-sensitivity measurement and reduce the measurement cost even when the sample is small and has a low concentration. It is in.

Another object of the present invention is to provide a method for measuring a protein for an inflammatory marker, which can perform measurement a plurality of times with the same sensor chip and can reduce the measurement cost.

[0009]

A first aspect of the present invention is an anti-C-film fixed to a metal thin film formed on the surface of a transparent substrate.
The test sample solution containing the C-reactive protein with respect to the reactive protein antibody is fed to specifically bind the C-reactive protein to the anti-C-reactive protein antibody and a prism having a transparent substrate adhered thereto. Light is radiated from one side toward the boundary between the transparent substrate and the metal thin film, and the surface plasmon resonance angle is detected based on the reflected light from the boundary, and the specific binding between the anti-C-reactive protein antibody and the C-reactive protein is detected. C-reactive protein in the test sample solution is quantitatively measured based on the displacement of the surface plasmon resonance angle caused by the above.

[0010] According to a second aspect of the present invention, a test sample solution containing a C-reactive protein is fed to an anti-C-reactive protein antibody fixed on a metal thin film formed on the surface of a transparent substrate. Anti-C-
After specifically binding the C-reactive protein to the reactive protein antibody, the anti-C-reactive protein antibody solution is fed to specifically bind the anti-C-reactive protein antibody to the C-reactive protein. And irradiating light from one side of the prism with the transparent substrate in contact with the boundary between the transparent substrate and the metal thin film, detecting the surface plasmon resonance angle by the reflected light from the boundary, and detecting the anti-C-reactive protein antibody. C-reactive protein in the test sample solution is quantitatively measured based on the displacement of the surface plasmon resonance angle due to the specific binding of C-reactive protein and anti-C-reactive protein antibody.

According to a fourth aspect of the present invention, the anti-C-reactive protein antibody immobilized on the metal thin film by detecting the displacement of the surface plasmon resonance angle and then feeding a withdrawal liquid onto the metal thin film. Characterized in that the C-reactive protein is removed from the sample to enable re-measurement.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below with reference to the drawings. Embodiment 1 In FIGS. 1 to 4, a sensor chip 3 of a surface plasmon resonance angle detection device 1 (hereinafter, referred to as an SPR device) embodying the method of the present invention has an optical refractive index of 1.4 to 2.0. A metal thin film 7 of gold (Au) or silver (Ag) is formed on the surface of a glass substrate 5. A semi-cylindrical or triangular prism 9 whose optical refractive index is substantially the same as that of the glass substrate 5 is closely attached to the glass substrate 5 opposite to the metal thin film 7 via a matching oil, a silicon rubber sheet or the like.

A laser beam irradiating device 11 is arranged on the left outer peripheral surface of the illustrated prism 9, and a light receiving device 13 is arranged on the right outer peripheral surface of the illustrated prism symmetrically. The laser light irradiation device 11 includes a laser light source 15 that outputs laser light,
The laser light output from the laser light source 15 is
And a first lens 17 that converges so as to irradiate at the boundary of the metal thin film 7 and over the entire measurement area described later.

The light receiving device 13 includes a glass substrate 5 and a metal thin film 7.
A second lens 19 for converting reflected light from the boundary of
A large number of light receiving cells 20a are arranged, and include a light receiving member 20 including a photodiode array, a CCD, or the like that outputs an electric signal according to the light intensity of the parallel light from the second lens 19.

The measurement region of the metal thin film 7 is divided into a sensing region 7a and a non-sensing region 7b, and an anti-CRP antibody 21 is fixed to the sensing region 7a by a chemical method. As the anti-CRP antibody 21, an anti-CRP monoclonal antibody or an anti-CRP polyclonal antibody is immobilized in the case of an inflammation test, and troponin, CK-MB (C
K-MM, CK-BB, CK = creatine kinase),
In the case of myoglobin etc. myocardial infarction test, anti-troponin antibody, anti-CK-MB antibody, anti-myoglobin antibody,
In the case of a blood coagulation test using FDP (Fibrinogen Degradation Products) or D-dimer, an anti-FDP antibody,
Antibody D dimer antibody is applied.

Anti-CRP against the surface of the sensing area 7a
An example of a method for immobilizing an anti-CRP antibody 21 such as a monoclonal antibody or an anti-CRP polyclonal antibody will be described.
As shown in FIG. 4, an antibody solution obtained by dissolving an anti-CRP antibody in an acetate buffer (pH: 5) such that the final concentration is 0.1 to 1 mg / ml is applied to the surface of the sensing region 7a of the metal thin film 7. Contact and immobilize by chemical bonding for 5-60 minutes.

In the sensing area 7a, the anti-CR
Surface or non-sensing region 7b where P antibody is not fixed
On the surface of casein buffer (pH: 7.4) containing bovine serum albumin (1 μg / ml) having a specific non-binding property for CRP for 30 minutes, and serum of CRP-free for about 30 minutes , 1 M ethanolamine hydrochloride (pH:
8.0 to 8.5) for about 10 minutes in order to contact each other and fix them to form a block film 7c.
Restricts the adhesion of CRP and other substances and contaminants in the CRP liquid 25 when the liquid is fed.

On the upper surface of the glass substrate 5, a cell block 23 having a flow cell 23a formed on the lower surface to cover the sensing area 7a and the non-sensing area 7b is formed via a matching oil or silicon rubber sheet (not shown). Adhered interchangeably.

Flow cell 23 in cell block 23
A supply nozzle 27 for supplying the CRP liquid 25 into the flow cell 23a is provided on the inflow side of the flow cell 23a, and a CR overflowing from the flow cell 23a is provided on the outflow side of the flow cell 23a.
A recovery nozzle 29 for recovering the P solution 25 is provided, and the CRP solution 2 is applied to the surface of the metal thin film 7 in the flow cell 23a.
5 is fed at a predetermined flow rate.

As the CRP solution 25, a recombinant CR is used.
P dissolved in PBS (-), a mixed sample containing CRP and other biological components, serum and plasma are applied.

Next, a method for measuring the protein for an inflammatory marker is shown in FIG.
This will be described with reference to FIG. CR to be measured in advance
After the glass substrate 5 in which the anti-CRP antibody 21 corresponding to the P is fixed to the sensing area 7a is brought into close contact with the prism 9, the cell block 23 is brought into close contact with the upper surface of the glass substrate 5, and the sensor chip 3 is set.

Next, pure water or a buffer solution is supplied from the supply nozzle 27 to fix the non-fixed anti-CRP adhering to the surface of the metal thin film 7.
After removing antibody 21 and other biological substances and contaminants, FIG.
The flow cell 23a is supplied through the supply nozzle 27 as shown in FIG.
The CRP solution 25 is supplied to the inside, and the CRP solution 25 overflowing from the flow cell 23a is collected through the collection nozzle 29, and the CRP solution 25 is fed over the surface of the metal thin film 7 for a predetermined time.

As shown in FIG. 6, the CRP contained in the CRP liquid 25 when the CRP liquid 25 is fed is changed to the sensing area 7a.
Non-sensing region 7 which specifically binds to the anti-CRP antibody immobilized on
b is recovered as it is without being fixed by the block film 7c.

Then, the CRP solution 2 into the flow cell 23a
At the time of liquid feeding of 5, the laser light from the laser light irradiation device 11 is
A boundary between the glass substrate 5 including the sensing region 7a and the non-sensing region 7b and the metal thin film 7 is irradiated, and the light reflected from the boundary is received by the light receiving device 13 to output an electric signal corresponding to the light intensity of the reflected light. .

Now, the CR which is fed in the flow cell 23a is
When the CRP in the P solution 25 specifically binds to the anti-CRP antibody immobilized on the sensing region 7a, the specific mass of CRP increases as compared with the case where only the anti-CRP antibody is used. The rate changes, and the light intensity of the reflected light increases.

In the non-sensing region 7b where the anti-CRP antibody is not fixed, the CRP solution 25
Because the binding of CRP and other biological components in it is blocked,
The light intensity of the reflected light from the location is substantially constant.

Then, the SPR angles from the sensing area 7a and the non-sensing area 7b are respectively detected, and the SPR angle from the non-sensing area 7b is set as a reference SRP angle.
The value obtained by subtracting the reference SPR angle from the non-sensing area 7b from the SPR angle from the sensing area 7a (ΔSPR
Based on the angle) and specifically bound to anti-CRP antibody 21
Quantify P.

This measuring operation is performed by a calibration curve (shown in FIG. 7) showing the ΔSPR angle measured in advance by a CRP having a known concentration.
, The CRP concentration of the CRP liquid 25, that is, the CRP is quantified based on the ΔSPR angle measured by the present measurement method.

This CRP tends to increase in the early stage of inflammation and decreases with the recovery of inflammation.
Based on the CRP concentration quantitatively measured by the R angle, the presence or absence of inflammation, the progress state, the size of the inflamed site, and the like are determined.

In the present embodiment, the change in the dielectric constant caused by the specific binding of the CRP as an analyte to the anti-CRP antibody immobilized on the sensing region 7a of the metal thin film 7 is represented by SP.
The CRP in the CRP liquid 25 is detected by detecting the R angle.
CRP can be quantified with high sensitivity even if is small and has a low concentration.

In this embodiment, the non-sensing area 7
A value Δ obtained by subtracting the reference SPR angle from the SPR angle from the sensing area 7a with the SPR angle from b as the reference SPR angle
Since the CRP in the CRP liquid 25 is quantitatively measured based on the SPR angle, measurement errors due to back noise can be reduced.

Embodiment 2 In FIGS. 8 and 9, the present embodiment is different from the method of Embodiment 1 in that the sensing region 7 in the metal thin film 7 is formed.
a in the CRP solution 25 with the anti-CRP antibody 21 immobilized on
After specific primary binding of RP, pure water
CRP solution containing CRP not specifically bound to anti-CRP antibody from above metal thin film 7 by feeding a buffer solution adjusted to a pH value suitable for antibody activity or a buffer solution containing a surfactant. 25 is removed.

Next, as shown in FIG. 8, an anti-CR solution adjusted to a final concentration of 20 to 100 μg / ml with a buffer solution adjusted to a pH value suitable for antibody activity on the metal thin film 7.
The secondary antibody solution 30 containing the P antibody 21 is fed, and as shown in FIG. 9, the CRP specifically bound to the anti-CRP antibody fixed on the sensing region 7a is
Is specifically secondary-bound as a secondary antibody.

At this time, similarly to the above, the laser light irradiating device 11 irradiates the laser light to the sensing region 7a and the non-sensing region 7b of the metal thin film 7 to reflect the reflected light from the boundary between the glass substrate 5 and the metal thin film 7. The light intensity is detected by the light receiving device 13 to detect a change in the SPR angle. At this time, as compared with the first embodiment, the CR specifically bound to the anti-CRP antibody
Further, the anti-CRP antibody 21 is specifically and secondarily bound to P to increase the change in the overall mass, and hence the change in the dielectric constant, to make the displacement of the SPR angle remarkable.

Then, the CRP contained in the CRP liquid 25 is quantified based on the ΔSPR angle obtained by subtracting the reference SPR angle based on the non-sensing area 7b from the detected SPR angle corresponding to the sensing area 7a.

The first embodiment detects the SPR angle based on the CRP that specifically binds to the anti-CRP
In contrast to the quantitative determination of CRP in Example 5, the present embodiment allows specific secondary binding of an anti-CRP antibody to specifically bound CRP, and the quantitative determination can be performed based on the SPR angle that greatly changes. Sensitivity can be increased.

Embodiment 3 In FIGS. 10 and 11, this embodiment is similar to Embodiment 1
The anti-CRP fixed to the sensing area 7a as shown in FIG.
After the CRP solution 25 is fed to the antibody to specifically bind the contained CRP, or as described in the second embodiment, the CRP specifically bound to the anti-CRP antibody immobilized on the sensing region 7a After further specific and secondary binding of anti-CRP antibody to
As shown in (2) shows a method for specific secondary binding of P antibody), a solution containing a surfactant component or a pH-adjusted acidic / alkaline buffer or high salt The separation liquid 32 composed of a concentration solution is fed, and CRP is removed from the anti-CRP antibody fixed in the sensing area 7a.
And the substance other than the CRP is forcibly released to return the sensing region 7a to the initial state where only the anti-CRP antibody is fixed. (Shown in FIG. 2)

Next, a different CRP solution 35 collected from the specimen to be tested is applied to the anti-CRP antibody 21 in the sensing area 7a in the same manner as in the first and second embodiments.
Specific binding of CRP contained in solution 35 and SP
The R angle is detected and quantified.

In this embodiment, the CRP contained in the CRP solution 35 collected from different samples can be re-quantified using one sensor chip, and the measurement cost can be reduced. (Shown in FIG. 6)

In the second embodiment, the CRP liquid 2
5 and the immobilized anti-C in Embodiment 3
When the CRP is detached from the RP antibody, the sensing light 7a and the non-sensing light 7b of the metal thin film 7 are irradiated with laser light from the laser light irradiation device 11 to detect reflected light, as in the case of quantifying the CRP. When the SPR angle returns to the initial value, it is determined that the cleaning and the separation have been completed.

Example 1 1 mg / m in 100 mM acetate buffer (pH: 5.0)
A solution prepared by dissolving 1 anti-polyclonal antibody was used and contact-fixed to the sensing region 7a.

The CRP solution 25 was prepared by dissolving a recombinant CRP prepared by the gene transfer technique in PBS (−), and was allowed to bind specifically to the anti-polyclonal antibody in the sensing region 7 a by feeding the solution to the metal thin film 7.

FIG. 11 shows the relationship between the ΔSPR angle and the CRP concentration. As the ΔSPR angle increases, the CRP concentration increases.

Example 2 100 μg in 100 mM acetate buffer (pH: 5.0)
An anti-polyclonal antibody was contact-fixed to the sensing region 7a using a solution prepared by dissolving the anti-polyclonal antibody at a concentration of / ml.

Recombinant CRP as CRP liquid 25
Was used in PBS (-), and specifically bound to an anti-polyclonal antibody. The recombinant CRP specifically and primarily bound to the anti-polyclonal antibody was secondarily bound to the anti-polyclonal antibody at 50 μg / ml as a secondary antibody.

FIG. 12 is a chart showing the relationship between the ΔSPR angle and the CRP concentration in the case where CRP was specifically primary-bound and the case where anti-polyclonal antibody was specifically secondary-bound. In the case of subsequent coupling, a large ΔSPR angle was obtained, and the quantitative measurement accuracy was improved.

Example 3 100 μg in 100 mM acetate buffer (pH: 5.0)
/ Ml of an anti-polyclonal antibody dissolved therein was used and contact-fixed to the sensing region 7a.

The CRP solution 25 is prepared by diluting a standard human serum with a known CRP concentration with PBS (−), and specifically binds the CRP in the human serum to the anti-polyclonal antibody. 50 μg / ml of the anti-polyclonal antibody is specifically secondarily bound to CRP in human serum specifically bound to the anti-polyclonal antibody.

FIG. 13 is a chart showing the relationship between the CRP concentration and the ΔSPR angle. As the CRP concentration increases, a larger ΔSPR angle is obtained, and the accuracy of quantitative measurement is improved.

Example 4 100 μg in 100 mM acetate buffer (pH: 5.0)
/ Ml of an anti-polyclonal antibody dissolved therein was used and contact-fixed to the sensing region 7a. CRP liquid 25
Was used to specifically bind to the anti-polyclonal antibody.

[0051] Next, a 50 mM glycine hydrochloride buffer (pH: 2.0) was fed as the detachment liquid 32 to detach the recombinant CRP specifically bound to the anti-polyclonal antibody immobilized on the sensing region 7a.

[0052] After the above specific primary binding, the same concentration of human standard serum 50 μg / ml anti-polyclonal antibody was specifically secondary-bound to the recombinant CRP.
The release liquid 32 was fed to separate the recombinant CRP from the anti-polyclonal antibody.

FIG. 14 shows the result of the above-mentioned detachment, and FIG. 15 shows the result of the above-mentioned detachment. In any case, when the detachment liquid 32 is fed at the position indicated by the arrow in FIG.
The PR angle returned to the value before CRP-specific binding.

[0054]

According to the present invention, an inflammation marker can be measured with high sensitivity using a small amount of a test sample at a low concentration. Further, even if the sample sample is small and has a low concentration, the measurement cost can be reduced by simply performing high-sensitivity measurement. Further, measurement can be performed a plurality of times by the same sensor chip, and the measurement cost can be reduced.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of an apparatus embodying a method for measuring an inflammatory marker protein.

FIG. 2 is an explanatory diagram showing a portion A of FIG. 1 in an enlarged manner.

FIG. 3 is an enlarged plan view of a sensor chip.

FIG. 4 is an explanatory view showing a fixed state of an anti-CRP antibody.

FIG. 5 is an explanatory diagram showing a state in which the CRP liquid is fed.

FIG. 6 is an explanatory diagram showing a specific binding state of CRP to an anti-CRP antibody.

FIG. 7 is a chart showing the relationship between CRP concentration and ΔSPR angle.

FIG. 8 is an explanatory diagram showing a state of feeding a secondary antibody solution.

FIG. 9 is an explanatory diagram showing a specific secondary binding state of an anti-CRP antibody to a specific primary bound CRP.

FIG. 10 is an explanatory diagram showing a state in which CRP is released from an anti-CRP antibody.

FIG. 11 is a chart showing a relationship between a ΔSPR angle and a CRP concentration according to the first embodiment.

FIG. 12 is a chart showing a relationship between a ΔSPR angle and a CRP concentration according to a second embodiment.

FIG. 13 is a chart showing a relationship between a ΔSPR angle and a CRP concentration according to a third embodiment.

FIG. 14 is a chart showing a CRP withdrawal state according to the fourth embodiment.

FIG. 15 is a chart showing a CRP withdrawal state according to the fourth embodiment.

[Explanation of symbols]

1-SPR device, 3-sensor chip, 5-glass substrate, 7-metal thin film, 7a-sensing region, 7b-non-sensing region, 9-prism, 11-laser irradiation device, 13-light receiving device, 21-resistance CRP antibody, 23-cell block, 25-CRP solution, 30-secondary antibody solution, 32-
Separation liquid, 35-different CRP liquid

Claims (6)

[Claims] An anti-C-reaction by sending a test sample solution containing a C-reactive protein to an anti-C-reactive protein antibody fixed on a metal thin film formed on the surface of a transparent substrate. C
Irradiating light toward the boundary between the transparent substrate and the metal thin film from one side of the prism to which the reactive protein is specifically bound and the transparent substrate is adhered, and detects the surface plasmon resonance angle by reflected light from the boundary; , Anti-C-reactive protein antibody and C
-A method for measuring a protein for an inflammatory marker, which quantitatively measures C-reactive protein in a test sample solution based on displacement of a surface plasmon resonance angle due to specific binding of a reactive protein. 2. An anti-C-reaction by sending a test sample solution containing a C-reactive protein to an anti-C-reactive protein antibody fixed on a metal thin film formed on the surface of a transparent substrate. C
-After the specific binding of the reactive protein, the anti-C-reactive protein antibody solution is fed to cause the specific secondary binding of the anti-C-reactive protein antibody to the C-reactive protein, and the transparent substrate adheres. Light is irradiated from one side of the prism toward the boundary between the transparent substrate and the metal thin film, the surface plasmon resonance angle is detected by the reflected light from the boundary, and the anti-C-reactive protein antibody and C-reactive protein are detected.
A method for quantitatively measuring C-reactive protein in a test sample solution based on displacement of surface plasmon resonance angle due to specific binding of a reactive protein and an anti-C-reactive protein antibody. 3. The cleaning liquid according to claim 2, wherein a cleaning liquid is fed onto the metal thin film on which the C-reactive protein is specifically and primary-bonded to the anti-C-reactive protein antibody to remove the excess test sample liquid. A method for measuring a protein for an inflammatory marker, in which an anti-C-reactive protein antibody solution is fed later to specifically and secondarily bind the anti-C-reactive protein antibody to the C-reactive protein. 4. The anti-C-reactive protein antibody immobilized on the metal thin film according to claim 1 or 2, wherein after detecting the displacement of the surface plasmon resonance angle, a liquid is withdrawn from the anti-C-reactive protein antibody fixed on the metal thin film.
-A method for measuring a protein for an inflammatory marker, which is capable of re-measuring by removing a reactive protein. 5. The method according to claim 1, wherein the C-reactive protein is troponin, CK-MB (C
K-MM, CK-BB, CK = creatine kinase),
A method for measuring a marker protein which is any of myoglobin. 6. The blood coagulation test according to claim 1, wherein the C-reactive protein is FDP (Fibrinogen Degradat
(Ion Products) and D-dimer.