JPH0233097B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring antigen-antibody reactions. More specifically, the present invention involves supporting an antibody or antigen on an insoluble carrier having a fine particle size, reacting the antigen and/or antibody, or a mixture thereof, and injecting the reaction mixture with a wavelength in the near-infrared region. The present invention relates to a method for quantitatively measuring an antigen-antibody reaction by irradiating light. In recent years, not only the medical field but also various fields such as biochemistry, hygiene, and epidemiology, the detection of trace substances, especially antigens and antibodies, not only qualitatively but also quantitatively has become an extremely important issue. . The present invention aims to solve this problem. Conventionally, there is a known method for semi-quantitatively observing antibodies or antigens by reacting latex carrying antibodies or antigens with antigens or antibodies on a glass plate and observing the state of agglutination with the naked eye. . In addition, in recent years, as a method for quantitatively measuring antigens and antibodies using the above-mentioned latex, a phenomenon has been developed in which latex particles are made to support antibodies or antigens, and when the antigens or antibodies are reacted with the latex particles, the latex particles aggregate. The following literature proposes a method for quantitatively measuring antigens or antibodies by measuring the rate of decrease in turbidity of the supernatant of such latex using visible light. (A) CROATICA CHEMICA ACTA42 (1970)
pp457−466 (B) European Journal of Biochemistry
Vol. 20, No. 4 (1971), pp553-560 The method proposed above attempts to quantify antigens or antibodies by measuring the rate of decrease in turbidity. It is necessary to use a very dilute latex, for example 0.007 to 0.028%, carrying a latex, to react with the antigen or antibody in a static state, and to remove impurities that affect turbidity from the specimen. etc. are necessary. However, as a result, in the above method, the antigen-antibody reaction rate inevitably decreases, the accuracy and reproducibility of the method for quantifying antibodies or antigens is poor, and the removal of the impurities sometimes requires extremely complicated operations. Therefore, the above method is particularly useful for blood or urine containing various substances, such as fibrinogen (Fg) and placental gonadotropin hormone (hCG), which require complex reagent preparation and which adversely affect the reaction. It is difficult to utilize the quantification of antigens that are unlikely to cause reproducible agglutination reactions with such substances. In addition, the following document, (C) Immunochemistry, Vol.12, pp349−351
(1975) irradiated the agglomerated latex particles with a laser beam, and calculated the average diffusion constant (), which is an index of the Brownian motion of the particles, which decreases in proportion to the size of the agglomerated particles, by calculating the scattered light of the laser beam. A method has been proposed for quantitatively measuring antibodies or antigens by determining from changes in the spectral width of . However, even in this method, the latex carrying the antibody or antigen is used at an extremely dilute concentration such as 0.001%, so the antigen-antibody reaction rate is low, and therefore the accuracy and reproducibility are only poor. However, there are disadvantages in that complicated calculations must be performed using spectral analysis, which requires complicated operations, and impurities in the specimen must be removed in advance. Therefore, this method has not yet been put into practical use. In addition, this document (C) states that the quantitative method using the turbidity method reported in the document (A) is extremely inaccurate (p. 350 of the same document).
Fig.2). Therefore, the present inventors invented a method and apparatus for rapidly quantifying antibodies and/or antigens contained in a specimen with high precision and good reproducibility, and previously filed Japanese Patent Application No. 51-97158. (hereinafter referred to as the "first application")
proposed in the specification. The invention of the earlier application proposed by the present inventors is to support antibodies or antigens on insoluble carrier particles with an average particle size of 1.6 microns or less, and to support the supported antibodies and/or antigens.
Alternatively, the antigen is reacted with the antigen or antibody or a mixture thereof in a liquid medium, and the reaction mixture is
having a wavelength selected from the range of 0.6 to 2.4 microns and at least 1.5 times the average particle size of the carrier particles;
The method for measuring an antigen-antibody reaction is characterized in that the absorbance of the reaction mixture is measured by irradiating light having a half-width or wavelength width of at least 0.03 microns. While continuing research on the invention of the earlier application, the present inventors discovered that the light irradiated to the reaction mixture was selected from monochromatic light or light or spectroscopy, which has been conventionally used for spectroscopic analysis. Even if the light is not close to monochromatic light, in other words, even if it is polychromatic light with multiple wavelengths, the measurement result of the absorbance of the polychromatic light will be substantially different from that obtained when monochromatic light is irradiated. Therefore, the antigen-antibody reaction can be detected with high precision and reproducibility by using high-intensity irradiation light without requiring an expensive and precise optical device or spectrometer as a light irradiation device. It has been discovered that rapid quantitative determination is possible, and the present invention has been achieved. Thus, according to the present invention, antibodies or antigens are supported on insoluble carrier particles having an average particle size of 1.6 microns or less, and the supported antibodies and/or antigens are
An antigen or an antibody or a mixture thereof is reacted in a liquid medium, and the reaction mixture has a specific wavelength range within the range of 0.6 to 2.4 microns, and when the reaction mixture is irradiated, the absorbance or absorbance increases over time. A method for measuring an antigen-antibody reaction, which comprises irradiating light containing polychromatic light and having a half-width or wavelength width of at least 0.03 microns, and measuring the absorbance or absorbance of the polychromatic light of the reaction mixture. is provided. According to the above-mentioned method of the present invention, extremely small amounts of antibodies and/or antigens can be detected with the same or better accuracy, which was previously possible only through radioimmunoassay (RIA). Quantitative measurements can be made much more quickly and safely. According to the above method of the present invention, not only multivalent antigens but also incomplete antigens such as haptens can be quantified with high precision, and furthermore, not only multivalent antigens but also incomplete antigens such as haptens can be quantified with high precision. , the antibody and/or antigen can be quantified using the inhibition reaction. According to the method of the present invention, furthermore, using a very small amount of the sample, it is rapidly assayed whether the concentration of the antibody or antigen contained in the sample is lower or higher than a certain level. You can also do that. Further features and advantages of the invention will become apparent from the description below. As already mentioned, in the method of the present invention, the average particle size is
The antibody or antigen is supported on insoluble carrier particles of 1.6 microns or less, the supported antibody and/or antigen is reacted with the antigen or antibody, or a mixture thereof in a liquid medium, and the reaction mixture is injected with particles of 0.6 to 2.4 micron. irradiating the reaction mixture with light including polychromatic light having a specific wavelength range within the range, showing an increase in absorbance or absorbance over time when irradiated with the reaction mixture, and having a half-width or wavelength width of at least 0.03 microns; (Hereinafter, this irradiated light will be referred to as irradiated light) is a method for measuring an antigen-antibody reaction, characterized in that the absorbance or absorbance of the polychromatic light of the reaction mixture is measured, and the irradiated light is As a method, the antigen-antibody reaction is quantified by not only using monochromatic light or selected light close to monochromatic light, but also using light containing polychromatic light having multiple wavelengths and measuring the absorbance of the polychromatic light. It has characteristics in points. The degree of agglutination reaction that occurs when latex particles carrying antibodies or antigens (such latex particles are usually also called sensitized carriers or sensitized latexes) is brought into contact with a test substance containing antigens or antibodies can be measured by In the conventional method of measuring the rate of decrease in turbidity of the supernatant of the sensitized latex, for example, the concentration of the sensitized latex is extremely diluted and the reaction is carried out in a static state, resulting in poor accuracy and reproducibility. As already mentioned, there are various drawbacks such as the need to remove impurities in the sample that affect It is more advantageous to carry out the reaction between the sensitizing latex and the antigen or antibody in the subject under non-static conditions, especially under active stirring, and also under conditions of a high concentration of the sensitizing latex. You can also do that. Thus, the present inventors have determined that in order to quantitatively detect the antibody-antigen reaction between the antibody or antigen supported on a fine particulate insoluble carrier and the antigen or antibody in a subject, (1) the average (2) The antigen-antibody reaction mixture has a specific wavelength range within the range of 0.6 to 2.4 microns, and the absorbance or absorbance when the reaction mixture is irradiated is used. (3) measuring the opacity of only the polychromatic portion of the illumination beam from the reaction mixture; It turned out to be extremely effective. This is because the degree or speed of the antigen-antibody reaction in the presence of the fine particulate insoluble carrier corresponds very closely to the opacity of the polychromatic light in the irradiation light beam. The extent or rate of progress of the antigen-antibody reaction will then depend on the amount (or concentration) of antibody and/or antigen contained in the subject, provided that this reaction is carried out under substantially constant conditions. It is clear that this will be supported. Therefore, according to the present invention, by using the above method, it is possible to quickly and accurately measure the concentration contained in a sample using a method that is completely different from the conventional methods of measuring turbidity and average diffusion constant. It becomes possible to quantify the antibodies and/or antigens that are detected. In the method of the present invention, as described above, the irradiation light has a specific wavelength range within the range of 0.6 to 2.4 microns and exhibits an increase in absorbance or absorbance over time when irradiated to the reaction mixture, and Light containing polychromatic light having a half-width or wavelength width of at least 0.03 microns is used. When an insoluble carrier (sensitized carrier) sensitized with an antigen or antibody and having an average particle size of 1.6 microns or less is allowed to react with the antibody or antigen in the test subject in the presence of a liquid medium, the antigen-antibody reaction is reduced. In both pre-stages, especially in the relatively early stage, the agglutination reaction of the sensitized latex progresses as the antigen-antibody reaction progresses, and as the agglutination reaction progresses, 0.6 It has been found that irradiation with polychromatic light of a suitable wavelength range within the range of ~2.4 microns increases the absorbance or extinction ratio of the polychromatic portion of the reaction mixture. Therefore, in the present invention, a wavelength range having a wavelength range of 0.6 to 2.4 microns and at which the absorbance or absorbance of the reaction mixture increases over time as the antigen-antibody reaction progresses is used. No matter what wavelength range the light has, as long as it includes colored light,
Can be used as irradiation light. The light that can be used in the method of the present invention can be determined by conducting simple preliminary experiments once the specific sensitizing latex to be used in the method and the antibody or antigen in the subject to be reacted with it are determined. This can be determined quite easily by a person skilled in the art. As long as the irradiation light used in the method of the present invention includes polychromatic light having the above characteristics, it may also include light components other than the polychromatic light, for example light components with wavelengths outside the range of 0.6 to 2.4 microns. can. What is important is that only "polychromatic light having a specific wavelength range within the range of 0.6 to 2.4 microns and showing an increase in absorbance or absorbance over time when irradiated to the reaction mixture" is used. The objective is to measure the absorbance or absorbance of the polychromatic light. Therefore, when using light consisting essentially only of the polychromatic light as the irradiation light, the absorbance or absorbance of the reaction mixture can be measured as is, while light components other than the polychromatic light can be measured as is. When using light that contains light as a source light for irradiation, (i) the light source light is divided into two parts in advance, and the light consisting essentially only of the selected polychromatic light is used as the irradiation light; irradiate the reaction mixture and measure its absorbance or absorbance; (ii) directly irradiate the reaction mixture with the light from the light source as irradiation light and analyze the transmitted light as irradiation light or spectroscopy; Measure the absorbance or absorbance of transmitted light consisting only of colored light; or (iii) directly irradiate the reaction mixture with the light source light as irradiation light and substantially sense only the polychromatic light in the transmitted light. By using a light sensing device, it is possible to measure the absorbance or absorptance of the light that is transmitted through the reaction mixture and consists essentially of the polychromatic light. In this way, the irradiation light can include light components other than the polychromatic light, and may also include light components with wavelengths outside the range of 0.6 to 2.4 microns, but light with wavelengths outside the range of 0.6 to 2.4 microns Components are components that do not actively contribute to the measurement of absorbance or extinction ratio in the method of the present invention, and in some cases may cause undesirable effects such as chemical changes in the reaction mixture, temperature increases, abnormal luminescence phenomena, etc. Generally speaking, the irradiation light should be
It is desirable that light components with wavelengths outside the range of 0.6 to 2.4 microns, especially visible light and ultraviolet light with wavelengths shorter than blue light, not be included in large amounts;
Advantageously, it is substantially free of light of wavelengths shorter than 0.6 microns, more preferably shorter than 0.8 microns. Furthermore, since light with a wavelength longer than 2.4 microns affects the temperature rise of the reaction mixture, it is generally desirable not to contain much light, and it is advantageous to contain substantially no light. Therefore, the irradiation light that can be particularly suitably used in the present invention is advantageously light mainly composed of polychromatic light having a wavelength range within the range of 0.6 to 2.4 microns, particularly 0.8 to 1.8 microns. The "polychromatic light" used in the present invention is polychromatic light composed of a plurality of substantially monochromatic lights, polychromatic light composed of a continuous spectrum, or a combination of both, and the polychromatic light has a range of 0.6 to 2.4 microns. ,Preferably
In the range of 0.8 to 1.8 microns, more preferably from 0.9 to
It is desirable to have a wavelength range in the range of 1.4 microns, and in terms of half-width or wavelength width, although not critical, generally at least 0.03 microns.
Suitably it has a half-width or wavelength width of microns, more preferably at least 0.05 microns. On the other hand, as described above, the present invention measures the change in the absorbance or absorbance of the polychromatic light due to the agglutination reaction of the sensitized latex, and utilizes the polychromatic light that shows an increase in the absorbance or absorbance over time. Therefore, any polychromatic light that exhibits this tendency can be used, but according to the present invention, such polychromatic light is at least 1.1 times the average particle size of the carrier particles, preferably at least 1.1 times the average particle size of the carrier particles. It has turned out to be particularly advantageous for the light to consist essentially of polychromatic light having a wavelength that is at least 1.5 times as long. Thus, any light source that provides the irradiation light can be used as long as it includes the irradiation light, and examples of the light source include tungsten lamps, xenon lamps, halogen lamps,
Examples include a Nernst grower, a nichrome heating wire, and a light emitting diode (LED). Among these, light from tungsten lamps, halogen lamps, xenon lamps, Nernst growers, etc., which are continuous spectrum light sources spanning visible and infrared light, is passed through a low pass filter.
This is preferable because it is possible to easily obtain irradiation light having a wide wavelength range that does not substantially include light with wavelengths shorter than 0.8 microns, for example, by simply applying a filter.
Furthermore, the above-mentioned light-emitting diode, for example, a Ga-As light-emitting diode, has a maximum wavelength of about 0.95 microns and a half-width of about 50 millimicrons, and can be used as irradiation light without any light or separation. This makes it a particularly advantageous light source. Conventionally, spectral analysis methods have been known that use infrared light with a wavelength of 2.5 microns or more or ultraviolet light with a wavelength of 0.4 microns or less to explore the structure or characteristics of the molecular structure of substances. , the near-infrared light in the range of 0.6 to 2.4 microns used in the present invention or the visible light rays close to this (hereinafter referred to as near-infrared light rays for convenience) has conventionally been considered to have little utility value. This is something that has not received much attention. However, according to the research of the present inventors, the above-mentioned near-infrared light rays can be applied to the basic medium of the subject containing the antigen or antibody, such as water, serum, urine, and saline, which is the object of the present invention. and that it is extremely permeable to aqueous media such as water and aqueous solutions, which are most commonly used as the base medium of the latex;
In particular, near-infrared light from 0.8 to 1.4 microns;
Light in the near-infrared range of 1.53 to 1.88 microns has extremely low absorption by aqueous media and is therefore fundamentally suitable for use in the present invention. Next, the insoluble carrier particles used in the present invention may be of any type as long as they have an average particle size of 1.6 microns or less. Insoluble carrier particles having an average particle size of more than 1.6 microns result in unstable uniformity of latexes containing such particles, making them unsuitable for the quantitative method of the present invention. Such insoluble carrier particles have an average particle size of 0.1 to 1.0 microns, especially 0.2 microns.
A range of 0.8 to 0.8 microns, especially 0.2 to 0.6 microns is preferred. According to the present invention, insoluble carrier particles (sensitized carrier) supporting antibodies or antigens and having an average particle size within the above range are reacted with antigens or antibodies in a subject at least in the presence of a liquid medium, and When the reaction mixture after the initiation of the reaction is irradiated with the light having a suitable wavelength in the range of 0.6 to 2.4 microns, the apparent reaction progress or reaction rate of the antigen-antibody reaction, especially in the early or middle stages of the reaction, can be improved. and the absorbance of the light or the rate of increase in the absorbance of the mixture correspond very well,
Moreover, the apparent progress rate or reaction rate of the reaction corresponds to the concentration of the antibody or antigen in the specimen.
Based on this principle, it becomes possible to quantitatively measure the concentration of antibodies or antigens in a subject. The absorbance (S%) referred to in the present invention is expressed by the following formula (1). S = I ₀ - I / I ₀ × 100 (%) ... (1) In the formula, I ₀ is the reaction mixture when the same system as the reaction mixture but without antigen and/or antibody is placed in the cell. , is the intensity of light transmitted through this, and I is the intensity of light transmitted through the reaction mixture when it is placed in the cell. As is clear from the above definition, the absorbance referred to in the present invention can also be referred to as a light non-transmittance or a light blocking rate. Further, the above absorbance corresponds to the value of absorbance (A) that can be measured using, for example, a spectrophotometer generally used for infrared absorption spectrum analysis, so it can be expressed by such absorbance for convenience. In the analysis method using infrared absorption spectroscopy, the above absorbance (A) is expressed by the following formula (2), A = logI ₀ /I ... (2) where the definitions of I ₀ and I are the same as in the above formula (1). , expressed as . Therefore, in the present invention, it is possible to measure the antigen-antibody reaction using either the absorbance of formula (1) or the absorbance of formula (2). As long as the measurements are carried out properly, the measurements obtained will show reliable agreement. The above absorbance (A) or absorbance (S) in the present invention
In short, it relates to the relative ratio of I ₀ /I, so if the basic medium of the subject _is a transparent liquid medium, the insoluble carrier particles carrying the antibody or antigen are Conveniently, a suspension containing the same may be diluted with, for example, water to a similar concentration. Incidentally, the transmittance (%) of a light beam in the range of 0.6 to 2.4 microns to 1 mm thick water is as shown in FIG. In FIG. 1, the horizontal axis shows the wavelength of the light beam, and the vertical axis shows the transmittance (%) of the light beam.
This results in a range of 0.6 to
Light rays with a wavelength of 1.4 microns are transmitted through water with almost no absorption, and rays with wavelengths in the range of 1.53 to 1.88 microns also pass through water to a considerable extent.
It can be seen that in the present invention, light beams having wavelengths in this range can basically be used. Also the first
According to the figure, for example, it is clear that light with a wavelength in the range of 2.1 to 2.35 microns also transmits about 20% through water, so although light with such a wavelength is not desirable, it is necessary to use a high-sensitivity photometer. It will be understood that it can be used by using Next, FIG. 2 shows the relationship between the absorbance (vertical axis) of polystyrene latex (solid content 1% by weight) and the wavelength of light (micron-horizontal axis) when a cell with a thickness of 2 mm is used. In Figure 2, curve A shows the change in absorbance of polystyrene latex with an average particle size of 0.481 microns (μ), and curve B shows the average particle size
The absorbance change of 0.804μ polystyrene latex is shown. In measuring the absorbance, the latex was diluted and measured, and the obtained absorbance value was multiplied by the dilution factor to obtain the absorbance of the latex. According to FIG. 2, the absorbance of the latex is extremely high for light rays with a wavelength of less than 0.6μ, and therefore the light opacity of the antigen-antibody reaction mixture can be changed using light rays of such a wavelength. It is extremely difficult to measure
In particular, for light rays with a wavelength of 1 μ or more, the absorbance by the latex itself is relatively small, so light rays with a wavelength of 0.8 μ or more, particularly 1 μ or more, are suitable for measuring the opacity. will be understood. Also, when comparing curve A and curve B in Figure 2,
It is observed that as the average particle size of the polystyrene particles in the latex increases, the absorbance of the latex increases accordingly. Therefore, it will be understood that latex particles having too large a particle size are unsuitable for the method of the present invention. As a result, according to the research of the present inventors, the insoluble carrier particles used in the present invention must have an average particle size of 1.6 microns or less, preferably those with an average particle size of 0.1 to 1 micron, especially 0.2 microns. Latex particles of ~0.8 micron have been found to be suitable. Next, in FIG. 5a (Example 1), FIG. 9 (Example 6), FIG. 6 (Example 2), etc., the light containing the polychromatic light is used as the irradiation light, and the object is examined after a certain period of time. This is a plot of the relationship between the antigen or antibody concentration in the liquid and the absorbance or absorbance, or the relationship between the time to reach a constant absorbance or absorbance and the antigen or antibody concentration in the test liquid, but the prior application described above As in the examples described in the specification, the absorbance or absorbance of the reaction mixture shows an extremely good correspondence with the reaction time (the degree of progress of the reaction), and the irradiation light is monochromatic or monochromatic. It is recognized that antigen-antibody reactions can be sufficiently quantified using polychromatic light without necessarily using close light. Thus, according to the present invention, a sensitized latex is prepared by supporting an antigen and/or antibody on insoluble carrier particles (latex) having the particle size as described above, and reacting the antibody and/or antigen in a subject with the sensitized latex. The absorbance of the reaction mixture was adjusted to 0.6 to 2.4μ,
The amount (or concentration) of the antigen and/or antibody in the subject is determined by measuring with light in a specific wavelength range, preferably in the range of 0.8 to 1.8μ, particularly 0.9 to 1.4μ.
It becomes possible to measure. The insoluble carrier particles used in the present invention include fine particles of an organic polymer substance that is substantially insoluble in the liquid medium used in the measurement of the present invention and has the above-mentioned average particle size, such as polystyrene, styrene-butadiene copolymer, etc. Latexes of organic polymers obtained by emulsion polymerization, such as individually dispersed coccus-type bacteria such as Staphylococcus and Streptococcus, M. marcescens or Rickettsia or cell membrane fragments, or inorganic materials such as silica, silica-alumina, and alumina. Oxides, other mineral powders, metals, etc. are used. In the present invention, such insoluble carrier particles (for example, latex particles) are made to carry (sensitize) an antibody or antigen capable of reacting with the antigen and/or antibody in the analyte to be measured. In this case, the antibody or antigen may be physically adsorbed and/or chemically adsorbed onto the carrier. Antibodies are composed of proteins, while antigens are composed of various substances such as proteins, polypeptides, steroids, polysaccharides, lipids, pollen, and dust. Many methods have already been proposed for supporting such antibodies or antigens, particularly antibodies, on insoluble carrier particles. If an insoluble carrier is to carry an incomplete antigen, in particular a hapten, it is advantageous to chemically modify the carrier, for example with a coupling agent, so that the antigen can be chemically adsorbed. In addition, as an insoluble carrier, for example, a latex of a polymer material having a functional group such as a sulfone group, an amino group, a carboxyl group, or a reactive derivative group thereof is used, and the antigen and/or antibody is chemically adsorbed onto the latex. You can also do that. Further, although water is most suitable as a liquid medium that can be used in the present invention, a mixture of water and a water-miscible organic solvent can also be used, and examples of the water-miscible organic solvent include methanol, ethanol, etc. alcohols; ketones such as acetone, etc. are suitable. The method of the present invention is completely different from the conventionally known turbidity measurement method or the measurement method of average diffusion constant using a laser beam. Provide conditions in which they can respond positively. For this reason, in the present invention, the concentration of insoluble carrier particles (for example, latex particles - hereinafter referred to as sensitized carrier particles) sensitized with the antibody or antigen is
Suspensions of 0.05% by weight or more are used, preferably 0.05-1% by weight, particularly preferably 0.2-0.6% by weight. As is clear from FIG. 2, if the concentration of the sensitized carrier particles becomes too large, the light transmittance of the suspension itself decreases, making it difficult to measure the absorbance or absorbance of the present invention. It is preferable that the concentration of the sensitized carrier particles in the suspension is as large as possible within the range where absorbance or absorbance can be measured, and by doing so, the sensitivity of quantitative measurement of antigen-antibody reaction can be increased. . Further, in the present invention, unlike the conventional method described above, it is preferable to react the sensitized carrier particles and the analyte containing the antigen and/or antibody under non-static conditions. For this purpose, it is advantageous to react both under stirring or shaking. Since both of these are generally reacted in a thin cell, it is advantageous to insert, for example, a stirrer that moves vertically or laterally into the cell to stir it. Of course, it is also possible to react the sensitized carrier particles and the above-mentioned analyte for a certain period of time under substantially constant conditions outside the cell, then put the reaction mixture into the cell and measure the absorbance or absorbance. Good, but the reaction conditions for each measurement,
In particular, in order to keep the reaction time constant, a cell is set in a photometric device, and the reaction between the sensitized carrier particles and the analyte is performed in this cell under substantially constant conditions without standing still, and the absorbance is measured. or by measuring the absorbance,
More accurate quantification can be performed. In this way, according to the present invention, the concentration of antigen and/or antibody in a subject can be determined not only in a manner that can be observed semi-quantitatively with the naked eye on a conventional glass plate, but also in a manner that can be observed semi-quantitatively with the naked eye on a conventional glass plate. Even trace amounts of antigens and/or antibodies that could be measured for the first time by (RIA) can be quantified with the same or higher accuracy. According to the present invention, in order to quantify antigens and/or antibodies in a subject (sample) containing unknown amounts of antigens and/or antibodies, standard samples containing a certain amount of specific antigens and/or antibodies are used. Prepare various diluted standard samples by diluting them at various ratios, and according to the present invention, react this with insoluble carrier particles sensitized with a certain amount of a specific antibody or antigen under certain conditions, The absorbance or absorbance of this reaction mixture is measured, and a standard curve (for convenience, Create a standard curve A). Next, using the same sensitized carrier particles used to create this standard curve, the unknown sample is allowed to react with the sensitized carrier under substantially constant conditions as in the case of its creation, and its absorbance or absorbance is By measuring and comparing this absorbance or absorbance with the standard curve A, the amount (or concentration) of the antigen and/or antibody in the unknown sample can be quantified. Alternatively, when creating the standard curve A, for example, a standard that shows the relationship between the reaction time required to reach a certain absorbance or absorbance and the amount (concentration) of the antigen or antibody in the standard sample used can be used. By creating a curve (hereinafter referred to as standard curve B for convenience), the sensitized carrier particles and the unknown sample can be reacted under substantially the same conditions as the standard curve to achieve the constant absorbance or absorbance. The amount (or concentration) of the antigen and/or antibody in the unknown sample can also be quantified by reading the reaction time required to reach the target. Thus, according to the invention: (A) measurement of the absorbance or extinction ratio of an unknown sample (in this case using the standard curve A); or (B) measurement of the reaction rate of the unknown sample, i.e. constant absorbance or extinction The amount (or concentration) of antigen and/or antibody in an unknown sample can be determined by either of the following: measuring the reaction time required to reach the desired rate (in this case using the standard curve B). As already mentioned, method (A) above can be used not only when the amount of antigen and/or antibody in an unknown sample is at a high concentration, but also when the concentration is so low that it can be measured for the first time by the RIA method. On the other hand, the reaction rate measurement method described in (B) above is highly accurate when the amount (concentration) of antigen and/or antibody in the specimen (unknown sample) is relatively large. can be quantified, but it has the advantage of being extremely simple to measure. According to the research of the present inventors, the standard curve A
may exhibit a gentler S-shape than a straight line in some cases, but there is no problem with the accuracy of quantification. The reason for the S-shape shown above is that the present inventors believe that when the concentration of the antigen and/or antibody is low, the reaction rate is involved, whereas when the concentration is high, the saturation phenomenon of the reactive active sites is involved. It is presumed that the following are involved. Of course, by appropriately selecting the conditions when creating the standard curve, the S
It is also possible to increase the linear portion of the curve and substantially utilize only this portion to quantify an unknown sample. The method of the present invention further utilizes the fact that the reaction rate is involved when the concentration of the antigen and/or antibody is low to substantially eliminate the antigen, antibody, or both contained in the test liquid. Contained in the liquid by reacting with the antibody and/or antigen supported on the carrier under certain conditions and measuring the increase in absorbance or absorbance of the reaction mixture within a certain period of time after the start of the reaction. It is also possible to quantify the antigen, antibody, or both. Details of this quantitative method can be found in the application (1) filed by the same applicant on the same date as the present application.
It is explained in detail in the specification of , so its explanation will be omitted here. As described above, the present invention is advantageous in bringing the sensitized carrier particles into contact with the analyte (sample) at as high a concentration as possible to cause the reaction. The optical path length is preferably 0.5 to 10 mm, particularly 1 to 5 mm. According to the present invention, when carrying out highly sensitive measurements of trace amounts of antigen or antibody-containing samples, such as those conventionally measured by the RIA method, in particular, (a) using antibodies or antigens with as high an equilibrium constant as possible; b) Especially as latex particles, the average particle size is 0.2.
~0.8μ and with as small a particle size distribution as possible, (c) The wavelength for measuring absorbance or absorbance is 0.8
~1.8μ, (d) the reaction time is relatively long, for example, from several minutes to several tens of minutes, and (e) the sensitized carrier latex is used at a high concentration within the range where the absorbance or absorbance can be measured. It is advantageous to use. Also, reaction rate measurement method (using standard curve B)
When quantifying an unknown sample by (f) use latex particles with a relatively large average particle size, (g) use latex with a large concentration of carrier particles within the range where absorbance can be measured, ( h) It is advantageous to use a relatively short reaction time, for example in the range of 5 seconds to 10 minutes, especially 10 seconds to 3 minutes, because highly accurate quantification can be carried out in a relatively short time. Note that the standard curve B used in this case, for example, has the time to reach a certain absorbance or absorbance on the vertical axis, and the concentration on the horizontal axis, and both the vertical and horizontal axes are expressed on a logarithmic scale. Curve B is a straight line, which is convenient. As described above, the present invention provides a method for measuring antigens and/or antibodies in a sample by using sensitized carrier particles and utilizing an agglutination reaction between the carrier particles and the antigens and/or antibodies in the sample. LA Law) was explained, but
The method of the present invention can also be applied to a sample quantitative method (LI method) that utilizes the above-mentioned agglutination inhibition reaction. By applying the method of the present invention to the LI method described above, incomplete antigens such as haptens can be quantified, for example. In this case, for example, the insoluble carrier particles used in the present invention may be loaded with an antigen, and the sensitized carrier particles may be reacted with a predetermined concentration of antigen (standard antigen solution) to produce a certain amount of antibody. The absorbance or absorbance of the resulting reaction mixture is measured. Thus, a standard curve C is created by varying the concentration of the standard antigen solution. Next, the unknown sample is reacted with the antibody at a certain concentration, and then this reaction mixture is reacted with the sensitized carrier. These reactions are carried out under substantially the same conditions as those in which the standard curve C was prepared.
By measuring the absorbance or absorbance of the reaction mixture of the sensitized carrier particles thus finally obtained and comparing this with the standard curve, the amount (concentration) of the antigen in the unknown sample can be determined. The LI method can be used to quantify the antibody in an unknown sample using the same method as the LI method described above, except that a certain antibody is supported on the insoluble carrier particles. For example, it is also possible to carry foreign antigens and antibodies and quantify the antigens and antibodies in an unknown sample. Thus, according to the present invention, for example: (1) Necessary blood tests of patients or blood donors associated with emergency surgery, such as type substances, Au or HB antigens;
(2) Detection of other contaminants and quantification of fibrin body degradation products (FDP), which have recently been shown to be useful in the prognosis management of renal transplant or renal failure patients; (2) Extremely useful in pregnancy diagnosis and prognosis management of chorioepithelialoma Quantification of placental gonadotropic hormone, which is considered to be important; (3) quantification of urinary estriol glucuronide, which is a metabolite of hCG and other follicular hormones, which are necessary for pregnancy management; and (4) quantification of urinary estriol glucuronide, which is said to be a labor-inducing substance. Quantification of oxytocin in the blood, (5) quantification of adrenal corticosteroids such as corticoids and aldosterone, and adrenocorticotropic hormone (ACTH), (6) quantification of insulin in diabetic patients, follicle-stimulating hormone in the anterior pituitary gland, and lutein formation. Quantification of hormones, follicular hormones, progestin, etc. (7) Furthermore, quantification of gastrin and secretin, which are gastrointestinal hormones, (8) Quantification of other allergic patients, syphilis patients, hemolytic streptococcal infections, rubella, collagen diseases It is possible to perform quantitative measurements of an extremely wide range of antigens and/or antibodies, such as detection and quantification of antibodies in body fluids of patients with autoimmune diseases and other infectious diseases. Furthermore, the method of the present invention can of course also be employed as a method for qualitatively or semi-quantitatively measuring such antigens and/or antibodies. Further, according to the present invention, the method of the present invention described above can be carried out using, for example, the apparatus described below. Devices that can be used in the present invention include: (a) an average particle size for supporting antibodies or antigens;
(b) an optical path length of 0.5 to accommodate a reaction mixture of an insoluble carrier particle of 1.6 microns or less; and (b) an antibody or antigen supported on the insoluble carrier particle and an antigen and/or antibody or a mixture thereof in a liquid medium. (c) having a wavelength range in the range of 0.6 to 2.4 microns;
and (d) a light irradiation device containing polychromatic light having a half-width or wavelength width of at least 0.03 microns; (e) a device responsive to the sensing device to determine the absorbance or absorbance of the polychromatic light of the reaction mixture; It is characterized by The basic structure of the measuring device described above is similar to that of conventionally known structural features, except that it has the structural features described in (a), (b), (c), (d), and (e) above. The structure can be similar to that of the absorbance measuring device. That is, an example of the basic structure of the device of the present invention is:
As shown in FIG. 3, the irradiation device consists of a light source 1 and an optical filter 2; a sample cell 3 for containing a sample for measuring an antigen-antibody reaction and a compensation cell 4 for containing a compensation control sample. ; photocells 5, 6 for sensing the amount of light transmitted through these cells and converting it into an electrical signal; an amplifier 7 for amplifying the electrical signal; and for displaying or recording the amplified electrical signal. It consists of a display or recording device 8. As the light source 1, the one described above can be used, and the light emitted from this light source 1 is filtered by an optical filter 2, if necessary, to a size of 0.6 to 2.4 microns, preferably 0.8 to 1.8 microns, particularly preferably 0.9 microns. The light is dispersed so that cells 3 and 4 are irradiated with polychromatic light having a specific wavelength range within the range of ~1.4 microns. Therefore, the optical filter 2 is selected from those having the ability to effectively disperse polychromatic light in the above wavelength range, and for example, a color filter that blocks light of 800 mm or less can be used as the optical filter. . Furthermore, a particular advantage of the present invention is that a Ga-As light emitting diode can be used as the light source 1, which makes it possible to avoid the use of expensive optical filters and prisms, and to reduce the production and maintenance costs of the device. Not only can it be made much cheaper, but it can also irradiate the cells 3 and 4 with much more powerful light than when the irradiation light is irradiated with light or spectroscopy, so there is no need for amplification using the amplification device 7. Various advantages are achieved, such as the ability to significantly reduce the On the other hand, as already mentioned, in FIG. 3, the optical filter 2 is omitted so that the light emitted from the light source 1 is directly irradiated onto the cells 3 and 4; An optical filter as described above may be inserted between the photocells 5 and 6. The sample cell 3 and the compensation cell 4 can each be made of transparent glass or synthetic resin (for example, acrylic resin), and can generally be box-shaped with a rectangular cross section. The optical path length of these cells is 0.5
~10 mm, preferably 1 to 5 mm, and advantageously the transparent surface has a transmittance of at least 30%, preferably more than 80%, for light in the wavelength range of 0.6 to 2.4 microns. It is. The sample cell 3 contains a reaction mixture in which the antibody or antigen supported on insoluble carrier particles is reacted with the antigen and/or antibody or a mixture thereof in a liquid medium, as described above with respect to the method of the present invention. Ru. On the other hand, the compensation cell 4 accommodates a control sample in which only antibodies or antigens supported by insoluble carrier particles are dispersed in a liquid medium. The light transmitted through these cells 3 and 4 is received by photocells 5 and 6, respectively, and converted into an electrical signal with an intensity corresponding to the amount of received light. The photocells 5 and 6 can be of any type as long as they have the function of converting the amount of light received into an electrical signal with a corresponding intensity, such as lead sulfide photoelectric elements or silicon photodiodes. etc. are advantageously used. The electrical signal thus converted by the photocell can be amplified by an amplifier 7 in the usual manner and recorded by a display or recorder 8. Furthermore, by incorporating a clock mechanism into the display or recorder 8, it is possible to automatically record the absorbance after a predetermined time or to record the time when a certain absorbance is reached. In a preferred embodiment of the apparatus of the present invention, the sample cell 3 can be provided with a stirring mechanism, and the stirring mechanism can be a stirring rod that moves within the cell or a micropropeller that rotates within the cell. can.
By using such a device, the antigen-antibody reaction between the analyte and the sensitized carrier particles can proceed under substantially constant conditions while promoting the reaction between the analyte and the sensitized carrier particles. Moreover, operations such as stopping the reaction immediately when a predetermined reaction time is reached, or accurately reading the reaction time up to a certain absorbance can be carried out very easily. Next, the present invention will be further explained by examples. Example 1 (1) Preparation of anti-hCG sensitized latex reagent 10 ml of anti-human placental gonadotropin (anti-hCG) antibody glycine buffer solution (concentration: 2 mg/ml)
1 ml of polystyrene latex (manufactured by Dow Chemical Company, solid content concentration: 10% by weight) with an average particle size of 0.220μ was added to the mixture, and the mixture was stirred at room temperature for 30 minutes.
Then, after heating to 40°C and stirring for an additional 30 minutes, centrifugation (12000 rpm) is performed for 50 minutes while cooling at 2 to 4°C. The precipitate was decanted, and the separated anti-hCG sensitized (carrier) latex particles were suspended in a bovine serum albumin solution (concentration: 0.2% by weight).
Prepare hCG-sensitized latex reagent. (2) Creation of calibration curve Anti-hCG-latex reagent prepared in (1) above
0.2ml of the standard hCG solution shown in Table-A below (in physiological saline containing 0.2% bovine serum albumin by weight).
hCG solution) in a small test tube, shake for 5 seconds to mix, then use an acrylic resin absorbance measuring cell (light path) equipped with an L-shaped stirring rod (1 mm diameter stainless steel rod) that moves up and down 2 mm long) and stir immediately at a speed of 160 times per minute. A regular tungsten lamp (12V, 8W) is used as the irradiation light source, and this is used in combination with an optical filter (D800 manufactured by Ditric Optics, USA) to irradiate the cell with light that excludes spectral components with a wavelength of 0.8 microns or less. did. The intensity of the irradiated light after it passed through the cell was measured using a silicon photocell (S874-8K manufactured by Hamamatsu Television Co., Ltd.), and the temporal change in absorbance was recorded with a pen recorder. The chart is shown in Figure 4. In the figure, the standard hCG solution is A, B, C, D, E, F in order from the diluted one.
Indicated by From FIG. 4, the absorbance value after 2 minutes was read, and the values are shown in Table A below. Furthermore, the corresponding absorbance value was determined by calculation and is also listed in Table-A. Furthermore, graphs in which these absorbances and absorbances are plotted against the corresponding hCG concentrations are shown in FIGS. 5a and 5b, respectively. Using these as a calibration curve, the amount of hCG in an unknown sample can be quantified as described in (3) below.

[Table] (3) Quantification of hCG in unknown samples Blood or urine is collected from the subject, and in the case of blood, serum is separated from it using a conventional method. Take 0.2 ml of the sample (shown in Table B) and shake it for 5 seconds in a small test tube with 0.2 ml of the anti-hCG latex reagent prepared in (1) above in exactly the same manner as described in (2) above.
Measure the absorbance after 2 minutes in the same manner as described in (2) above. Using the calibration curve obtained in (2) above, hCG
Quantify quantity. The results are shown in Table B below. For comparison, Table B shows the conventional radioimmunoassay (RIA) method (Ratky, SMet al.,
The measurement results obtained by Brit. J. Haematol. 30:145-149, 1975) are also shown.

[Table] Example 2 0.2 ml of anti-hCG-sensitized polystyrene latex (latex concentration: 0.5%) with an average particle size of 0.220μ prepared in exactly the same manner as in Example 1 (1) and the following Table-C Place 0.2 ml of standard hCG solution at the indicated concentration in a small test tube, shake for 5 seconds to mix, and then irradiate with tungsten light excluding spectral components of 0.8 microns or less in the same manner as described in Example 1 (2). The time required for the absorbance to reach 4% (time from the start of stirring) was measured. The results are shown in Table C below.

[Table] Figure 6 shows the above relationship in a graph on a logarithmic scale, where the horizontal axis shows hCG concentration and the vertical axis shows time (minutes). Using this FIG. 6 as a calibration curve, it was possible to quantify hCG in an unknown sample in exactly the same manner as in Example 1(3). Example 3 Antibiotics prepared in exactly the same manner as in Example 1(1) above
A standard hCG solution was prepared in the same manner as in Example 1(2) using the hCG-sensitized latex reagent and using the same equipment as in Example 1(2) except that the optical filter was removed. The absorbance was measured after 3 minutes. The results are shown in Table D below.

[Table] If the above results are plotted on a log-log graph with the concentration of hCG solution on the horizontal axis and the absorbance after 3 minutes on the vertical axis, a clean linear relationship can be obtained as shown in straight line A in Figure 7. show. Using the calibration curve thus obtained, it is possible to quantify hCG in an unknown sample. Example 4 Antibiotics prepared in exactly the same manner as in Example 1(1) above
The same equipment as used in Example 1(2) was used, except that hCG-latex reagent was used and a filter for removing spectral components with a wavelength of 0.8 microns or less was replaced from the front of the cell between the cell and the detector. , and reacted with hCG solutions of various concentrations, and the absorbance was measured 3 minutes after the reaction. The results are shown in Table E below.

[Table] If the above results are plotted on a log-log graph with the concentration of hCG solution on the horizontal axis and the absorbance after 3 minutes on the vertical axis, a clean linear relationship can be obtained as shown in straight line B in Figure 7. show. Using the calibration curve thus obtained, it is possible to quantify hCG in an unknown sample. Example 5 (1) Anti-fibrinogen antibody sensitized latex (anti-fibrinogen antibody)
Preparation of reagent (Fg sensitized latex) Add polystyrene latex (Dow sensitized latex) with an average particle size of 0.109μ to 10 ml of anti-human fibrinogen (Fg) antibody glycine buffer solution (concentration: 2 mg/ml).
Manufactured by Chemical Co., solid content concentration: 10% by weight) 0.5ml
and polystyrene latex with an average particle size of 0.234μ
Add 0.5ml and stir at room temperature for 30 minutes.
Then, after heating to 40°C and stirring for an additional 30 minutes, centrifugation (12,000 rpm) is performed for 50 minutes while cooling at 2 to 4°C. Decant the precipitate and separate the anti-Fg antibody-sensitized (supported) latex particles.
An anti-Fg-sensitized latex reagent having a concentration of 0.50% by weight of the sensitized latex particles is prepared by suspending the particles in a bovine serum albumin solution (concentration: 0.2% by weight). (2) Creating a calibration curve 0.2 ml of the anti-Fg-sensitized latex prepared as above and the standard Fg concentration shown in Table-F below.
The solution was mixed by shaking well for 5 seconds, and immediately irradiated with tungsten light excluding spectral components of 0.8 microns or less while performing vertical stirring using the same device as described in Example 1 (2). The time taken for the absorbance to reach 0.05 was measured. The results are shown in Table F below.

[Table] If the above results are plotted with the Fg concentration on the horizontal axis and the time until the absorbance reaches 0.05 on the vertical axis, a linear relationship will be obtained as shown in FIG. Example 6 Anti-hCG latex (average particle size 0.220μ) reagent prepared in the same manner as in Example 1(1) (latex concentration
0.25%) 0.15ml and the standard concentration shown in Table-G below.
Put 0.15 ml of hCG solution (dissolved in physiological saline containing 0.2% by weight bovine serum albumin) into a small test tube and shake for 5 seconds to mix.
Immediately place the cell in an acrylic resin cell (optical path length 4 mm) equipped with a small stirring rod with a rotation speed of 1200 rpm.
The absorbance is recorded while stirring at a speed of . At this time, a Ga-As light emitting diode (Monsanto ME7124 type, center wavelength: 940 nm, half width: 50 nm) was used as the illumination source to directly illuminate the cell, and a silicon photocell was used to measure the intensity of the light transmitted through the cell. (manufactured by Hamamatsu Television Co., Ltd., model S874-8K) was used. The absorbance measured 4 minutes after the start of stirring was read from the record, converted to absorbance, and plotted against the standard hCG concentration, and a graph is shown in FIG. Using this as a calibration curve, the hCG concentration in the unknown sample could be determined as in Example 1 (3).

[Table] Example 7 Except for using a polystyrene latex mixture with an average particle size of 0.109 micron and 0.220 micron.
In exactly the same manner as in Example 1 (1), anti-hCG-
Sensitized polystyrene latex (latex particle concentration: 0.5% by weight) is prepared. Anti-hCG-sensitized latex reagent thus obtained
0.2ml of standard hCG solution with the concentration shown in Table-H below.
ml was placed in a small test tube, and absorbance was measured by irradiating it with tungsten light excluding spectral components of 0.8 microns or less using the same apparatus as described in Example 1 (2). The results are shown in Table H below.

[Table] Based on the above measurement data, plot the above results on a graph with the concentration of the standard hCG solution on the horizontal axis and the absorbance after 6 minutes on the vertical axis to create a calibration curve as shown in Figure 10. . Example 8 Anti-hCG sensitized latex reagent prepared in Example 1 (1) (however, latex particle concentration: 0.33% by weight)
After mixing 0.1 ml of the standard hCG solution with the concentration shown in Table I below in a small test tube and shaking for 5 seconds, absorbance measurement was carried out using a small stirring bar with a rotary blade of 2.4 mm in diameter. The sample was placed in an acrylic resin cell (optical path length: 4 mm), and while stirring at a speed of 1200 rpm, the center wavelength was 0.95 microns and the wavelength width was
The absorbance is measured after 4 minutes of irradiation with continuous polychromatic light of 0.03 microns. The results are shown in Table I below.

[Table] Based on the above measurement data, plot the above results on a log-log graph with the concentration of the standard hCG solution on the horizontal axis and the absorbance after 4 minutes on the vertical axis, and create a calibration curve as shown in Figure 11. Create.

[Brief explanation of drawings]

Figure 1 shows the absorption spectrum of water measured using an absorbance measurement cell with an optical path length of 1 mm in the irradiation light wavelength range of 0.6 to 2.4 microns, and Figure 2 shows the absorption spectrum of water due to the difference in particle size of polystyrene latex. FIG. 3 is a graph showing the relationship between changes in particle size and changes in absorbance; FIG. 3 is a system diagram showing the basic structure of the device used in the present invention; FIG. 4 is a graph showing the relationship between changes in particle size and changes in absorbance; FIG.
Various concentrations of hCG were measured by using micron anti-hCG latex particles and irradiating the cell with polychromatic light, excluding spectral components with a wavelength of 0.8 or less, using a combination of a tungsten lamp and an optical filter as the irradiation light source.
This is a diagram showing the temporal change in the absorbance of a solution, and Figure 5a is a diagram that uses anti-hCG latex particles with an average particle size of 0.220 microns and combines a tungsten lamp and an optical filter as an irradiation light source to detect spectral components with a wavelength of 0.8 or less. Figure 5b is a calibration curve showing the change in absorbance of hCG solutions with various concentrations after 2 minutes of reaction, measured by irradiating the cell with polychromatic light. This graph shows the change in absorbance after 2 minutes of reaction of hCG solutions of various concentrations measured by using particles and using a combination of a tungsten lamp and an optical filter as the irradiation light source to irradiate the cell with polychromatic light excluding the spectrum with a wavelength of 0.8 microns or less. Figure 6 shows the calibration curve for anti-hCG.
- various concentrations of hCG using sensitizing latex reagents
The cell is irradiated with tungsten light that reacts with the solution and removes spectral components with a wavelength of 0.8 microns or less,
This is a calibration curve showing the time for the absorbance to reach 4%, and the straight line A in Figure 7 is the absorbance of the hCG solution measured by directly irradiating the light from a tungsten lamp as a light source without using an optical filter. Straight line B is a calibration curve for the absorbance of the hCG solution measured with an optical filter placed between the cell and the detector and a tungsten lamp as the light source.
Figure 8 shows that an anti-Fg-sensitized latex reagent is used to react with standard Fg, and the cell is irradiated with tungsten light from which spectral components of 0.8 microns or less have been removed.
This is a calibration curve showing the time for the absorbance to reach 0.05, and Figure 9 shows the results obtained by reacting with standard hCG solutions of various concentrations using an anti-hCG-sensitized latex reagent.
This is a calibration curve showing the absorbance measured using a Ga-As light emitting diode converted to absorbance.
The figure shows a standard using anti-hCG-sensitized latex reagent.
This is a graph showing the standard curve of absorbance measured by reacting with hCG solution and irradiating the cell with tungsten light excluding the 0.8 micron wavelength component.
React with the solution, wavelength 0.95 microns (half width 0.03
This is a calibration curve showing the absorbance after 4 minutes of antigen-antibody reaction in microns). In FIG. 3, 1 is a light source, 2 is a filter, 3 is a sample cell, 4 is a compensation cell, 5 and 6 are photocells, 7 is a width filter, and 8 is a recorder.

Claims (1)

[Claims] 1. Antibodies or antigens are supported on insoluble carrier particles having an average particle size of 1.6 microns or less, and the supported antibodies and/or antigens are reacted with the antigens, antibodies, or a mixture thereof in a liquid medium. , the reaction mixture has a specific wavelength range within the range of 0.6 to 2.4 microns, exhibits an increase in absorbance or absorbance over time when the reaction mixture is irradiated, and has a half-width or wavelength width of at least 0.03 microns. A method for measuring an antigen-antibody reaction, which comprises irradiating the reaction mixture with light containing polychromatic light and measuring the absorbance or absorbance of the polychromatic light of the reaction mixture. 2. The irradiating light consists primarily of polychromatic light having a wavelength within the range of 0.6 to 2.4 microns and exhibiting an increase in absorbance or absorbance over time when irradiated to the reaction mixture. the method of. 3. A method according to claim 1 or 2, wherein the polychromatic light has a wavelength within the range of 0.8 to 1.8 microns. 4. The method according to any one of claims 1 to 3, wherein the irradiation light does not substantially contain light with a wavelength shorter than 0.8 microns. 5. The method according to any one of claims 1 to 3, wherein the polychromatic light is polychromatic light having a half-width or wavelength width of at least 0.05 microns. 6 The reaction mixture is irradiated with light containing polychromatic light that has a specific wavelength range within the range of 0.6 to 2.4 microns and shows an increase in absorbance or absorbance over time when irradiated to the reaction mixture, and 2. The method according to claim 1, wherein the light transmitted through the mixture is spectrally analyzed, and the absorbance or absorbance of only the polychromatic light is measured. 7. The method of claim 1, wherein said polychromatic light consists essentially of polychromatic light having a wavelength at least 1.1 times the average particle size of said carrier particles. 8. The method of claim 1, wherein said polychromatic light consists essentially of polychromatic light having a wavelength at least 1.5 times the average particle size of said carrier particles. 9. Claim 1, which uses insoluble carrier particles with an average particle size in the range of 0.1 to 1.0 microns.
The method described in section. 10. The method of claim 1, wherein insoluble carrier particles having an average particle size in the range of 0.2 to 0.8 microns are used. 11. The method according to claim 1, wherein the carrier particles are a fine powder of an organic polymeric substance or a fine powder of an inorganic substance substantially insoluble in the liquid medium. 12 The organic polymer substance fine powder is a synthetic resin powder,
12. The method according to claim 11, wherein the material is bacteria or cell membrane fragments. 13. The method according to claim 11, wherein the organic polymer substance fine powder is polystyrene latex particles. 14. The method according to claim 11, wherein the inorganic substance fine powder is a metal, inorganic oxide, or mineral fine powder. 15. The method according to claim 11, wherein the inorganic substance fine powder is a fine powder of silica, alumina, or silica-alumina. 16. Claim 1, wherein the reaction between the insoluble carrier particles supporting the antibody or antigen and the antigen or antibody, or a mixture thereof, is carried out under conditions that promote contact between the carrier particles as much as possible. The method described in section. 17. The claimed invention, wherein the reaction between the insoluble carrier particles on which the antibody or antigen is supported and the antigen or antibody or a mixture thereof is carried out under substantially constant conditions that promote contact between the carrier particles. Range 1
The method described in section. 18. The method according to claim 16 or 17, wherein the reaction is carried out while stirring or shaking. 19 The antigen, antibody, or both contained in the test liquid is reacted with the antibody and/or antigen supported on the carrier under substantially constant conditions and for a substantially constant time, and the absorbance of this reaction mixture is determined. The method according to claim 1, wherein the antigen, antibody, or both contained in the test liquid is quantified by measuring absorbance. 20 The antigen, antibody, or both contained in the test liquid is reacted with the antibody and/or antigen supported on the carrier under substantially constant conditions, and the absorbance or absorbance of the reaction mixture is constant. quantifying the antigen, antibody, or both contained in the test liquid by measuring the time taken to reach the value of
A method according to claim 1. 21 The concentration of the carrier particles in the reaction mixture is 0.05
2. A method according to claim 1, wherein the carrier particles are used in a proportion such that the carrier particles are .about.1% by weight. 22 The concentration of the carrier particles in the reaction mixture is 0.1
22. A method as claimed in claim 21, characterized in that the carrier particles are used in a proportion of ~0.6% by weight. 23. The method of claim 1, wherein the liquid medium is water or a mixture of water and a water-miscible organic solvent. 24. Claim 1, which comprises reacting a suspension of carrier particles supporting a specific antibody or antigen with a diluted or concentrated sample solution containing the antigen or antibody. the method of. 25 First, the antigen or antibody is added to a test solution containing the antibody or antigen to be measured and allowed to react, and then a suspension of insoluble carrier particles supporting a specific antigen or antibody is added to this reaction mixture and allowed to react. A method according to claim 1, comprising: 26. The method according to claim 1, wherein the antibody or antigen is supported by the insoluble carrier particles by being physically and/or chemically adsorbed onto the insoluble carrier particles. 27 Claim 1, wherein the antibody or antigen is supported by the insoluble carrier particles by chemically bonding to the carrier particles via a coupling agent.
The method described in section.