JPS5896251A - Measuring method for antigen or antibody and reagent for measurement - Google Patents

Abstract

PURPOSE:To improve a method for measuring the absorbance of a reaction solution or the intensity of diffused light therein, by a method wherein nonsoluble carrier particles whose average particle sizes are less than 0.1 micron are made to carry antibodies or antigens and a light is applied thereon. CONSTITUTION:By applying a light of 0.2-0.52 microns on the reaction solution of antibodies or antigens supported by nonsoluble carrier particles of minute particle sizes, and of antigens or antibodies in a substance to be inspected, it is made possible to measure the progress of the reaction in a quantifying manner in high density of carrier particles. This is because water solution wherein said carrier particles are suspended transmits excellently the light within the abovementioned range, and because the degree of the progress of the reaction of the antigens with the antibodies in the abovementioned reaction mixture solution corresponds very well to the absorbance of the abovementioned irradiation light or the intensity of the diffused light thereof. Used as the nonsoluble carrier particles are particles of latex of organic macromolecules or those of inorganic oxide such as silica and alumina.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves reacting particles in which antibodies or antigens are supported on ultrafine insoluble carriers with antigens and/or antibodies, or a mixture thereof, and injecting this reaction mixture with visible light or ultraviolet light. The present invention relates to a method for quantitatively measuring an antigen or antibody by irradiating it with light, and a reagent for the measurement.

Conventionally, there has been known a method for semi-quantitatively observing antigens or antibodies by reacting latex carrying antibodies or antigens with antigens or antibodies on a glass plate and observing the state of aggregation by internal administration.

In addition, in recent years, the agglutination reaction has been measured by the decrease in turbidity of the reaction mixture using the above-mentioned latex agglutination method.
A method for quantitatively measuring antigens or antibodies has been proposed, but since the latex must be extremely diluted, it has poor accuracy and reproducibility as a method for quantifying antigens or antibodies, which poses a major practical limitation. I couldn't escape it. As a further improvement of this method, antibodies or antigens are supported on insoluble carrier particles, and the antigens or antibodies or a mixture thereof are reacted with the insoluble carrier particles. A method has been proposed (Japanese Patent Application Laid-Open No. 53-24015) in which the absorbance is measured by irradiating the light with a light source called

In the measurement method described in the above-mentioned Japanese Patent Application Laid-open Publication No. 2003-12000, near-infrared light must be used to determine the absorption darkness, and a special device for this purpose is required. For this reason, it is not possible to perform the measurement described in the above-mentioned Japanese Patent Application Laid-Open Publication No. 2003-120013 using a normal spectrometer or nephelometer.

Alternatively, latex particles with an average particle size of 0.1 micron or more are sensitized with antibodies or antigens, the sensitized latex is mixed with a test substance containing the antigen or antibodies, and the agglutination of the latex is measured by turbidimetry. In the conventional Tsuki method, because the light transmittance of the latex suspension in the visible light and/or ultraviolet light region is extremely poor, measurement cannot be performed unless the latex concentration is extremely low, and coagulation and sedimentation may occur. As a result, reproducibility and accuracy were poor, which caused practical difficulties.

Therefore, an insoluble carrier such as latex particles carrying an antibody or antigen is reacted with the analyte at a high concentration as possible, and the progress of the reaction can be monitored using an ordinary spectrometer or comparable needle without using light in a special wavelength range. It has been desired to measure using light in the visible light range.

The object of the present invention is to eliminate the need for the above-mentioned special equipment or light source of a special wavelength, and to remove antibodies or antigens in a subject from a
-1 We want to provide a method for rapid measurement with good reproducibility.

The present invention also makes it possible to measure antibodies or antigens in extremely small amounts using conventional spectrometers, nephelometers, etc., which was previously only practically possible using radioimmunoassay (RIM). Alternatively, the present invention provides a method for quickly, safely, and simply quantifying the amount with higher accuracy.

The present invention provides a quantitative method that can also measure incomplete antigens such as potassium, hormones, and drugs.

The present invention further provides a method for quantifying antibodies or antigens using not only antibody and/or antigen agglutination reactions but also their inhibition reactions.

The inventors of the present invention conducted intensive studies to find a measurement method that improves the drawbacks of the conventional method, and surprisingly found that 0.
A reaction solution obtained by reacting a highly concentrated solution of particles in which an antibody or antigen is supported on an insoluble carrier having an ultrafine particle size of less than 1 μm with a solution containing the antigen and/or antibody or a mixture thereof is produced by the reaction with normal visible light. Not only can it be analyzed by
The present invention was completed by discovering that the method has high reproducibility.

The reason why the reproducibility of analysis is poor when using ultrafine particles is that sedimentation is less likely to occur even when the reaction progresses and one particle becomes larger, resulting in improved reproducibility. it is conceivable that.

That is, the present invention allows insoluble carrier particles having an average particle size of less than 0.1 micron to carry an antibody or antigen, and reacts the carried antibody or antigen with the antigen or antibody or a mixture thereof in a liquid medium. , this reaction solution KO,2
A II method for determining antigens or antibodies characterized by irradiating light with a wavelength of ~0.52 microns and measuring the absorbance or scattered light intensity of the reaction solution, and having an average particle size of less than 0.1 micron. The object of the present invention is to provide a reagent for measuring an antigen or antibody, which is made of particles in which the antibody or antigen is supported on an insoluble carrier particle.

According to IjIIk of the present invention, a reaction solution of an antibody or antigen supported on ultrafine insoluble carrier particles with an average particle size of less than 0.1 micron and an antigen or antibody in a subject is
0.52? By irradiating with Chron light, it is possible to quantitatively measure the progress of the reaction at a high carrier particle am. This is because an aqueous solution in which the carrier particles are suspended transmits light in the above-mentioned range well and responds extremely well to the above-mentioned range of light.

The insoluble carrier particles used in the present invention are substantially insoluble in the liquid medium used when carrying out the measurements of the present invention, and have an average particle size of less than 0.1 micron, more preferably 0.1 micron.
．． Fine particles of less than 1 micron to 0.06 micron, for example, polystyrene, organic polymer latex such as methyl methacrylate-methacrylic acid or styrene-methacrylic acid copolymer, or inorganic oxide particles such as silica or alumina are used. Among them, fine particles of organic polymer substances bind antibodies and/or antigens through chemical bonds.

It is highly preferable because it can be supported firmly and in large amounts.

When using the above-mentioned insoluble carrier particles in the measurement of the present invention, it is desirable to use them in a monodisperse state with uniform particle diameters as much as possible.

In the present invention, when insoluble carrier particles (e.g. latex particles) are loaded with antibodies or antigens that can react with antigens and/or antibodies in the specimen to be determined, the antibodies or antigens are physically attached to the carrier. It may be formed into several layers, or it may be chemically bonded to an organic polymer carrier having a functional group using a coupling agent, but chemical bonding is more preferable from the viewpoint of stability.

In the present invention, by using insoluble carrier particles having an extremely fine particle size, the light transmittance of the suspension of the particles to light having a wavelength of 0.2 to 0.52 microns can be extremely improved. , using light having a wavelength of 0.2 to 0.52 μm, preferably 0.35 to 0.52 μm, and measure the concentration of axillary particles at a high concentration of 0.1 wt. It became possible.

However, if the 11 degree angle of the wrinkled carrier particles becomes too large, an agglutination reaction (non-41IJ) due to causes other than antigen-antibody reactions may occur.
(lI-like agglutination reaction) can no longer be ignored, leading to deterioration of measurement sensitivity. In order to quantify with high sensitivity, accuracy, and reproducibility in a short reaction time, it is preferable to use a suspension of 0.1 to 2 weight - more preferably 0.5 to 2 weight -1.

In addition, in the present invention, it is possible to allow the reaction to proceed under static conditions after mixing the sensitized carrier particles and the analyte. For good results, mix the reactants under stirring or shaking! It is more preferable to widen the i. For stirring or shaking the reaction mixture, it is possible to use, for example, customary magnetic turbulence stirrers.

The absorbance or scattered light intensity may be fixed by allowing the reaction between the sensitized carrier particles and the analyte to occur outside the cell under substantially fixed conditions for a fixed period of time, and then placing the reaction mixture into the cell. , place a cell in a spectroscopic needle or nephelometer, allow the reaction to proceed in the cell under substantially constant conditions, and measure the absorbance or scattered light intensity after a certain time of reaction; It is also possible to allow the reaction to proceed under certain conditions and measure the absorbance or scattered light intensity at fixed time intervals or continuously to determine the rate of change in absorbance or scattered light intensity over time.In particular, the rate of change in absorbance or scattered light intensity over time can be determined. By keeping the rate of change constant, highly sensitive and highly accurate quantification can be performed in a shorter time.

In order to quantify the antigen or antibody contained in a subject (sample) according to the present invention, K uses a standard sample containing a certain amount of a certain antigen or antibody, and various diluted standard samples obtained by diluting this at various magnifications. According to the present invention, this is reacted with a certain amount of insoluble carrier particles sensitized with a certain antibody or antigen under certain conditions, and the absorbance or scattered light intensity of this reaction mixture is measured to determine the wrinkle antigen or antibody. Create a standard curve showing the relationship between amount (concentration) and absorbance or scattered light intensity. Next, use the same sensitized carrier particles that were used to create this standard III. The amount of antigen or antibody in the unknown sample can be determined by reacting the unknown sample with the sensitized carrier under substantially the same conditions as above, and comparing and checking the absorption and scattered light intensity with the standard curve. (Isodo)
can be quantified.

Alternatively, when creating the standard curve, a standard curve is created that shows the relationship between the rate of change in absorbance or scattered light intensity over time and the amount (concentration) of antigen or carrier in the standard sample used. By doing so, the amount (concentration) of antigen or antibody in an unknown sample can be quantified in the same manner as in the above method.

Example 5t 40 equipped with stirring blade, cooling pipe, and nitrogen air flow pipe
Add 21 water and 5J9 methylcellulose to a flask, dissolve the methylcellulose at 60°C, and then cool to room temperature.

While stirring, add 200 # of acrylic acid, 800 g of methyl methacrylate, 200 g of ethylene glycol diacrylate, 4II of n-butyl mercaptan, and 10011 of methacrylic acid in this order.

After replacing the upper space of the reaction solution with nitrogen, it is heated to 80° C. under a nitrogen stream. A solution of 5 g of azobisisobutyronitrile dissolved in 100 cc of methanol was slowly added so that the polymerization reaction temperature did not exceed 95° C., and the polymerization was continued for 5 hours. After the reaction is completed, cool
The copolymer particles are washed with water for -1 min and dried at 60°C overnight.

Put the dry powder 2001 into a 2t flask equipped with a stirrer, add 2501 water, 50 JI diethylene glycol heptanoethyl ether, and 51 concentrated ammonia water (289b).
was added, the flask was heated to 70°C, and stirred vigorously for 6 hours. Cooling and filtration gave a bluish clear dispersion. As a result of examination using an electronic ponghui mirror, the particle size of the particles in the dispersion was 0.02 to 0.08 microns, and the average particle size was 0.06? It was Kron.

Anti-human IgG was bound to these latex particles using the carbodiimide method, separated from unreacted 1'g G, and then suspended in a bovine blood alzomine (0.1% by weight) bath solution. A gG-immobilized latex reagent was prepared.

The anti-human EGG-immobilized Utex obtained in this way
．． Diluted standard IgG bath solutions of various concentrations were prepared by diluting 2117tl-KujunganggG solution (0.1q/d).
8i1i1 and reacted for 5 minutes at room temperature while stirring with a magnetic turbulence stirrer. After that, the absorbance at a wavelength of 0.4 microns was measured using a spectrometer, and the IgG
Taking the fII liquid once on the horizontal axis, @, length 0.4? The absorbance of the test liquid (normal human blood diluted 1000 times was used) was measured using the same method as described above. - The engineering gG@ degree was calculated from the line. The results are shown in Table 1.

For comparison, results obtained using the conventional radioisotope & 61J method (RIM method) are also shown.

Table 1 Patent applicant: Asahi Medical Co., Ltd.

Claims (1)

[Claims] t An antibody or an antigen is supported on insoluble carrier particles with an average particle size of less than 0.1 micron, and the carried antibody or antigen is reacted with the antigen, the antibody, or a mixture thereof in a liquid medium. Antigen or antibody measurement method 2, characterized in that the reaction solution is irradiated with light in the wavelength range of 0.2 to 0.52 microns and the absorbance or scattered light intensity of the reaction solution is measured. A reagent for measuring antigens or antibodies consisting of particles in which antibodies or antigens are supported on insoluble carrier particles with a particle size of less than 0.1 micron.