JP3266960B2 - Immunoassay using colloidal gold particles - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an improved immunoassay using colloidal gold particles.

[0002]

BACKGROUND OF THE INVENTION In immunoassays, an agglutination reaction analysis method for mannan and the like have been reported as a method using colloidal gold particles as a carrier and utilizing a color change caused by an antigen-antibody reaction generated on the colloidal gold particles. (Horisber
Ger, Rossert et al., J. Histochem. Cytochem., 25.295-305 (1
977), JP-A-57-86051, etc.).

[0003] However, the above-mentioned method has a problem that the reaction speed is slow and the prozone phenomenon is remarkable. Therefore, it cannot be said that the method is a practical method.

[0004] An immunoassay for performing a desired measurement by measuring turbidity and scattered light caused by an antigen-antibody complex resulting from an antigen-antibody reaction, and a method for measuring agglutination of latex particles sensitized with an antibody to an object to be measured. In immunoassays such as the latex agglutination reaction, which performs the intended measurement based on the degree, various measures are taken to promote the antigen-antibody reaction or to effectively measure the trace components by suppressing the nonspecific reaction. Additives such as high molecular compounds such as polyethylene glycol, dextran, methylcellulose and polyvinylpyrrolidone, and polyanions such as dextran sulfate, heparin, polystyrene sulfonic acid, hyaluronic acid, chondroitin sulfate (JP-A-57-182169, JP-A-57-182169) No. 2-238361). However, whether these additives can be used to solve the above-mentioned problems in the immunoassay using colloidal gold particles as described above or not is completely unknown.

[0005]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems in the immunoassay using colloidal gold particles. An object of the present invention is to provide an immunoassay using colloidal gold particles, which can be applied to an apparatus, is rapid, has high sensitivity, and has a wide measurement range.

[0006]

According to the present invention, a gold colloid particle (hereinafter abbreviated as a sensitized gold colloid particle) to which an antibody (or an antigen) for a substance to be measured is bound and the substance to be measured have an average molecular weight of about 20,000 or more. Of the polyanion or a salt thereof is 0.4
The present invention relates to an immunoassay method comprising reacting in a solution containing 2.52.5 (W / W)%, and quantifying or semi-quantifying a substance to be measured based on a change in absorbance of the resulting colloidal gold particles.

That is, the present inventors have conducted intensive studies to achieve the above object, and as a result, in conventional immunoassay methods such as latex agglutination reaction, the promotion of antigen-antibody reaction or suppression of nonspecific reaction has been suppressed. High molecular compounds such as polyethylene glycol, dextran, methylcellulose, polyvinylpyrrolidone, etc., and polyanions such as dextran sulfate, heparin, polystyrenesulfonic acid, hyaluronic acid, chondroitin sulfate, etc., used for effective measurement of trace components Among the additives, such as the molecular weight of about 20,000
Only the polyanion or a salt thereof significantly accelerates the antigen-antibody reaction in the immunoassay using the sensitized gold colloid particles, in other words, significantly increases the absorbance change based on the agglutination reaction of the sensitized gold colloid particles. As a result, the present invention has been completed.

The polyanion used in the present invention has an average molecular weight of about 20,000 or more, preferably about 20,000 to
Preferred examples include 1,000,000 of dextran sulfate, heparin, polystyrene sulfonic acid, hyaluronic acid, chondroitin sulfate and the like.

The salts are not particularly limited as long as they do not inhibit the promoting action of the antigen-antibody reaction. For example, ammonium salts such as salts with alkali metals such as lithium, sodium and potassium And the like.

The polyanion used in the present invention or a salt thereof (hereinafter abbreviated as polyanions of the present invention).
If the average molecular weight is too large, the viscosity of the test solution containing the colloidal gold particles will be high, making it difficult to handle.If the average molecular weight is too small, the effect of promoting the antigen-antibody reaction will be reduced, and sufficient effects will not be obtained. Care should be taken as problems arise.

The method of adding the polyanion of the present invention to the reaction system when measuring the substance to be measured according to the present invention is not particularly limited. For example, the polyanion of the present invention is previously added to a test solution containing sensitized gold colloid particles. How to add,
A preferred example is a method in which the polyanions of the present invention are added to a liquid for diluting a specimen containing a substance to be measured.

The concentration of the polyanion used in the present invention is not particularly limited as long as it is a concentration at which an antigen-antibody reaction is promoted. If the concentration is too low, the effect of promoting the antigen-antibody reaction is reduced. No effect is obtained, and if the concentration is too high, non-specific reactions will be promoted and problems such as the inability to carry out the intended measurement will occur.
To 2.5 (W / W)%, preferably 0.5 to 2.0 (W / W)%.

The substance to be measured in the immunoassay of the present invention is not particularly limited as long as an antibody (or antigen) against the substance to be measured can bind to colloidal gold particles. Specifically, for example, proteins such as albumin, hemoglobin, myoglobin, transferrin, protein A, and C-reactive protein (CRP), such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and ultra-low-density lipoprotein Nucleic acids such as lipid proteins such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) such as enzymes 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, Fab and F (ab) ₂ parts) such as fibrinogen, fibrin degradation products (FDP), prothrombin and thrombin Blood coagulation-related factors, for example, antibodies such as anti-streptolysin O antibody, antiviral antibody, and rheumatoid factor.

As the colloidal gold 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. Sc)
i., vol. 241, 20, 1973) may be used. Further, the particle size of the colloidal gold particles is not particularly limited, but is usually 20 nm to 90 nm, preferably 30 nm to 70 nm.

The sensitized gold colloid 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.) and the target antibody (or antigen) by a conventional method (J. Hi.
stochem.Cytochem., vol. 25, 1187-1200, 1977, Experien
tia, vol. 31, 1147, 1975, etc.). More specifically, the colloidal gold particles and 1 mg of the colloidal gold particles are usually 0.5 to 100 μm.
g, preferably 1 to 50 μg, of an antibody (or antigen) against the substance to be measured is allowed to react in an appropriate buffer for 5 to 30 minutes at room temperature, and then a dispersant such as Carbowax 20M is added. It can be easily obtained by fractionating 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 type, concentration, pH, etc. of the buffer used in the above reaction are not particularly limited as long as they do not inhibit the binding reaction between the colloidal gold particles and the antibody (or antigen) to the substance to be measured. Not done.

The antibodies used in the present invention include:
The antibody may be any antibody as long as it is an antibody against the substance to be measured, and is not particularly limited. That is, the usual method, for example, "Introduction to immunological experiments,
Second print, Naora Matsuhashi, Institute of Scientific Publishing, 1981 ”, etc., by immunizing animals such as horses, cows, sheep, rabbits, goats, rats, mice, etc. Polyclonal antibodies, or also according to the conventional method, ie, the cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975), using cells from the mouse tumor line and the analyte. Any monoclonal antibody produced by a hybridoma obtained by fusing with spleen cells of a mouse previously immunized may be used, and these may be used alone or in an appropriate combination thereof. Needless to say, these antibodies may be used as F (ab ') ₂ , Fab' or Fab after digestion with enzymes such as pepsin and papain, if necessary.

The present invention relates to a sensitized gold colloid particle obtained as described above and a sample containing a substance to be measured, for example, a sample derived from a living body such as blood, plasma, serum, cerebrospinal fluid, urine, or feces. And appropriately diluted with a buffer or the like, appropriately mixed in the presence of the polyanions of the present invention in the concentration range as described above, and measuring the change in absorbance of the gold colloid particles resulting from the resulting antigen-antibody reaction, By applying the result to a previously prepared calibration curve representing the relationship between the change in the absorbance of the colloidal gold particles and the amount of the substance to be measured, it can be easily carried out.

If the change in absorbance is less than a certain value, it is determined to be negative, and if it is more than a certain value, it is determined to be positive.

The change in absorbance in the present invention may be determined, for example, as follows. (1) The difference between the absorbance of the solution containing the sensitized gold colloid particles before the addition of the measurement target substance and the absorbance of the reaction solution after a lapse of a certain time after reacting the sensitized gold colloid particles with the measurement target substance is referred to as an absorbance change. I do. (2) 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 (particularly the maximum rate of change) is defined as the change in absorbance. (3) The absorbance of the reaction solution at a certain time after the start of the reaction between the sensitized gold colloid particles and the substance to be measured is defined 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 defined as the change in absorbance.

In the present invention, the wavelength for measuring the change in absorbance is not particularly limited as long as the change in absorbance can be measured, and the wavelength around 500 to 550 nm near the maximum absorption wavelength of the gold colloid particles is used. This is desirable because the measurement sensitivity can be increased. Needless to say, the absorbance change may be obtained by two-wavelength photometry using the main wavelength and the sub-wavelength.

In the present invention, it goes without saying that the change in absorbance may be visually observed, and the substance to be measured may be semi-quantified based on the result.

According to the present invention, the reactivity of the polyanions of the present invention is promoted by the action of the polyanions, so that the reaction time can be reduced and the sensitivity can be improved. In particular, when visually observing a sample near the detection limit concentration, the difference between negative and positive becomes clearer, so that the operator's skill, which has occurred in the conventional immunoassay using latex agglutination reaction, It is possible to reduce an error at the time of determination due to a low degree.

In the case where a conventional immunoassay utilizing latex agglutination is to be carried out using an analyzer, efficient measurement was difficult without a dedicated machine. Since changes due to antigen-antibody reactions can be measured with high sensitivity as changes in absorbance,
The change can be measured by a usual method such as a colorimeter, a spectrophotometer, a microplate reader, a biochemical automatic analyzer, or the like.

Further, if the measurement is performed by the above-described apparatus,
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 degree of experience, proficiency, or physical condition of the judge, which have occurred in the visual judgment of the conventional method such as the latex agglutination method, etc. It is possible to avoid discrepancies in the determination due to factors. Further, since the present invention can be applied to a biochemical automatic analyzer, a large number of samples can be processed 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.

[0027]

【Example】

Embodiment 1 FIG. (1) Preparation of anti-human IgG monoclonal antibody-sensitized gold colloid II test solution To 17 μl of anti-human IgG monoclonal antibody solution (1 mg / ml, manufactured by Wako Pure Chemical Industries, Ltd.) and adjusted to pH = 6.0 with 0.2M K ₂ CO ₃ 10 ml of the thus obtained colloidal gold sol solution (manufactured by Janssen) was quickly mixed and allowed to stand at room temperature for 10 minutes. Then, 500 μl of a 1 (w / w)% carbowax 20 M solution was added to the reaction solution.
, And homogenized, and centrifuged at 7000 g for 20 minutes. The precipitate (50 mM sodium phosphate,
0.0005% gelatin, 0.02% carbowax 20M, pH
6.5) was added to give OD ₅₄₀ = 5.0, which was used as an anti-human IgG monoclonal antibody-sensitized gold colloid reagent solution. (2) Investigation of the reaction accelerator in the immunoassay using the gold colloid reagent solution The following experiment was performed in the presence of various additives. 40 μl of a 2.5 μg / ml human IgG solution was added to each well of a 96-well plate, and 100 μl of an anti-human IgG monoclonal antibody-sensitized gold colloid test solution containing 0.6 (W / W)% of a predetermined additive was added thereto.
Λ ₁ by Tmax-J (Ver.2.01J, manufactured by Wako Pure Chemical Industries, Ltd.)
= 540 nm, λ ₂ = 405 nm, automix once, measurement time 10 m
in, microplate reader UVmax (Molecular Device Co., Ltd.) with conditions set to measurement interval 12 sec.
The two-wavelength negative kinetic was measured. Table 1 shows the results of measuring the change in absorbance (ΔOD ₅₄₀ ) 5 minutes after the start of the measurement.

[0028]

[Table 1]

As is apparent from Table 1, polyethylene glycol, which is used in conventional immunoassays to effectively measure a trace component by suppressing an antigen-antibody reaction or suppressing a nonspecific reaction, Among methylcellulose and sodium chondroitin sulfate, only chondroitin sodium significantly enhances the antigen-antibody reaction in the immunoassay using colloidal gold particles, in other words, significantly increases the absorbance change based on the aggregation reaction of colloidal gold particles. You can see that.

Embodiment 2 FIG. (1) Preparation of anti-human fibrinogen polyclonal antibody-sensitized gold colloid reagent solution Anti-human fibrinogen polyclonal antibody solution (1 mg
/ ml, manufactured by Wako Pure Chemical Industries, Ltd.) in 32 μl with 0.2 M 3- (N-morpholino) propanesulfonic acid (MOPS) buffer.
Gold colloid sol solution adjusted to 6.6 (Jansen) 1
0 ml was quickly mixed and left at room temperature for 10 minutes. Thereafter, 500 μl of a 1% (w / w) carbowax 20 M solution was added to the reaction solution, and the mixture was uniformly mixed.
After centrifugation for 20 minutes, the resulting precipitate was dispersed in a dispersion (0.2M MO
Contains PS, 0.02% carbowax 20M, 0.1% sodium nitride. The solution adjusted so that OD ₅₄₀ = 10.0 at pH 7.4) was used as an anti-human fibrinogen polyclonal antibody-sensitized gold colloid reagent solution. (2) Measurement of human fibrinogen 50 μg / ml human fibrinogen solution (containing 1.5 (W / W)% of a predetermined polyanion) in each well of a 96-well plate.
of the anti-human fibrinogen polyclonal antibody-sensitized gold colloid reagent solution, and add S
A microplate reader UVmax set to λ ₁ = 540 nm, λ ₂ = 650 nm, automix once, measurement time 10 min, measurement interval 30 sec. By OFTmax-J (Ver.2.01J, manufactured by Wako Pure Chemical Industries, Ltd.) (Molecular Devices Co., Ltd.) to perform two-wavelength negative kinetic measurement. The obtained measurement results were further analyzed by SOFTmax-J to determine the difference (ΔE) between the absorbance two minutes after and 10 minutes after the start of the measurement. Table 2 shows the obtained results. Table 2 shows the results when no polyanion was added and the results when a polyanion salt having a molecular weight of less than about 20,000 was used as comparative examples.

[0031]

[Table 2]

The average molecular weight of each polyanion shown in Table 2 was determined by using high performance liquid chromatography (HPLC). HPLC measurement conditions are as follows. -Equipment: SHIMADSU LC-6A (manufactured by Shimadzu Corporation). -Column: YMC-Pack Diol-300 (manufactured by YMC Corporation). -Eluent: 0.2 M phosphate buffer (pH 6.9).・ Eluent flow rate: 1.0 ml / min. -Column temperature: 35 ° C.・ Detection: Differential refractive index detector (Shodex RIse-61, Showa Denko
Co., Ltd.).・ Molecular weight marker: Shodex Standard P-82 Pullulan
(Manufactured by Showa Denko KK). As is clear from the results in Table 2, when the polyanions of the present invention were used, fibrinogen could be measured with high sensitivity, and even if the polyanions had a molecular weight of about 2
It is understood that the effect of the present invention cannot be obtained when the molecular weight is less than 0,000.

Embodiment 3 FIG. Hemoglobin was subjected to colorimetric analysis using a commercially available gold colloid agglutination reaction reagent (ImmunoGold Hem: sold by Wako Pure Chemical Industries, Ltd.). (Colloidal gold reagent) One bottle of Immunogold Hem gold colloid reagent (for 2.5 ml) was dissolved in 5.0 ml of the gold colloid reagent solution of the same kit to obtain a gold colloid reagent. (Hemoglobin standard solution) Hemoglobin solution (50 μm containing 0.5 μg / ml of hemoglobin and 0.5% of bovine serum albumin)
M Chondroitin sulfate C ・ Na in phosphate buffer (pH 7.4)
A salt (average molecular weight: about 45,000, manufactured by Wako Pure Chemical Industries, Ltd.) added to a predetermined concentration was used as a hemoglobin standard solution. (Procedure) 50 μl of hemoglobin standard solution was added to each well of a 96-well plate, and 100 μl of the above gold colloid reagent solution was further added. SOFTmax-J (Ver.2.01J, Wako Pure Chemical Industries, Ltd.)
Λ ₁ = 540 nm, λ ₂ = 650 nm, one automix, measurement time 20 min, measurement interval 20 sec., Using a microplate reader UVmax (manufactured by Molecular Devices) to perform two-wavelength negative kinetic measurement. (Result) FIG. 1 shows a time course showing the relationship between the obtained absorbance and the measurement time. In FIG. 1,-□-indicates that chondroitin sulfate C.Na salt was not added,-○-indicates that chondroitin sulfate C.Na salt concentration was 0.36% during reaction, and-△-indicates that reaction was not performed. When the chondroitin sulfate C · Na salt concentration at the time is 1.07%,-×-is when the chondroitin sulfate C · Na salt concentration at the time of the reaction is 1.43%, and-+-is chondroitin sulfate C · Na salt at the time of the reaction. The time course when the concentration is 1.78% is shown respectively. As is clear from the results in FIG. 1, when the chondroitin sulfate C.Na salt concentration is low, the effect of promoting the antigen-antibody reaction is hardly observed.

[0034]

As described above, the present invention requires the skill of the visual latex agglutination and the introduction of a special-purpose machine to measure automation, which the conventional latex agglutination method has. The present invention provides an immunoassay method using colloidal gold particles that is quick, highly sensitive, and has a wide measurement range that is easy to determine visually, can be applied to conventional automatic analyzers, and solves all the problems such as becoming. is there. According to the present invention, the action of a polyanion or a salt thereof promotes an antigen-antibody reaction, thereby shortening the reaction time and improving the sensitivity.In particular, a sample near the detection limit concentration is visually observed. In this case, the difference between the negative and the positive becomes clearer, so that the invention has a remarkable effect in that the determination error can be reduced, and is a great invention that contributes to the industry.

[Brief description of the drawings]

FIG. 1 shows a time course representing the relationship between the absorbance obtained in Example 3 and the measurement time.

[Explanation of symbols]

In FIG. 1,-□-indicates chondroitin sulfate C / Na salt was not added, and-○-indicates chondroitin sulfate C during the reaction.
-When the Na salt concentration is 0.36%,-△-is when the chondroitin sulfate C-Na salt concentration during the reaction is 1.07%, -x- is when the chondroitin sulfate C-Na salt concentration during the reaction is 1.43% of the case,
-+-Indicates that the chondroitin sulfate C · Na salt concentration during the reaction was 1.7
The time course in the case of 8% is shown respectively.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/543 541 G01N 33/531 G01N 33/553

Claims (7)

(57) [Claims] A colloidal gold particle (hereinafter abbreviated as “sensitized colloidal gold particle”) to which an antibody (or an antigen) for a substance to be measured is bound and a substance to be measured have an average molecular weight of about 20,0.
The reaction is carried out in a solution containing 0.4 to 2.5 (W / W)% of a polyanion or a salt thereof, and the substance to be measured is quantified or semi-quantified based on the resulting change in absorbance of the gold colloid particles. Immunoassay. 2. The polyanion or a salt thereof has an average molecular weight of:
2. The method of claim 1, wherein the amount is about 20,000 to 1,000,000. 3. The method according to claim 1, wherein the polyanion or a salt thereof is chondroitin sulfate or a salt thereof. 4. The absorbance change is determined by measuring 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 a lapse of a predetermined time after the reaction of the sensitized gold colloid particles with the substance to be measured. The analysis method according to any one of claims 1 to 3, which is a difference from: 5. The analytical method according to claim 1, wherein the change in absorbance is a change in absorbance of the reaction solution after the start of the reaction between the sensitized gold colloid particles and the substance to be measured. 6. The analysis method according to claim 1, wherein the change in absorbance is the absorbance of the reaction solution after a lapse of a predetermined time from the start of the reaction between the sensitized gold colloid particles and the substance to be measured. 7. The method according to claim 1, wherein the change in absorbance is a difference between two measurements of the absorbance of the reaction solution at an appropriate interval after the reaction between the sensitized gold colloid particles and the substance to be measured. Analysis method described in 1.