JPS622163A - Measurement of bodily liquor component - Google Patents

Abstract

PURPOSE:To enable assay of a bodily liquor component simply and at a high accuracy, by performing a second antigen reaction to make unreacted antibody left in the first antigen-antibody reaction react with a label antigen. CONSTITUTION:An antibody 2 is attached to a carrier 1 to form a regent 6. The reagent 6 thus obtained is added to a sample body liquor to perform a first antigen reaction between the reagent and an antigen with an unknown concentration contained in the sample body liquor. After the reaction, a labelled antigen 3 coupled to a fluorescent pigment 4 is added to a reaction liquid to cause a second antibody reaction with the unreacted antibody. After the reaction, the intensity of fluorescence, forward scattered light and side scattered light in the reaction liquid is measured to determined the integrated value of the intensity of fluorescence from the intensity measured to obtain the amount of reaction with the antigen 3 from the integrated value. Then, an second antigen reaction is caused using the same antigen 3 to determine a reference amount of reaction with which the amount of reaction previously determined is compared to obtain the concentration of the antigen in the sample bodily liquor. The concentration of the antibody can be determined by replacing the antigen with the antibody in the measuring method as mentioned above.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a method for measuring body fluid components using antigen-antibody reactions.

Background of the Invention Nephelometry and counting methods are known as methods for optically measuring body fluid components by attaching antibodies or antigens to particulate carriers and causing antigen-antibody reactions. In the former, a sample containing an antigen (or antibody) is added to a solution containing a carrier to which antibodies (or antigens) are attached (sensitized), and particles are aggregated by immunological agglutination based on a specific antigen-antibody reaction. This is a method of capturing changes in the turbidity of a solution by changes in transmitted and/or scattered light, and as aggregation progresses, the turbidity of the solution decreases. On the other hand, the latter method is a method found in Japanese Patent Applications No. 58-219753 and No. 59-100090 filed by the present applicant, in which the reaction solution subjected to the aggregation reaction in the same manner as above is poured into a flow cell. The number of aggregated particles is distinguished from non-agglomerated particles using a particle detection curve, and the measured value of the sample is obtained from the respective counting ratios. In addition to these methods, the single radial immunodiffusion method (SRID method), which uses immunological precipitation reactions, and the enzyme immunoassay method (E
IA method), RIA method, etc. are conventionally well known.

Problems to be Solved by the Invention In methods that utilize particle aggregation, such as the nephelometric method and counting method, even if the desired antigen-antibody reaction occurs, it cannot be detected unless particle aggregation occurs. As a result, sensitivity is poor and there is a risk that aggregation may occur due to other factors, resulting in measurement errors.

In addition, other measurement methods require a long time for measurement and require special care in processing before and after measurement because radioactive substances, enzymes, etc. are used.

The main object of the present invention is to provide a method for measuring body fluid components that eliminates the above-mentioned problems and can quantify body fluid components in a short time and with high precision.

Another object of the present invention is to provide a method for measuring body fluid components that can simultaneously quantify a plurality of body fluid components.

Means for Solving the Problems The method for measuring body fluid components of the present invention includes the steps of preparing a reagent by attaching an antibody (or antigen) to a carrier, and adding the reagent to a sample body fluid to be contained in the sample body fluid. A step of performing a first antigen-antibody reaction with an antigen (or antibody) at an unknown concentration, and a step of adding a labeled antigen (or labeled antibody) to the reaction solution after the reaction to form an unreacted antibody (or unreacted antigen) and a second antigen. a step of conducting an antibody reaction; a step of determining the amount of the labeled antigen (or labeled antibody) reacted with the reagent from the reaction solution obtained in the second antigen-antibody reaction; or antibody) alone with the reagent, and quantifying the antigen (or antibody) concentration in the sample body fluid from the amount of change.

Further, another measurement method of the present invention includes the step of creating a reagent in which antibodies (or antigens) for different antigens (or antibodies) are attached to multiple types of carriers having different particle sizes;
A step of adding the reagent to the sample body fluid to perform a first antigen-antibody reaction with each of the plurality of antigens (or antibodies) at unknown concentrations contained in the sample body fluid, and adding the plurality of antigens to one reaction solution after the reaction. a step of adding labeled antigens (or labeled antibodies) corresponding to each to perform a second antigen-antibody reaction with the unreacted antibodies (or unreacted antigens); (or labeled antibody) with the reagent, and compare the obtained reaction amount with the standard anti-9 amount obtained by reacting only the PJ recognition antigen (or labeled antibody) with the reagent. and quantifying the antigen (or antibody) concentration in the sample body fluid from the amount of change.

The present invention will be described in detail below, but for convenience of explanation, it will be assumed that an antibody is attached to the carrier and the amount of antigen in a sample body fluid is measured. On the contrary, the same method can be applied to the case where an antigen is attached to a carrier and the amount of antibody in a sample body fluid is measured.

The carrier may be a polymer latex having a uniform particle size, which is suspended in a stable dispersion medium to prevent mutual aggregation. When the particle size of the latex is non-uniform, when calculating the integrated fluorescence intensity from the fluorescence intensity and the scattered light intensity as described later, the forward scattered light intensity, which indicates the particle diameter, varies, making data analysis difficult. This is to analyze item information based on particle size. The polymer latex includes polystyrene, carboxylated polystyrene, carboxylated polystyrene having an amino group,
Polyvinyltoluene, styrene-butadiene copolymer,
Carboxylated styrene-butashev copolymer, styrene-divinylbenzene copolymer, vinyltoluene-tert-butylstyrene copolymer, polyester, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, acrylonitrile-butadiene-styrene copolymer, poly Vinyl acetate acrylate, polyvinylpyrrolidone.

Examples include latex made of synthetic polymer latex particles such as vinyl chloride-acrylate copolymer, and also latexes whose surfaces are treated with nonionic surfactants and the like can also be used. In addition to latex, synthetic polymer substances (such as bentonite and kaolin) are also available.

collodion, etc.) can also be used.

As the antibody attached to the surface of the carrier particles, an antibody that reacts only with a specific antigen is used.

As such antibodies, monoclonal antibodies can be suitably employed, and are obtained by culturing cells obtained by cell fusion of antibody-producing cells and tumor cells. This monoclonal antibody binds only to a single antigenic determinant, so if an antibody has only one determinant on its surface that can bind to it, the antibody that has already bound to one antigen will no longer be able to bind to another antigen. It does not bind to antigens, so when a monoclonal antibody is bound to the particle surface of a carrier, the particles do not aggregate with each other using the antigen as a mediator.

On the other hand, polyclonal antibodies are a group of antibodies that are reactive with many antigenic determinants of an antigen, and when such antibodies are used, there is a possibility that the carrier particles aggregate with each other via the antigen. be.

Such aggregation is undesirable because it causes measurement errors. However, if the antigen itself has only one epitope, it will have the same effect as a monoclonal antibody and particles will not aggregate, so polyclonal antibodies can also be used. Further, as another means for preventing particles from coagulating with each other, there is a method of utilizing the zeta potential of latex or other synthetic polymer substances, for example. That is, when the zeta potential of the particle surface is -4 QmV or more, the repulsive force between the particles becomes strong and a stable dispersion medium is created, whereas when it is less than -40 mV, the repulsive force is weak and the particles spontaneously aggregate. Thereupon, by adjusting the zeta potential of the particles, aggregation of the particles via the antigen is prevented. Even with such a method, the binding between the antibody and free antigen on the particle surface is not adversely affected.

In addition, various methods can be employed as long as they can maintain the particles in a stable state and prevent the particles from aggregating due to antigen-antibody reactions.

After the antibody on the surface of the carrier reacts with the antigen in the sample body fluid, the labeled antigen that binds to the unreacted antibody is, for example, FI.
Examples include antigens to which a fluorescent dye such as TC (fluorescein isothiocyanate) is attached. However, when manipulating the label, care must be taken to avoid loss of antigenicity.

According to the present invention, a first antigen-antibody reaction is performed in which an antigen contained in a sample body fluid is reacted with an antibody attached to a carrier, and then a labeled antigen is added to the unreacted antibody remaining without reacting with the antigen in the sample. Since the second antigen to be reacted causes an antibody reaction,
In this case, the amount of reaction between the labeled antigen and the antibody is measured, and the change It (decreased amount) from the standard reaction amount when only the labeled antigen is reacted is determined, and this corresponds to the amount of antigen contained in the sample body fluid. The antigen concentration can be easily and accurately quantified.

To measure the reaction amount, the integrated fluorescence intensity is determined from the fluorescence intensity based on the labeled antigen to which a fluorescent dye is attached and the scattered light intensity of the particles. This integrated fluorescence intensity corresponds to the amount of labeled antigen that reacted, and from this the amount of antigen in the sample can be determined. The scattered light intensity and fluorescence intensity of the particles can be measured, for example, by flowing the reaction solution through a flow cytometer. As the flow cytometer, for example, the one described in US Pat. No. 4,325,706 can be used.

Next, a more detailed explanation will be given based on FIGS. 1 to 3. FIG. 1 is an explanatory diagram for determining the reference integrated fluorescence intensity by allowing an excess amount of labeled antigen to act on the antibody bound to the surface of the carrier. In the figure, 6 is a reagent according to the present invention, which is a carrier 1 such as latex and antibody 2 bound to the carrier 1.

Antibody 2 reacts only with specific antigens. The labeled antigen 3 used is one to which a fluorescent dye 4 has been bound in advance, and each reacts with each antibody 2 and binds to all antibodies 2.
After the reaction, the reaction solution is poured into a flow cytometer and the fluorescence intensity is measured.
The forward scattered light intensity and the side scattered light intensity are each measured. At this time, since the solution contains an excess of unreacted labeled antigen 3, the reaction solution must be diluted to the extent that the influence of background fluorescence can be ignored. In addition, care must be taken to prevent the carriers 1 from bonding to each other. An integrated value of fluorescence intensity is determined from the measured fluorescence intensity and scattered light intensity. This integrated value is indicated by peak 5 in the three-dimensional graph shown in FIG. 1, and the volume of this peak 5 becomes the integrated fluorescence intensity. This value is approximately constant if the particle count is constant. That is,
The amount of antibody 2 bound to one carrier 1 is constant, and the amount of labeled antigen 3 bound to it is also almost constant, so when a certain number of particles are counted, the total room light 1) (total The amount of labeled antigen (labeled antigen amount) is also approximately constant, and even if there are variations in the amount of labeled antigen 3 bound to each carrier 1, if a sufficiently large number of labeled antigens are counted, the total amount will be constant. Note that the small peak 9 is a peak of free fluorescent dye that does not bind to the antibody 2.

When determining the antigen concentration in a sample body fluid, the reagent 6 is added to the sample body fluid to cause a first antigen-antibody reaction with the antigen 7 contained in the sample body fluid, as shown in FIG. Next, a second antigen-antibody reaction is caused using the same labeled antigen 3 as described above. Therefore, the amount of labeled antigen 3 bound to antibody 2 (reaction amount, peak 8 shown in FIG. 2) decreases by the amount of antigen contained in the sample. Therefore, the calculated integrated fluorescence intensity changes depending on the amount of antigen in the sample, and since the amount of change has a constant relationship with the amount of antigen, the antigen concentration can be quantified from this.

Furthermore, as shown in FIG. 3, when antibodies against different antigens are attached to multiple types of carriers 1a, lb, lc... having different particle sizes, a large number of antigens 8a, 8b contained in one sample body fluid can be attached. , 8c... can be measured simultaneously. Each peak 5a, 5b, 5c shown in the graph of FIG.
...indicates the amount of change in each antigen.

In Figures 1 to 3, the integrated fluorescence intensity is shown in three-dimensional graphs, but in this case, the diameter of the particle is shown by the forward scattered light intensity in the X direction, and the fluorescence intensity in the Z direction is Since the integrated fluorescence intensity can be calculated using the above, the measurement of the side scattered light intensity may be omitted.The side scattered light intensity is related to the properties of the particles, especially the surface condition of the particles.

Furthermore, instead of labeling with a fluorescent dye, an antigen labeled with an enzyme may be used to measure the amount of enzyme after the reaction.

Example The method of the present invention will now be explained in detail by way of example.

Example: Rabbit immunoglobulin G (rabbit TgG) was adsorbed onto polystyrene latex with a particle size of 0.75μ. i.e. 4 per ml of 0.5% latex solution.
A sample was prepared in which 0 μg of rabbit IgG was adsorbed. Next, this was reacted with anti-rabbit IgG in the sample at various concentrations, and then with labeled anti-rabbit IgG labeled with a fluorescent dye (FITC). Here, rabbit IgG serves as an antigen, and anti-rabbit IgG serves as an antibody. After 0 reactions, the fluorescence intensity of the reaction solution was measured using a flow cytometer (AR-IT manufactured by the present applicant) shown in FIG. The average fluorescence intensity with respect to the TgG concentration was determined, and the calibration curve shown in FIG. 5 was created from this. The average fluorescence intensity is the fluorescence intensity per latex, and is calculated by ten counts of cumulative fluorescence intensity.

As shown in FIG. 4, the flow cytometer guides light from an Ar ion laser (50 mW) to a flow cell 10, receives forward scattered light B through a pinhole plate 1) with a photodetector 12, and generates electricity. It is converted into a signal and sent to the amplifier 13. On the other hand, the fluorescence and side scattering light C emitted from the flow cell 10 pass through the first and second color filters 14, 15, the pinhole plate 16, the blue reflection Gichroic Fumiko-17 and the red reflection (580 nm) dichroic mirror. 18 reflects red fluorescence and line fluorescence. Each reflected light passes through the light receiving filter 20.21 and the light receiving portion 22, 2.
3.24 (photomultiplier tube, PMT). The line fluorescence signal (540-580 nm) D is sent to the amplifier 13. For each signal sent to the amplifier 13, the data analysis section 25 measures the fluorescence intensity of each particle.

By creating the calibration curve, the concentration of the sample can be determined by comparing the fluorescence intensity of a sample with an unknown amount of anti-heron IgG against the calibration curve.

Effects of the Invention According to the present invention, aggregation occurs due to antigen-antibody reaction and side-scattered light is generated by the total reflection mirror 19.

This method has the advantage that antigen (or antibody) in a sample can be quantified easily and with high precision without using any equipment.

Furthermore, by using reagents in which different antigens (or antibodies) (or antigens) are attached to multiple types of carriers with different particle sizes, it is possible to simultaneously measure multiple body fluid components.

[Brief explanation of drawings]

1 and 2 are explanatory diagrams showing the method of this invention, FIG. 3 is an explanatory diagram showing another method in this invention, FIG. 4 is an explanatory diagram of a flow cytometer in an example of this invention, and FIG. The figure is a graph showing a calibration curve. DESCRIPTION OF SYMBOLS 1...Carrier, 2...Antibody, 3...Labeled antigen, 6...
...Reagent, 7...Antigen Figure 4

Claims (6)

[Claims] (1) A step of creating a reagent with an antibody (or antigen) attached to a carrier, and adding the reagent to a sample body fluid to perform a first antigen-antibody reaction with an unknown concentration of the antigen (or antibody) contained in the sample body fluid. After the reaction, a step of adding a labeled antigen (or labeled antibody) to the reaction solution and causing a second antigen-antibody reaction with the unreacted antibody (or unreacted antigen); a step of determining the reaction amount of the labeled antigen (or labeled antibody) with the reagent from the obtained reaction solution; and a step of determining the reaction amount of the labeled antigen (or labeled antibody) with the reagent. A method for measuring body fluid components, comprising the step of comparing the amount of antigen (or antibody) in a sample body fluid with the amount of change in the amount of the antigen (or antibody) in the sample body fluid. (2) The labeled antigen (or labeled antibody) specifically reacts with the antibody (or antigen) attached to the carrier (
The method for measuring body fluid components according to claim (1), wherein a fluorescent dye is attached to a fluorescent dye (or an antibody). (3) Claim No. 1, wherein the reaction amount of the labeled antigen (or labeled antibody) is calculated from the cumulative fluorescence intensity determined from the scattered light intensity and fluorescence intensity of particles contained in the reaction solution.
Method for measuring body fluid components described in section. (4) The method for measuring body fluid components according to claim (1), wherein the antibody attached to the carrier is a monoclonal antibody. (5) The method for measuring body fluid components according to claim (1), wherein the carrier is latex particles having a uniform particle size. (6) A step of creating a reagent in which antibodies (or antigens) for different antigens (or antibodies) are attached to multiple types of carriers with different particle sizes, and adding the reagent to the sample body fluid to be contained in the sample body fluid. A step of performing a first antigen-antibody reaction with multiple types of antigens (or antibodies) at unknown concentrations, and after the reaction, adding labeled antigens (or labeled antibodies) corresponding to each of the multiple types of antigens to the reaction solution. A step of conducting a second antigen-antibody reaction with the reacted antibody (or unreacted antigen), and calculating the amount of reaction of each labeled antigen (or labeled antibody) with the reagent from the reaction solution obtained in the second antigen-antibody reaction. The step of determining the antigen (or antibody) in the sample body fluid by comparing the obtained reaction amount with the standard reaction amount obtained by reacting only the labeled antigen (or labeled antibody) with the reagent and determining the amount of change in the reaction amount.
A method for measuring body fluid components, the method comprising: quantifying the concentration.