阅读说明：本技术 抗体、使用其的复合材料、检测装置和方法 (Antibody, composite material using same, detection device, and detection method ) 是由 池内江美奈 于 2019-07-12 设计创作，主要内容包括：本发明涉及包含氨基酸序列的抗体,其中氨基酸序列按N至C方向包含以下结构域：N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C,其中FR指构架区氨基酸序列并且CDR指互补决定区氨基酸序列；CDR1包含由SEQ ID NO:1代表的氨基酸序列；CDR2包含由SEQ ID NO:2代表的氨基酸序列；和CDR3包含由SEQ ID NO:3代表的氨基酸序列。所述抗体能够与流感病毒的核内蛋白质结合。本发明还涉及含有所述抗体结合至选自固相支持物和标记物质中的至少一种的复合材料、包含所述复合材料和检测器的检测装置、以及基于复合材料和分析物中所含核内蛋白质的抗原-抗体反应检测物理量变化的检测方法。(The present invention relates to antibodies comprising an amino acid sequence comprising the following domains in the N to C direction: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, wherein FR denotes a framework region amino acid sequence and CDR denotes a complementarity determining region amino acid sequence; CDR1 comprises the amino acid sequence represented by SEQ ID NO. 1; CDR2 comprises the amino acid sequence represented by SEQ ID NO. 2; and CDR3 comprises the amino acid sequence represented by SEQ ID NO. 3. The antibody is capable of binding to an influenza virus nuclear protein. The present invention also relates to a composite material containing the antibody bound to at least one selected from the group consisting of a solid support and a labeling substance, a detection device comprising the composite material and a detector, and a detection method for detecting a change in a physical quantity based on an antigen-antibody reaction of the composite material and an intranuclear protein contained in an analyte.)

1. An antibody comprising an amino acid sequence, wherein the amino acid sequence comprises the following domains in the N to C direction: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, wherein

FR refers to framework region amino acid sequence and CDR refers to complementarity determining region amino acid sequence;

CDR1 comprises the amino acid sequence represented by SEQ ID NO. 1;

CDR2 comprises the amino acid sequence represented by SEQ ID NO. 2; and

CDR3 comprises the amino acid sequence represented by SEQ ID NO. 3.

2. The antibody of claim 1, wherein the antibody is capable of binding to an intranuclear protein of influenza a virus.

3. The antibody of claim 1, wherein the antibody is a single domain antibody.

4. The antibody of claim 1, wherein the influenza a virus is selected from influenza a virus subtypes H1N1 (a/lentil/YS/2011 pdm), H1N1 (a/hokkaido/6-5/2014 pdm), H5N1 (a/duck/hokkaido/Vac-3/2007), H7N7 (a/duck/hokkaido/Vac-2/2004), H1N1 (a/polio/8/34/kaempferia), H1N1 (a/duck/bird/723/1980), H1N1 (a/pig/hokkaido/2/81), H2N3 (a/dk/hoaido/17/01), H2N9 (a/duck/hong kong/278/78), H3N2 (a/duck/hoaido/5/77), At least one of H3N8 (a/duck/mongolia/4/03), H4N6 (a/dk/czech/56), H5N2 (a/duck/pennsylvania/10218/84), H5N3 (a/duck/hong kong/820/80), H6N5 (a/shearwater/s, australia/1/72), H7N2 (a/duck/hong kong/301/78), H7N7 (a/seal/massachusetts/1/1980), H9N2 (a/duck/hong kong/448/78), H9N2 (a/turkey/wisconsin/1966), H11N6 (a/duck/glaland/1/1956), and H12N5 (a/duck/labeda/60/76).

5. The antibody of claim 1, wherein

FR1 contains the amino acid sequence represented by SEQ ID NO. 4;

FR2 contains the amino acid sequence represented by SEQ ID NO. 5;

FR3 contains the amino acid sequence represented by SEQ ID NO. 6; and is

FR4 contains the amino acid sequence represented by SEQ ID NO. 7.

6. A composite material comprising the antibody of claim 1, wherein the antibody is bound to at least one selected from the group consisting of a solid support and a labeling substance.

7. The composite of claim 6, wherein the single domain antibody is bound to a solid support; and the solid support is selected from the group consisting of a plate, a bead, a disk, a tube, a filter, and a membrane.

8. The composite of claim 6, wherein the single domain antibody is bound to a labeling substance; and the labeling substance is selected from a fluorescent substance, a luminescent substance, a dye, an enzyme, and a radioactive substance.

9. A detection device, comprising: the composite material of claim 6; and a detector;

wherein the detector detects a change in the physical quantity based on an antigen-antibody reaction of the nuclear protein contained in the composite material and the analyte.

10. The detection method comprises the following steps:

(a) contacting the composite of claim 6 with an analyte; and

(b) the change in the physical quantity is detected based on the antigen-antibody reaction of the nuclear protein contained in the composite material and the analyte.

1. Field of the invention

The present invention relates to an antibody capable of binding to an influenza virus intranuclear protein, a composite material using the same, a detection device, and a method.

2. Description of the related Art

Patent document 1 and patent document 2 disclose antibodies each capable of binding to influenza virus. At least part of the antibodies disclosed in patent document 1 and patent document 2 are derived from alpaca. Patent document 1 and patent document 2 are incorporated herein by reference.

List of citations

Patent document

Patent document 1: U.S. Pat. No. 9,771,415

Patent document 2: U.S. Pat. No. 9,868,778

Summary of The Invention

An object of the present invention is to provide a novel antibody capable of binding to an intranuclear protein of influenza virus, a composite material using the same, a detection device, and a method.

The invention is an antibody comprising an amino acid sequence, wherein the amino acid sequence comprises the following domains in the N to C direction: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C, wherein

FR refers to framework region amino acid sequence and CDR refers to complementarity determining region amino acid sequence;

CDR1 comprises the amino acid sequence represented by SEQ ID NO. 1;

CDR2 comprises the amino acid sequence represented by SEQ ID NO. 2; and

CDR3 comprises the amino acid sequence represented by SEQ ID NO. 3.

The present invention provides novel antibodies capable of binding to the nuclear protein of influenza virus. The invention also provides composites comprising the novel antibodies. The present invention also provides a detection device and a detection method using the novel antibody.

Brief Description of Drawings

FIG. 1A is a vector diagram of various genes contained in a gene library used to link VHH antibodies.

FIG. 1B shows a detail of the vector diagram shown in FIG. 1A.

FIG. 2 shows a vector map for expression of VHH antibodies.

FIG. 3A is a graph showing the result of SPR evaluation of the binding ability to a recombinant intranuclear protein of a VHH antibody (concentration: 0.39nM) comprising the amino acid sequence represented by SEQ ID NO. 8.

FIG. 3B is a graph showing the result of SPR evaluation of the binding ability to a recombinant intranuclear protein of a VHH antibody (concentration: 0.78nM) comprising the amino acid sequence represented by SEQ ID NO. 8.

FIG. 3C is a graph showing the result of SPR evaluation of the binding ability of a VHH antibody (concentration: 1.56nM) comprising the amino acid sequence represented by SEQ ID NO. 8 to a recombinant intranuclear protein.

FIG. 3D is a graph showing the result of SPR evaluation of the binding ability to a recombinant intranuclear protein of a VHH antibody (concentration: 3.125nM) comprising the amino acid sequence represented by SEQ ID NO. 8.

FIG. 3E is a graph showing the result of SPR evaluation of the binding ability of a VHH antibody (concentration: 6.25nM) comprising the amino acid sequence represented by SEQ ID NO. 8 to a recombinant intranuclear protein.

FIG. 3F is a graph showing the result of SPR evaluation of the binding ability of a VHH antibody (concentration: 12.5nM) comprising the amino acid sequence represented by SEQ ID NO. 8 to a recombinant intranuclear protein.

FIG. 4A is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO:8 with influenza A virus H1N 1A/lentil/YS/2011 pdm.

FIG. 4B is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H1N 1A/Hokkaido/6-5/2014 pdm.

FIG. 4C is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H5N 1A/Duck/Hokkaido/Vac-3/2007.

FIG. 4D is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H7N 7A/Duck/Hokkaido/Vac-2/2004.

FIG. 4E is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H1N 1A/puerto Rico/8/34/Xinesan.

FIG. 4F is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H1N 1A/Duck/bird/723/1980.

FIG. 4G is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H1N 1A/pig/Hokkaido/2/81.

FIG. 4H is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H2N 3A/dk/Hokkaido/17/01.

FIG. 4I is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H2N 9A/Duck/hong Kong/278/78.

FIG. 4J is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H3N 2A/Duck/Hokkaido/5/77.

FIG. 4K is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H3N 8A/Duck/Mongolia/4/03.

FIG. 4L is a graph showing the measurement results of the cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H4N 6A/dk/Czech/56.

FIG. 4M is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H5N 2A/Duck/Pa/10218/84.

FIG. 4N is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H5N 3A/Duck/hong Kong/820/80.

FIG. 4O is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H6N 5A/shearwater/S. Australia/1/72.

FIG. 4P is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H7N 2A/Duck/hong Kong/301/78.

FIG. 4Q is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H7N 7A/seal/Massachusetts/1/1980.

FIG. 4R is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H9N 2A/Duck/hong Kong/448/78.

FIG. 4S is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO 8 with influenza A virus H9N 2A/turkey/wisconsin/1966.

FIG. 4T is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H11N 6A/duck/England/1/1956.

FIG. 4U is a graph showing the measurement results of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO. 8 with influenza A virus H12N 5A/Duck/Alberta/60/76.

FIG. 4V is a graph showing the results of measurements of cross-reactivity of a VHH antibody comprising the amino acid sequence represented by SEQ ID NO 8 with influenza B virus B/Hokkaido/M2/2014.

Detailed description of the embodiments

The antibodies of the invention are capable of binding to influenza a virus. In particular, the antibodies of the invention are capable of binding to the nuclear proteins of influenza a virus. As disclosed in patent document 1, an antibody capable of binding to influenza virus comprises a single domain amino acid sequence comprising the following domains in the N to C direction:

N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C

wherein

FR refers to the framework region amino acid sequence and CDR refers to the complementarity determining region amino acid sequence.

In the present invention, CDR1 comprises the amino acid sequence represented by GSIFSPNV (SEQ ID NO: 1).

In the present invention, CDR2 comprises the amino acid sequence represented by ITLGEST (SEQ ID NO: 2).

In the present invention, CDR3 comprises the amino acid sequence represented by NAGPILERVGPY (SEQ ID NO: 3).

Desirably, CDR1, CDR2 and CDR3 are represented by SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively. In this case, it is more desirable that FR1, FR2, FR3 and FR4 comprise the amino acid sequences represented by QVQLVESGGGLVQAGGSLRLSCIAS (SEQ ID NO:4), MGWYRQAPGKPRELVAA (SEQ ID NO:5), NYADSVKGRFTISRSNAENTVYLQMDSLKPEDTAVYYC (SEQ ID NO:6) and WGQGTQVTVSS (SEQ ID NO:7), respectively.

In other words, it is desirable that the antibody of the present invention comprises the amino acid sequence represented by QVQLVESGGGLVQAGGSLRLSCIASGSIFSPNVMGWYRQAPGKPRELVAAITLGESTNYADSVKGRFTISRSNAENTVYLQMDSLKPEDTAVYYCNAGPILERVGPYWGQGTQVTVSS (SEQ ID NO: 8).

The antibody comprising the amino acid sequence represented by SEQ ID NO. 8 has NO antigenic cross-reactivity with influenza viruses other than influenza A viruses. An example of an influenza virus other than influenza a virus is influenza b virus.

The antibody of the present invention can be used in a detection device or in a detection method for detecting an intranuclear protein of influenza A virus. In this case, the antibody of the present invention can be used in a state of being bound to another material in a composite material (for example, in a state of being bound to at least one selected from a solid support and a labeling substance in a composite material in which the antibody of the present invention has been bound to the solid support).

The shape and material of the solid phase support are not limited as long as the solid phase support is a support insoluble in the solvent of the reaction system for antigen-antibody reaction. Examples of the shape of the solid support are a plate, a bead, a disk, a tube, a filter and a membrane. Examples of materials for the solid support are polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene or polymethyl methacrylate, metals such as gold, silver or aluminum or glass. As a method for binding the antibody to the solid support, a known method such as physical adsorption, covalent binding, ion bonding or crosslinking is used.

For example, a labeling substance such as a fluorescent substance, a luminescent substance, a dye, an enzyme, or a radioactive substance is used. As a method for binding an antibody to a labeling substance, a publicly known method such as a physical adsorption method, a covalent binding method, an ion bonding method, or a crosslinking method is used.

In a detection method using the antibody of the present invention, a composite material containing the antibody is contacted with an analyte. Subsequently, the change in the physical quantity is detected based on the antigen-antibody reaction of the intranuclear protein of influenza A virus contained in the analyte and the antibody contained in the composite material. Examples of physical quantities are luminous intensity, chromaticity, light transmittance, turbidity, absorbance or radiation dose. As specific examples of the detection method, publicly known methods such as enzyme immunoassay, immunochromatography, latex agglutination, radioimmunoassay, fluorescence immunoassay, or surface plasmon resonance spectrometry are used.

The detection apparatus using the antibody of the present invention includes a detector for detecting any one of the physical quantities that change based on the antigen-antibody reaction. The detector comprises publicly known means such as a photometer, spectroscope or dosimeter.