JP2008191143A - Biotinylated nanoparticle for immunological assay - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tool capable of alignment-exhibiting densely antibodies on a surface without generating a steric hindrance, in a biodegradable nanoparticle, and allowing precise immunological assay via biotin, and an immunological assay using the same. <P>SOLUTION: This biotinylated nanoparticle for the immunological assay is a nano size of particle formed by taking a lipid double membrane into a protein having self-organizing ability and has an antibody coupling part in the protein having the self-organizing ability, and the protein having the self-organizing ability is labeled with the biotin. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a biotinylated nanoparticle for immunological measurement in which particles having a nano-sized diameter are biotinylated and an immunological measurement method.

  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.

  Methods for improving the sensitivity of immunological measurements have been energetically studied. For example, antibodies used for immunological measurements are labeled with biotin, and a complex of avidin and enzyme, or a complex of streptavidin and enzyme. Sensitization of immunological measurement by binding the body is known and widely used in research fields.

  In recent years, methods for improving the sensitivity of immunological measurements using nano-sized materials have been studied.For example, hepatitis B virus surface antigen (HBsAg) protein is converted into intracellular lipids by self-assembly. A substance sensing method using hollow nanoparticles formed by incorporating a double membrane is disclosed (see Patent Document 1). There is disclosed a technique in which a substance recognition molecule such as an antibody is presented on the surface of the biodegradable hollow nanoparticle and applied to substance sensing. This discloses immunoassay using nano-sized particles in which a protein having a self-organizing ability is formed by incorporating a lipid bilayer. However, it is still a problem to display antibodies on the surface of nano-sized particles at high density without steric hindrance, and no solution is disclosed. In addition, there is no description of high-sensitivity immunoassay using biotin for labeling nano-sized particles formed by incorporating a lipid bilayer with a protein having self-organization ability. There is also no description of a method for increasing the number of and the effect thereof.

On the other hand, an example in which an antibody is presented on the surface of a hollow nanoparticle containing a hepatitis B surface antigen protein via an antibody binding site is known (see Patent Document 2). That is, the invention relates to a drug delivery system and does not enable immunological measurement.
Ulrike, K. et al., Journal of Histochemistry and Cytochemistry, 2001, 49, p 623-630. International Publication No. 2005/095968 Pamphlet JP 2004-2313 A

In view of the above situation, in the present invention,
1) Biodegradable 2) The antibody can be arranged and displayed on the surface at high density without steric hindrance 3) Biotinylated nano for immunoassay enabling highly sensitive immunoassay via biotin The problem is to provide particles.

  As a result of intensive studies on a technique for solving the above problems, the present inventors have found that a protein having a self-organizing ability is a nano-sized particle formed by incorporating a lipid bilayer, and has a self-organizing ability. It has been found that the above problem can be solved by using biotinylated nanoparticles for immunological measurement in which a protein having an antibody has an antibody binding site and a protein having self-organization ability is labeled with biotin. Invented.

The present invention relates to the following biotinylated nanoparticles for immunological measurement, immunological measurement methods using the particles, and use of the particles for immunological measurement.
1. A nano-sized particle formed by incorporating a lipid bilayer into a protein having self-organization ability, and the protein having self-assembly ability has an antibody binding site and has self-assembly ability Biotinylated nanoparticles for immunological measurement, wherein the protein is labeled with biotin.
2. Item 2. The biotinylated nanoparticle for immunological measurement according to Item 1, wherein the antibody binding site is a ZZ tag.
3. Item 3. The biotinylated nanoparticle for immunological measurement according to Item 1 or 2, wherein the protein having the ability to self-assemble is a hepatitis B virus surface antigen (HBsAg) protein.
4). Item 4. The biotinylated nanoparticle for immunological measurement according to any one of Items 1 to 3, wherein biotin is bound to a lysine residue or N-terminal amino acid residue of the protein.
5. Item 5. The biotinylated nanoparticle for immunological measurement according to any one of Items 1 to 4, wherein a complex of an enzyme and a biotin-binding protein is bound, and an antibody is bound to an antibody binding site, which is recognized by the antibody. An immunological measurement method for measuring a substance to be measured using the enzyme.
6). Item 6. The immunological measurement method according to Item 5, wherein the substance to be measured is an antibody.
7). Item 7. The immunological measurement method according to Item 5 or 6, wherein the biotin-binding protein is avidin, streptavidin, or neutravidin.
8). A nano-sized particle formed by incorporating a lipid bilayer into a protein having self-organization ability, and the protein having self-assembly ability has an antibody binding site and has self-assembly ability Use of biotinylated nanoparticles whose protein is labeled with biotin for immunological measurements.

  By using the biotinylated nanoparticles for immunological measurement of the present invention for immunological measurements such as Western analysis and ELISA that are currently widely used, it becomes possible to perform more sensitive immunological measurements. .

  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 relates to “a nano-sized particle formed by incorporating a lipid bilayer into a protein having self-assembly ability, wherein the protein having self-assembly ability has an antibody binding site, The present invention relates to a biotinylated nanoparticle for immunological measurement in which a protein having an organizing ability is labeled with biotin.

  The “biotinylated nanoparticle for immunological measurement” in the present invention refers to a biodegradable biotinylated nanoparticle used for immunological measurement, and a protein having self-organization ability takes in a lipid bilayer. A nano-sized particle formed by the above, wherein a protein having a self-assembling ability has an antibody binding site, and a protein having a self-assembling ability is labeled with biotin.

  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 substance to be measured is not particularly limited, and examples thereof include biological substances such as DNA, RNA, proteins, sugar chains, 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 bound to Digoxigenin or Fluorescein is hybridized to nucleic acids on various membranes or resin-embedded tissue sections, and antibodies against Digoxigenin or Fluorescein are used. Examples include Southern hybridization, in situ hybridization, and northern hybridization that include a step of detecting the target DNA or RNA. 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 (Radioimmunoassay radioimmunoassay), FIA (Fluorescence immunoassay fluorescence immunoassay), FLISA (Fluorescence-Linked Immunosorbent Assay solid-phase fluorescence immunoassay), ELISPIL-t-SmIpI-Lt (EmSimI-Lonp Measurement, immunochromatography and the like.

  The kind of the substance to be measured in the present invention is preferably an antibody, more preferably a blood antibody related to a disease such as allergy or infection.

According to the purpose, immunological measurement in the present invention,
1) A direct method in which a target substance is directly recognized by an antibody, biotinylated nanoparticles are bound to the antibody, and biotin is detected.
2) An indirect method in which a target substance is recognized by an antibody, an antibody bound to the target substance is recognized by a labeled or unlabeled secondary antibody, biotinylated nanoparticles are bound to the secondary antibody, and biotin is detected. ,
3) Competition method,
4) A secondary antibody sandwich that captures the target substance with an immobilized antibody, recognizes it with another labeled or unlabeled secondary antibody, binds biotinylated nanoparticles to the secondary antibody, and detects biotin. Law,
5) Capturing the target substance with the immobilized antibody, recognizing the target substance with another antibody, and recognizing the target substance with another labeled or unlabeled tertiary antibody A three-antibody sandwich method that recognizes, binds biotinylated nanoparticles to a tertiary antibody, and detects biotin;
Etc. are selected.

  The antibody to be bound to the biotinylated nanoparticle used in the above 1) to 5) is preferably an antibody having a high binding force with the antibody binding site of the biotinylated nanoparticle, mouse-derived IgG, rabbit-derived IgG, human-derived IgG, Dog-derived IgG is preferably used.

  In the immunoassay method of the present invention, biotinylated nanoparticles for immunoassay may be added at the same time as the antibody, and after adding the antibody to the antibody binding site of the nanoparticle in advance, it is added as a conventional antibody. Alternatively, the nanoparticles may be added after the antibody is added. Preferably the antibody and nanoparticles are added simultaneously or the nanoparticles are added prior to adding the antibody.

  When the antibody and the nanoparticles are added simultaneously, the amount of biotinylated nanoparticles for immunological measurement is about 0.01 to 10 times the number of molecules of the antibody bound to the particles, preferably about 0.1. Use a molecular number of about 05 to 1 times. When the amount of biotinylated nanoparticles used (number of molecules) is too small compared to the amount of antibody bound to the particles (number of molecules), the effect of increasing the sensitivity due to the particles is reduced. The background becomes high.

  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”, virion-constituting proteins obtained from various viruses can be applied. Specifically, hepatitis B virus (Hpatitis B virus: HBV), hepatitis C virus (Hepatitis C virus), micro virus (micro virus), phage virus (adeno virus), adenovirus (adeno virus), hepadna Virus (hepadna virus), parvovirus (papovirus), retrovirus (retro virus), reovirus (coronavirus), coronavirus (corona virus) polyhedrossis virus), calicivirus, norovirus, sapovirus, etc. Proteins and membrane proteins, surface antigen protein or polyhedral protein and the like, are exemplified.

  “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 (HBsAg) 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 of the HBsAg protein, the production amount of nano-sized particles is increased. And can improve stability.

  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 having self-organizing ability”, it is expressed in eukaryotic cells such as yeast, insect cells, mammalian cells (eg CHO cells), This protein self-assembles while taking up the lipid bilayer of the endoplasmic reticulum of the host eukaryotic cell, and can efficiently produce particles having a physical strength and a nano-sized diameter. 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 increased. 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. Since the “protein having self-organization ability” has an antibody binding site, the surface of the biotinylated nanoparticle for immunological measurement in the present invention has the effect of self-assembly of “protein having self-assembly ability”. It is possible to align antibody binding sites at high density. As a result, antibodies used for immunological measurement can be aligned and fixed at a high density on the surface of the nanoparticles, so that highly sensitive and highly accurate immunological measurement is possible.

  The antibody binding site in the present invention is a polypeptide capable of binding to or interacting with a region other than the antibody antigen recognition site, and more preferably a binding site with the antibody Fc region. The antibody binding site in the present invention is preferably the full length or part of any of Fc receptor, protein A, protein G, protein A / G, protein L, and ZZ tags, and a variant thereof, more preferably an antibody The binding site is a ZZ tag. A protein having self-organization ability has a ZZ tag as an antibody binding site, and a nano-sized particle formed when the protein having self-assembly ability incorporates a lipid bilayer is denoted as ZZ particle. To do.

  In the present invention, the ZZ tag indicates a region in which the “binding site with the antibody Fc region” of protein A, called the Z region, is tandem. The amino acid sequence of the ZZ tag is the following repetitive sequence or a variant thereof. AQHDEAVDNKFNKEQQNAFYILHLPNLNEEEQRNAFIQSLKDDPSQSANLLLAEAKKLNDAQAPKVDNKFNKEQQNAFEYEILLPLPNLNEEQRNAFIQSLKDDPSQSANLLLAAPAKNLDAQ sequence The ZZ tag is preferably used from the viewpoint of low molecular weight and no steric hindrance. By using a polypeptide called a ZZ region (ZZ tag) or a variant thereof, it becomes possible to display antibodies on the nanoparticle surface at a high density without steric hindrance, making immunoassay more sensitive. It becomes possible to do. That is, in the biotinylated nanoparticle for immunological measurement of the present invention, the “protein having self-assembly ability” has a ZZ tag as an antibody binding site, so that the antibody used for immunological measurement is placed on the nanoparticle surface. Without steric hindrance, it becomes possible to align and fix at a higher density, so that more sensitive and highly accurate immunological measurement is possible.

  The ZZ tag sequence has as many as 12 lysine sequences in 122 amino acid sequences and is rich in amino groups in the side chain. On the other hand, the surface antigen protein of hepatitis B virus, which is a protein having a self-assembling ability described in Patent Document 1, has only three lysine groups, and even if the N-terminal amino acid residue is included, Only 4 places have amino groups. Therefore, when biotinylation is performed using an amino group as a target, biotinylation is possible only at four places at the maximum. On the other hand, by having a ZZ tag sequence, the number of lysine increased from 3 to 15, and biotinylation targeting 16 amino groups including the N-terminal amino acid residue occurred efficiently, resulting in A large amount of biotin is localized on the surface of the nanoparticle. As a result, the biotin-binding protein-enzyme complex binds more than when it does not have a ZZ tag, and the sensitivity of immunological measurement is improved compared to the case without a ZZ tag. To do.

  The nanoparticles of the present invention form one particle with about 100 proteins having self-assembly ability. Therefore, the degree of biotin or enzyme labeling per particle of the present invention is about 100 times the amount of labeling per protein having self-assembly ability.

  The degree of biotinylation per particle of the present invention is 1 to 15, preferably 2 to 12, more preferably 3 to 10 per molecule of protein having self-assembling ability.

  The number of labels such as enzymes per particle of the present invention is 0.1 to 10, preferably 0.1 to 5, more preferably 0.1 to 1 molecule of protein having self-assembly ability. One.

  If there are too few biotin residues bound per particle of the present invention, the number of labels such as enzymes bound to the particle will decrease, and the sensitivity will decrease. On the other hand, if the number of biotin is too large, it may affect the binding between the antibody and the substance to be measured that is recognized by the antibody.

  If the number of labels such as an enzyme bound per particle of the present invention is too small, the measurement sensitivity decreases, and if the number of labels such as an enzyme is too large, the antibody binds to the substance to be measured that is recognized by the antibody. May be affected.

  The biotin-binding protein only needs to be able to link a biotinylated protein having a self-assembling ability and a biotinylated labeling substance such as a biotinylated enzyme. Avidin, streptavidin, and neutravidin are preferably exemplified. In addition, there are a wide range of biotin-binding proteins that can bind to multiple biotins, such as streptavidin, neutravidin (NeutrAvidin), and fusion proteins in which multiple avidins are bound (which may be chemically bound by a linker). Is included.

  The particles of the present invention have a small amount of particles that do not affect the ability of the antibody to recognize the substance to be measured even though the antibodies are bound at high density to the antigen-binding site of the protein having the ability to self-assemble. This enables highly sensitive measurement of the target substance.

  Thus, the fact that a protein having a self-organizing ability has a ZZ tag as an antibody binding site makes it possible to display an antibody at a high density without steric hindrance, and to biotinylate targeting an amino group. Efficiently occurs and has the effect of increasing the number of biotin labeled. This greatly improves the sensitivity of immunological measurement. The present inventor has confirmed that the antibody binding ability is maintained even when one or more biotins are bound to the Lys residue of an antibody binding site such as a ZZ tag.

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. In addition, the fusion site may be present at any location from the N-terminal to the C-terminal as long as it does not lose its ability to self-assemble, but it is preferably present outside when the particles are formed.
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.

  The biotinylated nanoparticle for immunological measurement of the present invention is formed by incorporating a lipid bilayer with a protein having self-organization ability, so that a substance other than the target substance can be biotin for immunological measurement. It is possible to suppress nonspecific adsorption and interaction with activated nanoparticles, and to improve the effect and accuracy of reducing noise in immunological measurement. In addition, the target substance has an effect of suppressing noise by directly adsorbing and interacting with biotinylated nanoparticles for immunological measurement without using an antibody. This is important in immunological measurement and is the most important factor that determines the accuracy of detection.

  The biotinylated nanoparticles for immunological measurement 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 an important property in improving sensitivity in immunological measurement, enabling high-density alignment and high-density display of antibodies. 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 are no particular limitations on the species of the present invention that produce nano-sized particles formed by incorporating a lipid bilayer with a protein having a self-assembling ability, and it can be produced by either a eukaryote or a prokaryote. However, in order to increase the self-organization ability, production by eukaryotes is 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 nano-sized particles of the present invention.

  The method of “labeling with biotin” in the present invention is not particularly limited, and a commercially available biotinylation reagent that labels an amino group, thiol group, carboxyl group, hydroxyl group or the like as a target can be used. Preferably, a protein having self-assembly ability is labeled with biotin, and more preferably, the ZZ tag sequence is rich in lysine residues, so that the amino group of the lysine residue and the N-terminal amino acid residue Reagents targeting the amino group of are used. Thereby, the amount of biotin labeling can be increased. Moreover, you may use the reagent which has a spacer which provides hydrophilic property and a freedom degree.

  The “complex of enzyme and biotin-binding protein” in the present invention refers to a complex of an enzyme used for immunological measurement and a biotin-binding protein, and the complex may be formed by binding with a crosslinking agent. A complex may be formed via biotin. A complex may be formed as a fusion protein. A complex in which a biotinylated enzyme is bound to a biotin-binding protein such as avidin, streptavidin, or NeutrAvidin is preferable.

  The enzyme in the present invention is not particularly limited as long as it is an enzyme used in detection of immunological measurement, and peroxidase, alkaline phosphatase, luciferase, glucose oxidase, beta galactosidase, and the like are used. Preferably, horseradish-derived peroxidase (HRP) is used.

  In the present invention, an antibody that binds to an antibody-binding site means an immunoglobulin that is either naturally produced or produced 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 that binds to the antibody binding site may be labeled with an enzyme, biotin, or the like.

  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. 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. More preferably, alkaline phosphatase, horseradish peroxidase, or biotin is labeled.

  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 biotinylated nanoparticle for immunological measurement of the present invention and the antibody is preferably a covalent bond, preferably for the purpose of preventing the antibody from dropping off in the course of immunological measurement, More preferably, it is a covalent bond by a crosslinking agent.

  The biotinylated nanoparticles for immunological measurement of the present invention may have a label in addition to biotin. There is no particular limitation on the labeling of biotinylated nanoparticles for immunological measurement, as long as the binding ability with the antibody is not lost and the label can be detected.

  As an example of labeling of biotinylated nanoparticles for immunological measurement, biotin-binding proteins such as avidin, streptavidin, NeutrAvidin are used. Alternatively, indirect labeling of nanoparticles in which the nanoparticles are labeled with biotin and biotin is labeled with a complex of biotin-binding protein and enzyme can also be used.

In the present invention, a variant of an antibody, a variant of “protein having self-organization ability”, a variant of an antibody binding site, a variant of a ZZ tag, or a variant of a biotin-binding protein is 1) a gene for these proteins. Engineering points or those with multiple amino acid substitutions 2) Part of the protein is scraped from the entire length, or a new protein sequence is added 3) Part of the protein by nucleic acid, sugar, lipid, compound, etc. Which has been modified,
4) an antibody variant capable of recognizing the same substance as the antibody before the mutation;
5) A mutant having the ability to incorporate a lipid bilayer and form nanoparticles by self-assembly, similar to the “protein having self-assembly ability” before the mutation occurs,
6) A mutant having the ability to interact with the Fc region of an antibody, as well as the antibody binding site and ZZ tag before the mutation occurs,
7) A variant that retains the binding ability to biotin as well as the biotin-binding protein before the mutation occurs.
In order to prepare such a mutant, a primer for performing deletion, substitution or addition of amino acids on a circular plasmid containing a gene expressing the protein and a QuikChange site-directed mutagenesis kit, or a QuikChange multisite-directed mutagenesis Kit, or QuikChange XL site-directed mutagenesis kit (Stratagene), etc., is used to perform 12 to 18 cycles of PCR, 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 modification of a part of the protein with nucleic acid, sugar, lipid, compound, etc., specifically, serine or threonine residue in protein by phosphorylase, asparagine by glycosylation enzyme, Or, examples include serine or threonine residue glycosylation, or reduction / alkylation of cysteine residues with reduction / alkyl reagents. 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 biotinylated nanoparticles for immunological measurement of the present invention includes various western blotting, immunohistochemical staining, immunoelectron microscopic observation, EIA, ELISA, RIA, FIA, FLISA, ELISPOT, immunochromatography, immunochromatography. It is effective for electron microscope observation and flow cytometry.

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

Reference example 1
(Expression of ZZ-tagged nanoparticles (ZZ particles) by genetically modified yeast)
Based on the method described in Examples of JP-A-2004-002313, hepatitis B surface antigen protein fused with a ZZ tag as an antibody binding site is expressed in yeast, and nanoparticles having a ZZ tag (ZZ particles) are obtained. Produced. Dissolved in PBS and stored at 4 ° C. until use. This was used as a nanoparticle for immunological measurement that was not biotinylated.

Reference example 2
(Expression of nanoparticles without ZZ tag (wild type nanoparticles) by genetically modified yeast)
Based on the method described in the Examples of JP-A-2001-316298, hepatitis B virus surface antigen protein was expressed in yeast to produce nanoparticles having no antibody binding site or ZZ tag (wild-type nanoparticles). Dissolved in PBS and stored at 4 ° C. until use.

Example 1
(Biotinylation reaction of ZZ particles and wild type nanoparticles, biotin labeling)
The ZZ particle solution described in Reference Example 1 and the wild-type nanoparticles described in Example 2 were concentrated to a concentration of 600 μg / ml or more using an ultrafiltration filter (Millipore). 100 μl of a ZZ nanoparticle / wild-type nanoparticle solution was biotinylated at room temperature for 30 minutes according to the attached protocol using EZ-Link Sulfo-NHS-Biotinylation Kit (manufactured by Pierce), and the amino group of lysine residue and N The terminal amino group was labeled with biotin. Free unreacted biotin was removed using Zeba ^™ Desert Spin Columns (Pierce) according to the attached protocol. The following experiments were performed using biotinylated ZZ particles obtained by biotinylating ZZ particles as biotinylated nanoparticles for immunological measurement, and using wild-type biotinylated nanoparticles obtained by biotinylating wild-type nanoparticles as controls. .

Example 2
(Biotin quantification of biotinylated nanoparticles for immunological measurement and wild-type biotinylated nanoparticles)
Biotin quantification was performed using EZ ^™ Biotin Quantitation Kit (Pierce) according to the attached protocol. The protein was quantified using a QuantiPro BCA Assay Kit (manufactured by SIGMA) according to the attached protocol. The absorbance was measured using a spectrophotometer multi-scan spectrum (manufactured by Thermolab System). The molecular weight of the hepatitis B virus surface antigen protein fused with the ZZ tag as the antibody binding site was 48 kDa, and the molecular weight of the hepatitis B virus surface antigen protein without the ZZ tag was 44 kDa, and the number of biotin labels per protein was evaluated. 6 molecules of biotin were labeled per molecule of hepatitis B virus surface antigen protein fused with a ZZ tag, whereas 3 molecules of biotin per molecule of hepatitis B virus surface antigen protein without a ZZ tag. It was labeled. From this result, it was confirmed that biotin labeling efficiency was improved by using a ZZ tag containing many lysine residues having an amino group in the side chain as an antibody binding site. About 100 molecules of hepatitis B virus surface antigen protein, which is a protein having a self-organizing ability, form one particle. Therefore, it is considered that each biotinylated nanoparticle for immunological measurement has about 600 biotins. On the other hand, it is considered that each wild-type biotinylated nanoparticle has about 300 biotins.

Example 3
(Immunoassay using biotinylated nanoparticles for immunological measurement, wild-type biotinylated nanoparticles, and nanoparticles for immunological measurement not labeled with biotin)
20 μg / ml of Ovalbumin (manufactured by SIGMA) solution as an antigen was serially diluted in two steps to about 19.5 ng / ml with PBS in a multiwell plate, and 100 μl was allowed to stand at 4 ° C. overnight to solidify. After washing with PBST solution containing 0.05% Tween 20 three times, 400 ng / ml of mouse monoclonal antibody against Ovalbumin (Abcam) diluted with PBST containing 1/4 volume of Block Ace was added as the primary antibody, and room temperature was added. Shake for 1 hour. After washing with PBST three times, an enzyme-labeled antibody diluted with PBST containing 1/4 volume of 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. It was. At this time, nothing was added to the enzyme-labeled antibody solution, and ZZ particles prepared in Example 1 were added as nanoparticle for immunological measurement at 2.4 μg / ml, biotinylation prepared in Example 1 ZZ particles added with 2.4 μg / ml as biotinylated nanoparticles for immunological measurement, wild-type biotinylated nanoparticles prepared in Example 1 as biotinylated nanoparticles having no antibody binding site, 2.4 μg / ml The addition of ml was performed simultaneously. After the enzyme-labeled antibody reaction, the plate was washed 3 times with PBST. ABC reagent (manufactured by PIERCE), which is a complex of biotinylated HRP and avidin as a complex of enzyme and avidin, was added to the well according to the attached protocol, and reacted at room temperature for 30 minutes. After washing 5 times with PBST, a color reaction was performed by adding TMB reagent. After stopping the reaction with 2N sulfuric acid, the absorbance at 450 nm was measured. A spectrophotometer multi-scan spectrum (manufactured by Thermolab System) was used for the measurement (FIG. 1). The magnification of detection sensitivity was calculated from the antigen concentration when the absorbance gave a certain value. As a result, compared to when no nanoparticles were used, the sensitivity was not improved at all when wild-type biotinylated nanoparticles, which are biotinylated nanoparticles without antibody binding sites, were used. When the measurement nanoparticles were used, the detection sensitivity was improved about 15 times. On the other hand, when biotinylated nanoparticles for immunological measurement were used, the detection sensitivity was improved by 30 times or more. That is, by using biotinylated nanoparticles for immunological measurement, the sensitivity was further improved by 2 to 4 times compared to the case of using biotinylated nanoparticles that were not biotinylated. This indicates that sensitivity was improved as a result of increasing the biotinylation efficiency of a protein having self-organization ability by using a ZZ tag with high biotin labeling efficiency as an antibody binding site.

Example 4
(Immunoassay using biotinylated nanoparticles for immunological measurement, wild-type biotinylated nanoparticles, and nanoparticles for immunological measurement not labeled with biotin)
The experiment described in Example 3 was performed using a biotin-labeled antibody instead of the enzyme-labeled antibody. Specifically, 20 μg / ml of Ovalbumin (manufactured by SIGMA) solution as an antigen was serially diluted in two steps to approximately 19.5 ng / ml with PBS in a multiwell plate, and 100 μl was allowed to stand at 4 ° C. overnight to form a solid layer. Made it. After washing with PBST solution containing 0.05% Tween 20 three times, 400 ng / ml of mouse monoclonal antibody against Ovalbumin (Abcam) diluted with PBST containing 1/4 volume of Block Ace was added as the primary antibody, and room temperature was added. Shake for 1 hour. After washing three times with PBST, 1.5 μg / ml of a biotin-labeled antibody diluted with PBST containing ¼ volume of block ace and a biotin-labeled Rabbit anti-mouse IgG antibody (manufactured by SIGMA) is shaken at room temperature for 1 hour. Reacted. At this time, nothing was added to the biotin-labeled antibody solution, and ZZ particles prepared in Reference Example 1 were added as nanoparticle for immunological measurement at 2.4 μg / ml, biotinylated prepared in Example 1 ZZ particles added with 2.4 μg / ml as biotinylated nanoparticles for immunological measurement, wild-type biotinylated nanoparticles prepared in Example 1 as biotinylated nanoparticles having no antibody binding site, 2.4 μg / ml The addition of ml was performed simultaneously. After the biotin-labeled antibody reaction, the plate was washed 3 times with PBST. ABC reagent (manufactured by PIERCE), which is a complex of biotinylated HRP and avidin as a complex of enzyme and avidin, was added to the well according to the attached protocol, and reacted at room temperature for 30 minutes. After washing 5 times with PBST, a color reaction was performed by adding TMB reagent. After stopping the reaction with 2N sulfuric acid, the absorbance at 450 nm was measured. A spectrophotometer multi-scan spectrum (manufactured by Thermolab System) was used for the measurement (FIG. 2). The magnification of detection sensitivity was calculated from the antigen concentration when the absorbance gave a certain value. As a result, compared to when no nanoparticles were used, the sensitivity was not improved at all when wild-type biotinylated nanoparticles, which are biotinylated nanoparticles without antibody binding sites, were used. When the measurement nanoparticles were used, the detection sensitivity was improved about twice. On the other hand, when the biotinylated nanoparticles for immunological measurement were used, the detection sensitivity was improved 4 times or more. That is, by using biotinylated nanoparticles for immunological measurement, the sensitivity was further improved by 2 to 4 times compared to the case of using biotinylated nanoparticles that were not biotinylated. This shows that the sensitivity was improved as a result of increasing the biotinylation efficiency of a protein having a self-organization ability by using a ZZ tag with high biotin labeling efficiency as an antibody binding site.

Example 5
(Immunological measurement of disease-related blood antibodies using biotinylated nanoparticles for immunological measurement)
100 μl of Ovalbumin (manufactured by SIGMA) solution diluted with PBS (100 μl each) was added to the multiwell plate and allowed to stand overnight at 4 ° C. for solid phase immobilization. After discarding the solution, 200 μl of ¼ volume of Block Ace diluted with PBST was added to the well, followed by blocking at room temperature for 1 hour. After washing 3 times with PBST, 250 μg / ml of anti-ovalbumin mouse IgE antibody (manufactured by AbD serotec) diluted with human serum was added to each well at 100 μL and allowed to stand at room temperature for 1 hour for reaction. Next, after washing three times with PBST, 5 μg / ml of HRP-labeled anti-mouse IgE rabbit antibody (Cosmo Bio) solution diluted with PBST containing ¼ volume of Block Ace was added to each well as 100 μl as an enzyme-labeled antibody solution. Each was added and allowed to react at room temperature for 1 hour. At this time, nothing was added to the enzyme-labeled antibody solution, and ZZ particles prepared in Reference Example 1 were added as nanoparticle for immunological measurement at 2.4 μg / ml, biotinylation prepared in Example 1 Simultaneously adding 2.4 μg / ml of ZZ particles as biotinylated nanoparticles for immunological measurement. After the enzyme-labeled antibody reaction, the plate was washed three times with PBST, and an ABC reagent (PIERCE), which is a complex of biotinylated HRP and avidin, was added to the well as a complex of enzyme and avidin according to the attached protocol, and 30 minutes at room temperature. Reacted. After washing 5 times with PBST, a color reaction was performed by adding TMB reagent. After stopping the reaction with 2N sulfuric acid, the absorbance at 450 nm was measured. For the measurement, a spectrophotometer multi-scan spectrum (manufactured by Thermolab System) was used. The increase in detection sensitivity was calculated by comparing the color values at 250 pg / mL. As a result, the average color development value when the nanoparticles were not used was 0.42, whereas it was 1.37 when the immunoassay nanoparticles were used, and the detection sensitivity was improved about 3 times. On the other hand, when biotinylated nanoparticles for immunological measurement were used, it was 1.56, and the detection sensitivity was improved about 3.7 times or more. That is, by using biotinylated nanoparticles for immunological measurement, the sensitivity was further improved by about 1.2 times compared to the case of using biotinylated nanoparticles that were not biotinylated. This indicates that sensitivity was improved as a result of increasing the biotinylation efficiency of a protein having self-organization ability by using a ZZ tag with high biotin labeling efficiency as an antibody binding site.

Comparative Example 1
(Immunological measurement of antibody titer using a commercial kit)
DS mouse IgE ELISA (manufactured by Sumitomo Dainippon Pharma Co., Ltd.) and Levis OVA-IgE mouse (manufactured by Shiba Goat Co., Ltd.) were selected as commercially available measurement kits for mouse anti-ovalbumin IgE, and n = 24 according to the attached protocol. The detection limit of each was determined by measurement at. Mouse anti-ovalbumin IgE antibody (AbD serotec) was used as a standard solution. The determination of the detection limit of anti-ovalbumin IgE was defined as a detection limit value at a concentration showing an average value −2SD that does not overlap with the average value + 2SD when the IgE concentration was zero. For comparison with the present invention, the amount of specimen used for measurement by DS mouse IgE ELISA is 100 μl.

The mouse anti-ovalbumin IgE detection limit value of DS mouse IgE ELISA was 2 ng / ml, and the CV value at the detection limit value was 10% (FIG. 3).
The detection limit value of mouse anti-ovalbumin IgE in Levis OVA-IgE mice was higher than 2 ng / ml, and 2 ng / ml could not be detected.

Example 6
(Immunoassay of antibody titer using immunoassay nanoparticles not labeled with biotin)
Mouse anti-ovalbumin IgE was measured using immunoassay nanoparticles not labeled with biotin. Each detection limit was determined by measurement at n = 24. Mouse anti-ovalbumin IgE antibody (AbD serotec) was used as a standard solution. The determination of the detection limit of anti-ovalbumin IgE was defined as a detection limit value at a concentration showing an average value −2SD that does not overlap with the average value + 2SD when the IgE concentration was zero.

  100 μl of Ovalbumin (manufactured by SIGMA) solution diluted with PBS (100 μl each) was added to the multiwell plate and allowed to stand overnight at 4 ° C. for solid phase immobilization. After discarding the solution, the plate was washed 3 times with PBST, and the washing solution was discarded. Anti-ovalbumin mouse IgE antibody (manufactured by AbD serotec) 2000 to 0 pg / mL diluted with 1/4 volume of Block Ace diluted with PBST was added to each well at 100 μL and allowed to stand at room temperature for 1 hour for reaction. Next, after washing three times with PBST, 5 μg / ml of HRP-labeled anti-mouse IgE rabbit antibody (Cosmo Bio) solution diluted with PBST containing ¼ volume of Block Ace was added to each well as 100 μl as an enzyme-labeled antibody solution. Each was added and allowed to react at room temperature for 1 hour. At this time, the enzyme-labeled antibody solution was added simultaneously with nothing added and with the addition of 2.4 μg / ml of the ZZ particles prepared in Reference Example 1 as immunological measurement nanoparticles. After washing 5 times with PBST, a color reaction was performed by adding TMB reagent. After stopping the reaction with 2N sulfuric acid, the absorbance at 450 nm was measured. For the measurement, a spectrophotometer multi-scan spectrum (manufactured by Thermolab System) was used. When the immunoassay nanoparticles not labeled with biotin were used, the detection limit value of mouse anti-ovalbumin IgE was 250 pg / ml, and the CV value at the detection limit value was 8.1%. The detection limit value of mouse anti-ovalbumin IgE when not using immunoassay nanoparticles was 500 pg / ml, and the CV value at the detection limit value was 7.4%.

Example 7
(Immunoassay of antibody titer using biotinylated nanoparticles for immunological measurement)
Mouse anti-ovalbumin IgE was measured using the biotinylated nanoparticles for immunological measurement of the present invention. Each detection limit was determined by measurement at n = 24. Mouse anti-ovalbumin IgE antibody (AbD serotec) was used as a standard solution. The determination of the detection limit of anti-ovalbumin IgE was defined as a detection limit value at a concentration showing an average value −2SD that does not overlap with the average value + 2SD when the IgE concentration was zero.

  100 μl of Ovalbumin (manufactured by SIGMA) solution diluted with PBS (100 μl each) was added to the multiwell plate and allowed to stand overnight at 4 ° C. for solid phase immobilization. After discarding the solution, the plate was washed 3 times with PBST, and the washing solution was discarded. Anti-ovalbumin mouse IgE antibody (manufactured by AbD serotec) 250-0 pg / mL diluted with 1/4 volume of Block Ace diluted with PBST was added to each well at 100 μL and allowed to stand at room temperature for 1 hour for reaction. Next, after washing three times with PBST, 5 μg / ml of a biotin-labeled anti-mouse IgE rabbit antibody (Cosmo Bio) solution diluted with PBST containing ¼ volume of block ace was used as an enzyme-labeled antibody solution, 100 μl in each well. Each was added and allowed to react at room temperature for 1 hour. At this time, a biotin-labeled antibody solution was added at the same time as nothing added and 2.4 μg / ml of biotinylated ZZ particles prepared in Example 1 as biotinylated nanoparticles for immunological measurement. . After washing three times with PBST, a biotinylated HRP-avidin complex ABC reagent (PIERCE) was added to the wells as a complex of enzyme and avidin according to the attached protocol, and allowed to react at room temperature for 30 minutes. After washing 5 times with PBST, a color reaction was performed by adding TMB reagent. After stopping the reaction with 2N sulfuric acid, the absorbance at 450 nm was measured. For the measurement, a spectrophotometer multi-scan spectrum (manufactured by Thermolab System) was used. When using biotinylated nanoparticles for immunological measurement, the detection limit value of mouse anti-ovalbumin IgE was 40 pg / ml (FIG. 4), and the CV value at the detection limit value was 7.1%. The detection limit value of mouse anti-ovalbumin IgE when not using biotinylated nanoparticles for immunological measurement was 250 pg / ml.

  As a result, when the nanoparticles for immunological measurement were not used, the measurement limit value was reduced by a factor of 4 compared to the commercially available kit. When using nanoparticles for immunological measurement, the measurement limit value was reduced by a factor of 8 compared to a commercially available kit. When ABC reagent that is a complex of enzyme and avidin is used without using immunological measurement nanoparticles or biotinylated nanoparticles for immunological measurement, the measurement limit value is reduced by 8 times compared to a commercially available kit. did. Furthermore, when an ABC reagent which is a complex of biotinylated nanoparticles for immunological measurement, enzyme and biotin-binding protein was used, the measurement limit value was reduced by 50 times compared to a commercially available kit.

FIG. 1 is a diagram showing the effect of biotinylated nanoparticles for immunological measurement in immunological measurement. FIG. 2 is a diagram showing the effect of biotinylated nanoparticles for immunological measurement in immunological measurement. FIG. 3 is a diagram showing the detection limit of a commercially available kit. FIG. 4 is a diagram showing that the detection limit is lowered when a biotinylated nanoparticle for immunological measurement is used by binding a complex of an enzyme and a biotin-binding protein.

Claims (8)

A nano-sized particle formed by incorporating a lipid bilayer into a protein having self-organization ability, and the protein having self-assembly ability has an antibody binding site and has self-assembly ability Biotinylated nanoparticles for immunological measurement, wherein the protein is labeled with biotin. The biotinylated nanoparticle for immunological measurement according to claim 1, wherein the antibody binding site is a ZZ tag. The biotinylated nanoparticle for immunological measurement according to claim 1 or 2, wherein the protein having self-organization ability is hepatitis B virus surface antigen (HBsAg) protein. The biotinylated nanoparticle for immunological measurement according to any one of claims 1 to 3, wherein biotin is bound to a lysine residue or an N-terminal amino acid residue of the protein. The biotinylated nanoparticle for immunological measurement according to any one of claims 1 to 4, wherein a complex of an enzyme and a biotin-binding protein is bound, and an antibody is bound to an antibody binding site, and is recognized by the antibody. An immunological measurement method in which a measurement target substance is measured using the enzyme. The method for immunological measurement according to claim 5, wherein the substance to be measured is an antibody. The immunoassay method according to claim 5 or 6, wherein the biotin-binding protein is avidin, streptavidin, or neutravidin. A nano-sized particle formed by incorporating a lipid bilayer into a protein having self-organization ability, and the protein having self-assembly ability has an antibody binding site and has self-assembly ability Use of biotinylated nanoparticles whose protein is labeled with biotin for immunological measurements.

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