JP2007155691A - Substance measuring method, and measuring composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance measuring method using a highly sensitive protein array using a fluorescent substance or the like. <P>SOLUTION: A simple and practical measuring system requiring no highly sensitive and complicated amplifying operation step is provided by using the protein array with a biological active substance such as an antibody, an antigen and lectin immobilized on a support having a three-dimensional polymer compound capable of holding stably biological activity with high density, and by using a quantum dot fluorescent substance extremely stable in optical detection in combination therewith. A detection spot and a fluorescent signal obtained by the system are stable and uniform to provide a highly reliable measured result. A plurality of items in a disease marker or disease preventive marker is simultaneously measured by a detecting reagent using the protein array and quantum dot fluorescence. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for measuring a specific substance present in a biological sample, a food sample, an environmental sample, etc., and a reagent composition used in those methods.

  Measurement systems using proteins such as antibodies, receptors, and lectins are currently used in a wide variety of fields such as medicine, pharmacy, biology, and environmental engineering, and are indispensable as measurement and evaluation systems for substances. ing. These measurement systems often measure a single object, but an efficient evaluation method that can simultaneously measure a plurality of objects using a small amount of an object sample is desired. In recent years, protein arrays and antibody arrays (Schena et al., Nielsen et al., Wang et al.), Etc., have been reported as evaluation methods that satisfy these requirements.

  “Schoena, Protein Array (2004) Jones & Bartlett Publisher” (Schena M. et al. Protein Array (2004) Jones and Bartlett Publishers).   “Camharm Patty edited by Protein Microarray Technology (2004) Wiley VCH Verlag Lamb GmbH & Co. KGaA” (Protein Microarray Technology (2004) Wiley-VCH Verlag GmbH & Co. KGaA).   Wan et al. Special Table 2005-504309

  Membranes made of optical slides or polymer materials are often used for protein array applications. As the slide, a FAST slide (S & S Bioscience, Schleicher and Schuell Bioscience GmbH) coated with nitrocellulose on the surface, a SuperProtein slide (TeleChem International Inc., Telecom International Inc.) coated with a hydrophobic polymer, and black. There is a Maxisorp slide coated with a polymer (Nalge Nunc Inc.). In addition, a glass slide based on which amino groups, carboxyl groups, and thiol groups are introduced on the surface so as to be chemically crosslinkable with proteins is also commercially available.

  For these various slides, protein arrays are prepared by preparing spots of various shapes by using various arrayers / spotters for protein solutions or by using techniques.

  These protein arrays include, for example, measurement targets related to apoptosis, measurement targets related to cell signals, etc. for each research field, or a set of measurement targets (markers) related to specific diseases and viral infections, or a plurality of them. It is configured in the form of an ellipse and a configuration is assumed.

Commercially available protein arrays can be broadly classified as follows: 1) An antibody array that is an exhaustive antibody array and is used for differential analysis (Clontech Laboratories Inc .: Clontech ^™ Ab Microarray 500, etc.), 2) There are antibody arrays (manufactured by Invitrogen Corp .: Cartesian Array ^™ AKit Human cytokine, etc.) that can measure a plurality of items. There are various types such as an array (Sakai-Kato et al.) And a lectin array (Pyrobello et al.).

Better et al., Journal Cell Biochem Sample 39, 79-84 (2002) (Vetter D. et. Al., J. Cell Biochem. Suppl. (2002) Vol. 39, pp 79-pp 84). Chiari et al., Proteomics 5: 3600-3603 (Chiari M. et. Al., Proteomics (2005) Vol. 5, pp 3600-pp 3603) Sakai-Kato et al., Analytical Chemistry 77, 7080-7083 (2005) (Sakai-Kato, K. et. Al., Anal. Chem. (2005) Vol. 77, pp 7080-pp 7083) Pyrobello et al., Chembiochem 6: 985-989 (2005) (Pilello KT. Et. Al., Chembiochem. (2005) Vol. 6, pp985-pp989)

  Labeling substances used in protein arrays include fluorescein, rhodamine, Cy-5, Cy-3 or Alexa, fluorescent materials such as Quantum Dot (Empedcres et al.), Tag peptides such as FLAG, biotin modification There are enzymes such as biotin, alkaline phosphatase and peroxidase.

Empedo Cres et al. Special Table 2003-522926

  Furthermore, a method has been reported in which alkaline phosphatase is used as a labeling enzyme and biotinylated tyramide is immobilized around the area of alkaline phosphatase to be detected and sensitized with fluorescently labeled streptavidin (Geho et al.).

  Geho et al. Bioconjugate Chem 16 559-566 (2005) (Geho D. et. Al. Bioconjugate (2005) Vol. 16, pp559-pp566)

  The main thing of protein array slides is that the absolute volume capable of adsorbing proteins is small, and it is difficult to maintain the activity of proteins such as antibodies unless a stabilizer is used. In addition, slides with amino or carboxyl groups introduced on the surface often bind randomly to the protein or ligand of the antibody or the amino group of the ligand via a bivalent cross-linking group. There is a problem in which amino group or the like is used. An amino group or the like at a specific site directly has an extremely great influence on the antigen binding activity and the like. Even if the binding activity is not directly reduced, depending on the binding site, the binding to the measurement target may be reduced sterically, and an uncontrolled cross-linking mode is not preferable for the production of a protein array.

  In some cases, the thiol group and amino group on the slide surface are controlled for cross-linking with proteins. The number of thiol groups that can be generally used in proteins to be immobilized is limited (in particular, antibodies, (Fab) 2, (Fab), (Fab ') 2, (Fab'), etc.) After the amino group is treated with a maleimide reagent or the like, it can be bonded to the amino group on the slide surface. It is also possible to fix the free (reduced) thiol group of the protein and the reduced thiol group on the slide surface by oxidative condensation. When a thiol group is used, there is little possibility of reducing the activity of the original protein or the like by the immobilization step. However, when preparing (Fab) 2, (Fab), (Fab ') 2, (Fab') from antibody molecules (IgG), antigens are processed in the process of reduction, recovery of target samples, and purification. The binding activity decreases and the yield decreases. These complicated processing steps, especially when many types of antibodies are handled and a protein array is produced, are extremely serious obstacles when considering antibody lot management, quality control of processing steps, overall array performance management, etc. Become.

  For various slides described above, it is possible to construct spots such as circles and squares by using a protein solution with various arrayers / spotters or using a manual microarrayer (manufactured by Greiner BioOne). : Greiner bio-one GmbH Manual MicroArray for DNA & Protein Chip or PerkinElmer Piezo Array Biochip System: PerkinElmer INC. Piezoarray Biochip System).

  The antibody array used for differential analysis is an exhaustive antibody array, and the total amount of proteins contained in the sample to be compared is made equal, and then all of the proteins contained in each sample are mixed with two or more fluorescent dyes. It is necessary to label with (refer to the product manual of the aforementioned Clontech). Labeling with different fluorescent dyes can also result in altered protein binding.

  Arrays in which a plurality of antibodies are spotted (fixed) on the surface of a hydrophobic carrier (slide membrane) such as nitrocellulose are commercially available. As a measurement method, first, a sample solution (including an antigen to be measured) is bound with an immobilized antibody on an array, and then a secondary antibody solution is added to form a complex. Further, alkaline phosphatase or peroxidase-labeled antibody is reacted with this secondary antibody, and reacted with a primary antibody-antigen-secondary antibody-enzyme-labeled anti-secondary antibody complex and a predetermined chemically synthesized substrate, and the luminescence is emitted from the photographic film. Etc., and finally a signal spot is obtained. A schematic diagram of the measurement is shown in FIG. Alternatively, a method of visualizing as a visible pigment by an enzymatic reaction is also possible. However, due to the diffusibility of products produced by enzyme reactions, the difficulty of uniformly controlling enzyme reactions, the difficulty of making spot shapes uniform, etc., methods using these labeled enzymes are highly variable. It is a system. In addition, there are many cases where the final signal ranges overlap, and there is a case where the quantitativeness is questioned. In this measurement format, there is a limit to using a densely spotted array, and it is generally used as a macroarray rather than a microarray. Chemical arrays, peptide arrays, P450 arrays, lectin arrays, etc. have been reported academically, but there are many problems to be solved such as cost, reproducibility, stability, and measurement sensitivity in order to make a highly practical array. This is the current situation.

  Since fluorescein, rhodamine, Cy-5, and Cy-3, which are used as labeling substances, cause fading due to excitation light rapidly, it is difficult to maintain a fluorescence signal without time change necessary for analysis. This means that it is required to complete the measurement as soon as possible from the time when detection becomes possible. It also means that it is difficult to re-measure using the same slide or the like repeatedly. Fluorescent materials such as Alexa have improved light stability, but long exposures cause problems similar to those of the fluorescent dyes described above.

  Fluorescent materials based on quantum dots have high light stability and are stable even when irradiated with excitation light for a long time. In some cases, the quantum dot particles are coated with polyethylene glycol or the like to increase water solubility and reduce the background. However, in many cases, sufficient measurement sensitivity cannot be obtained even with the above-mentioned slide and water-soluble quantum dots. In order to obtain the required sensitivity, biotinylated tyramide is used with a labeling enzyme such as peroxidase. An amplification process of a physical signal is required, which is immobilized around a labeling enzyme and amplified with a signal, and then a streptavidin-labeled quantum dot is allowed to act on (see Geho et al.). For this reason, since an additional reaction step is required after the enzyme reaction is performed, the detection method is complicated and has many variations. In addition to the nonspecific reaction in the antibody reaction, it is necessary to consider not only the nonspecific reaction of biotinylated tyramide, but the final signal-to-noise ratio (S / N) may decrease.

  As a result of diligent efforts to solve the problems of the above-described protein array assay system, the present inventors can immobilize proteins in a quantitatively higher density than before, and at the same time retain the activity of proteins and the like. As a method for uniform signal formation, a fluorescent substance based on quantum dots that has extremely high optical stability is used as a detection system, and immobilization of physiologically active proteins has a three-dimensional structure including a hydrophilic structure. The present inventors have found that the method performed on the polymer gel is extremely suitable, and found that the problems of the conventional protein array can be solved, thereby completing the present invention. An example of a measurement system according to the present invention is shown as a schematic diagram in FIG.

That is, the present invention
1) Quantum dot fluorescence on a substrate that is physically or chemically immobilized in a three-dimensional gel compound with a protein that recognizes the substance to be measured or a substance selected from the whole, part, or similar of the substance to be measured The present invention relates to an assay method characterized by measuring a target substance in a sample using a compound as a label,
2) One or more of polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, polyethylene oxide, polyacrylic acid, polymethacrylic acid, polycarbonate, polylactic acid, polysaccharides, amino polysaccharides, complex sugars, nucleic acids, peptides, complex polymers The method according to 1), characterized in that a three-dimensional gel compound produced using a water-soluble polymer powder or an aqueous solution containing
3) One or more of polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, polyethylene oxide, polyacrylic acid, polymethacrylic acid, polycarbonate, polylactic acid, polysaccharides, aminopolysaccharides, complex sugars, nucleic acids, peptides, complex polymers Use a three-dimensional gel compound produced by crosslinking an aqueous solution of a water-soluble polymer containing benzene by exposing it to one or more of radiation, electron beam, gamma ray, radical generating reagent, and crosslinking reagent. 1) characterized in that
4) The substances to be measured are proteins, peptides, carbohydrates, lipids, glycolipids, low molecular compounds, and the method according to 1) to 3),
5) The method of 1) -4), wherein the protein that recognizes the substance to be measured is an antibody, a receptor, or a lectin,
6) The method according to 1) -5), wherein the immobilized substrate is a non-fluorescent glass, plastic, or metal substrate, or a glass, plastic, or metal substrate that does not interfere with measurement during fluorescence detection. Is,
7) The method according to 1) -6), wherein avidin or avidin-like substance and biotin are used in the measurement system,
And
8) It relates to the method 1) -7) characterized in that the physical signal is amplified in the measurement system,
9) relates to the method of 8), wherein a tyramide compound is used in the measurement system,
10) The method according to 1) -9), wherein a substrate having one or more array arrangements of two or more spots is used,
11) The method according to 1) -10), wherein a substrate having a reference spot in one or more array arrangements of two or more spots is used.
12) The method according to 1) -11), wherein a substrate having two or more kinds of reference spots is used in one or more array arrangements of two or more spots.
13) It relates to the method of 1) -12), wherein the diameter of the quantum dot fluorescent compound particles used is 0.1 to 200 nm,
14) It relates to the method of 1) -13) characterized in that a disease marker and a disease prevention marker are to be measured.
15) relates to a substrate for use in the method 1) -14),
16) It relates to a reagent composition for use in the method 1) -15).

  Glass, nylon, polycarbonate, polystyrene, metal plate or the like can be used as the immobilizing support substrate used in the present invention, and a substrate having a size of several centimeters × several centimeters or less is desirable. Although the thickness of a board | substrate can be set according to the hardness of a board | substrate, use of the thing about 0.1-5 mm with easy handling is preferable. Although the slide substrate may have various functional groups (amino group, carboxyl group, thiol group, etc.), it should avoid an obstacle to constructing a three-dimensional gel on the substrate. Further, it is necessary that the three-dimensional gel and the slide substrate are a combination of materials capable of optically detecting fluorescence after the reaction is completed. When using an optically transparent substrate, the fluorescence measurement may be performed by irradiating light from one direction of the substrate and detecting the resulting fluorescence at the top, but when using an optically opaque substrate such as a metal plate Light is emitted from one direction, and the resulting fluorescence is detected on the same side as the incidence.

The composition of the three-dimensional gel used in the present invention includes polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, polyethylene oxide, polyacrylic acid, polymethacrylic acid, polycarbonate, polylactic acid, polysaccharides, amino polysaccharides, complex sugars, and nucleic acids. -It is possible to use a three-dimensional gel compound produced by using a water-soluble polymer powder or aqueous solution containing one or more of peptides and complex polymers, including one or more of these. It is also possible to use a three-dimensional gel compound produced by crosslinking by exposing an aqueous solution of the water-soluble polymer to one or more of radiation, electron beam, gamma ray, radical generating reagent, and crosslinking reagent. . Examples of radical generating reagents include di-t-butyl peroxide, t-butyl hydroperoxide, azobisisobutyronitrile, azobisisovaleronitrile, benzophenone that is a photosensitizer, photophylline, rezaphyrin, and porphyrin dyes. , Metal complexes having a pyrrole ring, peroxides, and the like can be used. As the crosslinking agent, vinyl ether, aldehyde compounds, maleimide compounds such as EMCS, and the like can be used. Although these slides carrying a three-dimensional gel compound can be made by themselves under unique conditions, they can be prepared according to the previous reports of Travis et al. In addition, as a commercial product, for example, an HTA slide manufactured by Greiner BioOne or a hydrogel slide manufactured by PerkinElmer, Inc. can be used.

  Travis et al. Special Table 2003-524150   Shi et al. Special Table 2004-518138

  The three-dimensional gel can take a crosslinked structure inside, and the structure is important for controlling the size of pores inside the gel. After immobilization with the activity of the antibody, lectin / receptor / various antigens, etc., it is necessary that the pore size (20 to 1000 nm) allows each element other than the immobilization product to move freely during the reaction. The thickness of the three-dimensional gel can be selected between 1 μm and 500 μm. Proteins and the like can be spotted on a slide substrate by a hand or an automatic machine such as a protein arrayer, and a protein array can be prepared (manufactured by Greiner Bio-One, manual microarrayer: Greiner bio-one GmbH Manual Micro Arrayer). for DNA & Protein Chip or PerkinElmer Piezo Array Biochip System: PerkinElmer INC. Piezoarray Biochip System, Kaken Genetics DNA / antibody microarrayer, etc.). In the case of a hand, several spots to several hundred spots can be produced at a time by using an arrayer. The array can be manufactured in a contact format (a format in which the pins holding the sample are in direct contact with the substrate) or a non-contact format (a pin is not in contact with the substrate) depending on the performance of the arrayer / spotter. A plurality of arrays of several to 1,000 spots can be provided on the substrate according to the purpose.

3D gel can be coated by spotting (or coating) and fixing the required protein, and then dipping or coating in a solution containing animal-derived protein such as bovine serum albumin or gelatin and a polymer compound such as polyethylene glycol. Yes (this operation is also called a blocking operation). It can also be immersed or applied in a solution of sugar, amino acid, organic acid or the like. Glucose, saccharose, maltonose reducing sugars and non-reducing sugars, monosaccharides / polysaccharides can be used as sugars, glycine, glutamic acid etc. as amino acids, maleic acid, succinic acid, citric acid, malic acid etc. as organic acids Can be used. Furthermore, a solution containing a preservative such as sodium azide or procrine can be used in the same manner. The concentration of each reagent in the composition can be appropriately selected and set within a concentration range usually used in the field of immunoserology.
Each solution is preferably filtered and sterilized with a sterilizing filter or the like.

  The substrate or the like used in the present invention can be dried by a vacuum dryer, a vacuum dryer or an air drying operation. In consideration of antiseptic performance and the like, the work is preferably performed in a sterilized clean bench or clean room.

  As the quantum dot fluorescent material used in the present invention, semiconductor particles such as cadmium selenide and cadmium telluride can be used as they are, but particles with a hydrophilic polymer such as polyethylene glycol are used to suppress nonspecific reactions. It is preferable to coat the surface. Quantum dots have different emission wavelengths depending on their particle sizes, but those in the range of 0.1 nm to 200 nm can be used depending on the purpose. It is known that when the particle diameter is 1.5 nm or less, the ratio of so-called hole trapping phenomenon is high, and it exhibits white phosphorescence instead of fluorescence (Bauer et al.). It is necessary for various reactions in the gel that the particle size is not only smaller than the pore size of the polymer gel on the substrate but also the overall size when coupled with various proteins is smaller than the pore size. Quantum dots may be commercially available from Quantum Dot Corp. or Evident Technology.

  Bauer et al. Journal American Chemical Society 127 pages 15378-15379 (2005) (Bowers, MJ et al. J. Am. Chem. Soc. (2005) Vol. 127, pp 15378-15379).

  Proteins and quantum dots can be linked via various linkers such as a divalent cross-linking group. Moreover, it is also possible to link using the relationship between a protein and a protein having a specific binding strength to the protein or a ligand and a protein having a specific binding strength to the ligand. Examples of these include a complex of biotinylated mouse monoclonal antibody and quantum dot labeled streptavidin, a complex of rabbit polyclonal antibody and quantum dot labeled protein G, and the like.

  As a light source for detecting the quantum dots, it is sufficient that excitation necessary for fluorescence generation is possible, but a laser light source, an ultraviolet lamp, or the like can be generally used. The time required for irradiating the excitation light is not particularly limited because the quantum dots are stable to light irradiation, and continuous irradiation for several hours is possible. It is also possible to measure again after completion of the measurement.

  The antibodies used in the present invention can be those commercially available as research reagents and clinical diagnostics, and are prepared by immunizing mice, rats, rabbits, goats, cows, sheep, etc. by preparing an immunogen. be able to. As the immunogen, natural proteins, artificially processed proteins and peptides can be used. When producing antibodies against low molecular weight peptides, natural low molecules such as sugar chains and lipids, and synthetic chemical substances, proteins bound to serum albumin, hemocyanin or the like as haptens can be used as immunogens. A naturally occurring modification such as a glycated protein may be used. The antibody may be prepared according to the method of Harlow et al. Further, the antibody can be used not only in the complete form but also in the form of a fragment such as (Fab) 2 · (Fab) · (Fab ') 2 · (Fab'), or an antibody prepared by recombinant DNA technology, Antibody fragments (Fab, Fv, scFv, etc.) can also be used.

  Harlow et al. "Usage of Antibodies: Laboratory Manual" (1998) Cold Spring Harbor (Halling Antibodies: Laboratory Manual (1998) Cold Spring Harbor).

  As the protein other than the antibody used in the present invention, commercially available products and various biological samples such as serum and those collected and purified from living organisms prepared by recombinant DNA technology can be used. Their handling is preferably prepared by devising to maintain the activity necessary for interaction and binding. Further, depending on the purpose, not only a complete protein but also an incomplete protein in which sugar chains, lipids, and the like are dropped can be used. Furthermore, it is possible to use not only full-length proteins but also some fragments and peptides depending on the case.

  In the present invention, a low molecular chemical substance such as biotin or digoxigenin, a tag peptide or the like can be introduced into a protein such as an antibody and used at the time of detection. For example, a protein into which biotin has been introduced can be detected with quantum dot-labeled avidin or quantum dot-labeled streptavidin. Avidin and streptavidin can be either naturally derived or recombinant proteins. When a low molecular chemical substance or a tag peptide is introduced into a protein, it can be detected using a quantum dot-labeled antibody against them.

  In the present invention, the quantum dot signal can be measured without amplification, but an amplification system that generates biotinylated tyramide with an alkaline phosphatase label and a tyramide compound can also be used in combination. Since this system is extremely sensitive, a shorter time can be measured. A product of Dako A / S can be used as an amplification reagent for biotinylated tyramide.

  A plurality of spots on the protein array used in the present invention can be provided in a range of 2 to several thousand according to the purpose. The number of spots can be set according to the number of markers required for simultaneous evaluation of disease targets, the expected concentration range of each measurement target, etc., but this has a significant impact on protein array performance and manufacturing costs. Optimal array design should be implemented.

  The reference spot in the present invention refers to a spot for confirming the position of a positive spot, a negative spot or an array spot. These spots can be appropriately set and produced according to the number of objects to be measured and the purpose of measurement, but all of these types of reference spots are not essential in the present invention and can be selected. One or more reference spots are preferably installed for measurement standardization and management.

  In the present invention, various substances to be measured can be evaluated and measured. In particular, measurement of a disease marker group and measurement of a disease prevention marker are important. Disease-related markers include markers that specifically increase or decrease in amounts such as diabetes, kidney disease, cancer, hypertension, liver disease, myocardial infarction, rheumatism, allergies, atopy, stress, dementia, and infections (proteins, etc.) In other words, by evaluating a plurality of markers (multi-markers), it is possible to know the symptom / degree of the disease or the accuracy of the disease. For example, when subjected to oxidative stress, superoxide dismutase (SOD), glutathione transferase (GST), glutathione peroxidase (GPX), interleukin-18 (IL-18), 8- The amount of hydroxyguanosine (8-OHdG), hexanoyllysine and the like varies. These proteins vary during oxidative stress and during diseases such as diabetes. These markers include proteins in biological samples derived from diseased and healthy individuals, for example, SELDI-TOF-MS system (Ciphergen Biosystems Inc.) and two-dimensional electrophoresis system (Vita: WITA) It is possible to discover by comparing using GmbH) or the like. Several of these markers may be used at the same time. As long as each marker can be quantitatively secured, it is possible in principle to produce a specific antibody for the purpose of quantification without determining the amino acid sequence or structure.

  While disease marker abundance varies in a biological sample of a diseased patient, disease prevention markers can be observed even in normal conditions, indicating a risk as a pre-stage of disease onset. is there. The disease prevention marker refers to, for example, diabetes prevention marker, hypertension prevention marker, oxidative stress injury prevention marker and the like. If changes in disease prevention markers can be known as appropriate, it is possible to prevent or delay the onset of disease by, for example, changing or improving lifestyle habits or incorporating exercise therapy at an early stage. . This is not a cure, but is considered to be a good indicator for provoking prevention to healthy individuals. Considering the enormous costs spent by society as a whole on the disease, reducing the number of people who fall into disease by using preventive markers is adequate for the cost of measuring preventive markers.

  The reagent composition constituted by the present invention is mainly composed of a substrate on which a protein such as an antibody is immobilized, which is constituted as a protein array, for example, a sample diluted solution, a secondary antibody solution, a quantum dot labeled anti- (secondary antibody) antibody. It consists of solution, cleaning solution, measurement standard solution, etc. Each reagent can use research reagents and clinical diagnostic preparation techniques generally known in the field of immune sera and biotechnology.

  The fluorescence signal on the slide substrate or the like used in the present invention can be appropriately measured with a fluorescence microscope, a fluorescence slide scanner, or the like, and desired data can be acquired. The quantum dots can be excited with UV light or laser light, but for detection of excitation light, a necessary optical filter can be selected and used as appropriate according to the type of quantum dots.

  According to the present invention, stable signal changes corresponding to a plurality of substances to be measured can be captured simultaneously, each can be measured with high sensitivity, and a practical assay system can be provided. It is also possible to capture various types of signals as changes in color tone, enabling a variety of fluorescent multiplex assays.

  Embodiments of the present invention will be described below, but the present invention is not limited by the description of the examples, the methods, etc., and the disclosure of the present invention is equally applicable to essentially the same methods and reagent compositions. It has an impact.

The steps carried out in the present invention will be described below with an example in which antibodies are arrayed. 1) A substrate (slide glass) carrying a three-dimensional polymer gel on the surface is subjected to operations such as swelling, washing, and heating with pure water / buffer before spotting antibodies, and is suitable for immobilization. And 2) A mouse monoclonal antibody (primary antibody) solution against a specific antigen, which is prepared to a necessary concentration and composition in accordance with a predetermined array design using the above-described arrayer spotter, is spotted on the substrate in this state. 3) After spotting, hold for a certain period of time, bring the solution of blocking agent, protective agent and stabilizer into contact with the array, and leave or dry and use for measurement. 4) A hollow frame seal configured to secure a predetermined volume is attached to the periphery of the array. 5) Contact the blocking solution with the array before starting the measurement (in the frame seal. All the following steps are performed in this frame seal). In some cases, this step can be omitted. 6) The array containing the diluted biological sample is brought into contact with the array for a predetermined time. 7) Remove the solution containing the biological sample, add a predetermined volume of washing solution, and remove it after several tens of seconds to several minutes (washing operation). This operation is repeated a predetermined number of times. 8) A rabbit antibody (secondary antibody) solution against a specific antigen is brought into contact with the array for a predetermined time. 9) Repeat the cleaning operation a predetermined number of times.
10) A quantum dot labeled anti- (secondary antibody) solution is contacted with the array. 11) Repeat the washing operation a predetermined number of times. 12) The substrate (slide) is read with a fluorescence scanner or a fluorescence microscope, and the fluorescence signal (fluorescence intensity) generated at each spot is measured. It is preferable to prepare all measurement conditions such as exposure time.

  Changes and additions to the above series of operations can be made as appropriate, and these are considered as one variation of the present invention. For example, a combination of a secondary antibody and a quantum dot-labeled anti- (secondary antibody) antibody can be used by replacing the secondary antibody with a quantum dot directly labeled, and biotin is introduced into the secondary antibody. It is also possible to use quantum dot-labeled streptavidin.

  After pretreatment of PerkinElmer hydrogel slides according to the manufacturer's manual, anti-human SOD mouse monoclonal antibody (Lab Frontier: Lab Frontier: as shown in the array design in FIG. 3) by contact spotting method. Korea) was spotted at about 8 nL at each concentration, and then allowed to stand overnight. As a reference spot, mouse polyclonal antibody (mouse serum-derived IgG: manufactured by Sigma) and anti-human GST mouse monoclonal antibody, which have no binding to human SOD, were placed as negative controls. In addition, a rabbit antibody (Rabbit serum-derived IgG: manufactured by Sigma) is disposed as a positive control. One array of phosphate buffer (pH 7.2) (hereinafter abbreviated as BSA-PBS) containing 1% BSA and 0.15 M NaCl on a single array surrounded by a frame seal (MJ Research Inc .: MJ Research INC. USA) And blocking was carried out at room temperature for 1 hour. After removing the solution with a pipette, a standard solution of human SOD based on BSA-PBS (manufactured from human erythrocytes; Sigma) or human pool serum diluted 5 times with BSA-PBS (Chemicon International: Chemicon International: USA) ) 100 μL of solution was added onto one array surrounded by a frame seal. After the slide was kept in a moisturized chamber for 2 hours at room temperature, the solution was removed and washed three times with 100 μL of a phosphate buffer (pH 7.2) containing 0.15 M NaCl (hereinafter abbreviated as PBS). . Next, 100 μL of an anti-human SOD (Cu / Zn) rabbit antibody (Stressgen Biotechnology: Canada) solution (about 0.2 ng / mL) was added, and in the same manner as above, 2 μl at room temperature. Left to stand. After washing with 100 μL of PBS three times, a solution obtained by diluting a quantum dot-labeled (655) anti-rabbit IgG antibody (manufactured by Quantum Dot: Quantum Dot Corp .: USA) with BSA-PBS at 1: 200 was added in the chamber. It was allowed to stand at room temperature for 1 hour. After washing with 100 μL of PBS again three times, the sample was observed with a fluorescence microscope (Nikon research inverted microscope ECLIPSE TE2000E-U), the measurement image was transferred, and image analysis software Aqua-Lite Ver. The analysis was performed with 1.2.0.0 (manufactured by Hamamatsu Photonics). The acquired images are shown in [FIG. 4] (measurement of human SOD standard solution) and [FIG. 5] (measurement of human pooled serum). The background of the image is black, and each spot where a signal is generated is displayed in gray.

  [FIG. 3] From [FIG. 4] and [FIG. 3] [FIG. 5], no signal was observed in the spot portion where a mouse antibody having no binding to the measurement antigen, which is a negative control, was immobilized. A clear signal could be confirmed in the spot portion of the primary antibody.

  Using the same slide as the slide immobilized with the anti-human SOD mouse monoclonal antibody used in Example 1, one array surrounded by a frame seal was blocked in the same manner as in Example 1. After blocking, 100 μL of a solution obtained by diluting a quantum dot-labeled (655) anti-mouse IgG antibody (manufactured by Quantum Dot) to 1/200 in BSA-PBS was added, and left in a chamber for 1 hour at room temperature. Furthermore, it wash | cleaned 3 times by 100 microliters of PBS, and it observed with the fluorescence microscope similarly to Example 1, and performed image analysis. The image at this time is shown in FIG. The background of the image is black, and each spot where a signal is generated is displayed in gray.

  [FIG. 6] From the fact that it was found that all the primary antibodies were spotted on the slide according to the concentration, and all the portions where the mouse antibody was immobilized as a negative control in Example 1 could be detected. Together with the results of Example 1, it was determined that a human SOD sandwich assay was established on the protein array.

  In place of the anti-human SOD mouse monoclonal antibody which is the primary antibody of Example 1, a protein array was similarly prepared using an anti-human IL-18 mouse monoclonal antibody (manufactured by Medical and Biological Research Institute (MBL)). did. The mouse antibody of Example 1 was placed as a negative control as a reference spot. The array design is shown in FIG. Next, after blocking in the same manner as in Example 1, 100 μL of a standard solution of human IL-18 (recombinant human IL-18; manufactured by MBL) based on BSA-PBS was enclosed in a frame seal. Added on top. The slide was kept in a moisturized chamber, reacted at room temperature for 2 hours, and then washed 3 times with 100 μL of PBS. Subsequently, 100 μL of an anti-human IL-18 rat monoclonal antibody (MBL) solution (about 0.3 ng / mL) was added, and the mixture was allowed to stand at room temperature for 2 hours in a chamber. After washing with 100 μL of PBS three times, a solution obtained by diluting a quantum dot (655) -labeled anti-rat IgG antibody (manufactured by Quantum Dot) to 1/200 with BSA-PBS was added, and left at room temperature for 1 hour in a chamber. . After washing with 100 μL of PBS three times, the sample was observed with a fluorescence microscope in the same manner as in Example 1, and the measurement image was transferred and analyzed with image analysis software. The acquired image is shown in FIG. The background of the image is black, and each spot where a signal is generated is displayed in gray.

  [FIG. 7] From [FIG. 8], no signal was observed in the spot portion where the mouse antibody having no binding property, which is a negative control, was immobilized, and a clear signal was confirmed in the spot of the reactive primary antibody. did it.

  Using the same slide as the slide immobilized with the anti-human IL-18 mouse monoclonal antibody used in Example 3, one array surrounded by a frame seal was blocked in the same manner as in Example 1. After blocking, 100 μL of a solution obtained by diluting a quantum dot-labeled (655) anti-mouse IgG antibody (manufactured by Quantum Dot) to 1/200 in BSA-PBS was added, and left in a chamber for 1 hour at room temperature. Furthermore, it wash | cleaned 3 times by 100 microliters of PBS, and it observed with the fluorescence microscope similarly to Example 1, and performed image analysis. The image at this time is shown in FIG. The background of the image is black, and each spot where a signal is generated is displayed in gray.

  [FIG. 9] From the fact that it was found that all the primary antibodies were spotted on the slide according to the concentration, and as a negative control in Example 3, it was possible to detect all the parts where the mouse antibody was immobilized. Together with the results of Example 3, it was determined that a human IL-18 sandwich assay was established on the protein array.

  Among the assays of Example 1, the concentration of the human SOD standard solution was changed in the range of 10 to 2000 ng / mL, and the assay was performed using one array each. The relationship between the obtained fluorescence intensity (n = 9) and the SOD concentration at each concentration is shown in FIG. The concentration when the primary antibody is spotted is 400 ng / mL and 200 ng / mL.

  [FIG. 10] From FIG. 10, the SOD concentration and the fluorescence intensity correlated with each other at two levels of the primary antibody concentration. It was found that human SOD at a concentration of at least 10 ng / mL can be detected even when variations are taken into account.

  In the assay of Example 3, the concentration of the human IL-18 standard solution was changed in the range of 10 to 1000 ng / mL, and each array was used. The relationship between the obtained fluorescence intensity (n = 9) and IL-18 concentration at each concentration is shown in FIG. The concentration when the primary antibody is spotted is 400 ng / mL and 200 ng / mL.

  [FIG. 11] From FIG. 11, the IL-18 concentration and the fluorescence intensity correlated with each other at two levels of the primary antibody concentration. It was found that human IL-18 at a concentration of at least 10 ng / mL can be detected even when variations are taken into account.

  The present invention can be used by researchers and general consumers to evaluate and quantify various substances present in biological fluids, foods, environmental samples and the like alone or in combination. The present invention can be applied in a wide range of fields such as medicine, pharmacy, science, and engineering.

  The schematic diagram of the measurement system in the conventional protein array.   The schematic diagram (example) of the measuring system in the protein array by this invention.   The array design of the primary antibody of the protein array for human SOD measurement produced by this invention.   Image visualizing the presence of human SOD in a standard solution according to the present invention. (Background is black, signal is displayed in gray).   Image visualizing the presence of human SOD in human pooled serum according to the present invention. (Background is black, signal is displayed in gray).   An image visualizing the presence of an anti-SOD mouse monoclonal antibody (primary antibody) immobilized on a protein array. (Background is black, signal is displayed in gray).   The array design of the primary antibody of the protein array for human IL-18 measurement produced by this invention.   Image visualized human IL-18 in standard solution according to the present invention. (Background is black, signal is displayed in gray).   The image which visualized presence of the anti- IL-18 mouse monoclonal antibody (primary antibody) fix | immobilized by the protein array. (Background is black, signal is displayed in gray).   Relationship diagram between SOD concentration and fluorescence signal at each concentration when evaluated by the measurement system according to the present invention   Relationship diagram between IL-18 concentration and fluorescence signal at each concentration when evaluated by the measurement system according to the present invention

Claims (16)

  A quantum dot fluorescent compound is placed on a substrate on which a protein recognizing the measurement target substance or a substance selected from all, part, or similar of the measurement target substance is physically or chemically immobilized in a three-dimensional gel compound. An assay method comprising measuring a target substance in a sample as a label.   Polyvinyl pyrrolidone / polyethylene glycol / polyvinyl alcohol, polyethylene oxide / polyacrylic acid / polymethacrylic acid / polycarbonate / polylactic acid / polysaccharides / aminopolysaccharides / complex sugars / peptides / complex polymers containing one or more water The method according to claim 1, wherein a three-dimensional gel compound produced using a powder or aqueous solution of a functional polymer is used.   Polyvinyl pyrrolidone / polyethylene glycol / polyvinyl alcohol, polyethylene oxide / polyacrylic acid / polymethacrylic acid / polycarbonate / polylactic acid / polysaccharides / aminopolysaccharides / complex sugars / peptides / complex polymers containing one or more water It is characterized by using a three-dimensional gel compound produced by crosslinking an aqueous solution of a conductive polymer by exposing it to one or more of radiation, electron beam, gamma ray, radical generating reagent, and crosslinking reagent. The method of claim 1.   The method according to claims 1 to 3, wherein the substance to be measured is a protein, peptide, carbohydrate, lipid, glycolipid, or low molecular weight compound.   The method according to claim 1-4, wherein the protein recognizing the substance to be measured is an antibody, a receptor, or a lectin.   6. The method according to claim 1, wherein the fixed substrate is a non-fluorescent glass / plastic / metal substrate or a glass / plastic / metal substrate which does not interfere with the measurement when fluorescence is detected.   The method according to claim 1, wherein avidin or avidin-like substance and biotin are used in the measurement system.   The method according to claim 1, wherein a physical signal is amplified in the measurement system.   The method according to claim 8, wherein a tyramide compound is used in the measurement system.   10. The method of claim 1-9, wherein a substrate with one or more array arrangements of two or more spots is used.   11. The method of claim 1-10, wherein a substrate with a reference spot in one or more array arrangements of two or more spots is used.   12. The method of claim 1-11, wherein a substrate having two or more reference spots in one or more array arrangements of two or more spots is used.   The method according to claim 1, wherein the diameter of the quantum dot fluorescent compound particles used is 0.1 to 200 nm.   The method according to 1) -13), wherein a disease marker and a disease prevention marker are to be measured.   A substrate for use in the method of claims 1-14.   A reagent composition for use in the method of claim 1-15.

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