JP2007127626A - Nanoparticle for immunological measurement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a technique of immunological measurement capable of various types of labeling such as fluorescent labeling and enzyme labeling, therefore, highly versatile, having nonspecific signals, capable of highly sensitive detection, and highly safe to human bodies and the environment due to biodegradation. <P>SOLUTION: Nanoparticles for immunological measurement are formed by the capturing of a lipid bilayer by proteins having self-organizability, and the proteins having self-organizability has a portion bound to an antibody. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The invention of the present application relates to a nanoparticle for immunological measurement that is a nano-sized particle.

  Immunoassays such as ELISA (enzyme-linked immunosorbent assay), Western blotting, and tissue staining were established in 1941 by Coons et al. Establishment of a fluorescent antibody method for detecting plasma cell IgG with an antibody labeled with a fluorescent dye. And then the development of the enzyme antibody method by Nakane, Pierce et al. (1966). Peroxidase-anti-peroxidase; PAP method (1970), avidin-biotylated-peroxidase complex; ABC method (1981) ), Labeled streptavidin biotinylated antibody; LSAB method (1984), etc. have been developed (see Non-Patent Document 1). ).

  Immunological measurement involves reacting a specific antibody against an antigen, and skillfully combining further antigen-antibody reaction or chemical reaction to form an immune complex of the specific antibody and the labeling substance. Based on the principle of visualizing and quantifying the presence of an antigen by the fluorescence intensity of the labeled fluorescence. Even in a crude sample using an antibody, the antigen (target substance for detection; protein, peptide, nucleic acid, sugar chain, compound, etc.) ) Is an excellent research technique that can selectively stain only. This makes it possible to quantify the abundance without purifying the antigen that is the target of detection, and to identify the location of the antigen in the cell. Widely used in research fields.

  Immunological measurement is roughly classified into direct staining (direct method) and indirect staining (indirect method). The direct method is a method in which a primary antibody (an antibody recognizing an antigen) directly labeled with a labeling substance is reacted with an antigen, and a luminescence or fluorescent signal derived from the primary antibody bound to the antigen is detected. Therefore, there is an advantage that the reaction is completed in one step and the specificity is high. However, in this direct method, since the labeling substance must be labeled with the primary antibody, it is difficult to label with the labeling substance without impairing the activity of the antibody. There are some disadvantages.

  As a technique for improving the low sensitivity of the direct method, a fluorescent particle body having a diameter of 10 to 15 nm called a quantum dot (refer to Patent Document 1, Patent Document 2, and Non-Patent Document 2) is used. Alternatively, a method is known in which a streptavidin tag or the like is immobilized and particles bound to an antigen are detected by fluorescence emitted from the particles themselves. However, since the detection method is limited to fluorescence detection that requires an expensive detection device, the versatility is low. Furthermore, it is a non-biodegradable nanomaterial containing cadmium, selenium and the like, and there are concerns about environmental impact and safety to the human body (see Non-Patent Document 3).

  In addition, an example in which an immunoassay is performed using fluorescently labeled liposomes as biodegradable nanoparticles for immunological measurement is disclosed (see Non-Patent Document 4). However, the type of label is limited to the fluorescent label, and antibodies that recognize the target substance (antigen) cannot be aligned and displayed on the liposome at high density. In general, biological components such as liposomes are known to have low stability.

  In order to compensate for the stability of the liposome, an example in which a polymer is bound to stabilize the liposome and a non-specific signal is suppressed by the action of the polymer is disclosed (Patent Document 3). However, antibodies that recognize the target substance (antigen) cannot be aligned and displayed at high density.

  In general, the indirect method is used to compensate for the low sensitivity of the direct method. After reacting the primary antibody with the antigen, the labeled substance can be introduced into the antigen localized part by reacting with the secondary antibody labeled with the labeling substance (the antibody recognizing the primary antibody). Therefore, it takes time until detection. The secondary antibody labeling method is not limited to fluorescence, and can be used for various labeling such as enzyme labeling. Therefore, the secondary antibody labeling method is excellent in versatility. Further, the labeling amount can be increased by using ABC method, enzyme labeling polymer method, LSAB method and the like. Therefore, the advantage is that the sensitivity is higher than that of the direct method. However, this indirect staining method is inferior to the direct method in terms of specificity, such as increased non-specific binding by the secondary antibody and increased background (Non-patent Document 5). Also, it takes a long time to detect.

  On the other hand, hollow nanoparticles formed by hepatitis B virus surface antigen protein by incorporating intracellular lipid bilayers by self-organization are known, and examples of presenting antibodies on the surface of the hollow nanoparticles include Although known (see Patent Document 4), it is an invention related to the transport and introduction of a tissue-specific drug, that is, a drug delivery system, and does not enable immunological measurement.

Thus, there has been no example of developing an immunological measurement technique that allows various labels, is highly versatile, has a low non-specific signal, can be detected with high sensitivity, and is highly safe to the human body.
Ulike, K. et al., Journal of Histochemistry and Cytochemistry, 2001, 49, p623-630. Ozkan, M., Drug Discovery Today, 2004, Vol. 9, p1065-1071. Hoet, P. et al., Journal of Nanobiotechnology, 2004, Volume 2. Anup K. Singh et al., Biotechnology Progress, 1996, Vol. 12, p272-280. Masato Okada et al., Protein Experiment Note 3rd Edition, 2004, p118-119. Special Table 2002-544488 US Pat. No. 6,274,323 Japanese translation of PCT publication No. 2003-501631 JP 2004-2313 A

In view of the above situation, in the present invention,
1) A variety of labels such as fluorescent labels and enzyme labels are possible and highly versatile.
2) Low non-specific signal and high sensitivity detection,
3) It is biodegradable and highly safe for the human body and the environment.
The issue is to develop a method of immunological measurement.

  As a result of intensive studies on the method for solving the above problems, the present inventors have found that a protein having self-organization ability is formed by incorporating a lipid bilayer, and the protein having self-assembly ability has an antibody binding site. The present inventors have found that the above-mentioned problems can be solved by using the immunological measurement nanoparticles, which have led to the present invention.

  By using the immunoassay nanoparticles of the present invention for immunoassays such as Western analysis and ELISA, which are widely used at present, a variety of labels are possible, versatility, and non-specific signals are generated. Low and highly sensitive detection is possible, and immunological measurement with high safety to the human body can be performed.

Other terms and concepts in the present invention are defined in detail in the description of the embodiments and examples of the invention. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known literatures and the like, except for the technique that clearly shows the source. For example, genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, “Molecular Cloning-A Laboratory Manual”, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F .; M.M. et al. , “Current Protocols in Molecular Biology”, John Wiley & Sons, New York, N .; Y, 1995, and the like.

  The present invention is an immunoassay nanoparticle formed by incorporating a lipid bilayer into a “protein having self-assembly ability”, and the protein having self-assembly ability has an antibody binding site. .

  The immunological measurement in the present invention uses an antibody in detection of the presence or localization of a target substance and uses changes in absorption, luminescence, fluorescence, radiation, coloration, turbidity, etc. in the detection. The technique to do is shown. In the immunological measurement in the present invention, the type of the target substance is not particularly limited, and examples thereof include biological substances such as DNA, RNA, protein, sugar chain, cells, viruses, phages, and chemical substances. For example, as an example of immunological measurement using DNA or RNA as a target substance, DNA or RNA incorporating Digoxigenin, Fluorescein, or Biotin is hybridized to nucleic acid on various membranes or resin-embedded tissue sections, Digoxigenin, Fluorescein, Examples include Southern hybridization, in situ hybridization, and northern hybridization including a step of detecting the target DNA or RNA using an antibody against Biotin. Examples of immunological measurement using protein as a target substance include western blotting, immunohistochemical staining, immunoelectron microscopic observation, EIA (Enzyme immunoassay enzyme immunoassay), ELISA (Enzyme-Linked Immunosorbent Assay solid phase enzyme immunoassay) , RIA (Radioimmunosay radioimmunoassay), FIA (Fluorescenceimmunoassay fluorescence immunoassay), FLISA (Fluorescence-Linked Immunosorbent Assay solid phase fluorescence immunoassay), ELISPOT (ImmunoImmunoImmunoImmunoImmunosyminL). Examples of immunological measurements using cells, viruses, phages, chemical substances, etc. as target substances include immunohistochemical staining, immunoelectron microscopy, EIA (Enzyme immunoassay enzyme immunoassay), ELISA (Enzyme-Linked Immunosorbent Assay solid phase enzyme) Immunoassay), RIA (Radioimmunosay radioimmunoassay), FIA (Fluorescence immunoassay fluorescent immunoassay), FLISA (Fluorescence-Linked Immunosorbent Assay solid-phase fluorescent immunoassay), ELISPIt-mm-enzyme-SmP Measurement, immunochromatography and the like.

According to the purpose, immunological measurement in the present invention,
1) A direct method for directly recognizing and detecting a target substance by means of a labeled antibody,
2) An indirect method of recognizing a target substance with an antibody and recognizing and detecting an antibody bound to the target substance with a labeled antibody,
3) Competition method,
4) A two-antibody sandwich method in which a target substance is captured by an immobilized antibody and further detected by another labeled antibody,
5) A three-antibody sandwich method in which a target substance is captured by an immobilized antibody, a target substance is recognized by another antibody, and an antibody that has recognized the target substance is detected by a labeled antibody.
6) A labeled particle method for detecting particles in which nanoparticles are directly or indirectly labeled and bound to an antibody that recognizes a target substance is selected.

  In the present invention, the “protein having self-organization ability” is a protein having a property of self-assembly by protein-protein interaction such as hydrophobic interaction. A protein that has the property of self-assembling by protein-protein interaction can be confirmed by forming multimers between proteins by changing the temperature, pH, salt concentration, and solvent of the solution in which the protein is dissolved. it can.

  As the “protein having self-organizing ability”, virus-derived proteins such as virus particle constituent proteins obtained from various viruses can be applied. Specifically, hepatitis B virus (Hepatitis B Virus: HBV), hepatitis C virus (Hepatitis C virus), Micro virus, phage virus, adeno virus, hepadna virus, reversal, p , Corona viruses, Bombyx mori cytoplasmic polyhedrovirus, Calicivirus, Norovirus, Sapovirus, and other constituent proteins, membrane proteins, surface antigen proteins, and polyhedron proteins.

  “Proteins having self-organizing ability” are, for example, natural proteins derived from animal cells, plant cells, insect cells, viruses, phages, bacteria, fungi, etc., genetically engineered proteins, and various synthetic proteins. Etc. As the “protein having self-organizing ability”, a virus-derived protein, more preferably a protein derived from hepatitis virus, more preferably a protein derived from hepatitis B virus is used. Most preferably, hepatitis B virus surface antigen protein is used. At this time, as long as the self-organization ability is not lost, it may be a mutant having various mutations. By deleting a part of the amino acid in the preS region, the production amount and stability of nano-sized particles are reduced. Can be increased.

  In the present invention, the “protein having the ability to self-assemble” is preferably a virus-derived protein, more preferably a surface antigen protein of a virus (particularly hepatitis B virus). By using a surface antigen protein of a virus (particularly hepatitis B virus) as a “protein with self-organizing ability”, this protein causes self-organization while taking up a lipid bilayer in a eukaryotic cell. In particular, it is possible to efficiently produce nano-sized particles having a high strength. By expressing this protein in yeast, it is possible to produce nanoparticles of several to several tens of mg / L culture, so that highly efficient particles can be produced, and the cost of particle production is extremely low. Can do. This is important for industrial use of the particles.

  The nanoparticles produced by the virus surface antigen protein are physically very stable and can maintain their morphology even at high temperatures such as 60 to 80 ° C., 8 M urea, or various surfactants. The stability of such particles can be applied to various immunological measurement methods, so it is highly versatile, high in storage stability, easy to handle, and removes other impurities during measurement. Alternatively, it is possible to add a washing treatment for reducing non-specific binding (for example, washing with a denaturing agent or a surfactant is possible), so that the detection accuracy can be improved. Therefore, the use of nanoparticles produced by the virus surface antigen protein can be expected to greatly expand the range of immunological measurement methods.

  The “protein having self-assembly ability” in the present invention has an antibody binding site.

  The antibody binding site in the present invention is a polypeptide that can bind to or interact with a region other than the antigen recognition site of an antibody. The antibody binding site in the present invention is preferably the full length or a part of any of Fc receptor, protein A, protein G, protein A / G, protein L, and ZZ tag, more preferably “antibody Fc region possessed by these”. Binding site "or a variant thereof.

  The antibody binding site may be hydrophobically bound to the “protein having self-assembly ability” or may be covalently bound by chemical bonding or the like. The antibody binding site is preferably fused to a “protein having self-organization ability” as a fusion protein. Further, the fusion site may be present at any position from the N-terminal to the C-terminal as long as it does not lose its ability to self-assemble.

  In the immunoassay nanoparticles of the present invention, the antibody binding site is preferably a “binding site with the antibody Fc region”. In the immunological measurement nanoparticle of the present invention, more preferably, the binding site with the antibody Fc region is a ZZ tag.

  In the immunological measurement nanoparticles of the present invention, preferably, the “binding site with the antibody Fc region” is an antibody binding site of protein A or protein G.

  In the present invention, an antibody binding site binds an antibody with less steric hindrance, and more preferably, a ZZ region (ZZ tag) in which a “binding site with an antibody Fc region” of protein A called Z region is tandem. Or a variant thereof, and the following repetitive sequences or variants thereof. VDNKFNKEQQNAFYEILHLPNLNEEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPVVNKNKFNQQNAFYEILHLPNLNEEEQRNAFIQSLKDDPKSQSANLLLAEAAKKLNDAQAPK.

In the nanoparticle for immunological measurement of the present invention, the “protein having self-assembly ability” has an antibody binding site, so that the antibody-binding site is defined by the self-assembly effect of the “protein having self-assembly ability”. It is possible to align with high density, and as a result, antibodies used for immunological measurement can be aligned and fixed at high density, so that highly sensitive and highly accurate immunological measurement is possible.
In the present invention, the lipid bilayer is composed of two lipid layers having a thickness of 5 to 20 nm, and the polar head of the amphiphilic lipid is in contact with the hydrophilic solvent in each layer. The non-polar hydrocarbon portion faces the inside of the double layer. Examples of lipid bilayer membranes include biological membranes in living cells such as cell membranes, nuclear membranes, endoplasmic reticulum membranes, Golgi membranes, and vacuolar membranes, or artificially prepared liposomes. Among these, a lipid bilayer membrane derived from the endoplasmic reticulum membrane is more preferable. As the lipid bilayer membrane, those derived from eukaryotes such as animal cells, plant cells, fungal cells, insect cells and the like are preferable, and yeast-derived lipid bilayer membranes are particularly preferably used.

  In the present invention, the nanoparticle is a particle of 20 to 500 nm. The nanoparticles of the present invention are particles having a diameter of preferably 50 to 200 nm, more preferably 80 to 150 nm. The nanoparticles of the present invention are preferably hollow nanoparticles having a hollow space inside, and the space is not necessarily a gas-based space, and may be a solution-based space.

  As described above, the present invention is an immunological assay in which a protein having self-organization ability is formed by incorporating a lipid bilayer, and the protein having self-assembly ability has an antibody binding site. Nanoparticles.

  When the nanoparticles for immunological measurement of the present invention are formed while incorporating the lipid bilayer, substances other than the target substance adsorb and interact nonspecifically with the nanoparticles for immunological measurement. This can be suppressed and the effect and accuracy of reducing immunological measurement noise can be improved. In addition, the target substance has an effect of suppressing noise by directly adsorbing and interacting with immunological measurement nanoparticles without using an antibody. This is very important in immunological measurement and is the most important factor that determines the accuracy of detection.

  The immunoassay nanoparticles of the present invention are preferably composed mainly of proteins, lipids and sugars, and the main components are proteins.

  The ability of a protein having an antibody binding site to self-assemble is capable of high-density alignment and high-density display of antibodies, and is an important property in improving sensitivity in immunological measurement. In addition, having the ability to self-assemble indicates that other contaminating proteins are eliminated during the self-assembly process, which improves the accuracy of immunological measurements (suppresses non-specific signals). It is an important property. Furthermore, by incorporating a lipid bilayer membrane, non-specific adsorption and interaction can be suppressed, and the accuracy of immunological measurement can be improved (non-specific signal can be suppressed).

  There is no particular limitation on the species that produces the nanoparticles for immunological measurement of the present invention, and production by any eukaryotic organism or prokaryotic organism may be used. Is more preferable, and production by yeast cells is more preferable. Infectious processes such as viruses and phages, and recombinants are also within the range of species that produce the immunoassay nanoparticles of the present invention.

  In the nanoparticle for immunological measurement of the present invention, preferably, an antibody binds to an antibody binding site, and the binding between the antibody binding site and the antibody is a covalent bond by a cross-linking agent.

  In the present invention, an antibody means an immunoglobulin that is naturally produced or is synthesized in whole or in part. All derivatives and mutants thereof that maintain antigen recognition ability are also included. The antibody can be monoclonal or polyclonal. An antibody having an Fc region is preferable, and an antibody capable of binding to any of Fc receptor, protein A, protein G, protein A / G, protein L, and ZZ tag is more preferable.

  The antibody Fc region in the present invention is a fragment having no antigen recognition ability obtained by digesting an antibody with papain, and is generally a dimer of the C-terminal fragment of the H chain of the antibody.

  The binding mode between the “protein having self-organizing ability” and the antibody in the present invention is not particularly limited as long as the antigen recognition ability of the antibody is not lost. Ion bonds, hydrophobic bonds, hydrogen bonds, metal bonds, or disulfides It may be a covalent bond such as a bond, or a combination thereof. The binding between the antibody binding site of the nanoparticle for immunological measurement of the present invention and the antibody is preferably a covalent bond, and more preferably for the purpose of preventing the antibody from dropping off during the immunological measurement process. Is a covalent bond by a crosslinking agent.

  The cross-linking agent in the present invention refers to one that functions to covalently bond a “protein having self-assembly ability” and an antibody, and binds using an amino group, a carboxyl group, a thiol group, a hydroxyl group, or the like. There is no particular limitation as long as the reagent is commercially available and the antigen recognition ability of the antibody is not lost.

  The cross-linking agent can be strongly bound to an antibody binding site of the “protein having self-assembly ability” even if it is a kind of antibody that exhibits weak binding or interaction. Furthermore, it is possible to prevent the antibody from dropping off during the washing operation in the immunological measurement process. Examples of crosslinking agents include cyanogen bromide compounds, compounds with carbodiimide groups, compounds with aldehyde groups, compounds with imide ester groups, compounds with maleimide groups, compounds with N-hydroxysuccinimide ester groups, and haloacetyl groups A compound, a compound having a pyridyl disulfide group, an allyl azide compound, and the like are used alone or in combination. Since the cross-linking agent preferably reacts only with a primary amine under mild conditions that do not easily cause a decrease in antigen recognition ability, a compound having an imide ester group is provided, and more preferably a DMA (Dimethyl adipimide) compound, A DMP (Dimethyl phenylimidate) compound, a DMS (Dimethyl sublimidate) compound, and a DMBP (Dimethyl 3,3′-dithiobipropionimidate) compound are used.

  The nanoparticles for immunological measurement of the present invention are preferably labeled with nanoparticles.

  There is no particular limitation on the labeling of the nanoparticle for immunological measurement of the present invention, and any label can be used as long as it does not lose its ability to bind to an antibody. The labeling may be performed on the outer surface or the inner surface of the nanoparticle, or may be performed on the inside of the particle. As a method for labeling the inside of the particle, fusion with a liposome whose inside or surface has been labeled in advance, electroporation, pressure change, temperature change and the like are used alone or in combination.

  Examples of nanoparticle labeling include, for example, labeling with enzymes such as alkaline phosphatase, peroxidase, luciferase, glucose oxidase, beta galactosidase, and mutants thereof, and are also capable of forming nanoparticles. Fusion with “having protein” may be used. Fluorescein, Rhodamine, Texas Red, Dansyl, Lucifer Yellow VS, Umbelliferyl, rare earth chelate, Cy2, Cy3, Cy5, fluorescein isothiocyanate (FITC), TRITC, ALEXA (registered trademark) (Molecular Probe), quantum dots, etc. The label may be a fluorescent substance. Alternatively, it may be a label with a radioactive substance such as H3, C14, N15, P32, S35, Co57, Se75, or I125. Furthermore, labeling with biotin, avidin, streptavidin, gold particles, iron particles, magnetic particles, or magnetic beads may be used. A labeling substance can be easily selected according to various environments, and a plurality of labels can be used in combination. From the viewpoint of versatility and safety, alkaline phosphatase, horseradish peroxidase, fluorescein, fluorescein isothiocyanate, ALEXA (registered trademark), biotin, and gold particles are preferably used. Nanoparticles are labeled with biotin, and indirect labeling of nanoparticles that labels biotin with a complex of avidin and enzyme can also be used.

  The nanoparticle for immunological measurement of the present invention is preferably labeled with an antibody.

  The label of the antibody in the present invention is not particularly limited as long as it does not lose the antigen recognition ability of the antibody and can be detected. For example, it may be labeled with an enzyme such as alkaline phosphatase, peroxidase, luciferase, glucose oxidase, beta galactosidase or a variant thereof, such as fluorescein, rhodamine, Texas red, dansyl, lucifer yellow VS, umbelliferyl, rare earth chelate, It may be labeled with a fluorescent substance such as Cy2, Cy3, Cy5, fluorescein isothiocyanate (FITC), TRITC, ALEXA (registered trademark) (Molecular Probe), or quantum dots. Alternatively, it may be a label with a radioactive substance such as H3, C14, N15, P32, S35, Co57, Se75, or I125. Furthermore, labeling with biotin, avidin, streptavidin, gold particles, iron particles, magnetic particles, or magnetic beads may be used. A labeling substance can be easily selected according to various environments, and a plurality of labels can be used in combination. From the viewpoint of versatility and safety, alkaline phosphatase, horseradish peroxidase, fluorescein, fluorescein isothiocyanate, ALEXA (registered trademark), biotin, and gold particles are preferably used.

  The immunoassay nanoparticle of the present invention has an antibody-binding site, for example, the immunoassay nanoparticle can bind to a labeled antibody, so that the target substance is directly recognized by the labeled antibody. In the direct method, which is an immunological measurement method for detection, it is possible to increase the sensitivity, which has been difficult in the past, while suppressing non-specificity.

  The immunoassay nanoparticles of the present invention are preferably formed by self-organization of proteins in eukaryotic cells, and by using genetic engineering techniques, various surfaces and inner surfaces can be used. Various protein and peptide probes can be shared. Moreover, since the protein having the ability of self-organization according to the present invention forms a particle, it self-assembles while maintaining a certain direction, so that the antibody recognition site is also fixed to the particle in a state aligned in a certain direction. .

  Further, the nanoparticle for immunological measurement of the present invention can be used to routinely produce several to several tens of mg of particles in 1 L culture of yeast simply by expressing “a protein having self-assembly ability” in eukaryotic cells such as yeast. Production is possible. This means research or industrial significance. Below, the usefulness of the nanoparticle for immunoassay of this invention is listed.

  1) It takes a complicated process to produce nanoparticles using the principle of self-organization utilizing the amphipathic properties of lipids, such as nanoparticles and liposomes that are chemically synthesized and made by elaborate dispersion technology. Often, skill is required to routinely form uniform particles. However, the immunoassay nanoparticles of the present invention can form very uniform nanoparticles in cells simply by culturing cells such as yeast under certain conditions, and are purified using an affinity column. This makes it easy to obtain pure nanoparticles.

  2) In addition, chemical synthesis and liposomes must be stocked with the materials necessary for particle production each time, and it is extremely difficult to procure uniform materials consistently, so the quality of particles is influenced by the batch. Easy to receive. In particular, the production of nanoparticles generally requires advanced techniques and is time consuming and expensive. In contrast, the immunoassay nanoparticles of the present invention can store the yeast and cell seeds that produce the particles semipermanently by freezing, etc., and if necessary, store the seeds on the medium. It is possible to always produce nanoparticles of the same quality by growing them with

  3) In particular, nanoparticles as bioresearch materials such as immunological measurements often require a process of encapsulating or presenting biomaterials such as proteins at the time of production. It must be handled with care because it reacts very sensitively to the environmental factors in which it is placed and loses its function. In this respect, the immunoassay nanoparticles of the present invention can cause the yeast (or other eukaryotic cells) that produce the particles to produce proteins that are presented or encapsulated in the particles, and thus impair the function. It can be incorporated into the particles (surface presentation or inclusion).

  4) In addition, when incorporating proteins into particles, it is necessary to procure purified protein each time for other particles and add it when producing the particles. This greatly affects the quality stability and cost of the particles. Become a factor. In contrast, the immunoassay nanoparticles of the present invention produced using eukaryotic cells do not need to be separately purified or supplied with proteins to be incorporated into the particles. Particles can be created. In summary, the nanoparticles of the present invention can be stored semipermanently and easily by producing them by utilizing self-assembly of proteins in cells, producing stable quality particles at low cost and high efficiency. it can. In particular, this method is useful because proteins such as enzymes are often used in the immunological measurement field, which is a problem to be solved by the present invention.

In the present invention, a variant of an antibody or a variant of a “protein having self-organization ability” or a variant of an antibody binding site, a variant of a “binding site with an antibody Fc region”, a variant of a ZZ tag, a mutation of an enzyme The body is
1) These proteins are genetically engineered or have multiple amino acid substitutions,
2) A part of the entire length of the protein is removed or a new protein sequence is added.
3) Some proteins modified with nucleic acids, sugars, lipids, compounds, etc.
Because
4) an antibody variant capable of recognizing the same substance as the antibody before the mutation;
5) A variant having the ability to incorporate a lipid bilayer and form nanoparticles by self-assembly, similar to “a protein having self-assembly ability”,
6) Antibody binding site, “binding site with antibody Fc region”, mutant having the ability to interact with Fc region of antibody as well as ZZ tag,
7) A variant that changes the substrate in the same manner as the enzyme.
In order to produce such a mutant, a primer for performing deletion, substitution or addition of amino acids on a circular plasmid containing a gene that expresses the protein and a QuikChange site-directed mutagenesis kit, or QuikChange multi site-directed mutagenesis Kit, or QuikChange XL site-directed mutagenesis kit (STRATAGENE), etc., is used to perform PCR for 12 to 18 cycles, and the product is cleaved with restriction enzyme DpnI and transformed into E. coli. Is possible.

  Furthermore, random mutagenesis, site-directed mutagenesis, gene homologous recombination, or polymerase chain amplification (PCR) can be performed alone or in appropriate combination. For example, a method of replacing cytosine bases with uracil bases by chemical treatment with sodium bisulfite, a method of reducing the accuracy of nucleotide incorporation during DNA synthesis by performing PCR in a reaction solution containing manganese Various commercially available kits for introducing specific mutations can also be used. For example, edited by Sambrook et al. [Molecular Cloning-A Laboratory Manual, 2nd edition] Cold Spring Harbor Laboratory, 1989, edited by Masaaki Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., Ltd., 1988, Ehrlich, HE. Chapter [PCR Technology, Principles and Applications of DNA Amplification] It can be carried out according to the method described in the book of Stockton Press, 1989, etc., or by modifying those methods. In addition, as a part of the protein is modified with nucleic acid, sugar, lipid, compound, etc., specifically, serine in the protein by phosphorylase, or phosphorylation of threonine residue, asparagine by glycosylation enzyme, Or, it can be exemplified by the addition of a sugar chain of serine or threonine residue, or reduction / alkylation of cysteine residue by reduction / alkyl reagent. Or mix sugar chains, add phosphorylation enzymes and glycosylation enzymes, maintain the optimal conditions (temperature, pH, salt concentration, etc.) for the enzymes to work, and reduce the amount of protein in the solution. Dithiothreitol or mercaptoethanol is added to a final concentration of 5 mM, reacted at a temperature near 60 ° C. and at pH neutral or higher for 1 hour. It is possible by reaction over 1 hour at room temperature was added iodoacetamide 5~15mM concentration as kill agents. Of course, various modifications to the protein other than the above-described modifications are possible.

  The immunological measurement method using the nanoparticle for immunological measurement of the present invention includes various Western blotting, immunohistochemical staining, immunoelectron microscope observation, EIA, ELISA, RIA, FIA, FLISA, ELISPOT, immunochromatography, immunoelectron microscope. It is effective for observation and flow cytometry.

  The invention is illustrated in more detail by the following examples. The present invention is not limited to the following examples.

  As shown in Reference Example 2 described later, by expressing the HBV surface antigen L protein (hepatitis B virus surface antigen protein) in genetically modified yeast, lipids derived from the yeast endoplasmic reticulum from the expressed HBV surface antigen L protein It has been found and reported that ellipsoidal nanoparticles having a minor axis of about 20 nm and a major axis of about 150 nm are formed in which a large number of the same proteins are embedded in a double membrane (J. Bio. Chem., Vol. 267, No. 3, 1953-1961, 1992). Since such particles do not contain any HBV genome or other HBV proteins, they do not function as viruses and are extremely safe for the human body. In addition, it is characterized by extremely high stability with respect to pH, temperature, surfactant and the like.

  Furthermore, by cloning, it is possible to always produce nanoparticles with a uniform quality "protein with self-organization ability" and lipid bilayers, and yeast that is easy to handle can be used as a production system. It is also excellent in terms of productivity and production cost.

Reference example 1
(Synthesis and purification of C-terminal polyhistidine-tagged CAT protein in E. coli)
CAT protein fused with 6 histidines at the C-terminus was synthesized in E. coli BL21 using an expression vector. A single colony cultured at 37 ° C. for 16 hours on an LB plate was picked with a sterilized toothpick and pre-cultured at 37 ° C. in 10 ml of L-Broth (LB medium) supplemented with kanamycin. The preculture was added to 1 L LB medium supplemented with kanamycin and cultured at 37 ° C. for 2 to 3 hours. IPTG was added to a final concentration of 1 mM, and further cultured at 37 ° C. for 5 hours. Centrifugation was performed at 3000 rpm for 5 min at 4 ° C. to precipitate the cells, and the precipitated cells were washed with 60 ml of 50 mM Tris-HCl pH 8.0 and centrifuged again to precipitate the cells. After suspending 60 ml of 50 mM Tris-HCl pH 8.0 solution in the cells, the cells were disrupted using a digital sonicator (manufactured by BRANSON) while cooling in ice according to the attached protocol. The supernatant (soluble fraction) and precipitate (insoluble fraction) after centrifugation at 9000 rpm for 15 min at 4 ° C. were fractionated. The CAT protein in the E. coli lysate soluble fraction was passed through a nickel sepharose column (manufactured by Amersham Biosciences) equilibrated with a 50 mM Tris-HCl (pH 8.0) solution to adsorb the polyhistidine-tagged CAT protein. . The column was washed with 100 ml of 50 mM Tris-HCl (pH 8.0) solution, and then further washed with 100 ml of 50 mM imidazole / 50 mM Tris-HCl (pH 8.0) solution. Elution was performed with 50 ml of a 500 mM imidazole / 50 mM Tris-HCl (pH 8.0) solution, and a fraction containing a polyhistidine-tagged CAT protein was collected. Thereafter, the mixture was dialyzed overnight against 1000 times the amount of PBS and sterilized by filter.

Reference example 2
(Expression of ZZ-tagged nanoparticles (ZZ particles) by genetically modified yeast)
JP, 2004-002313, A Nanoparticle having a ZZ tag was prepared based on the method described in the examples.

Example 1
(Immunological measurement using nanoparticles with ZZ tag tag and labeled antibody)
The C-terminal polyhistidine-tagged CAT protein 4,500 ng / ml prepared in Reference Example 1 was serially diluted in two steps to about 141 ng / ml with PBS in a multi-well plate and allowed to stand overnight at 4 ° C. for solidification. It was. After washing three times with PBS, 3 μg / ml of FITC-labeled antibody against C-terminal polyhistidine tag (FITC-labeled anti-histidine tag antibody) was added in PBS containing 1/4 vol. Block Ace at room temperature for 1 hour. Shake. At this time, a sample to which 4 μg / ml of ZZ particles were added and a sample to which 4 μg / ml of FITC-labeled ZZ particles were added were simultaneously processed for comparison. For FITC labeling, EZ-Label Fluorescein Labeling Kit manufactured by PIERCE was used, and FITC labeling was performed according to the attached protocol. After shaking for 1 hour, the plate was washed 3 times with PBST, and the fluorescence of FITC was measured with a fluorescence plate reader. The addition of ZZ particles improved the detection sensitivity by about 8 times. Further, the addition of FITC-labeled ZZ particles improved the detection sensitivity by about 10 times (FIG. 1). FIG. 3 is a diagram showing the effect of ZZ particles in immunological measurement as shown in this example. FIG. 3 shows a case in which ZZ particles (3 in FIG. 3) are added to the labeled antibody in comparison with the detection system of the antigen (1 in FIG. 3) using only the labeled antibody labeled with the labeling substance (2 in FIG. 3). It shows that the labeled antibody recognizing the antigen (1 in FIG. 3) binds to the ZZ particle and the labeled antibody not recognizing the antigen, thereby improving the detection sensitivity.

Example 2
(Immunological measurement using labeled nanoparticles with ZZ tag and antibody)
The C-terminal polyhistidine-tagged CAT protein solution 4,500 ng / ml prepared in Reference Example 1 was serially diluted in two steps to approximately 141 ng / ml with PBS in a multi-well plate, and allowed to stand at 4 ° C. overnight to solidify. I let you. After washing 3 times with PBS, in PBS containing 1/4 vol. Block Ace, add 3 μg / ml each of the antibody against the FITC-labeled C-terminal polyhistidine tag or the antibody against the unlabeled C-terminal polyhistidine tag at room temperature. Shake for 1 hour. Those treated in the same manner with a solution containing no antibody were used for comparison. After washing with PBST three times, 4 μg / ml each of FITC-labeled ZZ particles and unlabeled ZZ particles were added in PBS containing 1/4 vol. Block Ace and shaken for 1 hour. Simultaneously treated with a solution containing no particles for comparison. For FITC labeling, EZ-Label Fluorescein Labeling Kit manufactured by PIERCE was used, and FITC labeling was performed according to the attached protocol. After shaking for 1 hour, the plate was washed 3 times with PBST, and the fluorescence of FITC was measured with a fluorescence plate reader.
Detection sensitivity was improved by adding labeled ZZ particles. Moreover, the detection sensitivity was further improved by the cooperation of the FITC-labeled antibody and the labeled ZZ particles (FIG. 2). At this time, no signal was observed in the comparison target containing no antibody, and therefore no non-specific signal due to ZZ particles was observed. FIG. 4 is a diagram showing the effect of ZZ particles in immunological measurement as shown in this example. FIG. 4 shows a comparison with the detection system of the antigen (1 in FIG. 4) using only the labeled antibody labeled with the labeling substance (2 in FIG. 4), and the antigen (4 in FIG. 4). ) And labeled ZZ particles (3 in FIG. 4) are added, the detection sensitivity is improved by binding of the unlabeled antibody and the labeled ZZ particles.

Example 3
(Immunological measurement using nanoparticles with ZZ tag and enzyme-labeled antibody)
560 ng / ml of the C-terminal polyhistidine-tagged CAT protein (antigen) solution prepared in Reference Example 1 was serially diluted in two steps to approximately 0.27 ng / ml with PBS in a multiwell plate, and 100 μl was allowed to stand at 4 ° C. overnight. And solidified. After washing with PBST three times, 250 ng / ml of a mouse monoclonal antibody (Invitrogen) against C-terminal polyhistidine tag diluted with PBST containing 1/4 vol. Block Ace was added as a primary antibody and shaken at room temperature for 2 hours. That ’s it. After washing 3 times with PBS, enzyme labeled antibody diluted with PBST containing 1 / 4vol. Block Ace, HRP labeled Rabbit anti-mouse IgG antibody (manufactured by SIGMA) 0.5μg / ml, 1μg / ml, 2μg / ml, 4μg / ml, 8 μg / ml, 16 μg / ml, and 32 μg / ml were shaken at room temperature for 1 hour. At this time, the effect of the ZZ particles was examined by adding 1.5 μg / ml of the ZZ particles to each enzyme-labeled antibody solution. After the enzyme-labeled antibody reaction, the plate was washed 3 times with PBST, and the color developed by TMB and sulfuric acid was measured by absorbance (450 nm). The increase in detection sensitivity due to ZZ particles was calculated from the antigen concentration when the absorbance (450 nm) gave 1.5. Compared to the case where ZZ particles are not used, the addition of ZZ particles improved the detection sensitivity by a maximum of about 5 times. At this time, the background caused by the adsorption of the ZZ particles on the substrate was hardly observed. Moreover, it can be read from the graph that the low concentration region can be measured while maintaining the linearity (FIGS. 5 and 7). FIG. 7 shows a comparison between a detection system in which an antigen (1 in FIG. 7) is detected by a labeled antibody labeled with a primary antibody (5 in FIG. 7) and a labeling substance (2 in FIG. 7), and ZZ particles (FIG. 7). 7-3) indicates that the detection sensitivity is improved by binding of the labeled antibody and the ZZ particles. The molecular weight of the ZZ particles was 4400 kDa, the molecular weight of the enzyme-labeled antibody was 200 kDa, and the influence of the molar ratio of the enzyme-labeled antibody and the ZZ particles on the sensitivity enhancement factor was examined (FIG. 6).

Example 4
(Immunological measurement using nanoparticles with ZZ tag and enzyme-labeled antibody)
Ovalbumin (manufactured by SIGMA) (antigen) solution 500 μg / ml was diluted in two steps sequentially with PBS to about 0.12 ng / ml in a multi-well plate, and 100 μl was allowed to stand at 4 ° C. overnight to solidify. After washing 3 times with PBST, 400 ng / ml of mouse monoclonal antibody against Ovalbumin (Abcam) diluted with PBST containing 1/4 vol. Block ace was added as a primary antibody and shaken at room temperature for 2 hours. After washing with PBS three times, an enzyme-labeled antibody diluted with PBST containing 1/4 vol. Block ace and an HRP-labeled Rabbit anti-mouse IgG antibody (manufactured by SIGMA) solution 2 μg / ml were shaken at room temperature for 1 hour. . At this time, the effect of the ZZ particles was examined by adding 2.4 μg / ml of the ZZ particles to the enzyme-labeled antibody solution. After the enzyme-labeled antibody reaction, the plate was washed 3 times with PBST, and the color developed by TMB and sulfuric acid was measured by absorbance (450 nm). The increase in detection sensitivity due to ZZ particles was calculated from the antigen concentration when the absorbance (450 nm) gave 1.5. Compared with the case where no ZZ particles were used, the detection sensitivity was improved about 15 times by the addition of ZZ particles. At this time, the background caused by the adsorption of the ZZ particles to the substrate was hardly observed (FIG. 8).

FIG. 1 is a diagram showing the effect of ZZ particles in immunological measurement. FIG. 2 is a diagram showing the effect of ZZ particles in immunological measurement. FIG. 3 is a diagram showing the effect of ZZ particles in immunological measurement. FIG. 4 is a diagram showing the effect of ZZ particles in immunological measurement. FIG. 5 is a diagram showing the effect of ZZ particles in immunological measurement. FIG. 6 is a diagram showing the optimum molar ratio of ZZ particles to enzyme-labeled antibody. FIG. 7 is a diagram showing the effect of ZZ particles in immunological measurement. FIG. 8 is a diagram showing the effect of ZZ particles in immunological measurement.

Explanation of symbols

1 antigen 2 labeling substance 3 ZZ particle 4 antibody 5 primary antibody

Claims (6)

A nanoparticle for immunological measurement, wherein a protein having self-organization ability is formed by taking in a lipid bilayer, and the protein having self-assembly ability has an antibody binding site. The nanoparticle for immunological measurement according to claim 1, wherein the antibody binds to the antibody binding site, and the binding between the antibody binding site and the antibody is a covalent bond by a cross-linking agent. The nanoparticle for immunological measurement according to claim 1 or 2, wherein the nanoparticle is labeled. The nanoparticle for immunological measurement according to any one of claims 1 to 3, wherein the antibody is labeled. The nanoparticle for immunological measurement according to any one of claims 1 to 4, wherein the protein having self-organization ability is a virus-derived protein. An immunological measurement method using the immunological measurement nanoparticles according to claim 1.