JP2005504309A - Methods and arrays for detecting biomolecules - Google Patents

Abstract

An array and method for detecting one or more biomolecules, the method generally comprising providing a first support on which one or more reagents are immobilized; Providing a second support on which the species or two or more ligands are immobilized, and contacting the reagent immobilized on the first support with the ligand immobilized on the second support; Or two or more reagents bind to one or more ligands, and after separating the first support from the second support, the one or more combined reagents are second supported. Maintaining binding with one or more ligands in the body. In one preferred method, the protein is sufficiently strong with the support that the protein can be dissociated from the support under certain conditions, such as after binding to other proteins immobilized on another support. Combined. For example, antibody arrays formed in accordance with the present invention can be used to detect protein expression in protein lysates and can be used in immunostaining to reveal the presence and location of proteins in cells. Is possible.

Description

[0001]
The present invention relates generally to novel methods and arrays for detecting a plurality of proteins, and more particularly to methods and arrays for detecting the expression, activity and function of a plurality of proteins.
[0002]
The availability of a large number of biological reagents such as hundreds of thousands of cloned DNA sequences, a large number of antibodies and recombinant proteins, and millions of compounds obtained by recombinant chemical synthesis, The development of technology using these reagents in pharmaceutical development was promoted. Special arrays of biological reagents have been designed that are addressable, in which each reagent is placed in place so that each reagent can later be identified by position. For example, in a DNA array, a large number of cDNAs or oligos can be immobilized at predetermined positions, respectively, and later identified by the positions. DNA arrays are used in large-scale hybridization assays for applications such as gene expression monitoring (Schena et al., 1995, Science 270: 467-470; DeRisi et al., 1996, Nature Genetics 14: 457 -460). An array of DNA clones in an expression vector is used to express the encoded protein in mammalian cells (Ziauddin and Sabatini, 2001, Nature 411, 107-110).
[0003]
In a protein array, many proteins can each be immobilized on a support at a predetermined location and subsequently identified by this unique location. Two types of protein arrays are particularly used: antibody arrays and recombinant protein arrays. Each of these contains multiple antibodies and multiple recombinant proteins. Because antibody arrays can bind to cellular proteins, they are particularly useful for revealing proteins with in vivo activity. This technique thus allows the study of the properties of multiple cellular proteins in a single assay. In particular, antibody arrays have been used to study in vivo protein-protein interactions, protein post-translational modifications and protein expression patterns (US Pat. No. 6,197,599).
[0004]
In addition, cells, tissues, lipids, polymers, drugs and other chemicals are prepared for large-scale screening assays in medical diagnostics, drug discovery, molecular biology, immunology and toxicology (Kononen et al. , Nature Medicine, 4: 844-7, 1998).
[0005]
Proteins are important components of cells, are the main players in various cellular processes, and are the target of most medicines. The entire human genome encodes approximately 40,000 proteins. Although any cell may contain DNA encoding all proteins, only some of these are normally expressed. Cell lines normally express about 10,000 proteins, and many more are expressed in tissues. The protein expression pattern of the cell determines the shape and function of the cell, and abnormal protein expression causes disease. Thus, one major task of proteomics is to identify proteins expressed at any source.
[0006]
Proteins (having the same primary amino acid) can exist in different forms in the cell primarily due to post-translational modifications. In many cases, only special post-translationally modified proteins are activated and directly related to cellular processes, so detection of the presence of these activated proteins in cells is Enables the provision of valuable information in the process.
[0007]
There are many different protein post-translational modifications such as phosphorylation, glycosylation, and ubiquitination. And they play an important role in regulating protein activity. Phosphorylation at serine, threonine or tyrosine residues is an important mechanism of signal transduction. Abnormal protein phosphorylation contributes to many human diseases. Among the methods for detecting protein phosphorylation, metabolic labeling of cells having radioisotopes and immunodetection with antibodies to phosphoproteins are most widely used. However, these methods are usually only applicable to the analysis of one or several proteins at a time. Antibodies specific for phosphorylated amino acids, such as PY20 and 4G10, can reveal multiple phosphorylated proteins, but these alone cannot identify individual phosphorylated proteins. A novel method for simultaneously detecting the presence or absence of multiple phosphorylated proteins or other modified proteins is highly desirable for information transfer research and clinical diagnosis.
[0008]
Quantification of protein expression is applied in various fields, including biomedical research, disease diagnosis, and identification of therapeutic markers and targets, and in the profile of cellular responses to toxins and drugs. In basic biomedical research, it is usually desirable to know what proteins are expressed in specific cells or under specific conditions. Then, by comparing protein expression profiles between different cell types, those proteins whose expression and activity characterize a particular cell type can be identified. Specific proteins are specifically activated in many signaling pathways, and detection of these active proteins, such as phosphorylated proteins, can provide important information in the activation of specific signaling pathways.
[0009]
Many diseases alter protein expression, and abnormal protein expression is often responsible for these diseases. Thus, determination of protein expression profiles and comparison of expression profiles between normal and abnormal biological samples are useful for understanding disease mechanisms. Detection of the presence of a protein is also useful for clinical diagnosis. For example, testing for the presence of several viral proteins instead of only one viral protein in a blood sample is a more reliable diagnostic method for viral infection. The protein profile would be very useful in distinguishing normal cells from malignant metastatic cancer cells that are early cancer and real murderers. In addition, protein expression profiles are useful in key areas of drug development, such as drug target selection, toxicology, and identification of surrogate markers of drug response.
[0010]
It has been a long-standing goal of molecular biologists to develop techniques that can quantify the expression level of every individual protein and the different forms of each protein in a biological sample in a reliable and reproducible manner. However, it has proved extremely difficult to achieve this. Traditionally, one or a few proteins can be detected by immunological methods such as Western blotting and enzyme-linked immunosorbent assay (ELISA). Two-dimensional gel electrophoresis can be used to analyze proteins expressed in a sample. However, this requires complex procedures and requires determining the identity of the protein displayed on the two-dimensional gel, which is difficult to achieve for most proteins. In recent years, protein arrays have been used to study protein expression patterns. In one strategy (US Pat. No. 6,197,599; Haab, et al., Genome Biol. 2, research0004.1-0004.13, 2001), antibody arrays are incubated with protein samples, and after incubation and washing, the Proteins specifically bound to each antibody in the array are detected.
[0011]
Immunochemical staining is a versatile technique to determine both the presence and location of antibodies (Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Press, 1988), and this information is used in biomedical research and clinical It is a huge value in medicine. Many of the current methods that use the step of incubating the antibody solution with the cells only allow the cells to be stained with one or several antibodies at a time. These methods are not suitable for applications that require examination of the expression and subcellular localization of many different proteins. Therefore, for such purposes, new methods are needed that can stain cells with a number of different antibodies.
[0012]
The object of the present invention is to allow the position of the reagent to be maintained on the array support and when the reagent is placed in contact with an interaction partner immobilized on another support, It is to provide an array of biological reagents that allows dissociation from the support.
[0013]
It is also an object of the present invention to provide a method for forming and using a novel array of biological reagents that detect one or more proteins at a time from other ligands. Preferred methods of the present invention are adapted to various support materials and immobilization methods for forming and using the array.
[0014]
Furthermore, the object of the present invention is to detect an array of biological reagents in order to detect proteins and their properties, in particular to detect and compare the presence and subcellular location of one or more proteins in a cell. It is to provide a method and an array that make it possible to use.
[0015]
The methods and arrays of the present invention are designed to substantially expand the use of biological arrays in the field of protein and other biomolecule detection. For example, an array of antibodies preferably immobilized on a solid first support is contacted with a protein sample immobilized on a second support such that one or more antibodies bind to their respective antigens. However, after binding, these antibodies are dissociated from the first support while maintaining association with the antigen on the second support.
[0016]
Suitable methods of the invention for detecting one or more biomolecules generally comprise providing a first support on which one or more reagents are immobilized, one or more Providing a second support on which two or more ligands are immobilized, and contacting the reagent immobilized on the first support with the ligand immobilized on the second support, A step of binding two or more reagents to one or two or more ligands, a step of separating the first support from the second support, and one or more reagents in the second support. Detecting. The reagent is preferably immobilized on the first support with a strength sufficient to immobilize the reagent in the contacting step and to allow the bound reagent to be dissociated from the first support in the separation step. It becomes. The reagent immobilized on the first support may contain one type or two or more types of antibodies.
[0017]
Before the ligand is immobilized on the second support, the gel electrophoresis is based at least in part on molecular weight, gel electrophoresis on the basis of at least part of the isoelectric point, at least partly on molecular weight and isoelectricity. Separated from each other using a variety of methods including, but not limited to, two-dimensional gel electrophoresis based on one or both points and immunological methods such as using one or more antibody arrays. obtain. Said reagent may be selected from the group consisting of antibodies, recombinant proteins, DNA, RNA, oligonucleotides, carbohydrates and small chemicals. These reagents may be immobilized at one or more predetermined positions on the first support. If the selected reagent is one or more antibodies, the antibodies can be specific for post-translationally modified proteins such as phosphorylated proteins.
[0018]
One or more reagents may be immobilized at one or more predetermined positions on the support, respectively. The number of different types of reagents on any arbitrary support is preferably 2-50, 5-100,000, 200-10,000, 50-1,000, 5-500, 5-100, and 5-50.
[0019]
The first and second supports may comprise a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride, glass or synthetic resin, and derivatives thereof.
[0020]
Another preferred method of the present invention for detecting one or more biomolecules in a cell generally comprises: (a) the antibody can be identified by the location at which the antibody is immobilized. Immobilizing one or more antibodies supplied at a predetermined position on one support to the first support; (b) placing the cells on the second support; (c) Contacting the antibody on the first support with the cell on the second support to allow the antibody to bind to any corresponding antigen in the cell; (d) the first After separating the support from the second support, one or more of the bound antibodies maintain binding to the corresponding antigen; and (e) the cells in the second support. Detecting the antibody bound to the antigen therein Tsu and a flop. This method can utilize cells immobilized on the second support, and the cells can comprise one or more tissue sections.
[0021]
The antibody can be specific for a post-translationally modified protein such as a phosphorylated protein. The number of different types of reagents that can be immobilized in one or more predetermined locations can include 5-100,000, 200-10,000, 50-1,000, 5-500, and 2-50. . The support may comprise a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride, glass, or synthetic resin and derivatives thereof. Proteins such as antibodies can be immobilized on the support in one or more shapes selected from the group consisting of circular, elongated and polygonal shapes.
[0022]
The first and second supports can include an array. The array can be formed using a variety of materials including, but not limited to, a large number of whole or partial capillaries.
[0023]
The antibody can be immobilized by applying one or more intermediates selected from the group consisting of modified protein A and modified protein G to the support. Protein A or protein G is modified to change the binding affinity. One or more of the antibodies are utilized for binding to at least one constant region configured to link to one or more of the modified proteins and one or more antigens It may have at least one variable region possible.
[0024]
The detection step preferably comprises determining whether one or more of the antibodies are present on one or both supports, and one or more positions of the antibodies after the separation step. One or two selected from the group consisting of: identifying one or more quantities of said antibody; and identifying one or more types of antibody The above detection steps are included.
[0025]
Another preferred method of the invention for detecting one or more biomolecules includes (a) immobilizing one or more ligands on a second support, and (b) one species. Or immobilizing to the first support one or more reagents adapted to interact with two or more of the ligands, and (c) one or more of the reagents 1 Contacting said ligand with said ligand to allow binding to a species or two or more of said ligands; (d) cross-linking one or more of said reagents to one or more of said ligands (E) in order to allow the one or more of the reagents bound to one or more of the ligands to be dissociated from the first support, the first support is Separating from the second support; and (f And detecting one or more of the reagents in the second support.
[0026]
One or more of the ligands and the reagents may be cross-linked using at least partially one or more aldehydes, preferably but not limited to formaldehyde and glutaraldehyde.
[0027]
One preferred embodiment of an array of the invention for use in detecting biomolecules is generally adapted to at least temporarily immobilize one or more reagents and one or two. Including a first support adapted to be placed in contact with a second support on which the ligand is immobilized, one or more of the reagents are one or more of the reagents It is allowed to bind to a ligand and subsequently dissociates from the first support and is allowed to maintain binding to the ligand on the second support.
[0028]
The support may include various materials including, but not limited to, one, two or more materials among nylon, nitrocellulose, polyvinylidene difluoride, glass or synthetic resin, and derivatives thereof. For example, the support may include one or more nylon membranes, or all or part of one or more capillaries. One or more of the reagents immobilized on the support may be immobilized in one or more shapes selected from the group consisting of a circular shape, an elongated shape, and a polygonal shape.
[0029]
The present invention relates to a novel array of biological reagents and methods of forming and using the array. The method includes the steps of providing a first support for immobilizing one or more reagents, providing a second support for immobilizing one or more ligands, and the reagents Contacting the ligand with one or more reagents binding to the ligand and, after binding, separating the support, the bound reagent maintains binding to the ligand. And detecting and / or comparing various biological targets.
[0030]
One of the methods allows a reagent immobilized on a solid support to bind to an interaction partner that is immobilized on another support. In particular, the antibody array is formed so as to be dissociable from the support after the immobilized antibodies bind to the respective antigens immobilized on the second support. Each antibody / antigen binding occurs at a predetermined position.
[0031]
The term “reagent” as used herein refers to any biologically relevant molecule such as antibodies, recombinant proteins, synthetic peptides, DNA, RNA, nucleotides and small chemicals.
[0032]
The term “ligand” as used herein refers to any biomolecule that interacts with one or more reagents.
[0033]
The term “array” as used herein refers to a device that includes, but is not limited to, a solid support and a plurality of reagents or ligands. For example, the antibodies can be immobilized to the support at each predetermined location so that each antibody can be identified by a particular location on the support.
[0034]
As used herein, the term “immobilization” refers to the restraint of the reagent on the support, thus limiting the operation of the reagent on the support. For example, when the antibody is immobilized on a support, the antibody is attached to the support and is not dissociated from the support, and the operation of the antibody on the support is also limited. However, under some conditions, as described in the present invention, the immobilized reagent can be dissociated from the support. The physical and chemical properties of this immobilization determine whether the immobilized reagent can dissociate from the support and how effective the dissociation is.
[0035]
As used herein, the term “support” means, for the specification and claims, a structure on which a biological reagent is deposited and immobilized. In a preferred embodiment, the support is a rigid plate (glass or synthetic resin) or membrane formed from, but not limited to, nitrocellulose, nylon, polyvinylidene difluoride (PVDF) or derivatives thereof. The membrane is easier to handle and the reagent can be easily immobilized. Glass or synthetic resin plates provide a rigid support and are required in some applications.
[0036]
Preferably, the solid supports are pretreated so that the biological reagents deposited on them can be fixed with a strength suitable for the particular application. One method of solid support treatment is to coat with a polymer layer that will then interact with the biological reagent by non-specific non-covalent bonding. For example, polymers including polylysine or polyethyleneimine can be used to coat glass slides or coverslips used for biomolecule immobilization.
[0037]
For example, Lehnrach et al. (Hybridization fingerprinting in genome mapping and sequencing, genome analysis, Vol. 1, Davies and Tilgham, Eds, Cold Spring Harbor Press, pp. 39-81, 1990) and Brown et al. (US Pat. No. 5,807,522). Several techniques, such as those described by No.) can be used to deposit and immobilize multiple biological reagents on a solid support. Each of the aforementioned papers is incorporated by reference in its entirety. For example, several nanoliters of antibody in aqueous solution can be printed on a glass slide using a robotic array. Thus, an array of biological reagents is formed by depositing multiple reagents on a flat solid support, one or several reagents at a time, and each reagent in place.
[0038]
Immobilization of antibodies can be done by adsorption (Trevan, 1980, Immobilized Enzymes: an introduction and their application in biotechnology. Wiley, Chichester). The adsorptive power involved can be non-specific hydrophobic or ionic interactions. Primarily, the adsorbent materials used include, but are not limited to, ion exchangers such as clay, charcoal, hydroxyapatite, and most often DEAE-Sephadex.
[0039]
Entrapment is another method for immobilizing antibodies (Trevan, 1980, Immobilized Enzymes: an introduction and their application in biotechnology. Wiley, Chichester). This entrapped antibody is not attached to the polymer, and these free diffusions are merely suppressed. One widely used matrix is a polyacrylamide gel. In certain preferred embodiments, the capillary facilitates arraying and immobilization of biological reagents. The term “capillary” is used to mean any fully or partially enclosed elongated structure that can contain and support a biological reagent. The capillaries are formed from materials such as synthetic resins and glass, which preferably do not interfere with the properties of the biological reagent. The height of the capillary will vary from a few micrometers to a few meters. Biological reagents are usually filled into capillaries in solution. After filling, the reagent solution is coagulated and the reagent is immobilized. The strength of immobilization can depend on any application.
[0040]
The reagent is immobilized directly or indirectly on the solid support. For example, the reagent is deposited directly at a high density on the support, which can be as small as a microscope slide. Similar techniques have been developed to form high density DNA microarrays (Shalon et al., Genome Reseatch, 1996 Jul; 6 (7): 639-645). The reagent can also be indirectly immobilized on the support. For example, protein A or protein G or mutants thereof can be first printed on the support as an intermediate. The antibody is then immobilized on a support by interaction with protein A or G. One advantage of the present invention is that when the antibody constant region is linked to protein A or G, the variable region of the antibody (antigen-binding domain) is fully exposed and available for binding to the antigen. That is. Another advantage is that protein A or G can be modified to alter the binding affinity of the antibody, so that when a carefully designed mutant of protein A or G is used, the antibody has the desired strength. It can be immobilized on the support. Thus, the antibody can be immobilized on the support on the one hand without losing positional information, and on the other hand can be separated from the support and bind to other ligands of higher affinity. Recombinant fusion proteins can be immobilized by interaction between the tags of these proteins and the ligand attached to the support. For example, a ligand (eg, glutathione or nickel) can first be covalently attached to a support, after which a recombinant fusion protein containing a tag (eg, GST or 6xHis) is interacted with the ligand. Immobilized on the support. Tags and ligands can be modified to change their affinity, and thus immobilization can have the desired strength.
[0041]
Antibodies are usually deposited on a support, usually in the form of circular dots. However, antibodies can also be deposited in other shapes. For example, the antibody can be immobilized in an elongated shape, for example, a rectangle that is several microns to several centimeters wide and several microns to several centimeters long. The antibody immobilized in such a form can be used to bind to the antigen after separation according to various methods.
[0042]
Antibodies specific for the antigen can be immobilized at specific locations on the support. In general, antibodies are immobilized at a location where each antibody will contact its own antigen when the antibody contacts a protein sample. Thus, a particular antibody can be immobilized at a particular location determined by the location of the antigen. For example, antigens are first separated by two-dimensional gel electrophoresis, and are moved and immobilized on a support. Each antigen is immobilized at a specific location determined by its molecular weight and isoelectric point. Therefore, each antibody is immobilized according to the position of the antigen on the support.
[0043]
The term “protein sample” as used herein refers to various proteins and protein mixtures. For example, this may be a lysate from a cell line or tissue. Alternatively, it may be an intact cell or a cell fixed to a support. The protein sample may be from different sources, for example, but not limited to, cultured cell lines, human or animal tissue, or blood.
[0044]
In certain preferred embodiments, the protein sample is a cultured cell or tissue section that can be placed and immobilized on a glass slide. Prior to use, these cells can be fixed and permeabilized to expose the protein. These fixed cells retain specific cell morphology, and many proteins remain at their original cell location. There are multiple ways to fix and permeabilize cells and tissues (Harlow and Lane, Antibodies, a laboratory manual, Cold Spring Harbor Press, 1988). Widely used fixatives are formaldehyde or glutaraldehyde solutions. Another widely used fixative solution alternatively contains methanol.
[0045]
Suspended cells such as lymphocytes settle on the support and are immobilized thereon. These are sectioned and placed on a support after being embedded in a medium such as paraffin, collagen or gelatin. Many known methods are available for preparing cell samples (Jones, TC, Ward, JM, Mohr, U. and Hunt, RD (editors), 1990, Hemopoietic System. Berlin, Springer-Verlag; Polak, JM and Van Noorden, S., 1997, Introduction to Immunocytochemistry. New York, Springer).
[0046]
In another preferred embodiment, the protein sample is prepared from cells or tissues by lysis. A typical lysis buffer contains a surfactant such as sodium dodecyl sulfate (SDS), Triton X-100. Immobilization of a protein sample may be covalent or non-covalent and immobilization methods are known in the art. The dimensions of the region where the protein sample is immobilized on the support will depend on the application and the volume and amount of the immobilized protein sample. For example, if a membrane is used as a support, if the membrane has a binding capacity of 10 mg protein per square centimeter, then up to 10 mg of protein lysate may be immobilized on a membrane with dimensions of 1 centimeter square. it can.
[0047]
The protein lysate can be uniformly placed on the support and immobilized thereon. However, for other applications, the protein can be first separated and then immobilized on a support. Many methods for protein separation are known in the art. For example, proteins may be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to molecular weight, or by two-dimensional electrophoresis according to both molecular weight and isoelectric point. After separation by electrophoresis, the protein can be moved and immobilized on the support.
[0048]
In another preferred embodiment, protein samples can be separated by immunological methods such as separation by antibody arrays. The protein sample is first incubated with the antibody array on the first support so that the antibody can capture and separate the antigen present in the protein sample. After washing away the unbound protein, the protein captured at each position is dissociated from the antibody and transferred to the second support and immobilized thereon. The interaction between the antibody and the first solid support can be considerably stronger than the interaction between the antibody and the antigen. Thus, conditions are required to disrupt the antibody-antigen interaction while leaving the antibody immobilization on the first support intact. Under such conditions, the antigen excluding the antibody can be transferred to and immobilized on the second support. In order to prevent the antibody from dissociating from the first solid support, the antibody is covalently immobilized on the support.
[0049]
In a typical transfer, a first support containing antibody and antigen is contacted with a second support. They are then placed in a buffer that can disrupt the interaction between the antigen and the antibody. At the same time, an electric current is applied so that the dissociated protein moves from the first support to the second support. After completion, the two supports are separated. This movement and immobilization can be performed simultaneously (proteins are moved and immobilized simultaneously) or sequentially (immobilized after the proteins are moved). And if immobilization is weaker than desired, the protein may be further immobilized, for example by covalent bonds. Methods for covalent immobilization of proteins on solid supports are known in the art.
[0050]
Once prepared, the protein sample is contacted with another antibody array. Preferably, the contacting is done such that each antibody contacts the corresponding antigen to allow specific binding. The antibodies in this array are dissociated from the support. Thus, after separation of the antibody array support and the protein sample support, antibodies that bind to the antigen will be present on the sample support. By detecting the antibody, the presence or absence and abundance of the antigen in the sample is detected.
[0051]
Part of the method is adapted such that an antibody immobilized on one support can bind to an antigen immobilized on the other support. In one preferred method, an antibody immobilized on one support is allowed to interact with an antigen immobilized on a second support by placing the two supports together in contact. The After a period of incubation, the two supports are separated. Depending on the interaction between the antibody and the antigen, some antibodies will dissociate from the first support and bind to the second support to which the antigen is immobilized. The strength of antibody immobilization on the first support is preferably weaker than the interaction between the antibody and the antigen, and is weaker than the strength of immobilization of the antigen on the second support. Conditions such as an electric field can be applied to facilitate the dissociation of the antibody from the support.
[0052]
After binding of the antigen and antibody, the antigen-antibody complex can be stabilized using covalent cross-linking. Crosslinking is preferably performed such that the antibody / antigen complex is crosslinked while the antibody is not crosslinked to the support. This is accomplished by selecting a cross-linking substance specific for the antibody and a special support. There are hundreds of known cross-linking substances, and various methods have been developed to use them to cross-link proteins (Wong, Shan S., Chemistry of protein conjugation and cross-linking. Boca Raton: CRC Press, 1993). For example, widely used cross-linking materials include aldehydes such as but not limited to formaldehyde and glutaraldehyde.
[0053]
The antibody bound to the antigen is then detected. Many known methods can be used for antibody detection. A common method is to use enzyme-conjugated secondary antibodies, such as horseradish peroxidase or alkaline phosphatase-conjugated anti-rabbit goat antibodies and anti-mouse goat antibodies. A fluorescently labeled secondary antibody may be used. Other techniques that can be used are immuno-PCR (Sano et al., 1992, Science 258, 120-122), rolling circle DNA amplification technology (Schweitzer et al., 2000, Proc. Natl. Acad. Sci. USA). 97, 10113-10119), and immunodetection amplified by T7 RNA polymerase (Zhang et al., 2001, Proc. Natl. Acad. Sci. USA, Vol. 98, 5497-5502).
[0054]
In one preferred embodiment of the array of the present invention, an array of antibodies is used to stain the cells, which is schematically illustrated in FIG. The cells are seeded on cover slips, cultured, fixed, and permeabilized to expose the antigen. The cell preparation is then incubated with the antibody array so that the antibody can bind to the corresponding antigen. After binding, the antibody array support and the cell support are separated. Some of the antibody will be transferred to the cell support due to interaction with the antigen. The amount of antibody bound and transferred will be related to the amount of antigen present in the cell.
[0055]
By detecting the presence and quantity of the antibody trapped on the cell support, the presence and quantity of the antigen in the cell can be determined. If antibodies are conjugated to enzymes, these antibodies can be revealed by enzymatic reactions. Alkaline phosphatase is widely used and known substrates will give an insoluble colored product after the reaction. Furthermore, if the antibody is labeled with a fluorescent molecule, the antibody may be observed directly by fluorescence microscopy. In many cases, fluorescent or enzyme-conjugated secondary antibodies can be used to determine the presence or absence of primary antibodies.
[0056]
For the purpose of revealing the presence and quantity of antigen, staining can be easily observed at low magnification. However, staining is preferably observed at high magnification to reveal other properties of the antigen, such as subcellular localization.
[0057]
Co-staining of two proteins (double staining) is a unique tool for studying two functionally related proteins. For example, evidence of protein interactions often includes proof that the proteins are co-localized in the same cellular structure. Two or more antibodies can be immobilized at the same location of the antibody array, so that two or more antigens can be detected at the same location simultaneously.
[0058]
Instead of antibodies, DNA probes can be sequenced and used to detect the presence or absence of specific DNA or RNA in cells. To do so, the DNA or RNA probe is immobilized on a solid support. Preferably, the immobilization of the probe is strong enough to maintain the probe position on the support, while preferably weaker than the interaction between the probe and the target, so when the probe binds to the target Can be dissociated from the support. This method has many advantages over in situ hybridization methods that usually only detect one or several DNA or RNA sequences. When an array of DNA or RNA probes formed in accordance with the present invention is used, the presence and location of multiple DNA or RNA sequences is detected at each predetermined location in a morphologically preserved tissue section or cell preparation. be able to. Methods for preparing DNA or RNA probes arranged on a support and methods for preparing tissues or cells are known in the art (Ian A. Darby (Editor), In Situ Hybridization Protocols (Methods in Molecular Biology , 123), by Humana Press; ISBN: 0896036863; 2nd edition, 2000).
[0059]
In another preferred embodiment, the antibody array is used to detect proteins in the protein lysate. To do so, the antibody array immobilized on the first support is incubated with the protein lysate immobilized on the second support. After a period of incubation, the antibody will bind to the corresponding antigen in the lysate immobilized on the second support. Thereafter, the first support is separated from the second support. Under certain conditions, the antigen-bound antibody will dissociate from the first support and remain on the second support to which the antigen is immobilized. The amount of antibody transferred to the second support will be proportional to the amount of antigen present in the protein lysate. Thus, detection of the amount of antibody on the second support will reveal the amount of antigen present in the protein sample.
[0060]
In many applications, proteins can be first separated and / or concentrated and it may be necessary to detect small amounts of protein. In one embodiment, proteins are first separated by one-dimensional SDS / PAGE, transferred to a membrane support, and each protein is immobilized on the support at a position determined by molecular weight. The lysate membrane is then contacted with the antibody array, and the antibodies are immobilized in an elongated shape at a specific location, so that each antibody will be able to contact the antigen at a specific location determined by the molecular weight of the antigen.
[0061]
Proteins may be separated by two-dimensional gel electrophoresis and transferred to a support such as a polyvinylidene difluoride (PVDF) membrane. The lysate membrane is then contacted with an antibody array, which contains a plurality of antibodies that can each be immobilized in place, and thus each antibody will contact the corresponding antigen.
[0062]
Proteins may be separated and concentrated by immunological methods. One way to achieve this is to use an antibody array. For example, a first antibody array can be incubated with a protein sample so that the protein is captured and separated by each antibody immobilized on the array. The antibodies in this first array are preferably strongly immobilized by covalent bonds. Thereafter, the antigen is dissociated from the antibody and moved and immobilized on the second support. This step can be performed by several known methods. The second antibody array is then used to detect the antigen. The antibodies in the second array are immobilized such that the antibodies can be dissociated from the array support after binding to the corresponding antigen. Antibodies in the first and second arrays against the same antigen can be immobilized at corresponding positions, which can be identical or different.
【Example】
[0063]
The following examples relate only to the drawings and are in no way intended to limit the invention. Although the examples describe the use of antibody arrays in accordance with the present invention, similar usage for arrays of biological reagents other than antibodies will be apparent to those familiar with the art. Such arrays of biological reagents include, but are not limited to, arrays of recombinant proteins, recombinant antibodies, single chain antibodies, nucleic acids, oligos, cDNA probes, carbohydrates, lipids, and small chemicals. For example, an array of immobilized cDNA probes can be used in situ hybridization to reveal the expression of multiple mRNAs in cells.
[0064]
(Example 1: Formation of antibody array)
Antibodies from commercial sources (approximately 0.5 μg / μL) were arrayed on nylon membranes (Amersham Life Science) using a robotic array. Approximately 40 ng of antibody in 80 nL solution was deposited with 0.6 mm solid pins at each spot spaced at 600 μm intervals. The antibody was immobilized by non-covalent bonding between the nylon membrane and the antibody. The antibody arrays were either used immediately or stored at 4 ° C. for less than 48 hours prior to use.
[0065]
(Example 2: Use of antibody array in immunochemical staining)
This example demonstrates the use of antibody arrays prepared by the methods disclosed for immunochemical staining. The antibody array formed in Example 1 with 200 antibodies against different cellular proteins was used.
[0066]
MDCK (Madin Darby Canine Kidney) cells were seeded on a culture dish and cultured for 2 days until fusion. They were then fixed with methanol / acetone (1: 1) at −20 ° C. for 10 minutes and rendered permeable. After washing with phosphate buffered saline (PBS), MDCK cells were contacted with the antibody array for about 1 hour. Cells were washed again with phosphate buffered saline, and alkaline phosphatase labeled secondary antibody was added for 30 minutes. After washing, staining was visualized by a color reaction with 5-bromo-4-chloro-indolyl-phosphatase (BCIP) and nitroblue tetrazolium (NBT) as substrates. The enzymatic reaction was stopped by washing off the substrate with PBS, and the image was obtained by scanning with a flat bed scanner (FIG. 2). Although cells at many locations were stained with antibodies, there was little antibody mixing as judged by the lack of staining in the area between immobilized antibodies. The normal pattern of stained spots matches the pattern on which the antibody is printed.
[0067]
(Example 3: Use of antibody array in fluorescent immunochemical staining)
In this example, about 5 ng of antibody in 10 nL solution was deposited using 0.3 mm solid pins at each spot on a nylon membrane with 300 μm spacing between adjacent spots. Thereafter, antibodies were used to stain A431 cells by the method schematically shown in FIG. 1 and using the method described in FIG. 2 except that a fluorescently labeled secondary antibody was used and staining was observed with a fluorescence microscope. Was used (FIG. 3). Low-magnification observation with a fluorescence microscope showed that each stained spot was approximately 300 μm in diameter and consisted of clusters of 400 cells (FIGS. 3a, 3c, 3e, 3g). There is a clear boundary between the stained and unstained areas. High magnification observations showed detailed staining patterns such as nuclear localization of IRF1 (FIG. 3b), cytoplasmic staining of 14-3-3β (FIG. 3d) and Ets-1 (FIG. 3f), and beta-catenin (FIG. 3h). ) Revealed cell-cell contact staining. Staining is indistinguishable from that obtained by standard immunostaining procedures.
[0068]
(Example 4: Antibody array formed in capillary tube)
In this example, an antibody array formed by capillaries is used to stain cells. Each antibody was mixed with a low melting point agarose solution and injected into a synthetic resin capillary having a height of 1 cm, an outer periphery of 2 mm, and a thickness of about 0.2 mm. After the gel became solid, the capillaries were bundled and traversed to produce an antibody about 1 mm high.
[0069]
The antibody array was then placed on a glass support and contacted with cells that were immobilized on another glass support to allow the antibody to bind to the antigen. The antibody bound to the antigen was detected by a fluorescently labeled secondary antibody and observed with a microscope as shown in FIG.
[0070]
(Example 5: Antibody array formed in capillaries for fluorescent immunostaining)
First, fluorescently labeled antibodies were mixed at high temperature with a low melting point agarose solution, and then these antibody solutions were injected into the capillaries to form a capillary array. The temperature was lowered so that these solutions solidified in the capillaries. These capillaries were cut to form a thin array less than 1 mm in height and placed on a synthetic resin solid support and adhered. In order for the antibody to be able to contact the antigen, the array was contacted with fixed cells placed on a coverslip. The cells and antibody array were then incubated for 2 hours at 37 ° C. Thereafter, the temperature was lowered to separate the array and the cells. The cells on the cover glass were then washed with PBS and observed with a microscope.
[0071]
(Example 6: Use of antibody array in double staining)
An antibody array with 100 spots was formed, each spot containing one or two antibodies. In spots where two different antibodies were immobilized, one of these antibodies was a mouse monoclonal antibody and the other was a rabbit polyclonal antibody. This array was used to stain A431 cells seeded on cover slips. After staining, A431 cells were incubated with Cy2-labeled anti-rabbit goat secondary antibody and Cy3-labeled anti-mouse goat secondary antibody. The presence and location of the antigen was examined by a fluorescence microscope (FIGS. 4a and 4b). Two corresponding antigens were observed where two antibodies were present. The two antibodies / antigens can be distinguished by different labels and different cellular localizations. For example, in FIG. 4, the nuclear localization of protein YY1 and the protein p130 ^cas Was shown separately (left and middle in FIG. 4a) and together (right in FIG. 4a and FIG. 4b).
[0072]
Example 7: Use of antibody array staining to compare protein expression between cell samples
Antibody arrays containing both mouse monoclonal and rabbit polyclonal antibodies against 240 proteins involved in various signal pathways were produced as described in the previous examples. The presence or absence of 240 proteins was then detected and compared between two human cancer cell lines widely used for research, A431 cells and ME180 cells. This result indicated that the two cell samples had significantly different protein expression patterns. Many of the 240 target proteins were differentially expressed in A431 and ME180 cells (FIG. 5). For example, A431 cells express more Cbl, cortactin, Neu, HSP70, JNK1, p53, Raf-1 and Stat1, while expressing less GSK-3α, Skp2p45, Plk3, and Stat5a than ME180 cells. The expression of these proteins was also examined by Western blotting (FIG. 6). And this result was obtained in two interrelated ways, suggesting that antibody array staining is an effective way to profile protein expression.
[0073]
(Example 8: Detection of pathway activation by antibody array)
Antibodies specific for activated proteins (phosphate proteins) were used to form antibody arrays, and these arrays were used to check for the presence of activated proteins in biological samples. The presence or absence of activated protein suggested that certain signaling pathways were activated in the sample.
[0074]
Example 9: Detection of specific proteins in protein lysates
In this example, 50 μg of bacterial lysate containing Stat1 recombinant protein was immobilized on a 1 cm 2 nitrocellulose membrane. The lysate membrane was then contacted with an antibody array containing a plurality of antibodies including Stat1 antibody in a nylon membrane.
[0075]
After 1-2 hours of incubation, some antibodies bound to the corresponding antigen in the lysate. When the lysate membrane and antibody array were physically separated, some antibodies dissociated from the antibody array support and bound to the lysate support due to interaction with the antigen. The amount of antibody bound to the lysate slide depends on the amount of antigen present in the lysate. The antibody was detected by HRP-conjugated secondary antibody and visualized by enhanced chemiluminescence (ECL) reaction.
[0076]
(Example 10: Detection of Stat1 protein by HRP-conjugated primary antibody)
This example is similar to Example 9 except that a horseradish peroxidase (HRP) conjugated Stat1 primary antibody is used such that an enzyme conjugated secondary antibody is not required for detection.
[0077]
(Example 11: Staining with an antibody array immobilized via a protein A mutant)
In this example, a recombinant protein A mutant with one antibody binding domain is first immobilized on a support and then the HRP conjugate antibody interacts with the protein A mutant forming an antibody array. It was fixed to the support via The antibody array thus formed was contacted with a protein lysate immobilized on another support. After 1 hour incubation, the antibody array was removed and the antibody bound to the protein sample support was detected via ECL reaction.
[0078]
(Example 12: Separation of protein sample by SDS / PAGE)
In this example, the A431 protein lysate was separated and transferred to a PVDF membrane by SDS / PAGE using curtain gel. An antibody array containing 12 antibodies against well-studied proteins was formed using a nylon membrane as a support and used in this assay. The antibody was immobilized in a rectangle and carefully placed on the array so that the antibody could bind to the corresponding antigen when the formed array was in contact with the protein lysate immobilized on the PVDF membrane. After binding of antibody and antigen, the nylon membrane support was removed from the PVDF membrane. Antibodies attached to the PVDF membrane through interaction with the corresponding antigen were detected using HRP-conjugated secondary antibody and revealed by ECL reaction. As shown in FIG. 7, several antibodies were detected as expressed in A431 cells. These are then detected in the PVDF membrane at the expected molecular weight positions. Antibodies deliberately immobilized at the wrong location give a relatively low signal, suggesting that this method effectively profiles protein expression.
[0079]
(Example 13: Separation of protein sample by two-dimensional gel electrophoresis)
This example is similar to Example 12 except that the protein is separated by two-dimensional gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane. Thereafter, the lysate film was brought into contact with an antibody array containing a plurality of antibodies and immobilizing the antibodies at predetermined positions so that each antibody was in contact with the antigen.
[0080]
(Example 14: Separation of protein sample by antibody array)
In this example, the proteins are first separated and concentrated by the antibody array. An antibody array was formed by covalently immobilizing the antibody to the support, which was then incubated with the protein sample such that the protein was captured and separated by each antibody immobilized on the array. Thereafter, the antigen was dissociated from the antibody and moved and immobilized on the sample support. Since the antibody was covalently immobilized, none or very little migrated to the sample support. A secondary antibody array was then formed such that antibodies of the same antigen were in the same corresponding positions as the antibodies were in the first antibody array. When the second antibody array contacted the sample support on which the antigen was immobilized, each antibody contacted and bound to the corresponding antigen. After binding, the array was separated from the sample support. As some antibodies bound to these antigens, the antibodies dissociated from the array support and bound to the sample support.
[0081]
Although the features of the present invention are shown in some of the figures, this is for convenience only and any feature may be combined with any or all other features according to the present invention . Other embodiments and variations will occur to those skilled in the art and are within the scope of the claims appended hereto.
[Brief description of the drawings]
[0082]
FIG. 1 schematically illustrates a preferred method of the invention for detecting multiple proteins.
FIG. 2 shows the results after using the preferred method and array of the invention for detecting multiple proteins. In this example, MDCK cells were stained with an array of 200 antibodies. This staining was observed by a color reaction with BCIP / NBT as a substrate mediated by alkaline phosphatase.
FIG. 3a shows the results after using the preferred method and array of the invention using fluorescent staining. 200 rabbit polyclonal antibodies were used. Staining of protein IRF1 at one position is shown on a 300 μm scale.
3b is a partially enlarged view of the image of FIG. 3a with a scale of 30 μm.
FIG. 3c shows the results after using the preferred method and array of the invention using fluorescent staining. 200 rabbit polyclonal antibodies were used. Staining of landmark molecule 14-3-3β at one position is shown.
3d is a partially enlarged view of the image of FIG. 3c.
FIG. 3e shows the results after using the preferred method and array of the invention using fluorescent staining. 200 rabbit polyclonal antibodies were used. Shown is staining of the cell adhesion protein β-catenin at one location.
3f is a partially enlarged view of the image of FIG. 3e.
FIG. 3g shows the results after using the preferred method and array of the invention using fluorescent staining. 200 rabbit polyclonal antibodies were used. Staining of transcription factor Ets-1 at one position is shown.
3h is a partially enlarged view of the image of FIG. 3g.
FIG. 4a: A431 cells were treated with rabbit anti-YY1 antibody (left green), mouse anti-p130 ^cas Antibody (center red), and YY1 antibody (right green) and p130 ^cas The results after using the preferred method and array of the present invention stained with an array containing both antibodies (right side red) at adjacent positions are shown. An anti-rabbit Cy2-labeled goat secondary antibody and an anti-mouse Cy3-labeled goat secondary antibody were used.
4b shows YY1 (green) and p130 shown in FIG. 4a. ^cas It is an enlarged view of double staining of (red).
FIG. 5 shows the results after using the preferred method and array of the present invention for detecting protein expression in two biological samples. Protein expression in ME180 cells is shown on the left and protein expression in A431 cells is shown on the right. An antibody array with 240 antibodies is used in this example. Staining was observed by a color reaction with BCIP / NBT as a substrate mediated by alkaline phosphatase.
FIG. 6 shows the results of using Western blotting to determine the differences in protein expression shown in FIG. The expression of 19 proteins in ME180 cells (left lane) and A431 cells (right lane) detected by Western blotting is shown. The proteins tested were A, Cbl (120 kD), B, Cbc2 (34 kD), C, Coractin (80 kD), D, Neu (185 kD), E, ERK1 (44 kD), F, Ets-1 (51 kD), G, GSK-3α (51 kD), H, HSP70 (70 kD), I, JNK1 (46 kD), J, Lyn (56 kD), K, NFkB p50 (50 kD), L, Skp2 p45 (45 kD), M, p53 ( 53 kD), N, Plk3 (70 kD), O, SH-PTP2 (60 kD), P, Raf-1 (74 kD), Q, Rb p107 (107 kD), R, Stat1 (84 kD), and S, Stat5a (95 kD) It is. The position corresponding to each antibody in FIG. Protein lysates of A431 and ME180 cells occupying equal culture areas are placed in each lane.
FIG. 7 shows the results after using the preferred method and array of the present invention to detect protein expression in protein lysates of A431 cells separated by SDS / PAGE. Antibodies against 12 proteins were immobilized at specific locations. The tested proteins were 1, IKKbeta, 2, E2F1, 3, ERK2, 4, 14-3-3, 5, Rbp107, 6, ERK1control (the antibody was immobilized at a position where no antigen was present). 7, Ets-1, 8, ERK1, 9, Stat1, 10, 14-3-3 control, 11, JNK1, 12, Stat2, 13, Lyn, and 14, p38. The position corresponding to each antibody is shown to the right of the corresponding spot.

Claims (53)

A method for detecting one or more biomolecules comprising:
Providing a first support on which one or more reagents are immobilized;
Providing a second support on which one or more ligands are immobilized;
Contacting one or more reagents with one or more ligands by contacting the reagent immobilized on the first support with a ligand immobilized on the second support;
Separating the first support from the second support;
Detecting one or more reagents in the second support;
A method comprising the steps of: The reagent is immobilized on the first support at a strength sufficient to immobilize the reagent in the contacting step and to allow the bound reagent to be dissociated from the first support in the separation step. The method of claim 1 wherein: The method according to claim 1, wherein the reagent comprises one or more antibodies. The method according to claim 1, wherein the reagent comprises one or more DNA probes. The method of claim 1, wherein the ligands are separated from each other before being immobilized on the second support. 6. The method of claim 5, wherein the ligands are separated from each other using gel electrophoresis based at least in part on molecular weight. 6. The method of claim 5, wherein the ligands are separated from each other using gel electrophoresis based at least in part on an isoelectric point. 6. The ligands are separated from each other using gel electrophoresis based at least in part on one or more molecular weights and isoelectric points, or both molecular weights and isoelectric points. The method described. 6. The method of claim 5, wherein the ligands are immunologically separated from each other. 10. The method of claim 9, wherein the ligands are separated from each other using one or more antibody arrays. The method according to claim 1, wherein the reagent is immobilized on the first support in one or more shapes selected from the group consisting of a circular shape, an elongated shape, and a polygonal shape. . The method of claim 1, wherein the reagent is selected from the group consisting of antibodies, recombinant proteins, DNA, RNA, oligonucleotides, carbohydrates and small chemicals. The method according to claim 1, wherein one or more of the reagents are immobilized at one or more predetermined positions on the first support. The method according to claim 1, wherein the reagents are respectively immobilized at predetermined positions on the first support. The method according to claim 14, wherein the reagent is an antibody. 16. The method of claim 15, wherein the one or more antibodies are specific for one or more post-translationally modified proteins. 17. The method of claim 16, wherein the one or more antibodies are specific for one or more phosphorylated proteins. The method according to claim 13, wherein 5 to 100,000 different types of reagents are respectively immobilized at one or more predetermined positions on the first support. 14. The method according to claim 13, wherein 200 to 10,000 different types of reagents are respectively immobilized at one or more predetermined positions on the first support. 14. The method according to claim 13, wherein 50 to 1,000 different types of reagents are respectively immobilized at one or more predetermined positions on the first support. The method according to claim 13, wherein 5 to 500 different types of reagents are respectively immobilized at one or more predetermined positions on the first support. The method according to claim 13, wherein 2 to 50 different types of reagents are respectively immobilized at one or more predetermined positions on the first support. The step of supplying the first support on which one or more reagents are immobilized is a step of applying one or more intermediates to the first support, The two or more intermediates are selected from the group consisting of protein A, protein G, and mutants thereof, and one or two or more of the reagents are used as one or more of the intermediates. Applying the one or more of the reagents to the first support to interact with an object. 2. The method of claim 1, wherein the first support comprises a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride, glass or synthetic resin, and derivatives thereof. 25. The method of claim 24, wherein the first support comprises a material selected from the group consisting of nylon and nylon derivatives. A method for detecting one or more biomolecules in a cell, comprising:
(A) Immobilizing one or more kinds of antibodies supplied at predetermined positions on the first support to the first support so that the antibodies can be identified by the position at which the antibodies are immobilized. When,
(B) placing the cells on a second support;
(C) contacting the antibody on the first support with the cell on the second support to allow the antibody to bind to any corresponding antigen in the cell;
(D) after separating the first support from the second support, one or more of the binding antibodies maintain binding with corresponding biomolecules;
(E) detecting the antibody bound to the biomolecule in the cell on the second support;
A method comprising the steps of: 27. The method of claim 26, wherein the cells are fixed to the second support. 27. The method of claim 26, wherein the cell is one or more tissue sections. 27. The method of claim 26, wherein the one or more antibodies are specific for one or more post-translationally modified proteins. 27. The method of claim 26, wherein the one or more antibodies are specific for one or more phosphorylated proteins. 27. The method according to claim 26, wherein 5 to 100,000 different reagents are respectively immobilized at one or more predetermined positions on the first support. 27. The method according to claim 26, wherein 200 to 10,000 different reagents are respectively immobilized at one or more predetermined positions on the first support. 27. The method according to claim 26, wherein 50 to 1,000 different reagents are respectively immobilized at one or more predetermined positions on the first support. 27. The method according to claim 26, wherein 5 to 500 different reagents are respectively immobilized at one or more predetermined positions on the first support. 27. The method according to claim 26, wherein 2 to 50 different reagents are respectively immobilized at one or more predetermined positions on the first support. 27. The method of claim 26, wherein the first support comprises a material selected from the group consisting of nitrocellulose, nylon, polyvinylidene difluoride, glass, synthetic resin, and derivatives thereof. 27. The method of claim 26, wherein the first support comprises a material selected from the group consisting of nylon and nylon derivatives. 27. The method of claim 26, wherein the reagent is immobilized on the first support in one or more shapes selected from the group consisting of a circular shape, an elongated shape, and a polygonal shape. 27. The method of claim 26, wherein the first support includes an array. 40. The method of claim 39, wherein the array comprises a plurality of total or partial capillaries. The step of immobilizing the antibody includes a step of applying one or more intermediates to the first support, and one or two or more of the antibodies are one or more of the intermediates. 27. The method of claim 26, comprising applying one or more of the antibodies to the first support to interact with the first support. 42. The method of claim 41, wherein the one or more intermediates are selected from the group consisting of protein A, protein G, and mutants thereof. The detecting step detects whether one or more of the antibodies are present on one or both supports, and after the separating step, determines one or more positions of the antibodies. One or more selected from the group consisting of: identifying; determining one or more quantities of the antibody; and identifying one or more types of antibodies 27. The method of claim 26, comprising a detecting step. A method for detecting one or more biomolecules comprising:
(A) immobilizing one or more reagents on the first support;
(B) immobilizing one or more ligands configured to interact with one or more of the reagents on a second support;
(C) contacting the reagent with the ligand to enable one or more of the reagents to bind to one or more of the ligands;
(D) crosslinking one or more of the reagents with one or more of the ligands;
(E) In order to dissociate one or more of the reagents bound to one or more of the ligands from the first support, the first support is used as the second support. A step of separating from,
(F) detecting one or more of the reagents or ligands;
Including methods. 45. The method of claim 44, wherein one or more of the ligands and reagents are cross-linked at least partially with one or more aldehydes. 46. The method of claim 45, wherein the one or more aldehydes are selected from the group consisting of formaldehyde and glutaraldehyde. An array used to detect one or more biomolecules,
A first support configured to immobilize one or more reagents, wherein the one or more ligands are disposed in contact with the second support to be immobilized. Comprising a first support configured in
One or more of the reagents may bind to one or more of the ligands, then dissociate from the first support and maintain binding to the ligands on the second support. An array characterized in that it is enabled. The first support includes one or more materials selected from the group consisting of nylon, nitrocellulose, polyvinylidene difluoride, glass, synthetic resin, and derivatives thereof. 47. The array according to 47. 48. The array of claim 47, wherein the first support comprises all or part of one or more capillaries. 48. The one or more kinds of the reagents are immobilized in one or more shapes selected from the group consisting of a circular shape, an elongated shape, and a polygonal shape. array. 48. The array of claim 47, wherein the one or more reagents comprise an antibody. 48. The array of claim 47, wherein one or more of the reagents comprise a DNA probe. 48. The array of claim 47, wherein the one or more reagents are selected from the group consisting of antibodies, recombinant proteins, DNA, RNA, oligonucleotides, carbohydrates and small chemicals.

Images