JP3338551B2 - Method for measuring human serum albumin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a trace amount of human serum albumin with high sensitivity. More specifically, the present invention relates to a method for highly sensitive measurement of trace albumin using a quartz oscillation type human serum albumin sensor.

[0002]

2. Description of the Related Art In recent years, attention has been paid to changes in oscillation frequency with respect to changes in surface mass of quartz oscillators, and quartz oscillators have been applied to the analysis of many substances in solutions. Recently, attempts have been made to immobilize antibodies on the surface and use them to detect various antigenic substances (Mura
matsu et al., Anal.Chimi.Acta, vol. 188, 257 (1986)
, Davis et al., Anal.Chem., Vol. 61, 1227 (198
9)).

For early diagnosis of diabetic nephropathy, it is effective to detect a trace amount of human serum albumin (HSA) excreted in urine as a marker. Conventionally, HSA
The concentration is measured by radioimmunoassay (RIA), enzyme immunoassay (EIA), or the like. However, these methods require the use of a labeled antibody or antigen, and the operation is complicated. And the measurement time is long.

On the other hand, there have been several reports on the detection of HSA using a quartz oscillator (Prusak-Socha).
czewski and JHTLuong, Anal.Lett., vol.23, 401
(1990), M. Muratsugu et al., Anal. Chem., Vol. 65,
2933 (1993)), however, there is still a need for improved sensitivity and repetitive response.

Therefore, the inventors of the present invention have intensively studied a method of detecting a trace amount of HSA using a quartz oscillator by amplifying a change in oscillation frequency by further increasing the change in surface mass to thereby improve the detection sensitivity. Then, in an HSA measurement method using an AT-cut element with a metal electrode as a quartz oscillator and a quartz oscillation sensor in which an anti-HSA antibody is adsorbed and immobilized on the electrode, a solution containing HSA was flowed to select HSA. Rather than immediately measuring the change in frequency after binding to the immobilized antibody, after binding HSA, a solution containing an anti-HSA antibody is further allowed to flow, and the HSA is sandwiched between the anti-HSA antibodies. It was found that, when the change in frequency was measured after binding to HSA, the detection sensitivity was significantly improved as compared with the case where the change was measured after binding to HSA. The present invention has been completed by further research based on this finding.

That is, an object of the present invention is to provide a high-sensitivity measurement method for HSA using an AT-cut element having a metal electrode as a crystal oscillator and using a crystal oscillation sensor in which an anti-HSA antibody is adsorbed and fixed to the electrode. Is to provide.

[0007]

That is, the gist of the present invention is as follows. (1) An AT-cut element with a metal electrode is used as a quartz oscillator, and an anti-human serum albumin antibody (anti-HS
A) is used to bind human serum albumin (HSA) in a test solution to the immobilized antibody using a quartz-crystal vibrating sensor to which the immobilized antibody is immobilized.
(2) a method for measuring human serum albumin, which comprises binding an antibody and then measuring the frequency; (2) the method according to (1), wherein the anti-HSA antibody adsorbed and immobilized on the metal electrode is a monoclonal antibody against HSA. Method, (3)
Another anti-HSA antibody that further binds to HSA is HSA
(4) The method according to (1) or (2) above, which is a polyclonal antibody against
(2) wherein the heat treatment is performed at 0 to 80 ° C.
Or (5) the method according to any one of (1) to (4), wherein the metal electrode is a gold electrode.

As a frequency measuring apparatus using a quartz oscillation type sensor using an AT-cut element with a metal electrode as a quartz oscillator used in the present invention, a known apparatus, for example, disclosed in Japanese Patent Application Laid-Open No. 3-115947. Are included. However, a flow cell type as shown in FIG. 1 can be more preferably used. That is, the crystal unit is incorporated in the flow cell such that only one side of the crystal unit (the side on which the antibody is immobilized) contacts the flowing solution, and oscillation is performed with the solution flowing.

[0009] Examples of the crystal oscillator, AT-cut, those of the vibration frequency 5~10MH _Z preferred. As electrodes,
Various types of electrodes such as a gold electrode, a silver electrode, and a platinum black electrode can be used. In the method of the present invention for adsorbing and immobilizing an anti-HSA antibody, a gold electrode is preferable from the viewpoint of reproducibility of measurement accuracy and the like.

The method for immobilizing the anti-HSA antibody on the gold electrode includes a method via protein A, a method via 2-mercaptoethylamine, a method via γ-aminopropyltriethoxysilane, etc. However, the method is not sufficiently satisfactory in any of the viewpoints of the amount of reaction, the response sensitivity at frequency, the stability of the immobilized anti-HSA antibody, and repeated use, and the method of direct adsorption immobilization is the most excellent.

The direct adsorption and immobilization method uses anti-HSA on a metal electrode.
In this method, the antibody solution is dropped and dried. Specifically, for example, 50-3000 to a gold electrode (80 mm ² )
ppm of anti-HSA antibody in PBS (+ 0.1% NaN ₃ ,
pH 7.2) This is carried out by dropping 10 μl of a solution and drying. By this method, the hydrophobic Fc portion of the anti-HSA antibody is hydrophobically bonded to the gold surface,
It seems that it is fixed by adsorption.

The drying may be carried out at room temperature,
When heated to ８０80 ° C. for about 5 to 60 minutes, only the relatively easily denatured Fc portion of the anti-HSA antibody is denatured and becomes easily hydrophobically bonded, while the Fv portion which is an antigen recognition site is not denatured. In some cases, the sensitivity to HSA is improved.

Examples of the anti-HSA antibody for immobilization used in the present invention include polyclonal antibodies such as mice, rabbits and goats against HSA, and monoclonal antibodies. Among them, for example, from the viewpoints of sensitivity, response reproducibility, etc. And mouse HSA monoclonal antibodies (Sigma, Zymmet, Japan Biotest Laboratory, Sedarane, etc.) are particularly preferred. This is due to the fact that the HSA monoclonal antibody is uniform, so that the binding strength to the gold electrode is small and the bond with HSA is strong and uniform.

In order to increase the surface mass of the crystal oscillator, another anti-HSA antibody is further bound to the trapped HSA. Such an anti-HSA antibody may be a polyclonal antibody (for example, Sigma, Cosmo Bio Inc.). Company)
Is preferred. This seems to be due to the requirement to bind to the unreacted site of the trapped HSA, and thus to the polyclonal antibody containing various antibodies more easily than the monoclonal antibody, and to increase the binding efficiency.

A small amount of H is measured using a quartz oscillation type HSA sensor.
To measure SA, a flow cell as shown in FIG.
The quartz oscillator on which the anti-HSA antibody is immobilized as described above is incorporated, and oscillation is performed in a state where the test liquid containing HSA is circulated on only one surface of the quartz oscillator. Preferably, the internal volume of the cell is usually 0.01 to 0.5 cm ^{3 in} the case of a minute measurement, and the flow rate of the solution is about 0.1 to 1.0 ml · min ⁻¹ . As the sensor signal, the amount of change in the resonance frequency due to the change in the weight of the quartz oscillator due to the antigen-antibody reaction is measured by a frequency counter at room temperature. Sample solution to be circulated (H
Solution containing SA), polyclonal antibody solution is usually 1m
l. HSA and polyclonal antibodies use phosphate buffered saline (pH 7.2) (PBS). In order to dissociate the bond between the HSA and the monoclonal antibody after the measurement, a disodium hydrogen phosphate-citrate buffer having a pH of 3.0 is used.

The principle of the measuring method of the present invention is as follows. First, PBS is circulated through a quartz oscillator on which a monoclonal antibody is immobilized, and the obtained stable frequency is defined as F ₀ . When the HSA solution is circulated, an antigen-antibody reaction occurs between the immobilized monoclonal antibody and HSA, and the weight of the surface of the quartz oscillator increases, so that the frequency becomes F _1.
To decrease. The amount of frequency decrease at this time is represented by ΔF ₁ = (F ₀
−F ₁ ). Then, when flowing through the polyclonal antibody solution, by further polyclonal antibody binds to the unreacted sites HSA bound to the monoclonal antibody, the frequency is reduced to F _2. Therefore, the sensitivity of the measuring method of the present invention is ΔF ₁ = (F ₀ −F ₁ ) and ΔF ₂
= (F ₀ −F ₂ ), which is the sensitivity of F ₁ −F ₂ amplified by the polyclonal antibody. Since the response in the second step (F ₁ -F ₂ ) is considered to be proportional to the amount of HSA bound to the immobilized antibody, the measurement sensitivity of HSA can be increased.

[0017]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples and the like. As Example 1 crystal oscillator using an AT-cut element with a gold electrode of the fundamental frequency 6MH _Z. The antibody was immobilized by adding an anti-HSA monoclonal antibody solution (500 ppm) on a gold electrode.
This was performed by dropping 0 μl and drying at 50 ° C. This device was assembled in the flow cell shown in FIG. 1 and oscillated with a carrier solution flowing only on one side of the device. The internal volume of the cell is 0.05 cm ³ , and the flow rate of the solution is
It was 0.44 ml · min ⁻¹ . First, 1m of PBS solution
The resonance frequency (F ₀ ) at the point where 1 is circulated and stabilized
Was measured. Then PBS containing 15 ppm HSA
1 ml of the solution was circulated and the resonance frequency (F ₁ ) was measured when it was stabilized. In addition, 1000 ppm of HS
1 ml of the polyclonal antibody solution to A was circulated and the resonance frequency (F ₂ ) was measured when it was stabilized.
The result is shown in FIG. First, observed responses of reduced frequency of about 100H _Z by circulation of 15ppm of HSA, further reduces the frequency by then circulated 1000ppm polyclonal antibody, about the entire 30
0H _Z above sensitivity was obtained.

Example 2 In order to reuse the quartz oscillator used in Example 1, PBS was circulated through the flow cell after measurement and washed, and then a disodium hydrogen phosphate-citrate buffer solution of pH 3.0 was used. Was circulated. Next, when the resonance frequency was measured while circulating the PBS, the resonance frequency was restored to the level before the measurement, as shown in FIG. Therefore, according to the method of the present invention, it is possible to repeatedly use the same fixed crystal resonator. In fact, the immobilized quartz resonator achieved stable sensitivity even after repeating the response to 100 ppm HSA for about 30 times in 4 days.

Example 3 In the same manner as in Examples 1 and 2, various concentrations of HS
The resonance frequency of the solution A was measured, and ΔF ₁ and ΔF
Asked for _two . The result is shown in FIG. HSA concentration is 0
In the range of 20 ppm, the HSA concentration is ΔF ₁ or ΔF ₂
It can be seen that a proportional relationship holds with any of the above. Also,
While [Delta] F ₁ per 1ppm is about 6H _Z, delta
For F ₂ is about 20H _Z, a further addition of a polyclonal antibody, it can be seen that sensitivity is amplified approximately three times or more. The normal value of the HSA concentration in human urine is about 10 ppm, and it can be said that the measurement method of the present invention is suitable for early diagnosis of diabetic nephropathy.

Comparative Example Bovine serum albumin (BSA) was circulated instead of HSA, and the resonance frequency was measured in the same manner as in Example 1. No change in resonance frequency was observed up to 1000 ppm in the experiment.

[0021]

According to the measuring method of the present invention, a very small amount of HS
A, in particular, a trace amount of HSA in urine can be measured simply and quickly. Therefore, it is possible to provide a useful means for early diagnosis of diabetic nephropathy by detecting a trace amount of human serum albumin (HSA) excreted in urine as a marker.

[Brief description of the drawings]

FIG. 1 shows an example of a cross-sectional view of a flow cell for a quartz oscillation type HSA sensor used in the measurement method of the present invention.

FIG. 2 is a diagram showing a frequency response of a sensor when an HSA solution and then a polyclonal antibody solution are allowed to flow. It also shows the recovery of the frequency when the acidic buffer was passed after the measurement.

FIG. 3 is a graph showing frequency reduction amounts (ΔF ₁ and ΔF ₂ ) and H obtained when a solution having various HSA concentrations is circulated.
It is a figure which shows the correlation with SA concentration.

[Explanation of symbols]

 Reference Signs List 1 sample solution inlet 2 sample solution outlet 3 gold electrode 4 crystal oscillator 5 silicon rubber sheet 6 copper lead wire 7 acrylic resin

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/53-33/379

Claims (5)

(57) [Claims] 1. An AT-cut element with a metal electrode is a quartz oscillator, and an anti-human serum albumin antibody (anti-H
A human serum albumin (HSA) in a test solution is bound to the immobilized antibody using a quartz-crystal vibrating sensor on which the SA antibody is adsorbed and immobilized, and the bound HSA is further bound to another anti-HS.
A method for measuring human serum albumin, comprising binding antibody A and then measuring the frequency. 2. The anti-HSA antibody adsorbed and immobilized on the metal electrode is a monoclonal antibody against HSA.
The described method. 3. Another anti-HSA further binding to HSA
The method according to claim 1 or 2, wherein the antibody is a polyclonal antibody against HSA. 4. The method according to claim 2, wherein the monoclonal antibody is heat-treated at 50 to 80 ° C. 5. The method according to claim 1, wherein the metal electrode is a gold electrode.