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WO2008053821A1 - Procédé de détection fluorométrique d'une réaction antigène-anticorps - Google Patents

Procédé de détection fluorométrique d'une réaction antigène-anticorps Download PDF

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Publication number
WO2008053821A1
WO2008053821A1 PCT/JP2007/071001 JP2007071001W WO2008053821A1 WO 2008053821 A1 WO2008053821 A1 WO 2008053821A1 JP 2007071001 W JP2007071001 W JP 2007071001W WO 2008053821 A1 WO2008053821 A1 WO 2008053821A1
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Prior art keywords
antigen
antibody
sample solution
labeled
fluorescence
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PCT/JP2007/071001
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English (en)
Japanese (ja)
Inventor
Hidetaka Nakata
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Olympus Corp
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Olympus Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • 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 for the method.
  • antigen-antibody reactions make it possible to detect various biomolecules inside and outside the living body. It is applied to diagnosis and screening of bioactive 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 (for body fluids obtained from living bodies). In this case, 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.
  • an antibody against the above-mentioned disease causative agent or a substance derived therefrom is present in the living body is determined by an antigen (or epitope) for the antibody (such as a body fluid obtained from the living body).
  • an antigen or epitope
  • the presence or absence of a disease-causing substance in a living body may be examined by detecting whether an antigen-antibody reaction has occurred.
  • 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.
  • the specific substance in a body fluid or the like obtained from a living body.
  • Patent Document 1 proposes a special measurement chip for detecting an antigen-antibody reaction using surface plasmon resonance.
  • Patent Document 2 a special sample for detecting an antigen-antibody reaction using an apparatus combining cell flowmetry and fluorescence measurement is disclosed.
  • 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.
  • the sample required for the measurement may be in a very low concentration and in a very small amount compared to the past (the amount used in one measurement is about several tens of times), and the measurement time is also long. It is greatly shortened (one measurement is about 10 seconds).
  • the method or apparatus for detecting an antigen-antibody reaction using the measurement technique for faint light as described above is more effective than conventional methods even when the number of specimens is large, such as for diagnosing diseases and screening for physiologically active substances. It is expected to be a powerful tool that can perform inspections promptly.
  • Patent Document 1 JP 2006-250668
  • Patent Document 2 JP 2005-098877
  • Patent Document 3 JP 2005-337805
  • one object of the present invention is to provide a method for detecting a multi-item or multi-sample antigen-antibody reaction simply and rapidly.
  • the method for detecting an antigen-antibody reaction of the present invention comprises a plurality of types of antigens or antibodies labeled with mutually different fluorescent dyes, the plurality of types of antigens or antibodies.
  • a process of preparing a mixed sample solution by mixing it with a test sample solution to be tested for the presence or absence of antibodies or antigens against antibody deviations, and at least one of the mixed sample solutions Based on the process of measuring the fluorescence intensity of the fluorescent dye and the time change of the measured fluorescence intensity, the antigen or antibody labeled with the fluorescent dye corresponding to the fluorescence intensity is bound to the molecule in the sample solution.
  • 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.
  • label with a 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. It is characterized by determining that it exists in the inside.
  • a plurality of types of antigens or antibodies labeled with a fluorescent dye mixed with a test sample solution as understood by those skilled in the art are antigens (that is, substances that can become antigens in any living body). If so, the substance to be detected whether or not 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. If is an antibody, the substance to be detected whether it is contained in the test sample solution is an antigen.
  • the antigen-antibody reaction is detected by measuring the fluorescence intensity of the fluorescent dye, and the antigen labeled with the fluorescent dye corresponding to the fluorescence intensity based on the change in the fluorescence intensity over time.
  • the amount of test sample and the processing time are greatly increased compared to traditional methods such as ELISA, because fluorescence analysis is used to determine whether antibodies bind to molecules in the test sample solution.
  • the test sample solution that is merely reduced is mixed with multiple types of antigens or antibodies labeled with different fluorescent dyes, so that Even if there are multiple substances whose presence or absence should be confirmed, the amount of sample to be tested is only the amount of sample necessary for one test.
  • 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. Whether or not to cause an antibody reaction, it is necessary to measure the fluorescence intensity of each fluorescent dye separately (excitation wavelength or light reception in the fluorescence detection device depending on the absorption wavelength or emission wavelength characteristics of the dye to be detected). The wavelength is selected.) The sample solution itself does not need to be replaced. If multiple types of fluorescent dyes can be excited with one wavelength of excitation light, use a device (filter, diffraction grating, etc.) that discriminates the wavelength as appropriate in the photodetector of the fluorescence measurement device! ! /
  • the fluorescence intensity of each of the fluorescent dyes labeled with a plurality of types of antigens or antibodies is measured, and each time change of the measured fluorescence intensity is measured. Based on this, it is determined whether or not the antigen or antibody labeled with a fluorescent dye corresponding to each of the fluorescence intensities is bound to a molecule in the test sample solution, and the fluorescent dye corresponding to each of the fluorescence intensities is used. When it is determined that the labeled 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.
  • the fluorescence measuring apparatus used can simultaneously excite and detect fluorescence of a plurality of types of fluorescent dyes having different absorption and emission wavelength characteristics, the fluorescence measurement of the fluorescent dyes may be performed simultaneously. Furthermore, the measurement time is shortened.
  • the fluorescence intensity is measured by measuring the fluorescence from one fluorescent molecule and analyzing the temporal change or fluctuation to observe the state or motion of the fluorescent single molecule. This is done by fluorescence analysis (single molecule fluorescence analysis technology).
  • fluorescence analysis single molecule fluorescence analysis technology
  • the fluorescence intensity is measured by “fluorescence correlation spectroscopy”. In that 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 the translational diffusion time calculated by fluorescence correlation spectroscopy based on the change in fluorescence intensity over time. It may be determined based on.
  • the fluorescence intensity can be measured by the “fluorescence intensity distribution analysis method”.
  • an antigen or an antibody labeled with a fluorescent dye corresponding to the fluorescence intensity is bound to a molecule in the sample solution.
  • the light intensity of the fluorescent light is 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 below).
  • Multiple types of fluorescently labeled antigens or antibodies must be bound to one molecule in the sample solution at the same time.
  • “One fluorescent emitter” is integrated in a solution that is not a single fluorescent molecule.
  • the fluorescence intensity is measured by “fluorescence depolarization”
  • the antigen labeled with a fluorescent dye corresponding to the fluorescence intensity is used.
  • the antibody is bound to a molecule in the sample solution May be determined based on the fluorescence polarization degree calculated by the fluorescence depolarization method based on the temporal change of the fluorescence intensity (in this case, a normal fluorescence depolarization measurement apparatus, not an apparatus that performs single molecule fluorescence analysis). May be.
  • the above-described present invention 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 screening of other antibodies. 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 labeled with different fluorescent dyes) is used.
  • the antigen or antibody may be a protein contained in food that becomes an antigen when an antigen-antibody reaction occurs.
  • the probe sample may be an antigen against an antibody contained in the body fluid of an allergic disease patient.
  • 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.
  • a body fluid sample obtained from serum or plasma or other living body may be used as the test sample solution.
  • a body fluid sample which is a test sample solution necessary for detecting the presence or absence of antigens or antibodies related to a plurality of diseases, is a very small amount. , And! /.
  • the sample solution may have 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 the autofluorescence does not get on the measurement. I prefer to do it.
  • the fluorescence measurement is a microplate having a plurality of wells and suitable for detecting an antigen-antibody reaction by a fluorometric analysis, and at least one of each of the plurality of wells is previously detected. It can be carried out advantageously by means of a microplate characterized in that two fluorescently labeled antigens or antibodies are dispensed. In the microplate mode, 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 comprising a plurality of types of antigens or antibodies labeled with different fluorescent dyes is dispensed to each of the plurality of wells.
  • a plurality of types of test antibody solutions can be systematically detected for a plurality of types of antigen-antibody reactions.
  • a plurality of types of antigens or antibodies labeled with different fluorescent dyes may be dispensed on a plurality of wells.
  • multiple types of antigen-antibody reactions are confirmed for one test sample solution by dispensing one test sample solution into a well in which different antigens or antibodies are dispensed.
  • the operation of changing the sample solution to be tested in the fluorescence measurement / analysis apparatus is further simplified. This feature is very advantageous when diagnosing the presence or absence of a disease or its causative substance, or screening for other antibodies or physiologically active substances.
  • the above-described microplate may be held in a state in which a fluorescently labeled antigen or antibody dispensed into a plurality of wells is dried. This enables long-term storage of the probe sample and eliminates the need to prepare a fluorescently labeled antigen or antibody for each test.
  • the user can perform a fluorometric analysis simply by adding the sample solution to the well when performing the test, which is very convenient. If 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.
  • the invention's effect [0014] According to the present invention, detection of multiple types of antigen-antibody reactions can be performed on a small amount of test sample using fluorescence measurement analysis, in particular, single-molecule fluorescence measurement analysis as understood by the above explanation. And can be executed quickly.
  • fluorescence measurement analysis in particular, single-molecule fluorescence measurement analysis as understood by the above explanation.
  • a treatment such as immobilizing the antigen or antibody on a microbead or a substrate is required.
  • the fluorescent labeling operation of the sample is necessary, but the fluorescence measurement and analysis are performed.
  • the test sample solution and the probe sample need only be mixed as they are, so that there is no loss of the sample in the operation such as fixing the sample.
  • the probe sample since it is not necessary to fix the probe sample to a substrate or the like, the possibility of sample denaturation is reduced and the reliability of the measurement result is increased.
  • the method of the present invention can be used with a small amount of sample in a short time, even when the number of specimens is large, such as diagnosis of diseases or various diseases and screening of physiologically active substances. It is expected to be used as a method for detection and identification of many types of antigen-antibody reactions that can be performed with high reliability.
  • FIG. 1 schematically shows the state of molecules in a sample during the treatment process of the embodiment.
  • FIG. 2 (A) is a schematic view of a microplate that is advantageously used in a preferred embodiment of the method of the present invention.
  • Fig. 2 (B) is a schematic diagram showing the state of the well during fluorescence measurement.
  • FIGS. 2 (C)-(E) are diagrams illustrating various modes of dispensing candidate antigens.
  • Fig. 2 (F)-(G) schematically shows the drying process of pre-dispensed antigen candidates.
  • FIG. 3 is a graph showing the result of translational diffusion time calculated from the autocorrelation function of fluorescence intensity obtained by fluorescence correlation spectroscopy of a sample in the example.
  • anti-A antibody represents anti-ovalbumin antibody and anti-B antibody represents anti / 3 lactoglobulin antibody.
  • A is the excitation wavelength 543nm and the received light wavelength 560-620nm
  • B is the excitation wavelength 633 ⁇ m
  • C is excitation wavelength 543nm and 633nm, receiving wavelength 565-595nm
  • D is excitation wavelength 543nm and 633nm, receiving wavelength 650-690nm This is the case.
  • FIG. 1 shows that an antigen-antibody reaction includes, for example, serum, plasma, urine, nasal discharge, tears, stool, tissue extract, and the like in a body fluid sample obtained or a test sample solution such as a culture solution.
  • FIG. 2 is a schematic diagram showing the state of molecules in a sample in the course of processing of a preferred embodiment of the method of the present invention for detecting existing antibodies.
  • Viruses, pathogenic proteins, tumors or antibodies derived therefrom.
  • the body fluid sample collected from the patient contains antibodies specific to the allergic disease (to allergens such as food, pollen, house dust, mold and mites). included.
  • allergens such as food, pollen, house dust, mold and mites.
  • 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 undergo an antigen-antibody reaction (or whether an antigen-antibody reaction itself will occur).
  • a probe sample for antibody detection a plurality of types of antigen candidates A1-A4 corresponding to antibody candidates whose presence or absence in the test sample solution should be confirmed are separately fluorescent.
  • the plurality of types of antigen candidates corresponding to the candidate antibodies to be confirmed are arbitrary physiologically active substances for those skilled in the art, such as proteins, nucleic acids, lipids, saccharides, and / or other living organisms. It can be a molecule or chemical!
  • the fluorescent labels of these antigen candidates are Depending on the type and characteristics of a substance that is an antigen candidate, this may be done by any method for those skilled in the art. 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, fluorescently labeled substances may be chemically synthesized by any method.
  • the fluorescent dye may be any fluorescent dye usually used in this field, such as TAMRA (carboxymethylrhodamine), TMRUetramethylrhodamine) Alexa647, Rhoaamine Green, Alexa488, but is not limited thereto. What is important is that the spectrum of the emission wavelength of the fluorescent dye added to the multiple types of antigen candidate substances mixed in one test sample solution is different, and the power of each of these fluorescences. It is necessary to be able to identify by selecting the wavelength. If it is not possible to identify the light receiving wavelength, it is only necessary to be able to identify the excitation wavelength (however, the light source of the fluorescence measuring device must be able to supply excitation light of each excitation wavelength! ).
  • the single molecule fluorescence analysis system MF20 may be used for the fluorescence measurement.
  • the movement states of the fluorescent dyes F1 to F4 are determined by any one of fluorescence correlation spectroscopy, fluorescence depolarization, and fluorescence intensity distribution analysis.
  • fluorescence correlation spectroscopy fluorescence correlation spectroscopy
  • fluorescence depolarization fluorescence depolarization
  • fluorescence intensity distribution analysis it will be described how the measurement result reflects whether or not the antigen candidate is bound to the antibody.
  • the fluorescent dyes Fl and F3 move together with the antigen and the antibody, and accordingly, in FIG. ? 2.
  • the speed of movement is significantly reduced and the translational diffusion time is increased. Therefore, the translational diffusion time indicates whether an antigen-antibody reaction has occurred for a certain antigen, and the presence or absence of antibodies to the antigens Al and A3 in the test sample solution is confirmed.
  • the fluorescence intensity distribution analysis method photons emitted from within a minute fluorescence observation region are detected (photon counting), and the frequency of detection of photons per unit time is statistically processed, so that The number density of fluorescent emitters in the fluorescence observation region and the fluorescence intensity per fluorescent emitter are calculated.
  • each fluorescent dye molecule that independently moved as a single fluorescent emitter before binding to the antibody. Since it is constrained and moves as a single fluorescent emitter, the number density of fluorescent emitters is reduced, while the presence of multiple fluorescent dye molecules in the antibody increases the fluorescence intensity per fluorescent emitter. To do.
  • a high-pass filter or a band-pass filter is inserted in the optical path to the light-receiving surface of the fluorescence photodetector, or only light of a specific wavelength is received by the photodetector using a diffraction grating or the like. It may be.
  • the configuration of the force and the wavelength discrimination may be made by any configuration for those skilled in the art. It is also possible to excite the dye with light of various wavelengths and identify the fluorescence of multiple types of dyes by the difference in absorption (excitation) wavelength characteristics of the dye.
  • the antigen candidate is bound to the antibody from the movement state of the fluorescent dye by fluorescence measurement and analysis, that is, the presence or absence of the antigen-antibody reaction is detected.
  • the fluorescent dye is moving together with the antibody from the numerical value of the result.
  • the ability to estimate whether or not the force can be S Fluorescence measurement / analysis may be executed for comparison before the antigen candidate is mixed with the test sample solution.
  • the test sample may be an antibody
  • the probe sample may be an antigen
  • the test sample may be an antigen
  • the probe sample may be an antigen
  • the probe sample may be an antibody
  • the probe sample may be an antibody.
  • the antigen needs to be so large that the movement of the antibody, which is the probe sample, changes significantly due to the antigen-antibody reaction.
  • a microplate 10 that is advantageously used in the present invention has a plurality of wells 12 whose upper surfaces are open, and a bottom member 14 of a plate made of glass or light-transmitting plastic.
  • the overall size of the microplate is about 10 cm in the plane, the capacity of one well is about 100, and about several tens of samples are injected in the measurement.
  • the excitation light of the device as shown in Fig. 2 (B) is transmitted through the bottom surface and collected in the well, where the excited dye The fluorescence passes through the bottom surface and is guided to a photodetector (not shown).
  • a different test sample solution may be injected into each well of the microplate. Also preferably, If the type of antibody to be detected in advance has been identified, for example, if it is intended to detect antibodies in body fluid samples of patients with autoimmune disease or allergic disease, prior to measurement The microplate is prepared in a state where the fluorescently labeled antigen candidates are dispensed into each well.
  • a plurality of types of antigen candidates labeled with different fluorescent dyes are pre-dispensed on each of the plurality of wells (see FIG. 2 (C)).
  • different test sample solutions are injected into each well, and a large number of test sample solutions can be inspected with one microplate.
  • different types of antigen candidates may be dispensed to each of the plurality of wells. For each well, one type of antigen candidate may be dispensed (Fig. 2 (D)), but multiple types of antigen candidates may be dispensed (Fig. 2 (E)).
  • the number of fluorescent dyes that can be distinguished in the same solution in a single fluorescence measurement is limited by the width of the emission wavelength range of the dyes (the dyes with large overlapping of emission wavelength spectra are the same). It is difficult to measure in solution.) 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 A combination of antigen candidates may be dispensed.
  • Preparing such a microplate in which antigen candidates are dispensed in advance is convenient because it is not necessary to prepare antigen candidates for each examination.
  • the antigen candidate does not lose its activity as an antigen even after drying, multiple types of antigen candidates (for example, frequently used antigen candidates) are dispensed to each well, then dried, It can be stored.
  • the preparation of the microplate may be performed as follows (FIG. 2 (F)).
  • the microplate into which the antigen candidate is dispensed is stored for a long time, it is stored in a light-shielded refrigerated state so as to avoid denaturation of the antigen candidate and the fluorescent dye.
  • the sample is returned to room temperature, and a test sample solution is injected into each well. After an appropriate reaction time, fluorescence is measured.
  • the following experiment was performed. It should be noted that the following examples illustrate the effectiveness of the present invention and are not intended to limit the scope of the present invention!
  • allergic diseases are the release of chemical mediators such as histamine from mast cells when antigens (allergens) that enter the body bind to IgE antibodies on the surface of mast cells. Symptoms that appear due to the action of chemical transmitters. Patients who develop allergic symptoms to a substance have antibodies that use 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 can be identified, which is very useful information for the prevention and treatment of allergies.
  • the measurement processing operation was as follows.
  • Ovalbumin (manufacturer MP Biomedicals, Inc.) uses TAMRA NHS-ester (Olympus) and / 3 lactoglobulin (manufacturer ICN BIOMEDICALS) uses ATT0633 NHS — ester (ATTO-TEC) Each was fluorescently labeled and purified.
  • the absorption and fluorescence wavelength peaks of TAMRA [5- (and-6) -Carboxytetramethylrhodamine] are 541 nm and 565 nm, respectively, and the absorption and fluorescence wavelength peaks of ATT0633 are 629 nm and 657 ⁇ m, respectively.
  • the autocorrelation function of fluorescence intensity was calculated, and the average of translational diffusion time was calculated.
  • Fig. 3 is a graph showing the results of the translational diffusion times of the four types of test solutions in each of cases (i) and (iii) above.
  • the translational diffusion time is the fluorescence intensity at the ⁇ excitation wavelength of 543 nm (Fig. 3 (A)) and the fluorescence intensity of the photodetector with the received wavelength of 565-595 nm in (iii) (Fig. 3 ( C) Measured from) '' Calculated! /, Which increases only in solutions containing only anti-ovalbumin antibodies, solutions containing both antibodies, and (ii) fluorescence at an excitation wavelength of 6 33 nm. Intensities (Fig.
  • FIGS. 3 (B) and (D) are considered to represent the movement state of the / 3 lactoglobulin molecule, and in the presence of the anti / 3 lactoglobulin antibody, The / 3 lactoglobulin molecule is shown to bind to the anti / 3 lactoglobulin antibody even when the antibody is present.
  • the reaction between an antigen and an antibody that can cause allergy is detected and caused. It should be understood that antigen identification is performed with a small amount of sample and at high throughput even when multiple types of antigen-antibody reactions occur in the test sample solution.

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Abstract

La présente invention concerne un nouveau procédé grâce auquel on peut détecter de manière pratique et rapide une réaction antigène-anticorps dans un certain nombre d'articles ou dans un certain nombre de spécimens. Ce procédé comprend le mélange de plusieurs types d'antigènes ou d'anticorps, qui ont été marqués respectivement par des colorants fluorescents différents les uns des autres, avec une solution d'essai témoin, la mesure de l'intensité fluorescente d'au moins un colorant fluorescent dans la solution, et le fait de déterminer si l'antigène ou l'anticorps ayant été marqué par le colorant fluorescent correspondant à l'intensité fluorescente est lié à une molécule dans la solution d'essai témoin sur la base d'un changement d'intensité fluorescente au cours du temps. Lorsque l'on détermine que l'antigène ou l'anticorps ayant été marqué par le colorant fluorescent correspondant à l'intensité fluorescente est lié à une molécule dans la solution d'essai témoin, on détermine que l'antigène ou l'anticorps ayant été marqué par le colorant fluorescent correspondant à l'intensité fluorescente est lié à une molécule dans la solution d'essai témoin et de ce fait il apparait une réaction antigène-anticorps. La présente invention concerne également une microplaque appropriée à ce procédé.
PCT/JP2007/071001 2006-11-02 2007-10-29 Procédé de détection fluorométrique d'une réaction antigène-anticorps Ceased WO2008053821A1 (fr)

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JP2006299697A JP2010256010A (ja) 2006-11-02 2006-11-02 蛍光分析による抗原抗体反応検出方法
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JP2001272404A (ja) * 2000-03-27 2001-10-05 Olympus Optical Co Ltd 蛍光相関分光法による抗原抗体反応
WO2005015212A1 (fr) * 2003-08-07 2005-02-17 Cellfree Sciences Co., Ltd. Reactif permettant de produire une puce a proteines
JP2005172460A (ja) * 2003-12-08 2005-06-30 Olympus Corp タンパク質とサンプルとの反応を検出する方法

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JP2001272404A (ja) * 2000-03-27 2001-10-05 Olympus Optical Co Ltd 蛍光相関分光法による抗原抗体反応
WO2005015212A1 (fr) * 2003-08-07 2005-02-17 Cellfree Sciences Co., Ltd. Reactif permettant de produire une puce a proteines
JP2005172460A (ja) * 2003-12-08 2005-06-30 Olympus Corp タンパク質とサンプルとの反応を検出する方法

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