JP2010256010A - Method of fluorometrically detecting antigen-antibody reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and rapidly detecting the antigen-antibody reaction of a number of items or a number of specimens. <P>SOLUTION: Various antigens or antibodies labeled by mutually different fluorescent coloring matters are mixed with a sample solution to be inspected and the fluorescent intensity of at least one fluorescent coloring matter in the sample solution to be inspected is measured to judge whether the antigen or antibody labeled by the fluorescent coloring matter corresponding to the fluorescent intensity is bonded to the molecule in the sample solution to be inspected on the basis of a timewise change in the fluorescent intensity. When the antigen or antibody labeled by the fluorescent coloring matter corresponding to the fluorescent intensity is determined to be bonded to the molecule in the sample solution to be inspected, the antigen or antibody labeled by the fluorescent coloring matter corresponding to the fluorescent intensity is determined to be bonded to the molecule in the sample solution to be inspected to produce antigen-antibody reaction. Further, the microplate fitted to this method is provided. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for detecting an antigen-antibody reaction, and more particularly to a method for detecting an antigen-antibody reaction by a single-molecule fluorescence analysis technique and a microplate that can be advantageously used in the method.

  As is well known in the field of biological science, medicine or pharmacy, antigen-antibody reaction can detect various biomolecules inside and outside the living body. Applied to screening of substances. For example, antibodies that react specifically with causative substances of various diseases such as bacteria, viruses, pathogenic proteins, tumors, etc., or substances derived from such causative substances, are used (in body fluids obtained from living bodies). By detecting whether or not an antigen-antibody reaction has occurred, the presence or absence of a causative substance in the living body or a substance derived therefrom is examined. Conversely, whether or not an antibody against the causative agent of the disease as described above or a substance derived therefrom is present in the living body is determined by an antigen (or epitope) against the antibody (in a body fluid obtained from the living body). ) In some cases, it is examined whether or not a causative substance of a disease exists in a living body by detecting whether or not an antigen-antibody reaction has occurred. Furthermore, in the case of some diseases such as allergies and autoimmune diseases, whether or not there is an antibody with a specific substance (food, pollen, house dust, mold, mite, etc.) as an antigen in the living body, The specific substance is examined by detecting whether or not an antigen-antibody reaction has occurred (in a body fluid obtained from a living body).

  As a method for detecting an antigen-antibody reaction, a biochemical method such as ELISA or Western blotting is generally used. However, these traditional methods require many processing operations for detection, take time, A large amount of sample is required. Therefore, in recent years, development of a method or apparatus that enables detection of an antigen-antibody reaction with a smaller amount of sample and in a shorter time has been underway. For example, Patent Document 1 proposes a special measurement chip for detecting an antigen-antibody reaction using surface plasmon resonance. Further, Patent Document 2 discloses a special sample for detecting an antigen-antibody reaction using an apparatus that combines cell flowmetry and fluorescence measurement.

In recent years, with the advancement of optical measurement technology using an optical system of an optical microscope, it has become possible to detect and measure weak light such as fluorescence of one photon or single molecule of fluorescence. Attempts are being made to detect antigen-antibody reactions using measurement techniques. As such an example, for example, Patent Document 3 shows that an antigen-antibody reaction is detected by fluorescence correlation spectroscopy or fluorescence polarization analysis using an optical system of a laser confocal microscope. According to such a method, the sample required for the measurement may be a very low concentration and a very small amount compared to the past (the amount used in one measurement is about several tens of μl), and the measurement time is greatly shortened. (One measurement is about 10 seconds). Therefore, the method or apparatus for detecting an antigen-antibody reaction using the weak light measurement technique as described above is more rapid than before, even when the number of specimens is large, such as for diagnosing diseases or screening for physiologically active substances. It is expected to be a powerful tool that can perform inspections.
JP 2006-250668 A JP 2005-098877 A JP 2005-337805 A

  By the way, when the detection method of antigen-antibody reaction is applied to the diagnosis of the presence or absence of a disease or its causative substance and the screening of other antibodies or antigens, it is often the case that a plurality of kinds of samples are included in a sample to be examined (test sample) In some cases, confirmation of the presence or absence of an antibody or antigen (a disease causative substance or a substance derived therefrom) is desired (for example, diagnosis of allergy). For example, according to the antigen-antibody reaction detection method using the fluorescence correlation spectroscopy or the fluorescence ellipsometry described in Patent Document 3 above, the confirmation test of individual antibodies or antigens can be performed in a small amount of test sample in a short time. However, even if there are a large number of test samples, especially in the clinical field, and there are a large number of antibodies to be tested, in order to complete all tests, Sample amount and inspection time are required. Therefore, even if it is necessary to confirm the antigen-antibody reaction of such multiple items or multiple samples, it would be advantageous to have a method or apparatus that can quickly perform a test with a small amount of test sample. Moreover, it is desirable that the preparation or processing operation in the examination of the antigen-antibody reaction is as simple as possible.

  Thus, one object of the present invention is to provide a simple and rapid method for detecting multi-item or multi-sample antigen-antibody reactions.

  According to one aspect of the present invention, the method for detecting an antigen-antibody reaction of the present invention comprises a plurality of types of antigens or antibodies labeled with different fluorescent dyes, and any one of the plurality of types of antigens or antibodies. A process of preparing a mixed sample solution by mixing with a test sample solution to be examined whether or not an antibody or antigen is contained, and a process of measuring the fluorescence intensity of at least one fluorescent dye in the mixed sample solution; Determining whether or not an antigen or an antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is bound to a molecule in the test sample solution based on the time change of the measured fluorescence intensity, When it is determined that an antigen or antibody labeled with a fluorescent dye corresponding to the measured fluorescence intensity is bound to a molecule in the test sample solution, it is labeled with a fluorescent dye corresponding to the fluorescence intensity. Antigen or antibody Judging that the antigen-antibody reaction has occurred by binding to molecules in the test sample solution, and determining that the antigen labeled with the fluorescent dye corresponding to the fluorescence intensity or the antibody against the antibody or the antigen is present in the test sample solution It is characterized by. As will be understood by those skilled in the art, “a plurality of types of antigens or antibodies” labeled with a fluorescent dye mixed with a test sample solution are antigens (that is, substances that can become antigens in any living body). For example, the substance to be detected whether it is contained in the test sample solution is an antibody, and “a plurality of types of antigens or antibodies” labeled with a fluorescent dye mixed with the test sample solution are antibodies. If present, the substance to be detected whether it is contained in the test sample solution is an antigen.

  According to the above configuration, the antigen-antibody reaction is detected by measuring the fluorescence intensity of the fluorescent dye, and based on the change in the fluorescence time, the antigen or antibody labeled with the fluorescent dye corresponding to the fluorescence intensity is detected. Since it is performed by fluorescence analysis to determine whether or not it is bound to a molecule in the solution, not only the amount of the sample to be tested and the processing operation time are greatly reduced, but also the sample to be tested is compared with the traditional method such as ELISA. Even if there are multiple items to be examined or substances to be confirmed by the method of mixing multiple types of antigens or antibodies labeled with different fluorescent dyes into the test sample solution, the amount of test sample is Only the sample amount necessary for one inspection is sufficient. Antigens or antibodies to be mixed into the test sample solution, that is, “probe” samples for searching for substances in the test sample solution are labeled with different fluorescent dyes. It is necessary to measure the fluorescence intensity of each fluorescent dye separately (selection of excitation wavelength or light reception wavelength in the fluorescence detector according to the absorption wavelength or emission wavelength characteristics of the dye to be detected) It is not necessary to replace the test sample solution itself. In the case where a plurality of types of fluorescent dyes can be excited with one wavelength of excitation light, an apparatus (filter, diffraction grating, etc.) that appropriately discriminates wavelengths in the photodetector of the fluorescence measurement apparatus may be used.

  Further, in the above configuration, the fluorescence intensity is measured for all of the fluorescent dyes labeled with a plurality of types of antigens or antibodies, and the fluorescence intensity is measured based on each time change of the measured fluorescence intensity. It is determined whether the antigen or antibody labeled with the corresponding fluorescent dye is bound to a molecule in the test sample solution, and the antigen or antibody labeled with the fluorescent dye corresponding to each of the fluorescence intensities is detected. When it is determined that it binds to a molecule in the test sample solution, the antigen or antibody labeled with a fluorescent dye corresponding to each of the fluorescence intensities binds to the molecule in the test sample solution and causes an antigen-antibody reaction. You may come to judge that. However, if the results to be detected are found during the sequential measurement and analysis of the fluorescence intensity of each of the fluorescent dyes mixed in the test sample solution, it is not necessary to perform the fluorescence measurement of all the dyes. It should be understood that it is good. In addition, in the fluorescence measurement device to be used, when excitation and detection of fluorescence of a plurality of types of fluorescent dyes having different absorption / emission wavelength characteristics can be performed simultaneously, fluorescence measurement of the fluorescent dye may be performed simultaneously. Furthermore, the measurement time is shortened.

  In the above-described method of the present invention, the fluorescence intensity is measured by measuring fluorescence from a single fluorescent molecule, analyzing the temporal change or fluctuation thereof, and observing the state or movement of the single fluorescent molecule ( Single molecule fluorescence analysis technique). For example, the fluorescence intensity may be measured by “fluorescence correlation spectroscopy”. In this case, an antigen or antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is bound to a molecule in the test sample solution. Whether or not there is may be determined based on a translational diffusion time calculated by fluorescence correlation spectroscopy based on a temporal change in fluorescence intensity. Further, the fluorescence intensity may be measured by “fluorescence intensity distribution analysis method”. In this case, an antigen or an antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is bound to a molecule in the test sample solution. May be determined based on the fluorescence intensity per fluorescent light emitter calculated by the fluorescence intensity distribution analysis method based on the temporal change of the fluorescence intensity (in this case, as described later, a plurality of types of Fluorescently-labeled antigen or antibody must be bound to one molecule in the sample solution at the same time. "One fluorescent emitter" is not a single fluorescent molecule, but moves together in solution. Fluorescence intensity may be measured by “fluorescence depolarization”, in which case an antigen or antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is detected. Bound to molecules in the sample solution Whether or not may be determined by the degree of fluorescence polarization calculated by the fluorescence depolarization method based on the change in fluorescence intensity over time (in this case, a normal fluorescence depolarization measurement device, not a device that performs single-molecule fluorescence analysis) May be.)

  The present invention described above is advantageously used when there are a plurality of antigen-antibody reactions to be examined, such as diagnosis of the presence or absence of a disease or its causative substance, and other antibody screening. Therefore, for example, when the antigen-antibody detection method of the present invention is used for diagnosis of allergic diseases caused by food, the probe sample used in the present invention (a plurality of types of antigens labeled with different fluorescent dyes) is used. Alternatively, the antibody may be a protein contained in food that becomes an antigen when an antigen-antibody reaction occurs. Further, the probe sample may be an antigen against an antibody contained in the body fluid of an allergic disease patient. In these cases, according to the present invention, it is possible to quickly determine which one of a plurality of foods or proteins contained therein or other allergens causes an allergic reaction, Inspection and diagnosis are possible. Further, the probe sample may be an antigen against an antibody contained in the body fluid of a patient with an autoimmune disease, whereby the type of autoimmune disease can be quickly identified. When the present invention is used for the examination / diagnosis of diseases as described above, a body fluid sample obtained from serum or plasma or other living body may be used as the test sample solution. It should be understood that, in this case, according to the characteristics of the present invention, the body fluid sample to be a test sample solution necessary for examining the presence or absence of antigens or antibodies related to a plurality of diseases may be a trace amount. It is. If there is a possibility that the test sample solution has autofluorescence such as serum, use a fluorescent dye that can be excited at a wavelength as long as possible and at least 540 nm or less so that autofluorescence does not enter the measurement. Is preferred.

  In carrying out the above method, the fluorescence measurement is a microplate having a plurality of wells suitable for detecting an antigen-antibody reaction by fluorometric analysis, and each of the plurality of wells is previously provided with at least one fluorescent label. It can be carried out advantageously by means of a microplate characterized in that the antigens or antibodies that have been dispensed are dispensed. In such a microplate embodiment, a plurality of types of antigens or antibodies previously labeled with different fluorescent dyes may be dispensed into the same well. That is, a probe sample composed of a plurality of types of antigens or antibodies labeled with different fluorescent dyes is dispensed into each of a plurality of wells. In this case, by dispensing a plurality of test sample solutions into separate wells, a plurality of types of antigen-antibody reactions can be detected systematically for a large number of test sample solutions. In another embodiment of the microplate, a plurality of types of antigens or antibodies labeled with different fluorescent dyes may be dispensed in a plurality of wells. In this case, multiple types of antigen-antibody reactions are confirmed for one test sample solution by dispensing one test sample solution into wells in which different antigens or antibodies are dispensed. In any case, since a large number of tests are performed on one plate, the operation of changing the test sample solution in the fluorescence measurement / analysis apparatus is further simplified. Such a feature is very advantageous when diagnosing the presence or absence of a disease or its causative substance and screening for other antibodies or physiologically active substances.

  The microplate may be held in a state where the fluorescently labeled antigen or antibody dispensed into a plurality of wells is dried. As a result, the probe sample can be stored for a long period of time, and there is no need to prepare a fluorescently labeled antigen or antibody for each test. The user can perform fluorescence measurement / analysis simply by adding the test sample solution to the well when performing the test, which is very convenient. When the test sample solution may have autofluorescence such as serum, it is preferable to use a fluorescent dye that can be excited at 540 nm or more.

  According to the present invention, as can be understood from the above description, detection of multiple types of antigen-antibody reactions can be quickly performed on a small amount of test sample using fluorescence measurement analysis, particularly single molecule fluorescence measurement analysis. it can. In the conventional methods for detecting various types of antigen-antibody reactions, in order to identify the type of antigen-antibody reaction, processing such as immobilizing an antigen or antibody on microbeads or a substrate is necessary. However, according to the present invention, since various types of antigen-antibody reactions are distinguished by the wavelength of the fluorescent dye, the sample is a sample specimen for fluorescence measurement and analysis, although the fluorescent labeling operation of the sample is required. Preparation of the mixed sample solution requires only that the test sample solution and the probe sample be mixed almost as they are. Therefore, there is no loss of the sample in the operation of fixing the sample and the probe sample is placed on the substrate or the like. Since it is not necessary to fix the sample, the possibility of denaturation of the sample is reduced, and the reliability of the measurement result is increased.

  Thus, the method of the present invention can be used to test a small amount of sample in a short time and with high reliability even when the number of specimens is large, such as diagnosis of diseases or various diseases and screening of physiologically active substances. Is expected to be used as a method for detecting and identifying various types of antigen-antibody reactions.

  Other objects and advantages of the present invention will become apparent from the following description of preferred embodiments of the present invention.

  The present invention will now be described in detail with reference to a few preferred embodiments with reference to the accompanying drawings.

Procedure for detection of antigen-antibody reaction FIG. 1 shows a test of a body fluid sample or a culture fluid obtained from a living body such as serum, plasma, urine, nasal discharge, tears, feces, tissue extract, etc. FIG. 3 is a schematic diagram showing the state of molecules in a sample during a treatment process in a preferred embodiment of the method of the present invention for detecting an antibody contained in a sample solution.

  Referring to FIG. 1, as shown in FIG. 1 (a), for example, in a body fluid sample of a patient with a certain disease, when the disease is caused, the causative substance (bacteria, virus, pathogenic protein) , Tumor) or antibodies derived therefrom. In the case of an allergic disease, the patient's bodily fluid sample contains an antibody (against allergens such as food, pollen, house dust, mold, mites, etc.) specific to the allergic disease. Alternatively, antibodies can be produced from any cultured cell. The type of disease is identified by identifying substances that specifically bind to those antibodies, that is, cause an antigen-antibody reaction. However, until the type of disease is identified, it is not known which substance will cause an antigen-antibody reaction (or whether an antigen-antibody reaction itself will occur). It may be necessary to check whether it wakes up. Therefore, in the present embodiment, as a probe sample for antibody detection, a plurality of types of antigen candidates A1-A4 corresponding to antibody candidates to be confirmed to be present in the test sample solution are respectively divided into different fluorescent dyes F1. -F4 added (fluorescently labeled) is prepared as a probe sample (FIG. 1 (b)), and these are mixed with a body fluid sample.

The plurality of types of antigen candidates corresponding to the antibody candidates to be confirmed may be any physiologically active substance for those skilled in the art, such as proteins, nucleic acids, lipids, saccharides, other biomolecules, or chemical substances. . The fluorescent labeling of these antigen candidates may be performed by any method for those skilled in the art depending on the type and characteristics of the substance that is the antigen candidate. For example, in the case of a protein, fluorescent labeling may be performed by a chemical labeling method that targets a specific group in the protein. As for other substances, a fluorescently labeled substance may be chemically synthesized by an arbitrary method. As the fluorescent dye, any fluorescent dye usually used in this field, for example, TAMRA (carboxymethylrhodamine), TMR (tetramethylrhodamine), Alexa647, Rhodamine
It may be Green, Alexa488, etc., but is not limited thereto. What is important is that the spectrum of the emission wavelength of the fluorescent dye added to a plurality of types of antigen candidate substances mixed in one test sample solution is different, and each of these fluorescences is measured in the fluorescence measurement described later. It is necessary to be in an aspect that can be identified by selection of the light receiving wavelength. If the light receiving wavelength cannot be identified, it is sufficient that the light can be identified by the excitation wavelength (however, the light source of the fluorescence measuring device needs to be able to supply excitation light of each excitation wavelength).

  Thus, when antigen candidates A1-A4 are mixed in the test sample solution (FIG. 1 (c)), if the corresponding antibodies are present, they specifically bind to the antibodies (antigen-antibody reaction, A1). , A3). On the other hand, when there is no antibody that can specifically bind, the antigen candidate floats alone in the solution (A2, A4). Therefore, the modes of Brownian motion in the solution are different between the dyes F1 and F3 of the antigen bound to the antibody and the dyes F2 and F4 not bound to the antibody. Such a difference in motility is reflected in a temporal change (fluctuation) in the fluorescence intensity of the fluorescent dyes F1-F4, so that whether or not an antigen candidate has bound to the antibody can be determined based on the temporal change in the fluorescence intensity. .

  For fluorescence measurement, typically, a single molecule fluorescence analysis system MF20 (Olympus) may be used. In such a system, the movement state of the fluorescent dye F is determined by any one of fluorescence correlation spectroscopy, fluorescence depolarization, and fluorescence intensity distribution analysis. Hereinafter, how each measurement method reflects whether or not an antigen candidate is bound to an antibody will be reflected in the measurement result.

  In fluorescence correlation spectroscopy, the speed of movement (translational movement) of molecules passing through a minute fluorescence observation region by Brownian motion is observed. The speed of the translational movement of the molecule is reflected in the shape of the autocorrelation function with the time of the measured fluorescence intensity as a variable. As an index of the translational speed of the molecule, the length of time (translational diffusion time) from the start of measurement until the autocorrelation function value is halved is used. Since the movement of the molecule becomes slower as the size of the molecule becomes larger, the translational diffusion time becomes longer. In the case of the present invention, when the antigen binds to the antibody as shown in A1 and A3 of FIG. 1 (c), the fluorescent dye F moves together with the antigen and the antibody, and accordingly, A2 and A4 of FIG. As compared with the above, the speed of movement of the fluorescent dye F is remarkably reduced, and the translational diffusion time becomes longer. Therefore, the translational diffusion time indicates whether an antigen-antibody reaction has occurred for a certain antigen, and confirms that antibodies against antigens A1 and A3 are present in the test sample solution.

  In fluorescence depolarization, as is known in the art, the speed of rotational Brownian motion (rotation) of molecules is observed. The speed of the rotational movement of the molecule is reflected in the measured ratio between the intensity of longitudinally polarized light and transversely polarized light, or the degree of polarization. Since the rotation of the molecule becomes slower as the size of the molecule increases, the degree of polarization increases. In the case of the present invention, since the rotational Brownian motion of the fluorescent dye on the antigen bound to the antibody is slowed, the degree of polarization is increased, and thus it can be determined whether or not an antigen-antibody reaction has occurred for a certain antigen, and the antigen A1 , It is confirmed that an antibody against A3 is present in the test sample solution.

  In the fluorescence intensity distribution analysis method, photons emitted from the minute fluorescence observation area are detected (photon counting), and the frequency of detection of photons per unit time is statistically processed, so that the minute fluorescence observation area The number density of the fluorescent light emitters and the fluorescence intensity per fluorescent light emitter are calculated. In the case of the present invention, when a plurality of fluorescently labeled antigens bind to one antibody, such as the antigen A3, each fluorescent dye molecule that has independently moved as a single fluorescent emitter before binding is bound to the antibody. Therefore, the number density of the fluorescent light emitters is reduced, and the presence of a plurality of fluorescent dye molecules in the antibody increases the fluorescence intensity per fluorescent light emitter. Thus, for antigen A3, it is detected that an antigen-antibody reaction has occurred from a decrease in the number density of the fluorescent emitters and an increase in the fluorescence intensity per fluorescent emitter. Even in the case of the antigen A1 that binds only to the antibody, if the fluorescence intensity of the fluorescent dye of the antigen A1 changes due to binding to the antibody, it is possible to detect the presence or absence of binding. There is.

  In actual measurement, with respect to one test sample solution, the fluorescence intensity is measured in a manner in which the fluorescence of the fluorescent dyes F1-F4 can be identified, and the change with time is analyzed. For wavelength discrimination, a high-pass filter or band-pass filter is inserted in the optical path to the light-receiving surface of the fluorescent light detector, or only light of a specific wavelength is received by the light detector using a diffraction grating or the like. It may be. Such a wavelength discrimination configuration may be made by any configuration for those skilled in the art. Alternatively, the dye may be excited with light of various wavelengths, and the fluorescence of a plurality of types of dyes may be identified based on differences in absorption (excitation) wavelength characteristics of the dye.

  Thus, it is determined whether or not the antigen candidate is bound to the antibody from the movement state of the fluorescent dye by the fluorescence measurement / analysis, that is, the presence or absence of the antigen-antibody reaction is detected. In the above method, even if the fluorescence measurement / analysis is executed only at the stage where the antigen candidate and the test sample solution are mixed, whether or not the fluorescent dye moves together with the antibody from the numerical value of the result. However, fluorescence measurement / analysis may be performed for comparison at a stage before the antigen candidate is mixed with the test sample solution. In the above embodiment, the test sample is an antibody and the probe sample is an antigen. However, the test sample may be an antigen and the probe sample may be an antibody. However, in that case, it is necessary that the antigen is so large that the motion state of the antibody as the probe sample changes significantly due to the antigen-antibody reaction.

Anti-antibody reaction detection microplate By the way, in the fluorescence measurement / analysis described above, when the microplate schematically shown in FIG. 2 (A) is used, various test sample solutions can be more efficiently obtained. Measurement and analysis can be performed. Referring to FIG. 1, a microplate 10 that is advantageously used in the present invention has a plurality of wells 12 whose upper surface is open, and a bottom member 14 of a plate made of glass or plastic that transmits light. The overall size of the microplate is about 10 cm in a plane, the capacity of one well is about 100 μl, and a sample of about several tens μl is injected in the measurement. When the microplate is placed in the fluorescence measuring device, as shown in FIG. 2B, the excitation light of the device passes through the bottom surface and is collected in the well, where the excited dye The fluorescence passes through the bottom surface and is guided to a photodetector (not shown).

  In the present invention, when a microplate is used for fluorescence measurement, a different test sample solution may be injected into each well of the microplate. Also, preferably, when the type of antibody to be detected in advance is specified, for example, when it is intended to detect an antibody in a body fluid sample of a patient with an autoimmune disease or allergic disease Prior to measurement, a fluorescently labeled antigen candidate is prepared in a state dispensed to each well.

  As one mode of dispensing the antigen candidates on the microplate, a plurality of types of antigen candidates labeled with mutually different fluorescent dyes are dispensed in advance in each of the plurality of wells (FIG. 2 (C)). . In this case, different test sample solutions are injected into each well, and a large number of test sample solutions can be inspected with one microplate. As another aspect, different types of antigen candidates may be dispensed into each of the plurality of wells. For each well, one type of antigen candidate may be dispensed (FIG. 2D), but a plurality of types of antigen candidates may be dispensed (FIG. 2E). The number of fluorescent dyes that can be distinguished in the same solution in a single fluorescence measurement is limited by the range of the emission wavelength range of the dye (the spectrum of the emission wavelength is large). It is difficult to measure.) Therefore, when it is desired to detect a number of antigen-antibody reactions in a single sample solution exceeding the types of dyes available in the same solution, separate antigen candidates or separate The combination of antigen candidates may be dispensed.

  If such a microplate in which antigen candidates are dispensed in advance is prepared, it is not necessary to prepare the antigen candidates for each examination, which is convenient. In addition, if the antigen candidate does not lose its activity as an antigen even after drying, it can be dried and stored after dispensing multiple types of antigen candidates (for example, frequently used antigen candidates) to each well. It may be like this.

Such a microplate may be prepared, for example, as follows (FIG. 2 (F)).
1. The antigen whose reaction is to be detected is fluorescently labeled and purified.
2. 5 μl of 10 nM fluorescently labeled antigen is added to each well of the microplate and dried by natural drying, pressure drying, freeze drying or the like.
In addition, when the microplate into which the antigen candidate is dispensed is stored for a long time, the microplate is stored in a light-shielded refrigerated state so as to avoid denaturation of the antigen candidate and the fluorescent dye. At the time of use, it is returned to room temperature, a test sample solution is injected into each well, and after an appropriate reaction time, fluorescence measurement is performed.

  In order to verify the effectiveness of the present invention described above, the following experiment was performed. It should be understood that the following examples illustrate the effectiveness of the present invention and do not limit the scope of the present invention.

  It was confirmed that two antigen-antibody reactions involved in allergic diseases, which are typical examples of antigen-antibody reactions, are detected in one test sample solution by the method of the present invention. To put it simply, allergic diseases are the release of chemical mediators such as histamine from mast cells by binding antigens (allergens) that have entered the body to IgE antibodies on the surface of mast cells. Symptoms that appear due to the action of chemical transmitters. A patient who develops allergic symptoms to a certain substance has an antibody that uses that substance as an antigen. Therefore, if it is possible to detect which substance an antibody is present in the body fluid of such a patient, the allergen is specified, which is very useful information for the prevention and treatment of allergies. In the experiment below, two types of allergic diseases, ovalbumin contained in egg white known to cause food allergy symptoms and β-lactoglobulin contained in milk, were fluorescently labeled. An experiment was conducted to detect an antigen-antibody reaction as an antigen.

The measurement operation was as follows.
1. Ovalbumin (manufacturer MP Biomedicals, Inc.) is fluorescently labeled using TAMRA NHS-ester (Olympus) and β-lactoglobulin (manufacturer ICN BIOMEDICALS) is labeled with ATTO633 NHS-ester (ATTO-TEC). Purified. The absorption / fluorescence wavelength peaks of TAMRA [5- (and-6) -Carboxytetramethylrhodamine] are 541 nm and 565 nm, respectively, and the absorption / fluorescence wavelength peaks of ATTO633 are 629 nm and 657 nm, respectively.
2. Thus, the fluorescence-labeled antigen was dissolved in phosphate buffer + 0.05% Tween 20, and a probe solution containing both antigens was prepared so that the final concentration of each antigen was nM. Then, only anti-ovalbumin antibody (manufacturer MP Biomedicals, Inc.), anti-β-lactoglobulin antibody (manufacturer BETHYL LABORATORIES) only, and a solution containing both antibodies (test sample solution) were mixed, respectively, Was adjusted to 50 μL and left at 37 ° C. for 30 minutes. As a control, a probe solution mixed with a solution containing no antibody was also prepared.
3. With respect to the four types of test solutions thus prepared (including those for control), the single molecule fluorescence analysis system MF20 (Olympus) was used, and the translational diffusion time of the fluorescent molecules was measured by fluorescence correlation spectroscopy. The measurement was performed in three ways: (i) when excited at a wavelength of 543 nm, (ii) when excited at a wavelength of 633 nm, and (iii) double excited at a wavelength of 543 nm and a wavelength of 633 nm. The wavelength ranges received by the photodetector at the time of measurement are as follows.
(I) When the excitation wavelength is 543 nm: 560-620 nm
(ii) When the excitation wavelength is 633 nm: 660-710 nm
(iii) Excitation wavelengths 543 nm and 633 nm (double excitation) ... 565-595 nm and 650-690 nm
In the case of (iii), two photodetectors were used so that light having a wavelength in the range of 565-595 nm was incident on one of them and light having a wavelength in the range of 650-690 nm was incident on the other. The laser intensity is 100 μW, each sample is measured 5 times for 10 seconds, and the autocorrelation function of the fluorescence intensity is calculated and translated for each by the program for fluorescence correlation spectroscopy prepared in MF20. The average diffusion time was calculated.

  FIG. 3 is a graph showing the results of translational diffusion times of the four kinds of test solutions in each of the cases (i) to (iii). Referring to the figure, the translational diffusion time is calculated based on (i) the fluorescence intensity at the excitation wavelength of 543 nm (FIG. 3A) and (iii) the fluorescence intensity of the photodetector having a light reception wavelength of 565-595 nm (FIG. 3 (C)), it was increased only in the solution containing only the anti-ovalbumin antibody and the solution containing both antibodies, and (ii) the fluorescence intensity at the excitation wavelength of 633 nm (FIG. 3). (C)) and (iii) measured and calculated from the fluorescence intensity (FIG. 3 (D)) of the photodetector having a light receiving wavelength of 650-690 nm, both of the solution containing only anti-β-lactoglobulin antibody, Increased only in solutions containing antibodies. The fluorescent dye TAMRA added to ovalbumin has an emission wavelength (peak) of 565 nm, and the fluorescent dye ATTO633 added to β-lactoglobulin has an emission wavelength (peak) of 657 nm. ) And (C) are considered to represent the motion state of the ovalbumin molecule, and in the presence of the anti-ovalbumin antibody, the ovalbumin molecule is It shows binding to anti-ovalbumin antibodies. Similarly, the results of FIGS. 3 (B) and (D) are considered to represent the motion state of β-lactoglobulin molecule. In the presence of anti-β-lactoglobulin antibody, other antibodies Even when present, β-lactoglobulin molecules have been shown to bind to anti-β-lactoglobulin antibodies.

  These results show that a specific antigen-antibody reaction can be achieved by selecting excitation and emission wavelengths appropriately even when multiple types of antibodies are mixed in the test sample solution and multiple types of antigen-antibody reactions are occurring. Indicates that it can be confirmed. Further, as shown in the results of the measurement under the condition of the simultaneous excitation of the dye (iii) (FIGS. 3C and 3D), even when the fluorescent label of the antigen is simultaneously excited. This shows that a specific antigen-antibody reaction can be selectively confirmed among a plurality of types of antigen-antibody reactions by appropriately selecting the emission wavelength.

  Thus, as illustrated in the above examples, according to the present invention, the reaction between an antigen and an antibody that can cause allergies is detected, and the identification of the causative antigen is performed in the test. It should be understood that even when a plurality of types of antigen-antibody reactions occur in the sample solution, the reaction is performed with a small amount of sample and at a high throughput.

FIG. 1 schematically shows the state of molecules in a sample during the process of the embodiment. FIG. 2 (A) is a schematic view of a microplate advantageously used in a preferred embodiment of the method of the present invention. FIG. 2B is a diagram schematically showing the state of the well during the fluorescence measurement. FIGS. 2C to 2E are diagrams for explaining various aspects of dispensing of antigen candidates. FIGS. 2 (F)-(G) schematically show the drying process of antigen candidates dispensed in advance. FIG. 3 is a graph showing the result of the translational diffusion time calculated from the autocorrelation function of the fluorescence intensity obtained by the fluorescence correlation spectroscopy of the sample in the example. In the figure, anti-A antibody represents an anti-ovalbumin antibody and anti-B antibody represents an anti-β-lactoglobulin antibody. (A) is an excitation wavelength of 543 nm and a light reception wavelength of 560-620 nm, (B) is an excitation wavelength of 633 nm and a light reception wavelength of 660-710 nm, and (C) is an excitation wavelength of 543 nm and 633 nm, and a light reception wavelength of 565. In the case of −595 nm, (D) is the case where the excitation wavelengths are 543 nm and 633 nm, and the light receiving wavelength is 650 to 690 nm.

Explanation of symbols

10 ... microplate 12 ... well 14 ... bottom surface of microplate

Claims (15)

A method for detecting an antigen-antibody reaction comprising:
A plurality of types of antigens or antibodies labeled with different fluorescent dyes are mixed with a test sample solution to be tested for the presence of antibodies or antigens against any of the plurality of antigens or antibodies. The process of preparing
Measuring the fluorescence intensity of at least one of the fluorescent dyes in the mixed sample solution;
Determining whether or not an antigen or antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is bound to a molecule in the test sample solution based on the time variation of the measured fluorescence intensity. And when it is determined that the antigen or antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is bound to a molecule in the test sample solution, the antigen or antibody is labeled with the fluorescent dye corresponding to the fluorescence intensity. It is determined that the antigen or antibody bound to the molecule in the test sample solution has caused an antigen-antibody reaction, and the antibody or antigen against the antigen or antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is the test sample solution. A method characterized by determining that it is included in the data.   The method according to claim 1, wherein the fluorescence intensity of each of the fluorescent dyes labeled with the plurality of types of antigens or antibodies is measured, and based on each time change of the measured fluorescence intensity, It was determined whether an antigen or an antibody labeled with a fluorescent dye corresponding to each of the fluorescence intensities was bound to a molecule in the test sample solution, and labeled with a fluorescent dye corresponding to each of the fluorescent intensities When it is determined that an antigen or antibody is bound to a molecule in the test sample solution, the antigen or antibody labeled with a fluorescent dye corresponding to each of the fluorescence intensities is a molecule in the test sample solution. And determining that an antigen-antibody reaction has occurred.   The method according to claim 1 or 2, wherein the fluorescence intensity is measured by fluorescence correlation spectroscopy, and an antigen or an antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is added to a molecule in the test sample solution. A method of determining whether or not they are bound based on a translational diffusion time calculated by the fluorescence correlation spectroscopy based on a temporal change in the fluorescence intensity.   3. The method according to claim 1, wherein the fluorescence intensity is measured by a fluorescence depolarization method, and an antigen or an antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is added to a molecule in the test sample solution. A method of determining whether or not they are combined based on a fluorescence polarization degree calculated by the fluorescence depolarization method based on a temporal change in the fluorescence intensity.   3. The method according to claim 1, wherein the fluorescence intensity is measured by a fluorescence intensity distribution analysis method, and an antigen or an antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is a molecule in the test sample solution. Is determined based on the fluorescence intensity per fluorescence emitter calculated by the fluorescence intensity distribution analysis method based on the time change of the fluorescence intensity.   3. The method according to claim 1 or 2, wherein a plurality of types of antigens or antibodies labeled with different fluorescent dyes mixed with the test sample solution are used as food that becomes an antigen when the antigen-antibody reaction occurs. A method characterized in that the protein is contained.   3. The method according to claim 1 or 2, wherein a plurality of types of antigens or antibodies labeled with different fluorescent dyes mixed with the test sample solution are antigens against antibodies contained in a body fluid of an allergic disease patient. A method characterized by being.   3. The method according to claim 1 or 2, wherein the plurality of types of antigens or antibodies labeled with the different fluorescent dyes mixed in the test sample solution are antigens against antibodies contained in a body fluid of an autoimmune disease patient. A method characterized by being.   3. The method according to claim 1, wherein the test sample solution is serum or plasma or a body fluid sample obtained from a living body.   3. The method according to claim 1 or 2, wherein the fluorescent label is excitable with light having a wavelength of 540 nm or more.   A microplate having a plurality of wells suitable for detecting an antigen-antibody reaction by fluorometric analysis, wherein at least one fluorescently labeled antigen or antibody is dispensed in advance in each of the plurality of wells A microplate characterized by that.   The microplate according to claim 11, wherein a plurality of types of antigens or antibodies previously labeled with different fluorescent dyes are dispensed into the same well.   12. The microplate according to claim 11, wherein at least one kind of antigen or antibody labeled with different fluorescent dyes separately in advance is dispensed into the plurality of wells.   14. The microplate according to claim 11, wherein the fluorescently labeled antigen or antibody dispensed into the plurality of wells is held in a dried state. 14. The microplate according to claim 11, wherein the fluorescent label can be excited by light having a wavelength of 540 nm or more.

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