Adenovirus immunoassay method and immunoassay device

Abstract

To provide: a monoclonal antibody with which adenoviruses contained in a sample to be tested can be rapidly and easily detected and measured with high sensitivity; and an adenovirus immunoassay method using the monoclonal antibody and an adenovirus immunoassay instrument including the monoclonal antibody.SOLUTION: The present invention provides: a monoclonal antibody that is subjected to an antigen-antibody reaction with adenovirus type 1, type 2, type 3, type 4, type 5, type 6, type 7, type 8, type 11, type 19, type 31, type 37, type 53, type 54, type 56, type 64, type 79, type 81, and type 85 subtype viruses, or an antigen-binding fragment thereof; and an immunoassay method using the monoclonal antibody and an immunoassay instrument including the monoclonal antibody.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method and an immunoassay device for adenovirus, and an anti-adenovirus antibody therefor. [Background technology]

[0002] Adenoviruses are known pathogens responsible for respiratory diseases such as acute febrile pharyngitis, pharyngoconjunctivitis, acute respiratory tract infections, and viral pneumonia; eye diseases such as acute follicular conjunctivitis and epidemic keratoconjunctivitis; digestive diseases such as infectious gastroenteritis; and urinary diseases such as urethritis. Currently, adenoviruses are classified into seven types, A through G, with over 80 types. While types 1 through 51 have been reported as serotypes, types 52 and beyond have been reported as genotypes based on complete nucleotide sequences (Non-Patent Document 1). When adenovirus infects humans, it exhibits a variety of clinical symptoms, with few specific pathologies, making it difficult to prove adenovirus infection based on clinical symptoms. Furthermore, adenoviruses are highly infectious, making it necessary to prove viral infection early to prevent outbreaks.

[0003] Immunochromatography and EIA using anti-adenovirus antibodies have been developed as rapid and simple methods for detecting adenovirus, but the positive rate in the ophthalmological field, where the amount of sample that can be collected is limited, is less than 60%. Therefore, there is a need for more sensitive rapid diagnostic methods or anti-adenovirus monoclonal antibodies that can be used for such methods.

[0004] The National Institute of Infectious Diseases' epidemic surveillance has tracked the occurrence of adenovirus-associated diseases, including pharyngoconjunctival fever, infectious gastroenteritis, and epidemic keratoconjunctivitis. It is known that acute respiratory disease and pharyngoconjunctival fever are caused by adenoviruses of species B, C, and E, infectious gastroenteritis by adenoviruses of species A, F, and G, and epidemic keratoconjunctivitis by adenoviruses of species B, D, and E (Non-Patent Document 2). Adenovirus type 3 of species B and adenovirus type 4 of species E are the most common causes of epidemic keratoconjunctivitis and pharyngoconjunctival fever, while adenovirus types 8, 19, and 37 of species D are also responsible for large outbreaks of epidemic keratoconjunctivitis in several countries, particularly in East Asia and Southeast Asia. Adenovirus types 8, 19, and 37 are well known epidemiological causes of nosocomial infections. Recently, nosocomial infections caused by adenoviruses have become a major social problem in public health, posing economic and ethical challenges for hospitals.

[0005] Currently, several monoclonal antibodies that react with adenovirus have been produced and reported. For example, a method for detecting adenovirus using a monoclonal antibody that reacts with a specific subtype of adenovirus has been reported (Patent Documents 1 and 2). Adenovirus detection kits that use monoclonal antibodies against adenovirus are also commercially available. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Seto D, et al., J Virol 85: 5701-5702, 2011 [Non-patent document 2] IASR Vol. 38 p.133-135: July 2017 issue [Patent Document 1] Patent Publication No. 2000-290298 [Patent Document 2] Patent Publication No. 2000-290299 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the monoclonal antibodies against adenovirus currently reported or produced are not sufficiently sensitive for detecting adenovirus and only react to a limited number of adenovirus subtypes. Therefore, there is a demand for monoclonal antibodies with higher sensitivity that react to a greater number of adenovirus subtypes. The present invention aims to provide a monoclonal antibody that can rapidly, easily, and highly sensitively detect and measure many subtypes of adenovirus contained in a test sample, and an immunoassay method and immunoassay device for adenovirus using the same. [Means for solving the problem]

[0008] As a result of extensive research into the above-mentioned problems, the present inventors discovered a monoclonal antibody capable of detecting many subtypes of adenovirus with high sensitivity, and thus completed the present invention.

[0009] That is, the present invention is Provided is a monoclonal antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with all subtypes of adenovirus, including types 1, 2, 3, 4, 5, 6, 7, 8, 11, 19, 31, 37, 53, 54, 56, 64, 79, 81, and 85, and that undergoes a stronger antigen-antibody reaction with type 8 adenovirus than with types 1, 2, 3, 5, 6, 7, 11, 19, 31, and 37 adenoviruses. [Effects of the Invention]

[0010] The present invention provides a monoclonal antibody that can rapidly, easily, and highly sensitively detect and measure many subtypes of adenovirus contained in a test sample, as well as an adenovirus immunoassay method and immunoassay device using the same. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of Western blotting performed in Example 3. [Figure 2] FIG. 1 shows the results of staining the gel with CBB after electrophoresis performed in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail.

[0013] <Monoclonal antibody or antigen-binding fragment thereof> The monoclonal antibody or antigen-binding fragment thereof of the present invention specifically binds to adenovirus, and specifically, undergoes an antigen-antibody reaction with all subtypes of adenovirus, including types 1, 2, 3, 4, 5, 6, 7, 8, 11, 19, 31, 37, 53, 54, 56, 64, 79, 81, and 85.

[0014] The monoclonal antibody or antigen-binding fragment thereof of the present invention has a basic structure consisting of a heavy chain and a light chain, and each of the heavy chain and light chain has a variable region capable of specifically binding to an antigen. H refers to the variable region of the heavy chain, and V L refers to the light chain variable region. The heavy and light chain variable regions each contain amino acid sequences of complementarity-determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, as well as framework regions (FRs). For example, a variable region contains three CDRs and three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4).

[0015] The monoclonal antibodies of the present invention include four-chain antibodies (e.g., two light chains and two heavy chains), recombinant antibodies, or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synhumanized antibodies, half antibodies, and bispecific antibodies). Furthermore, the class of the monoclonal antibody is not limited to IgG, but may also be IgM or IgY.

[0016] In the present invention, an antigen-binding fragment of a monoclonal antibody refers to a fragment in which only the antigen-binding site of the monoclonal antibody is isolated, and examples thereof include fragments having specific antigen-binding ability, such as Fab, Fab', F(ab')2, and single-chain antibody (scFv), which are produced by known methods.

[0017] (Method for producing monoclonal antibodies) The monoclonal antibodies of the present invention can be obtained using known immunological techniques by immunizing an animal with a complex or extract containing an adenovirus antigen, or the adenovirus antigen or a partial peptide thereof, and producing hybridomas using cells from the immunized animal. The length of the peptide used for immunization is not particularly limited, but a peptide of preferably 5 or more amino acids, more preferably 10 or more amino acids, can be used as an immunogen.

[0018] Immunogens can be obtained from the culture medium, or by incorporating adenovirus-encoding DNA into a plasmid vector and introducing it into host cells for expression. The adenovirus or its partial peptides used as immunogens can be expressed as fusion proteins with proteins such as those listed below, and used as immunogens either after purification or as is. Fusion proteins can be prepared using tags commonly used by those skilled in the art as "protein expression and purification tags," such as glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (TRX), Nus tag, S tag, HSV tag, FRAG tag, and polyhistidine tag. Fusion proteins with these tags are preferably cleaved using digestive enzymes to separate the adenovirus or its partial peptide from the remaining tag portions, followed by separation and purification before use as immunogens.

[0019] Preparation of monoclonal antibodies from immunized animals can be easily carried out by the well-known method of Kohler et al. (Kohler et al., Nature, vol. 256, pp. 495-497 (1975)). Specifically, antibody-producing cells such as spleen cells and lymphocytes are collected from the immunized animal and fused with mouse myeloma cells by standard methods to produce hybridomas. The resulting hybridomas are then cloned by limiting dilution or other methods, and monoclonal antibodies that undergo an antigen-antibody reaction with the antigen used to immunize the animal are selected from the monoclonal antibodies produced by each cloned hybridoma.

[0020] Monoclonal antibodies can be purified from ascites or culture supernatant using known immunoglobulin purification methods. Examples include fractionation by salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, and gel filtration. Purification can also be performed by affinity chromatography using a carrier bound to Protein A, Protein G, or Protein L, depending on the immunized animal species and the class of the monoclonal antibody.

[0021] <Immunoassay method>

[0022] In the present invention, the use of the above-mentioned monoclonal antibody or antigen-binding fragment thereof makes it possible to detect with high sensitivity all subtypes of adenovirus, including types 1, 2, 3, 4, 5, 6, 7, 8, 11, 19, 31, 37, 53, 54, 56, 64, 79, 81, and 85. Hereinafter, in the description preceding the examples, unless otherwise clear from the context, the term "monoclonal antibody" means "monoclonal antibody or antigen-binding fragment thereof."

[0023] In the present invention, adenovirus is detected by immunoassay of adenovirus utilizing the antigen-antibody reaction between the above-mentioned monoclonal antibody and adenovirus in a sample. The term "antigen-antibody reaction of a monoclonal antibody with adenovirus" means that the monoclonal antibody reacts specifically with adenovirus. "Specific" means that in a liquid system in which the antigen protein and the monoclonal antibody are mixed, the antibody does not undergo a detectable antigen-antibody reaction with components other than the antigen protein, or, even if it does undergo some kind of binding or association reaction, the reaction is clearly weaker than the antigen-antibody reaction between the antibody and the antigen protein.

[0024] In the present invention, any immunoassay method known to those skilled in the art, such as a competitive method, an agglutination method, a Western blot method, an immunostaining method, or a sandwich method, can be used.

[0025] The immunoassay method of the present invention is preferably a sandwich method. The sandwich method itself is well known in the field of immunoassay and can be carried out, for example, by immunochromatography or ELISA. These sandwich methods are all well known, and the method of the present invention can be carried out by the well-known sandwich method except for using the monoclonal antibody of the present invention described above.

[0026] The sandwich method uses two types of antibodies that recognize an antigen (an immobilized antibody immobilized on a solid phase and a labeled antibody), but in the method of the present invention, at least one of these two types of antibodies is the monoclonal antibody of the present invention described above.

[0027] In immunoassays using the sandwich method as the detection principle, any solid phase on which an antibody can be immobilized by known techniques can be used, and any known substance can be selected, such as a porous thin film (membrane) with capillary action, particulate matter, a test tube, or a resin plate. Furthermore, substances that can be used to label the antibody include enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, and colloidal particles. Among the immunoassays using the various materials mentioned above, lateral flow immunoassays using membranes are preferred, particularly from the viewpoint of simplicity and speed in clinical testing.

[0028] In the present invention, even when adenovirus is quantitatively or semi-quantitatively measured using a monoclonal antibody, quantification and semi-quantitation necessarily involve "measurement," and are therefore included in the "measurement" of the present invention. That is, in the present invention, the "measurement" of immunoassay includes all of quantification, semi-quantitation, and detection. [Example]

[0029] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0030] Example 1: Preparation of anti-adenovirus monoclonal antibodies 1. Adenovirus Antigen Preparation Adenovirus was infected into susceptible mammalian cells, and after culturing for several days, the culture medium of the adenovirus-infected cells was inactivated by ultraviolet irradiation.

[0031] 2. Preparation of Anti-adenovirus Monoclonal Antibodies BALB / c mice were immunized with the inactivated adenovirus antigen described in 1., and the spleens were removed from the mice after a certain period of time. These spleens were then fused with mouse myeloma cells (P3x63) using the method of Kohler et al. (Nature, vol. 256, pp. 495-497 (1975)), and several hybridoma cell lines producing anti-adenovirus antibodies were obtained. The cell line was intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites fluid was collected approximately two weeks later. IgG was purified from the ascites fluid by affinity chromatography using a protein A column, and multiple purified anti-adenovirus monoclonal antibodies were obtained.

[0032] In the following examples, two anti-adenovirus monoclonal antibodies, Antibody 1 and Antibody 2, were used, selected from the multiple anti-adenovirus monoclonal antibodies obtained, taking into consideration their reactivity and specificity.

[0033] Example 2: Immunoassay device for measuring adenovirus 1. Immobilization of anti-adenovirus antibodies onto nitrocellulose membranes A solution of the anti-adenovirus antibody (antibody 2) prepared in Example 1 diluted with a buffer solution and an anti-mouse IgG antibody were prepared, and the anti-adenovirus antibody was applied in a line shape to the sample pad side of a nitrocellulose membrane backed with a PET film, and the anti-mouse IgG antibody was applied in a line shape to the absorber side. The nitrocellulose membrane was then thoroughly dried under warm air to obtain an anti-adenovirus antibody-immobilized membrane.

[0034] 2. Immobilization of anti-adenovirus antibodies to colored polystyrene particles The anti-adenovirus antibody (antibody 1) prepared in Example 1 was covalently bound to colored polystyrene particles, and the colored polystyrene particles were then suspended in a suspension. The particles were then thoroughly dispersed by ultrasonic treatment to obtain anti-adenovirus antibody-bound colored polystyrene particles. In this specification, the particles obtained in this manner are referred to as anti-adenovirus antibody-immobilized particles.

[0035] 3. Coating and drying of anti-adenovirus antibody-conjugated colored polystyrene particles A predetermined amount of the anti-adenovirus antibody-immobilized particles prepared in 2 was applied to a glass fiber nonwoven fabric and thoroughly dried under warm air. In this specification, the pad obtained in this manner is referred to as a labeled antibody pad.

[0036] 4. Fabrication of Adenovirus Testing Device The anti-adenovirus antibody immobilized membrane prepared in 1 and the labeled antibody pads prepared in 2 and 3 were attached to other components (backing sheet, absorption band, sample pad) and cut to a width of 5 mm to form an adenovirus testing device.

[0037] 5. Confirmation of reactivity of the adenovirus test device The culture medium of cells infected with each type of adenovirus was diluted with buffer to prepare two-fold serial dilutions of each type of adenovirus. The prepared adenovirus dilution was added to the sample suspension (10 mM Tris (pH 8.0), 1 w / v% polyoxyethylene octylphenyl ether, 3 w / v% arginine, 3 w / v% BSA), and 50 μL of the solution was added dropwise to the adenovirus testing device prepared in step 4, followed by leaving it to stand for 5 minutes.

[0038] If color development can be visually confirmed at both the anti-mouse IgG antibody and anti-adenovirus antibody application positions, the result is judged as + (positive). If color development can be visually confirmed only at the anti-mouse IgG antibody application position, but not at the anti-adenovirus antibody application position, the result is judged as - (negative). If color development cannot be visually confirmed at the anti-mouse IgG antibody application position, the result is judged as invalid. The minimum adenovirus concentration determined as positive was taken as the minimum detection sensitivity, and the results are shown in Table 1.

[0039] [Table 1]

[0040] As shown in Table 1, it was confirmed that the immunoassay device using the anti-adenovirus antibody of the present invention reacts with many types of adenoviruses belonging to species A to D. In addition, reactions to subtypes 53, 54, 56, 64, 79, 81, and 85 were also confirmed.

[0041] 6. Confirmation of the specificity of the adenovirus testing device 50 μL of a sample suspension containing a virus that causes a respiratory infection was added dropwise to the adenovirus testing device prepared in 4 and allowed to stand for 5 minutes. If color development can be visually confirmed at both the anti-mouse IgG antibody and anti-adenovirus antibody application positions, the test is judged as +. If color development can be visually confirmed only at the anti-mouse IgG antibody application position, but not at the anti-adenovirus antibody application position, the test is judged as -. If color development cannot be visually confirmed at the anti-mouse IgG antibody application position, the test is judged as invalid. The results are shown in Table 2.

[0042] [Table 2]

[0043] As shown in Table 2, the immunoassay device using the anti-adenovirus antibody of the present invention reacts to adenovirus but does not show cross-reactivity with other viruses that cause respiratory infections, confirming that it reacts specifically to adenovirus.

[0044] 7. Performance Comparison of Adenovirus Testing Devices The minimum detection sensitivity of the adenovirus test device (this product) prepared in 4 was compared with that of a commercially available adenovirus kit. Adenovirus-infected cell cultures were diluted with buffer to prepare two-fold serial dilutions. 50 μL of the sample suspension containing the adenovirus dilution was added dropwise to the kit and left to stand for 5 minutes before reading. For commercially available kits, the test was performed according to the package insert using the specified amount of adenovirus dilution as the sample. The maximum dilution factor for adenovirus that resulted in a positive result using this product was set to "1," and the maximum dilution factor for a positive result using a commercially available adenovirus kit was used as the relative sensitivity, as shown in Table 3.

[0045] [Table 3]

[0046] As shown in Table 3, it was confirmed that the immunoassay device using the anti-adenovirus antibody of the present invention had the highest reactivity against type 2 adenovirus and was also the most reactive against other types of adenovirus, alongside Kit A.

[0047] Example 3: Antigen Recognition Site of Anti-adenovirus Monoclonal Antibody The antigen recognition site of the anti-adenovirus monoclonal antibody obtained in Example 1 was confirmed by Western blotting and LC-MS / MS.

[0048] 1. Preparation of Adenovirus Concentrate A549 cells were infected with adenovirus and cultured. On day 7 of culture, the adenovirus-infected cells were harvested and disrupted by sonication. The cell lysate was centrifuged to remove cell debris, yielding an adenovirus concentrate.

[0049] 2. Sample preparation without reduction treatment A two-fold dilution series of the adenovirus concentrate obtained in step 1 was prepared, and each reagent was added to a final concentration of 62.5 mM Tris-HCl (pH 6.5), 10 w / v% glycerol, 2.3 w / v% SDS, and 0.05% BPB (dye), and standard SDS-PAGE was performed without heat denaturation.

[0050] 3. Sample preparation with reduction treatment A two-fold dilution series of the adenovirus concentrate obtained in step 1 was prepared, and each reagent was added to a final concentration of 62.5 mM Tris-HCl (pH 6.5), 10 w / v% glycerol, 2.3 w / v% SDS, 0.05% BPB (dye), and 5% 2-mercaptoethanol. After heat denaturation at 95°C for 1 minute, the solution was subjected to standard SDS-PAGE.

[0051] 4. Western Blotting The electrophoresed gels obtained in steps 2 and 3 were transferred to a PVDF membrane. After blocking with skim milk, the membrane was thoroughly washed with PBS-Tween. An anti-adenovirus antibody adjusted to 3.8 μg / mL in PBS-Tween was allowed to react for 1 hour at room temperature. After thorough washing with PBS-Tween, an HRP-labeled anti-mouse antibody diluted 3000-fold was allowed to react for 1 hour at room temperature. After thorough washing with PBS-Tween, the signal was detected using a chemiluminescent detection reagent. The results of Western blotting are shown in FIG.

[0052] As shown in Figure 1, both of the two antibodies (antibody 1 and antibody 2) produced in Example 1 reacted strongly with a protein of approximately 200 kD (hexon trimer) contained in the sample obtained in Example 2 (without reduction treatment) (left panel). The reduction treatment is thought to have converted the hexon trimer into a monomer, and a very weak reaction was observed with a protein of approximately 100 kD (hexon monomer) in the sample obtained in Example 3 (with reduction treatment) (right panel). The gels obtained in 2 and 3 after electrophoresis were stained with CBB and the results are shown in FIG.

[0053] As indicated by the arrows in Figure 2, the major protein contained in the sample obtained in step 2 (without reduction) was approximately 200 kD (left panel), while that in the sample obtained in step 3 (with reduction) was 100-150 kD (right panel). The stained regions indicated by the squares in the figure were excised, hydrolyzed with trypsin, and analyzed for amino acid sequence by LC-MS / MS. Analysis of the resulting peptide fragments using Mascot (Ver. 2.5) (Matrix Science) and Scaffold (Proteome Software) revealed that the adenovirus hexon protein was the major component in both stained regions.

[0054] 1 and 2 show that both of the antibodies obtained in Example 1 react strongly with the hexon trimer protein and weakly with the monomeric hexon protein.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof which undergoes an antigen-antibody reaction with all subtypes of adenovirus, including types 1, 2, 3, 4, 5, 6, 7, 8, 11, 19, 31, 37, 53, 54, 56, 64, 79, 81 and 85, and which undergoes a stronger antigen-antibody reaction with type 8 adenovirus than with types 1, 2, 3, 5, 6, 7, 11, 19, 31 and 37 adenoviruses.

2. The antibody or antigen-binding fragment thereof described in claim 1, wherein the monoclonal antibody does not undergo antigen-antibody reaction with any of the viruses Coxsackievirus type A9, Coxsackievirus type B4, Coxsackievirus type B5, and Coxsackievirus type B6.

3. The antibody or antigen-binding fragment thereof described in claim 1, wherein the monoclonal antibody does not undergo antigen-antibody reaction with any of the viruses Echo virus type 2, Echo virus type 3, Echo virus type 4, Echo virus type 6, Echo virus type 9, Echo virus type 11, and Echo virus type 30.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody does not undergo antigen-antibody reaction with any of the following viruses: Herpes simplex virus type 1, Human Metapneumovirus type A, Human Metapneumovirus type B, Influenza virus A / New Caledonia / 20 / 99 (H1N1), Influenza virus A / Beijing / 262 / 95 (H1N1), Influenza virus A / New York / 55 / 2004 (H3N2), Influenza virus A / Hiroshima / 52 / 2005 (H3N2), Influenza virus B / Shanghai / 361 / 2002 (Yamagata), and Influenza virus B / Malaysia / 2506 / 2004 (Victoria).

5. The antibody or antigen-binding fragment thereof described in claim 1, wherein the monoclonal antibody does not undergo antigen-antibody reaction with any of the viruses Measles virus, Mumps virus, Parainfluenza virus type 1, Parainfluenza virus type 2, Parainfluenza virus type 3, Parainfluenza virus type 4, RS virus Long strain (type A), and RS virus CH-18 strain (type B).

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