JPH0694719A - Colorimetry analysis method using gold colloid particle - Google Patents

Abstract

PURPOSE:To obtain a colorimetry analysis method using a gold colloid particle which can be applied to an automatic analyzer etc. CONSTITUTION:A colorimetry analysis method is used for cause a gold colloid particle where an antibody (or an antigen) for an object to be measured is connected to react with the object to be measured, thus determining the object to be measured based on the generated absorbance change of the gold colloid particle. Problems due to a conventional method, for example, the disagreement, etc., of judgment caused by external factors such as the illumination at a measurement place and the type of a light source used for lighting or human factors such as the degree of experience and practice or the physical conditions of a judge, can be avoided and at the same time the object to be measured can be determined (completely).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an improved method of immunoassay using colloidal gold particles.

[0002]

BACKGROUND OF THE INVENTION Conventionally, there have been known methods for measuring an antigen-antibody reaction, such as a change in turbidity due to a reaction between an antigen and an antibody itself, a latex method using an insolubilized carrier, and a precipitation reaction in a gel on an agar plate. It is a reaction that causes turbidity in a solvent, and colorimetric analysis is difficult. In addition, since the agglutination reaction is judged by the naked eye, it must be qualitative or semi-quantitative, and it is fully possible that an error in measurement will occur depending on the individual. There are many problems with it.

In the immunoassay method, gold colloidal particles are used, and agglutination reaction analysis for mannan utilizing this color change (Horisberger, Rossert et al., J. Histoche).
m.Cytochem., 25.295-305 (1977)), and an immunoassay method using gold colloidal particles as a carrier (JP-A-57-86051). However, the immunoassay method using these colloidal gold particles has a disadvantage that the judgment of positive and negative in the vicinity of the detection limit is apt to be influenced by the subjectivity even if it is experienced, because the judgment is performed visually.

[0004]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and provides a colorimetric analysis method using colloidal gold particles, which can be measured by using an automatic analyzer or the like. That is the purpose.

[0005]

According to the present invention, colloidal gold particles (hereinafter abbreviated as sensitized gold colloidal particles) to which an antibody (or antigen) to a substance to be measured is bound and a substance to be measured are reacted with each other. It is an invention of a colorimetric method characterized by quantifying a substance to be measured based on a change in absorbance of colloidal particles.

The substance to be measured in the colorimetric method of the present invention is not particularly limited as long as the antibody (or antigen) to the substance to be measured can bind to the gold colloid particles, but Specifically, for example, proteins such as albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), such as high-density lipoprotein (HLD), low-density lipoprotein (LDL), ultra-low density lipoprotein, etc. Nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as alkaline phosphatase, lactate dehydrogenase, lipase and amylase, such as IgG,
Immunoglobulins such as IgM, IgA, IgD and IgE (or fragments thereof such as Fc portion, Fab portion and F (ab) ₂ portion), blood coagulation factors such as fibrinogen and thrombin, such as anti-streptolidine O Antibodies, antiviral antibodies, antibodies such as rheumatoid factor, etc. may be mentioned.

As the gold colloidal particles used in the present invention, commercially available ones may be used, but a conventional method, for example, a method of reducing chloroauric acid with sodium citrate (Nature Phys. Sci., V
ol. 241, 20, 1973 etc.) may be used. Further, the particle size of the gold colloidal particles is not particularly limited, but examples thereof include those in the range of usually 20 nm to 90 nm, preferably 30 nm to 70 nm.

The sensitized gold colloidal particles used in the present invention can be easily obtained, for example, as follows. That is, commercially available colloidal gold particles or a conventional method, for example, a method of reducing chloroauric acid with sodium citrate (Nature P
hys.Sci., vol. 241, 20, 1973, etc.) was used to prepare gold colloidal particles according to a conventional method (J. Histochem. Cytochem., vol. 25, 1187).
~ 1200, 1977, Experientia, vol. 31, 1147, 1975, etc.) can be easily prepared. More specifically, gold colloid particles and gold colloid particles 1 mg
On the other hand, usually 0.5 to 100 μg, preferably 1 to 50 μg of the antibody (or antigen) to the substance to be measured is reacted at room temperature for 5 to 30 minutes in an appropriate buffer, and then, for example, Carbowax 20M or the like. It can be easily obtained by adding a dispersant and collecting the target sensitized gold colloid particles by centrifugation or the like. The obtained sensitized gold colloid particles may be uniformly dispersed and stored in a solution containing a dispersant such as Carbowax 20M. The buffer solution used in the above reaction is not particularly limited in kind, concentration, pH and the like as long as it does not inhibit the binding reaction between the gold colloid particles and the antibody (or antigen) to the substance to be measured.

The present invention is directed to a sample containing the sensitized gold colloid particles obtained as described above and a substance to be measured, for example, a sample derived from a living body such as blood, plasma, serum, cerebrospinal fluid, urine, feces, or the like. Is appropriately diluted with a buffer or the like, and mixed appropriately, and the change in absorbance of the gold colloid particles resulting from the antigen-antibody reaction that occurs as a result is measured, and the result is calculated for the gold colloid particles prepared in advance. By applying it to a calibration curve that shows the relationship between the change in absorbance and the amount of the substance to be measured,
It can be easily implemented. In addition, if the change in absorbance is a certain value or less, it is negative, and if it is more than a certain value, it is positive.

The change in absorbance in the present invention may be obtained as follows, for example. (1) the absorbance of the solution containing the sensitized gold colloid particles before the addition of the measurement target substance, and the difference in the absorbance of the reaction solution after the sensitized gold colloid particles and the measurement target substance are reacted for a certain period of time as the absorbance change To do. (2) The absorbance change rate (particularly the maximum change rate) of the reaction solution after the reaction between the sensitized gold colloid particles and the substance to be measured is started is defined as the absorbance change. (3) The absorbance of the reaction solution after a certain period of time has elapsed from the start of the reaction between the sensitized gold colloid particles and the substance to be measured is taken as the absorbance change. (4) After the reaction between the sensitized gold colloid particles and the substance to be measured is started, the absorbance of the reaction solution is measured twice at appropriate intervals, and the difference is taken as the absorbance change.

In the present invention, the wavelength for measuring the change in absorbance is not particularly limited as long as it is a wavelength capable of measuring the change in absorbance, but is in the vicinity of 500 to 550 nm, which is the maximum absorption wavelength of the gold colloid particles. It is desirable because the measurement sensitivity can be increased. Also, it goes without saying that the change in absorbance may be obtained by two-wavelength photometry using the main wavelength and the sub wavelength.

According to the present invention, since a change caused by an antigen-antibody reaction can be measured as a change in absorbance, a measuring method utilizing the antigen-antibody reaction can be, for example, a colorimeter, a spectrophotometer, a microplate. It is possible to measure with an automatic analyzer or biochemical analyzer. Further, since it is possible to measure with the device as described above, the external factors such as the illuminance at the measurement location, the type of the light source used for illumination, and the like, which are caused by the conventional visual determination, and the experience of the determiner, It is possible to avoid discrepancies in judgments due to human factors such as proficiency or physical condition. Moreover, by applying the present invention to an automatic biochemical analyzer, it is possible to process a large number of samples in a short time.

Hereinafter, the present invention will be described in more detail with reference to examples of the present invention, but the present invention is not limited thereto.

【Example】

Example 1. (1) Preparation of Anti-Human IgG Monoclonal Antibody Sensitization Colloidal Test Solution 17 μl of anti-human IgG monoclonal antibody solution (1 mg / ml, manufactured by Wako Pure Chemical Industries, Ltd.) was added with 0.2 M K ₂ CO ₃ aqueous solution to p.
Gold Korodesol solution adjusted to H = 6.0 (manufactured by Jansen) 1
0 ml was mixed rapidly and left at room temperature for 10 minutes. Then, 500 μl of 1% (w / w) Carbowax 20M solution was added to the reaction mixture and mixed to homogeneity, followed by centrifugation at 7,000 g for 20 minutes, and the precipitate was dispersed (50 mM sodium phosphate, 0.0005% Anti-human Ig was prepared by adding keratin, 0.02% carbowax 20M pH6.5) so that OD ₅₄₀ = 5.0.
It was used as a G monoclonal antibody sensitized gold colloid test solution. (2) Preparation of calibration curve using gold corolide reagent 40 Human IgG solution of known concentration in each well of 96-well plate
Then, add 100 μl of the anti-human IgG monoclonal antibody-sensitized gold colloid reagent solution obtained in (1) above, and add SOF.
Λ ₁ by Tmax-J (Ver.2.01J, Wako Pure Chemical Industries, Ltd.)
= 540nm, λ ₂ = 405nm, once automix, measurement time 10min,
Two-wavelength negative kinetic measurement was carried out with a microplate reader UVmax (manufactured by Molecular Devices Co., Ltd.) under the condition that the measurement interval was set to 12 sec. The obtained measurement result is further S
Using OFTmax-J, lag time 12sec, Vmax point = 2
The maximum reaction rate Vmax was obtained by treating under the conditions of. A calibration curve showing the relationship between the obtained Vmax and the IgG concentration (μg / ml) is shown in FIG. As is clear from FIG. 1, it can be seen that a calibration curve having good linearity can be obtained.

Example 2. (1) Preparation of anti-CRP polyclonal antibody-sensitized colony test solution Anti-CRP polyclonal antibody (rabbit) solution (41 μg / m
l, Wako Pure Chemical Industries, Ltd.) 65 μl with 0.1 M potassium dihydrogen phosphate-0.05 M sodium tetraborate buffer to a pH of 6.8
10 ml of gold corodiesol solution (manufactured by Jansen) adjusted to
Were mixed rapidly and left at room temperature for 10 minutes. Then 500μ of 1% (w / w) Carbowax 20M solution in the reaction mixture
1) and mixed to homogeneity, followed by centrifugation at 7,000 g for 20 minutes, and the resulting precipitate was treated with a dispersion (50 mM sodium phosphate, 0.02% carbowax 20M, pH 7.4) to give OD ₅₄₀ = 5.0. The solution prepared as described above was used as an anti-CRP polyclonal antibody-sensitized gold reagent solution. (2) Preparation of calibration curve using gold colloid reagent 40 μl of known concentration CRP solution in each well of 96-well plate
Then, add 100 μl of the anti-CRP polyclonal antibody-sensitized gold color reagent solution described above, and add SOFTmax-J (Ver.2.
01J, Wako Pure Chemical Industries, Ltd.) λ ₁ = 540nm, λ ₂ = 650n
m, auto mix once, measurement time 10 min, measurement interval 20 sec.
Two-wavelength negative kinetic measurement was performed with a microplate reader UVmax (manufactured by Molecular Devices Co., Ltd.) set to the above conditions. A calibration curve showing the relationship between the obtained absorbance and the CRP concentration (μg / ml) is shown in FIG. As is clear from FIG.
It can be seen that a calibration curve having good linearity can be obtained.

Embodiment 3. (1) Preparation of anti-human fibrinogen polyclonal antibody sensitized gold coleide test solution Anti-human fibrinogen polyclonal antibody solution (1 mg
/ ml, Wako Pure Chemical Industries, Ltd.) 32 μl, 0.2M 3- (N
-With morpholino) propanesulfonic acid (MOPS) buffer
Gold Corodiesol solution (manufactured by Jansen) adjusted to pH = 6.6 1
0 ml was mixed rapidly and left at room temperature for 10 minutes. Then, 1% (w / w) Carbowax 20M in the reaction solution
Add 500 μl of solution and mix evenly, 7,000 g
After centrifuging for 20 minutes, the resulting precipitate is dispersed (0.2M
MOPS, 0.02% carbowax 20M, 0.1% sodium nitride, pH 7.4)
The anti-human fibrinogen polyclonal antibody-sensitized gold colony test solution was prepared so that OD ₅₄₀ = 10.0. (2) Preparation of calibration curve using gold corolide reagent 40 μl of human fibrinogen solution of known concentration was placed in each well of a 96-well plate, and 100 μl of the anti-human fibrinogen polyclonal antibody-sensitized gold colloid test solution was added to each well, and SOFTmax-J (Ver.2.01J, Wako Pure Chemical Industries, Ltd.
Λ ₁ = 540 nm, λ ₂ = 650 nm, automix once, measurement time 10 min, measurement interval 30 sec., And a two-wavelength negative kinetic measurement was carried out with a microplate reader UVmax (manufactured by Molecular Devices). The obtained measurement results were further analyzed by SOFTmax-J, and the difference between the absorbance 2 minutes after the start of the measurement and the absorbance 10 minutes after the measurement was started. A calibration curve showing the relationship between this absorbance difference and the fibrinogen (Fgn) concentration (μg / ml) is shown in FIG. As is clear from FIG. 3, it can be seen that a calibration curve having good linearity can be obtained.

Embodiment 4. Commercially available gold colloid agglutination reaction reagent (ImmunoGold Hem: Wako Pure Chemical Industries, Ltd.)
Was used for colorimetric analysis of hemoglobin. (Gold colloidal test solution) 1 bottle of Immunogold Hem's gold colloid reagent (for 2.5 ml) was added to the same solution of the gold colloid reagent of the kit 5.
What was melt | dissolved in 0 ml was made into the gold-colloid test liquid. (Hemoglobin standard solution) Contains a predetermined concentration of hemoglobin
A 50 mM phosphate buffer solution (pH 7.4) was used as a hemoglobin standard solution. (Apparatus to be used) An automatic analyzer Super Z828 (manufactured by Nitteku Co., Ltd.) was partially modified and used. (Operation method) The above-mentioned automatic analyzer was set under the following measurement conditions to perform measurement.・ Sample volume 83 μl.・ Amount of gold test solution collected is 200 μl.・ Measurement wavelength 542/659 nm. -Measurement method 2-point end assay (measurement of the difference between the absorbance 2 minutes after the start of the reaction and the absorbance 9 minutes after the reaction was started.) (Results) A calibration curve showing the relationship between the obtained absorbance difference and the hemoglobin concentration is shown in FIG. Shown in. As is clear from FIG.
It can be seen that a good calibration curve can be obtained.

Example 5. Detection of hemoglobin in human feces (colloidal gold test solution) Same as in Example 4. (Sample) For 67 samples of fresh human feces, a sample extract prepared according to the instruction manual attached to Immunogold Hem was used. (Apparatus used and operation method) The same as in Example 4. (Result) Positive results with an absorbance difference of 0.06 or more, 0.
Tables 1 and 2 show the results of the judgment with negative values less than 06.

Reference Example 1. Hemoglobin was detected in 67 samples of the fresh human feces used in Example 5 by using Immunogold Hem. In addition, the measurement operation is performed by the standard operation method described in the item description attached to the kit,
The judgment was made visually by a trained inspector. The obtained results are also shown in Table 1.

Reference Example 2. The 67 samples of fresh human feces used in Example 5 were subjected to detection of hemoglobin using OC-hemodia'Eiken '(registered trademark of Eiken Chemical Co., Ltd.). The measurement operation was performed by the standard operation method described in the instruction manual attached to the kit, and the determination was visually performed by a trained inspector. The obtained results are also shown in Table 2.

[0021]

[Table 1]

[0022]

[Table 2] As is clear from the results of Tables 1 and 2, it can be seen that the judgment results obtained by the method of the present invention are in good agreement with those visually judged by a trained inspector.

[0023]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a colorimetric analysis method using colloidal gold particles which can be applied to an automatic analyzer and the like. Discrepancies in judgment caused by external factors such as the illuminance at the measurement location, the type of light source used for illumination, etc. and human factors such as the experience and proficiency of the judge or physical condition, etc. It is an invention that has a remarkable effect in that it is possible to avoid the above, and that it is possible to quantify (not semi-quantitatively) the substance to be measured, and it is a great invention that contributes to the art.

[Brief description of drawings]

FIG. 1 shows a calibration curve showing the relationship between Vmax and IgG concentration, which was obtained in Example 1.

FIG. 2 shows a calibration curve showing the relationship between absorbance and CRP concentration obtained in Example 2.

FIG. 3 shows a calibration curve showing the relationship between the difference in absorbance and the concentration of fibrinogen, which was obtained in Example 3.

FIG. 4 shows a calibration curve showing the relationship between the difference in absorbance and the hemoglobin concentration, which was obtained in Example 4.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Megumi Kumai 67-3 Higashi Sonoda-cho, Amagasaki City

Claims (5)

[Claims] 1. The absorbance of gold colloidal particles produced as a result of reacting gold colloidal particles (hereinafter abbreviated as sensitized gold colloidal particles) to which an antibody (or antigen) to the measurement target substance is bound, with the measurement target substance. A colorimetric method characterized by quantifying a substance to be measured based on changes. 2. The absorbance change is the absorbance of the solution containing the sensitized gold colloid particles before the addition of the substance to be measured, and the absorbance of the reaction solution after the sensitized gold colloid particles and the substance to be measured are reacted for a certain period of time. The analysis method according to claim 1, which is a difference from 3. The analysis method according to claim 1, wherein the change in absorbance is the rate of change in absorbance of the reaction solution after the reaction between the sensitized gold colloid particles and the substance to be measured has started. 4. The analytical method according to claim 1, wherein the change in absorbance is the absorbance of the reaction solution after a certain period of time has elapsed after the reaction between the sensitized gold colloid particles and the substance to be measured. 5. The analysis method according to claim 1, wherein the change in absorbance is a difference between the absorbance of the reaction solution measured twice at appropriate intervals after the reaction between the sensitized gold colloid particles and the substance to be measured is started. .