WO2023074524A1

WO2023074524A1 - Novel lateral flow assay

Info

Publication number: WO2023074524A1
Application number: PCT/JP2022/039105
Authority: WO
Inventors: 卓人東條; 真由美和田; 大貴西口; 彰人川原
Original assignee: 花王株式会社
Priority date: 2021-10-29
Filing date: 2022-10-20
Publication date: 2023-05-04
Also published as: JP2023067793A; CN118339454A

Abstract

The present invention provides a novel lateral flow assay. This lateral flow assay uses an antibody immobilized to a label to detect an antigen in a sample. The label is iron(III) oxide particles.

Description

Novel lateral flow assay

The present invention relates to a novel lateral flow assay.

Conventionally, immunoassays using proteins, especially antibodies and fragments containing their molecular recognition sites, have been used in the medical diagnostic field. For example, detection of antigenic proteins associated with specific diseases in clinical samples such as nasal swabs, blood, urine, etc., and clinical tests for the qualitative or quantitative detection of specific pathogenic microorganisms, viruses and chemical substances are known. It is Among immunoassays, the lateral flow assay makes it possible to visualize the presence of the antigen by visually observing the color development on the membrane due to the antigen-antibody reaction. Therefore, it is used not only in the medical diagnostic field but also in a wide range of fields such as clinical practice, veterinary medicine, agriculture, food industry, biological defense and sanitary environment.

The lateral flow assay is a clinical test method with excellent sensitivity and reproducibility, but there is a problem in terms of running costs, especially in fields other than medical diagnostics (agriculture, food industry, sanitary environment survey, etc.). . Although detection devices such as lateral flow assays for food allergen testing and lateral flow assays for living environment surveys are commercially available for non-medical diagnostic use, most of them are sold at a price range exceeding 1,000 yen per test. It is For example, consumers should use such devices to conduct surveys of food allergens and sanitary environments when necessary. there is One reason for this is that the lateral flow assay is basically disposable, and the expensive carrier for detection and protein for detection that perform the detection function cannot be reused. In particular, gold nanoparticles are frequently used as detection carriers for lateral flow assays (for example, Non-Patent Document 1). Price volatility is an issue. In addition, latex nanoparticles, colored silica nanoparticles, colored cellulose nanoparticles, etc. are also used, but since they are all nanoparticles manufactured using advanced technology, they are sold at the same price range as gold nanoparticles. Therefore, even if such a material is used, the problem that the carrier is expensive has not yet been solved.

On the other hand, a magnetic detection type lateral flow assay using iron oxide (II, III) (Fe ₃ O ₄ ) nanoparticles has been used. Since it is the main focus, the use of a dedicated detection device is a premise, which poses a problem for general consumers to use. In addition, although the lateral flow assay using iron (II, III) oxide nanoparticles can be confirmed visually (Non-Patent Document 2), the visibility is insufficient.

(Non-Patent Document 1) Tanaka, R. et al. A novel enhancement assay for immunochromatographic test strips using gold nanoparticles. Anal Bioanal Chem 385, 1414-1420, 2006
(Non-Patent Document 2) Connolly, R. &O' Kennedy, R. Magnetic lateral flow immunoassay test strip development -Considerations for proof of concept evaluation. Methods 116, 132-140, 2017

The present invention relates to the following 1) and 2).
1) A lateral flow assay in which an antibody immobilized on a label is used to detect an antigen in a sample, the label being iron (III) oxide particles.
2) Reagents for lateral flow assays comprising antibodies immobilized on iron(III) oxide particles.

General structure of test strips for lateral flow assays. Comparison of detection line visibility of iron (III) oxide particles and iron (II, III) oxide particles.

Detailed description of the invention

As used herein, the term "lateral flow assay" refers to a labeled antibody that specifically binds to an antigen and is immobilized on a labeled antibody, and an antibody that specifically binds to an antigen and is immobilized on a membrane. Among the methods for visually detecting the presence of antigens contained in a sample using a capture antibody, this is a method in which the sample is developed in the horizontal direction with respect to the membrane. Here, the "label" means a substance that carries an antibody and functions as a visually detectable marker. Specifically, in the lateral flow assay, the labeled antibody and the antigen in the sample form a complex, and the complex is captured by the capture antibody immobilized on the membrane, and the complex accumulates and develops color. , can detect the presence of antigen in a sample. In the present specification, "immobilization" and "immobilization" refer to the state in which an antibody is supported on a carrier or membrane, or the state in which the antibody is supported.

As used herein, the target "antigen" is not particularly limited, and includes, for example, antigens derived from allergens, biomarkers, viruses, bacteria, fungi, protozoa, and the like. As used herein, the term "detection" can also be referred to as "measurement", and "antigen detection" includes proof of the presence or absence of an antigen and quantification of the antigen in the broadest sense. Must be construed and should not be construed as limiting in any way.

An allergen is a substance that enters the body from the outside through inhalation, piercing, ingestion, or contact and causes hypersensitivity or allergic reaction. Allergens include pollen of grasses (reeds, giant mothballs, giant sparrow, daffodil, sycamore, wheat, cornuca, bromeliad, corn sorghum, long grass, ragweed, broad house fescue, sarcophagus, etc.); , dandelion, wormwood, lesser sorrel, ragweed, ragweed, ragweed, plantain, mugwort, etc.); , beech, pine, willow, etc.); animal epidermis (duck feathers, cat dander, dog dander, cow dander, horse dander, rabbit epidermis, hamster epidermis, guinea pig epidermis, sheep epidermis, pig epidermis, goat epidermis, chicken dander); , goose feathers, budgerigars' feathers, budgerigars' droppings, mice, rats, etc.); (Acacia acarina, Dermatophagoides acarina, Dermatophagoides leopard mite, Green spider mite, Dermatophagoides leopard mite, etc.); Food (eggs, milk, wheat, buckwheat, peanuts, shrimp, crab, almonds, abalone, squid, salmon roe, oranges, cashew nuts, kiwifruit, beef, walnuts, sesame , salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yam, apples, gelatin, etc.);

Biomarkers are substances in the body, such as proteins and nucleic acids contained in body fluids such as blood, saliva, and sweat, urine, and feces. etc. Biomarkers include CK-MB, H-FABP, BNP, NT-proBNP, topolonin, myoglobin, albumin, ceruloplasmin, calbindin, clusterin, cystatin C, KIM-1, NGAL, Osteopontin, TFF3, TIMP-1, VEGF -A, L-FABP, Chorionic Gonadotropin, Interleukin, Amylase, NMP22, CEA, PSA, CYFRA21-1, SLX, CA125, SCC, NSE, ProGRP, CA19-9, CA19-9, AFP, PIVKA-II, AFP-L3, Span-1, DUPAN-2, CA50, BTA, CA15-3 and the like.

Viruses can be of all types, regardless of the type of nucleic acid (RNA, DNA) and the presence or absence of an envelope. SARS coronavirus; SARS coronavirus-2; respiratory syncytial virus; mumps virus; Lassa virus; dengue virus; Hepatitis A virus; Hepatitis E virus; Rhinovirus; Astrovirus; Rotavirus; Coxsackievirus; Enterovirus; ; human papillomavirus and the like. Bacteria include Pertussis, Diphtheria, Escherichia coli, Haemophilus influenzae, Helicobacter, Neisseria meningitidis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Group A Streptococcus, Group B Streptococcus, Staphylococcus aureus, Tetanus, Legionella, and Tuberculosis. bacteria, Mycoplasma, Vibrio parahaemolyticus, Salmonella spp., Escherichia coli, Campylobacter, Clostridium perfringens, Shigella, Bacillus cereus, Clostridium botulinum, Streptococcus mutans, and the like. Examples of fungi include Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, Aspergillus ustus, etc.), Blastomyces dermatitidis, etc., Candida albicans, etc. , Coccidioides fungi (e.g. Coccidioides immitis etc.), Cryptococcus fungi (e.g. Cryptococcus neoformans, Cryptococcus gattii etc.), Histoplasma fungi (e.g. Histoplasma capsulatum etc.), Paracoccidioides fungi (e.g. Paracoccidioides brasiliensis etc.), Sporothrix fungi ( For example, Sporothrix schenckii, etc.). Protozoa include Plasmodium, Leishmania, Cryptosporidium, Trypanosoma Gambia, Trypanosoma Rhodesia, Trypanosoma cruzi, Trichomonas, Toxoplasma, Babesia, Entamoeba histolytica, Giardia, and the like.

The present invention relates to providing a novel lateral flow assay that can be performed at a much lower cost than conventional methods.

In order to solve the above problems, the present inventors have investigated various substances as carriers for immobilizing detection antibodies in lateral flow assays, that is, as labeled substances for detection antibodies. Iron (III) oxide (Fe ₂ O ₃ ) particles have been found to be suitable as labels. In addition, immunoglobulin G (IgG) was immobilized on iron (III) oxide particles, and a heavy chain variable domain antibody derived from a heavy chain antibody expected to be inexpensively produced by microbial culture was immobilized on iron (III) oxide particles. In both cases, it was found that a lateral flow assay for visually confirming color development by iron (III) oxide particles could be constructed. Surprisingly, it was also found that the lateral flow assay using iron (III) oxide particles has overwhelmingly superior visibility compared to the case using iron (II, III) oxide particles.

According to the present invention, it has detection sensitivity equivalent to that of conventional lateral flow assays that use gold nanoparticles as labels, and enables lateral flow assays at significantly lower costs. Therefore, it is expected that the cost of the lateral flow assay device will be reduced, and the utilization of the lateral flow assay device will be promoted in a wide range of fields, both medical diagnostics and non-medical fields.

Samples to be subjected to the lateral flow assay of the present invention include samples that contain antigens or that may contain antigens. Such samples include tracheal swabs, nasal swabs, oral swabs, pharyngeal swabs, nasal washes, nasal aspirates, nasal discharge, snot, saliva, sputum, tears, blood, serum, urine, feces, tissues, cells. In addition to biological samples such as lysates of tissue or cells, food raw materials, processed foods, environmental samples, samples collected from hard or soft surfaces, and the like may be used. The sample may be subjected to the assay as it is or after crushing the sample, extracting the antigen, and recovering the solution containing the antigen, and the antigen of interest may be appropriately concentrated or diluted with water or buffer. may Preferably the sample is a liquid sample.

The method of the present invention is a lateral flow assay in which an antibody immobilized on a label is used to detect an antigen in a sample, and the label is iron (III) oxide particles.

Iron (III) oxide is a reddish brown solid represented by the chemical formula Fe ₂ O ₃ (CAS Registry Number: 1309-37-1). Iron (III) oxide is a water-insoluble and chemically stable compound, and is highly safe, as it has a track record of being used as a pigment. It is also inexpensive and available in large quantities.

The shape of the iron (III) oxide particles is not particularly limited, and may be, for example, spherical, needle-like, or spindle-like.
The average particle size of the iron (III) oxide particles is not particularly limited, but is preferably 10 nm or more, more preferably 50 nm or more, preferably 1000 nm or less, and more preferably 500 nm or less. A specific range of the average particle size of the iron (III) oxide particles is preferably 10 to 1000 nm, more preferably 50 to 500 nm. The average particle size of iron (III) oxide particles can be measured by a laser diffraction/scattering method.

As iron (III) oxide particles, for example, ferric oxide (particle diameter: 300 nm, Kojundo Chemical Laboratory Co., Ltd.), Iron (III) oxide (particle diameter: <5 μm, Sigma-Aldrich), etc. are commercially available. and these can be used.

The antibody immobilized on the iron (III) oxide particles may be any antibody that specifically binds to the antigen of interest, and may be either a polyclonal antibody or a monoclonal antibody. In addition, antibody fragments such as F(ab') ₂ , F(ab'), single-chain Fv (scFv), amino acid residues substituted with cysteine residues in VH and VL It may be a disulfide-bonded Fv (dsFv) or a polymer thereof, or a dimerized V region (diabody) in which the scFv is dimerized. Antibody fragments also include peptides containing a portion of an antibody, that is, peptides comprising a portion of the amino acid sequence that constitutes the antibody, as long as they specifically bind to the antigen of interest. The immunoglobulin class of the antibody is not particularly limited and may be any immunoglobulin class of IgG, IgM, IgA, IgE, IgD and IgY, preferably IgG. Antibodies may be any of non-human animal antibodies, human chimeric antibodies, humanized antibodies and human antibodies. Antibodies from non-human animals include, for example, antibodies from mice, rats, hamsters, guinea pigs, rabbits, goats, camels, llamas, and alpacas.

A commercially available polyclonal antibody or monoclonal antibody can be used as the antibody to be immobilized on the iron (III) oxide particles. Alternatively, polyclonal or monoclonal antibodies produced using known means can be used. Mammal-derived monoclonal antibodies include those produced from hybridomas and those produced by designing antibody genes or antibody fragment genes and using well-known genetic engineering techniques.
For example, for antibodies, a recombinant vector was constructed by inserting a DNA encoding an H chain variable region and a DNA encoding an L chain variable region downstream of a suitable vector promoter, and introduced into a host cell. The H chain variable region and the L chain variable region are produced from the transformant and linked with a ligatable peptide, or the DNA encoding the H chain variable region and the DNA encoding the L chain variable region are linked by a known linker. A single-chain recombinant antibody protein having antigen-binding ability ( scFv) (see MacCfferty, J. et al., Nature, 348, 552-554, 1990, Tim Clackson et al, Nature, 352, 642-628, 1991, etc.). Furthermore, it is also possible to produce an expressed product by combining a DNA encoding a variable region and a DNA encoding a constant region. In this case, the constant region may be the same as the antibody from which the variable region is derived, or may be derived from a different antibody.

Antibody-producing hybridomas can be produced basically using known techniques as follows. For example, antigenic peptides are optionally conjugated with suitable carrier proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin, etc. to enhance immunogenicity and immunize non-human mammals. It can be produced by An antigen peptide used as a sensitizing antigen (immunogen) can be produced by genetic engineering techniques or chemical synthesis.

The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with mammalian myeloma cells, which are the parent cells used for cell fusion. Typically, rodent animals such as mice, rats, hamsters and the like are used.

Immunization of animals with sensitizing antigens is carried out according to known methods. For example, it is carried out by intraperitoneally or subcutaneously injecting a sensitizing antigen into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like. , subcutaneously, intracutaneously, intraperitoneally, or the like to animals for temporary stimulation, and then repeat the same procedure as necessary. The dosage of the antigen is appropriately determined depending on the route of administration and animal species, but the usual dosage is preferably about 10 μg to 1 mg per administration. After such immunization and confirmation of an increase in the desired antibody level in the serum, blood is collected from the mammal and serum components are purified to obtain polyclonal antibodies. When purifying serum components, an affinity column or the like on which a sensitizing antigen is immobilized can be used.

When producing monoclonal antibodies, immune cells are taken out from mammals with elevated antibody levels, and cell fusion is performed. Preferred immune cells for cell fusion are splenocytes, among others.
As mammalian myeloma cells as the other parent cells to be fused with the immune cells, various known cell lines such as P3X63, NS-1, MPC-11 and SP2/0 are appropriately used.

The cell fusion of the immune cells and myeloma cells can be performed according to a known method, for example, the method of Keller et al. (Kohler et al., Nature, vol, 256, p495-497 (1975)). That is, in the presence of a cell fusion promoter such as polyethylene glycol (PEG with an average molecular weight of 1000 to 6000, 30 to 60% concentration), Sendai virus (HVJ), if desired, an adjuvant such as dimethyl sulfoxide is added, RPMI1640 culture solution Fusion cells (hybridomas) are formed by mixing immune cells and myeloma cells in a nutrient medium such as MEM medium.

Hybridomas formed by fusion are cultured in a selection medium such as a medium containing hypoxanthine, thymidine and aminopterin (HAT medium) for 1 to 7 days to separate unfused cells. The resulting hybridomas are further selected by the antibodies they produce. The selected hybridomas are cloned according to a known limiting dilution method to establish monoclonal antibody-producing hybridomas.
Known methods can be used to detect the activity of antibodies produced by hybridomas. Examples include ELISA, agglutination, and radioimmunoassay.

Monoclonal antibodies can be obtained from the obtained hybridoma by culturing the hybridoma according to a conventional method and obtaining the culture supernatant thereof, or by administering the hybridoma to a mammal compatible therewith to proliferate the A method of obtaining ascites is adopted.

Antibody purification can be performed using known purification means such as salting out, gel filtration, ion exchange chromatography, or affinity chromatography.

Alternatively, the antibody immobilized on iron (III) oxide particles may be a heavy chain variable domain antibody. Heavy chain variable domain antibodies include VHH antibodies and IgNAR antibodies. A VHH antibody is a small antibody consisting of a single domain (single domain) containing the variable region of a heavy chain antibody without a light chain produced by camelids. A VHH antibody has three antigen-binding loops (antigen complementarity determining regions; CDRs) like the H chain of an IgG antibody. Since the molecular weight of VHH antibody is as small as 1/10 that of IgG antibody, it can bind to epitopes even when normal IgG cannot bind to epitopes due to steric structural problems, and is modified with many sugar chains. Since it can also bind to the surface of virus particles, etc., it has a wide range of possible target molecules. Furthermore, VHH antibodies are excellent in acid resistance and heat resistance, and unlike IgG, they do not need to be produced in cultured cells, and can be produced in Escherichia coli, yeast, and the like. Therefore, it has the advantage of being easy to mass-produce and easy to purify. Furthermore, since the VHH antibody is composed of a single-chain peptide, it is easy to modify its function using techniques such as protein engineering or chemical modification. From these points of view, the antibody immobilized on the iron (III) oxide particles is preferably a heavy chain variable domain antibody, more preferably a VHH antibody.

The method for producing the heavy chain variable domain antibody is not particularly limited, and it can be easily produced by a technique known in the art. For example, it can be prepared by combining a solid-phase peptide synthesis method and a native chemical ligation (NCL) method, or by genetic engineering. For example, it is preferable to incorporate it into a plasmid), introduce it into a host cell, and produce it as a recombinant antibody.

Here, plasmids suitable for various host cells can be used as antibody expression plasmids. For example, pBR322, pBR325, pUC12, pUC13, pUC19 and other E. coli derived plasmids; pUB110, pTP5, pC194, pHY300pLK and other Bacillus subtilis derived vectors; pSH19, pSH15 and other yeast derived vectors; lambda phage and other bacteriophages; Viruses, adeno-associated viruses, lentiviruses, vaccinia viruses, baculoviruses and other viral vectors as well as appropriately modified vectors thereof can be used.

These expression plasmids have replication origins, selectable markers and promoters suitable for each plasmid, and optionally enhancers, transcription termination sequences (terminators), ribosome binding sites and polyadenylation signals. may have. Furthermore, in order to facilitate purification of the expressed polypeptide, the expression plasmid may have inserted therein a nucleotide sequence for fusing with a FLAG tag, His tag, HA tag, GST tag, or the like for expression.

Antibody-producing bacteria can be produced by introducing the above expression plasmid into desired bacteria by a desired method, such as electroporation, protoplast-PEG method, and the like. Examples of host cells used for recombinant antibody production include E. coli, Bacillus subtilis, Corynebacterium, various fungi, animal cells, plant cells and other cells, baculovirus/insect cells, yeast cells, and the like. can. Among these, Corynebacterium and Bacillus subtilis can be suitably used, and Bacillus subtilis with high productivity is more preferable.

When extracting the expressed antibody from the cultured bacterial bodies or cultured cells, after culturing, the bacterial bodies or cultured cells are collected by a known method, suspended in an appropriate buffer, and treated with ultrasonic waves, lysozyme and/or After disrupting the cells or cells by freezing and thawing, a soluble extract is obtained by centrifugation or filtration. In the case of secretory expression, the expressed antibody can also be obtained from the centrifugal supernatant of the culture medium. The peptide of interest can be obtained from the resulting extract by appropriately combining known separation/purification methods.

Known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, methods utilizing mainly molecular weight differences such as dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion A method utilizing a charge difference such as exchange chromatography, a method utilizing a specific affinity such as affinity chromatography, a method utilizing a hydrophobicity difference such as reversed-phase high-performance liquid chromatography, or isoelectric focusing. A method using a difference in isoelectric points such as is used.

In one example, when the antigen of interest is lysozyme contained in eggs, a specific example of the VHH antibody is a VHH antibody consisting of the amino acid sequence shown in SEQ ID NO: 13 (referred to as "1ZVY" in the examples below), the sequence Examples thereof include a VHH antibody consisting of the amino acid sequence represented by number 15 (referred to as "1ZVH" in Examples below). In another example, when the antigen of interest is the SARS-CoV-2 S1 protein, which is an antigen derived from SARS-CoV-2, a specific example of the VHH antibody is a VHH antibody consisting of the amino acid sequence shown in SEQ ID NO: 17. (referred to as "E9" in Examples below). In another example, when the antigen of interest is immunoglobulin G (IgG), a specific example of the VHH antibody is a VHH antibody consisting of the amino acid sequence represented by SEQ ID NO: 19 (“VHH28-His” in the examples below). ) and the like.

Antibodies can be immobilized on iron (III) oxide particles by physical adsorption or by chemical bonds such as covalent bonds via functional groups. Adsorption may also be performed by physical adsorption, but physical adsorption or adsorption using a material-adsorbing peptide or the like is preferably used from the viewpoint of operability. Physical adsorption or adsorption using a material-adsorbing peptide or the like can usually be carried out in a predetermined buffer solution. Examples of buffers include commonly used buffers such as phosphate buffer, Tris buffer, and Good's buffer. The pH of the buffer solution is preferably 6.0 to 9.5, more preferably 6.5 to 8.5, even more preferably 7.0 to 8.0. The antibody concentration is preferably 0.01 to 100 μg/mL, more preferably 0.1 to 20 μg/mL, even more preferably 1 to 10 μg/mL.
For antibody immobilization, the iron (III) oxide particles may be used as they are, or prior to antibody immobilization, they may be subjected to chemical immobilization to reduce non-specific immobilization of proteins other than those to be immobilized. Modification, for example, modification with PEG, NHS, carboxyl group, amine group, streptavidin, etc. may be performed in advance, but in the present invention, unmodified iron oxide that has not undergone such chemical modification before antibody immobilization (III) Particles are preferably used.
After antibody immobilization, the iron(III) oxide particles may be further blocked with a commonly used blocking agent. For blocking, proteins such as BSA, casein, and gelatin, and polymer compounds such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and PEG can be used.
Thus, the antibody immobilized on the iron (III) oxide particles, ie, the iron (III) oxide particle-labeled antibody, may be dispersed and stored in a storage reagent to prevent denaturation. As the denaturation inhibitor, proteins such as BSA, glycerin, sugar and the like are used.

In the lateral flow assay of the present invention, an iron (III) oxide particle-labeled antibody is used to detect the antigen in the sample. Specifically, the assay involves contacting an iron (III) oxide particle-labeled antibody with a sample. In one embodiment, the lateral flow assay of the present invention uses a reagent comprising an iron (III) oxide particle-labeled antibody, preferably an iron (III) oxide particle-labeled antibody, and a membrane on which a capture antibody is immobilized. It can be carried out.

Here, the capture antibody may be any antibody that specifically binds to the antigen of interest, and includes the same antibodies immobilized on the iron (III) oxide particles described above. From the viewpoint of detection sensitivity, it is preferable to use an antibody that recognizes an epitope different from that of the antibody immobilized on the iron (III) oxide particles as the capture antibody so that the antigen can be detected by a so-called sandwich assay.

Any material can be used as the membrane. For example, polyethylene, polyethylene terephthalate, polypropylene, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, ceramics, and the like formed into a membrane can be used. By appropriately selecting the pore size and structure of the membrane, it is possible to control the rate at which the complex formed from the iron (III) oxide particle-labeled antibody and the antigen develops on the membrane. A nitrocellulose membrane is preferably used as the membrane, and the pore size thereof is preferably 1 to 20 μm, more preferably 5 to 10 μm, from the viewpoint of development speed.

A capture antibody is immobilized on the membrane. The capture antibody is preferably immobilized to form a detection line on the membrane. Immobilization of the capture antibody to the membrane may be by physical adsorption, chemical bonding such as covalent bonding via a functional group, or adsorption using a material-adsorbing peptide or the like. However, from the viewpoint of operability, physical adsorption or adsorption using a material-adsorbing peptide or the like is preferably used. Immobilization of the capture antibody to the membrane can be performed by a known method. For example, by adjusting the capture antibody to a predetermined concentration and applying the solution in a line on the membrane using a device having a mechanism capable of moving the solution in the horizontal direction while ejecting the solution from a nozzle at a constant speed. done. At this time, the concentration of the capturing antibody is preferably 0.01-100 mg/mL, more preferably 0.1-10 mg/mL. Also, the amount of capture antibody immobilized on the membrane is preferably 0.5 to 2 μL/cm. The position on the membrane where the capture antibody is immobilized can be appropriately selected to match the design of the assay system.
In addition, the capture antibody solution described above can usually be prepared using a predetermined buffer solution. Examples of the buffer include commonly used buffers such as phosphate buffer, Tris buffer and Good's buffer. The pH of the buffer solution is preferably in the range of 6.0 to 9.5, more preferably 6.5 to 8.5, even more preferably 7.0 to 8.0.
After immobilization of the capture antibody, the membrane may be further blocked with a commonly used blocking agent. For blocking, proteins such as BSA, casein and gelatin, and polymers such as PVA, PVP and PEG can be used.
By drying the membrane coated with the capturing antibody, a membrane on which an appropriate amount of capturing antibody is immobilized can be obtained.

A control capture antibody may be further immobilized on the membrane. A control capture antibody is preferably immobilized to form a control line on the membrane. The control capture antibody may be any antibody that can bind to the iron (III) oxide particle-labeled antibody, and can be appropriately selected according to the type, origin, etc. of the iron (III) oxide particle-labeled antibody. A control line is provided to ensure the reliability of the assay. The method for immobilizing the control capture antibody on the membrane is the same as for the capture antibody described above. The position on the membrane where the control capture antibody is immobilized can be appropriately selected to match the design of the assay system, and is preferably downstream of the detection line.

An example of a specific assay is shown below. A sample solution is prepared by contacting an iron (III) oxide particle-labeled antibody with a sample, and the sample solution is added dropwise to the membrane on which the capture antibody is immobilized, or the membrane on which the capture antibody is immobilized is immersed in the sample solution. The sample liquid spreads on the membrane due to capillary action. When the target antigen is contained in the sample, the iron (III) oxide particle-labeled antibody and the target antigen in the sample form a complex, which is captured by the capture antibody on the membrane and the complex accumulates. As a result, the detection line develops a red color derived from the iron (III) oxide particles. On the other hand, when the sample does not contain the target antigen, the detection line does not develop color. Using such color development as an index, the presence or absence of the target antigen in the sample can be determined. That is, if color development is observed in the detection line, it can be determined that the sample contains the antigen of interest, and if color development is not observed in the detection line, it can be determined that the sample does not contain the target antigen. be able to. When the membrane has a control line, the control capture antibody captures excess iron oxide (III) particle-labeled antibody regardless of the presence or absence of the target antigen in the sample, and the iron oxide (III) particle-labeled antibody accumulates. As a result, the control line develops a red color derived from iron (III) oxide particles. Using such color development as an index, the validity of the assay can be evaluated. That is, if color development is observed in the control line, it can be determined that the assay was performed normally, and if color development is not observed in the control line, it can be determined that the assay was not performed normally.

In another embodiment, the lateral flow assay of the present invention can be performed using a common lateral flow assay reagent (test strip) consisting of a sample pad, a conjugate pad, a membrane, and an absorbent pad. . The general structure of the test strip is shown in FIG. In the present invention, the conjugate pad holds an iron (III) oxide particle-labeled antibody and the membrane has an immobilized capture antibody.

The sample pad is the part where the sample is dropped, and includes any substance and form that absorbs the sample while molded into the pad and allows the sample to pass through. Suitable materials for sample pads include, but are not limited to, glass fibers, acrylic fibers, hydrophilic polyethylene materials, dry paper, paper pulp, textiles, and the like. The sample pad can also have the function of a conjugate pad, which will be described later.

The conjugate pad holds an iron oxide (III) particle-labeled antibody, and when the sample passes through the conjugate pad, the iron (III) oxide particle-labeled antibody and the target antigen in the sample form a complex. It refers to the part that has the function to
Suitable materials for conjugate pads include, but are not limited to, paper, cellulose blends, nitrocellulose, polyesters, acrylonitrile copolymers, glass fibers or non-woven fibers such as rayon.

As the membrane, the same one as described above can be used. The membrane preferably includes a detection line to which capture antibodies are immobilized and a control line to which control capture antibodies are immobilized. The methods for immobilizing the capture antibody, the control capture antibody, or both on the membrane are as described above.

The absorbent pad is located at the most downstream position and is a part with liquid absorbency that controls the development of the sample by absorbing the sample developed on the membrane. Suitable materials for absorbent pads include, but are not limited to, filter paper.

The above reagent is a membrane with a sample pad, conjugate pad, and absorbent pad arranged and attached. The arrangement can be changed as appropriate, but the sample dropped onto the sample pad passes through the sample pad by capillary action, is transferred to the conjugate pad, and further passes through the conjugate pad by capillary action to the membrane. Each site is preferably arranged so that it can be transferred, spread out on a membrane, and absorbed into an absorbent pad. These are usually arranged on a solid support such as a plastic adhesive sheet. The solid phase support is preferably composed of a material that does not interfere with the development of the sample, and the adhesive component is also preferably a material that does not interfere with the development of the sample. It is also possible to laminate a polyester film or the like for the purpose of increasing the mechanical strength of the membrane and preventing evaporation (drying) of water during the assay. The reagent can be stored and mounted in an appropriate container (housing) in consideration of the size of the reagent, the method and position of adding the sample, the immobilization position of the capturing antibody, and the like. Such a stored and mounted state is called a "device".

An example of a specific assay is shown below. When the sample liquid is dropped onto the sample pad, the sample liquid passes through the sample pad and the conjugate pad sequentially due to capillary action, and develops on the membrane. When the target antigen is contained in the sample, the iron (III) oxide particle-labeled antibody retained on the conjugate pad and the target antigen in the sample form a complex, which is captured by the capture antibody on the membrane. The capture and accumulation of the complex causes the detection line to develop a red color derived from the iron (III) oxide particles. On the other hand, when the sample does not contain the target antigen, the detection line does not develop color. Using such color development as an index, the presence or absence of the target antigen in the sample can be determined. That is, if color development is observed in the detection line, it can be determined that the sample contains the antigen of interest, and if color development is not observed in the detection line, it can be determined that the sample does not contain the target antigen. be able to. When the membrane has a control line, the control capture antibody captures excess iron oxide (III) particle-labeled antibody regardless of the presence or absence of the target antigen in the sample, and the iron oxide (III) particle-labeled antibody accumulates. As a result, the control line develops a red color derived from iron (III) oxide particles. Using such color development as an index, the validity of the assay can be evaluated. That is, if color development is observed in the control line, it can be determined that the assay was performed normally, and if color development is not observed in the control line, it can be determined that the assay was not performed normally.

As shown in Examples below, when iron (III) oxide particles are used as a label for immobilizing an antibody that specifically binds to an antigen in a lateral flow assay, iron oxide (II, III) particles (the non-patented Visibility is overwhelmingly superior to the case of using document 2). Such an assay does not use the magnetism of the iron (III) oxide particles as an indicator, but rather uses the color development as an indicator, so a dedicated detection device is not required, and the antigen in the sample can be detected by a simple method of visual observation. can be done. Further, when iron (III) oxide particles are used as the label, a detection sensitivity equivalent to that of colloidal gold particles commonly used as the label can be obtained. Since iron (III) oxide particles are readily available at low cost, the cost of the label per assay is 1/100 or less compared to colloidal gold particles, and the cost of the lateral flow assay is remarkably high. Down is possible. Furthermore, if a VHH antibody, which can be mass-produced at a lower cost than a normal IgG antibody, is used as an antibody to be immobilized on iron (III) oxide particles, it is possible to improve mass productivity and further reduce costs.

This invention discloses the following aspects further regarding embodiment mentioned above.
<1> A lateral flow assay in which an antibody immobilized on a label is used to detect an antigen in a sample, wherein the label is iron (III) oxide particles.
<2> The lateral flow assay according to <1>, which comprises contacting an antibody immobilized on iron (III) oxide particles with a sample.
<3> The lateral flow assay according to <1> or <2>, which comprises capturing a complex formed from the antibody immobilized on the iron (III) oxide particles and the antigen in the sample with a capture antibody.
<4> The lateral flow assay described in <3>, wherein the capture antibody is immobilized on a membrane.
<5> The lateral flow assay according to any one of <1> to <4>, which comprises detecting the antigen using color development by iron (III) oxide particles as an indicator.
<6> The color development is caused by the accumulation of a complex formed from the antibody immobilized on the iron (III) oxide particles and the antigen in the sample, preferably immobilized on the iron (III) oxide particles. The lateral flow assay according to <5>, wherein a complex formed from the antibody obtained and the antigen in the sample is captured by the capture antibody and accumulated.
<5> or <5> or 6> Lateral flow assay as described.
<8> The lateral flow assay according to any one of <1> to <7>, wherein the iron (III) oxide particles have an average particle size of 10 to 1000 nm, preferably 50 to 500 nm.
<9> The lateral flow assay according to any one of <1> to <8>, wherein the iron (III) oxide particles are iron (III) oxide particles that have not been chemically modified for protein immobilization before antibody immobilization. .
<10> The antibody immobilized on iron (III) oxide particles is preferably an IgG antibody or a heavy chain variable domain antibody, more preferably a heavy chain variable domain antibody, still more preferably a VHH antibody, still more preferably a VHH antibody. is a VHH antibody consisting of the amino acid sequence of any one of SEQ ID NOs: 13, 15, 17 and 19, the lateral flow assay according to any one of <1> to <9>.
<11> The capture antibody is preferably an IgG antibody or a heavy chain variable domain antibody, more preferably a heavy chain variable domain antibody, still more preferably a VHH antibody, still more preferably SEQ ID NOs: 13, 15, 17 and The lateral flow assay according to any one of <3> to <10>, which is a VHH antibody consisting of the amino acid sequence shown in any one of 19.
<12> A reagent for lateral flow assay, comprising an antibody immobilized on iron (III) oxide particles.
<13> The reagent according to <12>, further comprising a membrane on which the capture antibody is immobilized.
<14> The reagent according to <12> or <13>, comprising a sample pad, a conjugate pad holding an antibody immobilized on iron (III) oxide particles, a membrane immobilized with a capture antibody, and an absorbent pad.
<15> The reagent according to any one of <12> to <14>, wherein the iron (III) oxide particles have an average particle size of 10 to 1000 nm, preferably 50 to 500 nm.
<16> The reagent according to any one of <12> to <15>, wherein the iron (III) oxide particles are iron (III) oxide particles that have not been chemically modified for protein immobilization before antibody immobilization.
<17> The antibody immobilized on the iron (III) oxide particles is preferably an IgG antibody or a heavy chain variable domain antibody, more preferably a heavy chain variable domain antibody, still more preferably a VHH antibody, still more preferably a VHH antibody. is a VHH antibody consisting of the amino acid sequence of any one of SEQ ID NOs: 13, 15, 17 and 19. The reagent according to any one of <12> to <16>.
<18> The capture antibody is preferably an IgG antibody or a heavy chain variable domain antibody, more preferably a heavy chain variable domain antibody, still more preferably a VHH antibody, still more preferably SEQ ID NOs: 13, 15, 17 and The reagent according to any one of <13> to <17>, which is a VHH antibody consisting of the amino acid sequence shown in any one of 19.

The present invention will be described in more detail below based on examples, but the present invention is not limited to these.

Example 1 Production of VHH by protease-deficient recombinant Bacillus subtilis 1
(1) Bacillus strains used Bacillus subtilis strains derived from Bacillus subtilis 168 strains were used. Deletion of extracellular protease genes (epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE, aprX) was carried out according to the method described in Japanese Patent No. 4485341 (deficient strains are Δepr, ΔwprA, Δmpr, ΔnprB, Δbpr, ΔnprE, Δvpr, ΔaprE, ΔaprX (strains lacking all are designated 168 Dpr9). Deletion of the sigF gene involved in sporulation was performed according to the method described in Japanese Patent No. 4336082 (deficient strains are described as ΔsigF). E.coli strains for gene construction are available from ECOS Competent E. coli DH5α strain (Nippon Gene) was used.

(2) Used medium LB medium: 1% Bacto ^™ Tryptone (Difco), 0.5% Bacto ^™ Yeast Extract (Difco), 1% sodium chloride. 1.5% agar was added to the plates. Tetracycline (50 ppm) was added as needed.
SMMP solution: Antibiotic Medium 3 (Difco) (35 g/L), sucrose (171.5 g/L), disodium maleate ( _3.2 g/L), _MgCl2.6H2O (4.06 g/L)
PEG solution: sucrose (85.75 g/L), disodium maleate (1.6 g/L), _MgCl2.6H2O ( _2.03 g/L), PEG8000 (400 g/L)
DM3 medium: 1% CMC (Kanto Kagaku), 0.5% Bacto ^™ Casamino Acids (Difco), 0.5% Bacto ^™ Yeast Extract (Difco), 8.1% disodium succinate/6H ₂ O, 0.5% Bacto™ Yeast Extract (Difco) 35% dipotassium hydrogen phosphate, 0.15% potassium dihydrogen phosphate, 0.5% glucose, 20 mM magnesium chloride, 0.01% BSA, 50 ppm tetracycline. 1% agar was added to the plates.
2x L-mal Medium: 2% Bacto ^™ Tryptone (Difco), 1% Bacto ^™ Yeast Extract (Difco), 1% Sodium Chloride, 7.5% Maltose Monohydrate, 7.5 ppm Manganese Sulfate, 15 ppm Tetracycline Reagent were manufactured by Fuji Film Wako Pure Chemical Industries, Ltd. unless otherwise specified.

(3) Construction of Gene Plasmid Construction of gene expression plasmid was performed by the following procedure. Artificially synthesized genes of SEQ ID NOS: 1 and 2 (Thermo Fisher) were mixed and B. It was introduced into the subtilis 168 Dpr9ΔsigF strain. Transformed B. A pK plasmid was extracted from the subtilis 168 Dpr9ΔsigF strain. A PCR fragment amplified using PrimeSTAR Max DNA polymerase (TaKaRa) and the primer set of SEQ ID NOs: 3 and 4 using the pK plasmid as a template; A PCR fragment of the SpoVG promoter (PspoVG) amplified using the primer set of SEQ ID NOS: 5 and 6 using the genomic DNA of S. subtilis strain 168 as a template, and the recombinant plasmid pHY-S237 described in JP-A-2014-158430 as a template. , PCR fragments of the S237 cellulase gene-derived secretory signal (S237pre) and the transcription terminator (Ts237) amplified using the primer sets of SEQ ID NOs: 7 and 8 and SEQ ID NOs: 9 and 10, respectively, were mixed and used with the In-Fusion HD Cloning Kit. (Takara) and transformed into E. coli to construct the PspoVG-S237pre-Ts237-pK plasmid. Using the PspoVG-S237pre-Ts237-pK plasmid as a template, a PCR fragment amplified using the primer set of SEQ ID NOs: 9 and 11 and PrimeSTAR Max, and SEQ ID NO: 12 (VHH antibody against lysozyme: gene encoding 1ZVY, translated amino acid sequence is SEQ ID NO: 13), SEQ ID NO: 14 (VHH antibody against lysozyme: gene encoding 1ZVH, translated amino acid sequence is SEQ ID NO: 15), SEQ ID NO: 16 (VHH antibody against SARS-CoV-2 S1 protein: gene encoding E9 , The translated amino acid sequence is SEQ ID NO: 17). Artificial synthetic genes (Thermo Fisher) are mixed, ligated using the In-Fusion HD Cloning Kit, and transformed into E. coli to construct an expression plasmid for each gene. bottom. The constructed plasmid was transformed into B. elegans according to the protoplast transformation method described below. It was introduced into the subtilis 168 Dpr9ΔsigF strain. Table 1 shows the primers used.

(4) Protoplast transformation method Plasmid introduction into Bacillus subtilis was performed by the protoplast method shown below. Various types of Bacillus subtilis stocked in glycerol were inoculated into 1 mL of LB liquid medium and cultured with shaking at 30° C. and 210 rpm overnight. On the next day, 10 μL of this culture solution was inoculated into a new 1 mL LB liquid medium, and cultured with shaking at 37° C. and 210 rpm for about 2 hours. This culture solution was collected in a 1.5 mL tube, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was removed. incubated. Then, centrifugation was performed at 3,500 rpm for 10 minutes, and the supernatant was removed and the pellet was suspended in 400 μL of SMMP. 33 μL of this suspension was mixed with various plasmids, and 100 μL of 40% PEG was added and vortexed. 350 μL of SMMP was added to this solution, mixed by inversion, incubated at 30° C. for 2 hours in an Eppendorf tube, and then spread on a DM3 agar medium plate. Transformant colonies were obtained by incubating DM3 agar plates at 30° C. for 2-3 days.

(5) Culture conditions A Bacillus subtilis strain carrying a plasmid was inoculated into 1 mL of LB medium containing 50 ppm tetracycline and shaken back and forth overnight at 30°C to prepare a preculture solution. 1% of the preculture was inoculated into 20 mL of 2×L-mal medium in an Erlenmeyer flask and cultured with shaking at 30° C. for 72 hours. The culture solution at the end of culture was centrifuged at 7,500 rpm for 5 minutes to collect the supernatant. Ni-NTA agarose beads (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the supernatant, and the His-tag binding domain antibody contained in the supernatant was purified according to the protocol of the kit. replaced.

Example 2 Lateral flow assay 1
(1) Method Nitrocellulose membrane used was FF120HP (cytiva lifesciences). A detection antibody was applied to a position 20 mm from the lower end of the membrane cut into 5 mm×100 mm. For the domain antibody (1ZVH or 1ZVY) produced in Bacillus subtilis in Example 1, a 1 mg/mL solution was added to commercially available IgG antibodies (Lysozyme Polyclonal Antibody (Cosmo Bio) and SARS/SARS-CoV-2 Coronavirus Spike Protein (subunit 1 ) For Polyclonal Antibody (Thermo Fisher Scientific) (hereafter SARS Polyclonal Antibody), draw a detection line with a 0.1 mg/mL solution using a paintbrush, and then leave it to dry at 37°C for 1 hour. rice field. The dried membrane was immersed in 3-fold diluted N101 (NOF Corporation) for 15 minutes, washed twice with deionized water, then immersed in a 3% sucrose solution and allowed to stand for 5 minutes. The membrane removed from the sucrose solution was dried overnight at room temperature.
Iron (III) oxide particles (300 nm, Kojundo Chemical) were suspended in Milli-Q water at 30 mg/mL. 200 μL of iron oxide (II, III) particles (10 nm, 5 mg/mL toluene, Wako Pure Chemical) were suspended in 1 mL of ethanol, centrifuged at 5,000 rpm for 15 minutes, and the supernatant was removed. 1 ml of ethanol was added to the precipitate, centrifuged at 5,000 rpm for 15 minutes, and the supernatant was removed. 1 mL of 20% ethanol was added to the precipitate, centrifuged at 5,000 rpm for 15 minutes, the supernatant was removed, and 200 μL of Milli-Q water was added to suspend the precipitate. 10 mM Tris-HCl (pH 8) was added to 6 μg of iron oxide (III) particles or iron oxide (II, III) particles, or 100 μL of gold particles (40 nm, 1 OD (530 nm abs max), G-40-20, Cosmo Bio). 500 μL was added and sonicated for 10 seconds (100 μg of iron (III) oxide particles for E9) (10 mM Tris-HCl (pH 8 or 9) when using gold particles). A domain antibody (1ZVH or 1ZVY: 10 µg, E9: 1 mg) produced by Bacillus subtilis or a commercially available IgG antibody (Lysozyme Polyclonal Antibody: 1 µg) was added and allowed to stand for 15 minutes. 200 μL of 10 mM Tris-HCl (pH 8) containing 1% BSA and 0.1% PEG20000 was added and allowed to stand for 15 minutes. After centrifugation at 5,000 rpm for 15 minutes, the supernatant was removed, 200 μL of 10 mM Tris-HCl (pH 8) containing 0.1% BSA and 0.5% Tween 20 was added, and sonication was performed for 10 seconds. (No Tween 20 when using Lysozyme Polyclonal Antibody or E9 on iron (III) oxide particle side, 0.1% BSA and 0.025% Tween 20 when using gold particles).
For assays using 1ZVH, 1ZVY or Lysozyme Polyclonal Antibody as antibodies, egg white lysozyme (Fujifilm Wako Pure Chemical Industries) 10 μg as antigens, E9 or SARS Polyclonal Antibody as antibodies for assays using SARS-CoV- 2 Spike Glycoprotein (S1), Sheep Fc-Tag (HEK293) (Native Antigen) (hereinafter SARS-CoV-2 S1) 1 μg of iron oxide (III) particle-labeled antibody or iron oxide (II, III) particle-labeled antibody added to the solution. For the assay using 1ZVH, 1ZVY or Lysozyme Polyclonal Antibody as the antibody, 10 μg of egg white lysozyme was added as the antigen to the solution of the gold particle-labeled antibody. A lateral flow assay was performed by allowing the solution to which the antigen was added to act on the membrane.

(2) Results In order to confirm the difference in detection visibility between iron (II, III) oxide particles and iron (III) oxide particles, a lateral flow assay was performed using 1ZVH on the detection line side and 1ZVY on the iron oxide particle side. rice field. The results are shown in FIG. Both iron (II, III) oxide particles and iron (III) oxide particles use the same amount of particles and antibodies/antigens. It exhibited a clear detection line.

In order to confirm that the lateral flow assay using iron oxide (III) particles can be applied to the detection of various antigens, a study was conducted by changing the antibodies on the iron oxide (III) particle side and the detection line side. Table 2 shows the results. This result showed that any antigen can be detected through visual recognition of the detection line regardless of the combination of IgG or domain antibody on the iron (III) oxide particle side and the detection line side. It has been confirmed that the detection line can also be visually recognized in a lysozyme detection system (corresponding to the upper three rows of Table 2) using gold particles as the label and 1ZVY and 1ZVH as the particle-side antibodies. In addition, the amino acid sequence homology between the domain antibodies used in this study was 68% for 1ZVY and 1ZVH, 63% for 1ZVY and E9, and 71% for 1ZVH and E9. It was also shown that the flow assay can be used independent of the domain antibody sequence.

Example 3 Production of VHH by protease-deficient recombinant Bacillus subtilis 2
(1) Bacterial strain used The same strain as in Example 1 (1) was used.

(2) Medium Used The same medium as in Example 1 (2) was used.

(3) Construction of Gene Plasmid Construction of gene expression plasmid was performed by the following procedure. SEQ ID NO: 18 (VHH antibody against IgG containing a His tag: gene encoding VHH28-His, translated amino acid sequence is SEQ ID NO: 19) using an artificial synthetic gene (GenScript) as a template, using KOD One DNA polymerase (TOYOBO) and sequence A PCR fragment amplified using the primer set of numbers 20 and 21 and a PCR fragment amplified using the primer set of SEQ ID NOs: 9 and 11 and PrimeSTAR Max (Takara) using the PspoVG-S237pre-Ts237-pK plasmid as a template. were mixed, ligated using an In-Fusion HD Cloning Kit (Takara), and transformed into E. coli to construct a VHH28-His gene expression plasmid.
In addition, using the artificially synthesized gene (GenScript) of SEQ ID NO: 18 as a template, a PCR fragment amplified using KOD One DNA polymerase and the primer set of SEQ ID NOS: 20 and 22, and the PspoVG-S237pre-Ts237-pK plasmid as a template. Then, the primer set of SEQ ID NOS: 23 and 11 and the PCR fragment amplified using KOD One DNA polymerase are mixed, ligated using the In-Fusion HD Cloning Kit, and transformed into E. coli to obtain VHH28-His - An expression plasmid for the FLAG gene was constructed. Table 3 shows the primers used.
The constructed plasmid was transformed into B. mutans according to the protoplast transformation method of Example 1 (4). It was introduced into the subtilis 168 Dpr9ΔsigF strain. The obtained Bacillus subtilis strain carrying the plasmid was cultured according to the culture conditions of Example 1(5) to produce domain antibodies.

Example 4 Lateral flow assay 2
(1) Method Nitrocellulose membrane used was FF120HP (cytiva lifesciences). A detection antibody was applied to a position 20 mm from the lower end of the membrane cut into 5 mm×100 mm. For the domain antibody (VHH28-His-FLAG) produced in Bacillus subtilis in Example 3, a 0.5 mg/mL solution was added to a commercially available IgG antibody (Anti-IgG (H+L), Cat, Rabbit-Poly (Bethyl Laboratories, A20 For -115A)), a 0.1 mg/mL solution was used to draw a detection line with a paintbrush, and then allowed to stand at 37°C for 1 hour to dry. The dried membrane was immersed in 5-fold diluted N101 (NOF Corporation) for 15 minutes, washed twice with deionized water, then immersed in a 3% sucrose solution and allowed to stand for 5 minutes. The membrane removed from the sucrose solution was dried overnight at room temperature.
Iron (III) oxide particles (300 nm, Kojundo Chemical) were suspended in Milli-Q water at 30 mg/mL. 20 μL of 30 mg/mL iron (III) oxide particles was added to 500 μL of 10 mM Tris-HCl (pH 8) and sonicated for 10 seconds. A domain antibody (VHH28-His: 3 μg) produced in Bacillus subtilis was added and allowed to stand for 15 minutes. 400 μL of 10 mM Tris-HCl (pH 8) containing 1% BSA and 0.1% PEG20000 was added and allowed to stand for 15 minutes. After centrifugation at 5,000 rpm for 15 minutes, remove the supernatant, add 500 μL of 10 mM Tris-HCl (pH 8) containing 0.1% BSA and 0.025%-0.05% Tween 20, and sonicate. was performed for 10 seconds. For the antigen, 1 μg of ChromPure Cat IgG, whole molecule (Jackson ImmunoResearch Laboratories Inc.) was added to the iron (III) oxide particle-labeled antibody solution. A lateral flow assay was performed by allowing the solution to which the antigen was added to act on the membrane.

(2) Results In order to confirm that the lateral flow assay using iron (III) oxide particles can be applied to the detection of Cat IgG, a study was conducted by changing the antibody on the detection line side. Table 4 shows the results. This result indicates that Cat IgG can be detected through visual recognition of the detection line regardless of the combination of IgG or domain antibodies on the detection line side.

Claims

A lateral flow assay in which an antibody immobilized on a label is used to detect an antigen in a sample, the label being iron (III) oxide particles.
The lateral flow assay according to claim 1, comprising contacting the antibody immobilized on iron (III) oxide particles with the sample.
The lateral flow assay according to claim 1 or 2, comprising detecting the antigen using color development by iron (III) oxide particles as an indicator.
The lateral flow assay according to any one of claims 1 to 3, wherein the iron (III) oxide particles have an average particle size of 10 to 1000 nm.
The lateral flow assay according to any one of claims 1 to 4, wherein the antibody immobilized on the iron (III) oxide particles is a VHH antibody.
A reagent for lateral flow assays containing antibodies immobilized on iron (III) oxide particles.
The reagent according to claim 6, further comprising a membrane on which capture antibodies are immobilized.
The reagent according to claim 6 or 7, comprising a sample pad, a conjugate pad holding antibodies immobilized on iron (III) oxide particles, a membrane immobilized with capture antibodies, and an absorbent pad.
The reagent according to any one of claims 6 to 8, wherein the antibody immobilized on the iron (III) oxide particles is a VHH antibody.