Method and instrument for immunoassay of adenovirus

Abstract

The present invention provides a monoclonal antibody capable of detecting and measuring adenovirus contained in a test sample quickly, simply, and with high sensitivity, and an immunoassay method and an immunoassay instrument for adenovirus using the same. [Solution] The present invention provides a monoclonal antibody or its antigen-binding fragment that undergoes an antigen-antibody reaction with a polypeptide having the amino acid sequence from positions 21 to 944 of the amino acid sequence shown in SEQ ID NO: 1, and an immunoassay method and immunoassay device using the same.

Description

[Technical field]

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

[0002] Adenoviruses are known as pathogens for respiratory diseases such as acute febrile pharyngitis, pharyngoconjunctivitis, acute airway inflammation, 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 to G, and there are more than 80 types. Up to type 51, they were reported as serotypes, but from type 52 onwards, they have been reported as genotypes based on the determination of the entire base sequence (Non-Patent Document 1). When adenovirus infects humans, it exhibits a variety of clinical symptoms and has few specific symptoms, so it is difficult to prove adenovirus infection from clinical symptoms. In addition, adenoviruses are highly infectious, and it is necessary to prove viral infection early in order to prevent mass infection.

[0003] Methods for rapid and simple detection of adenovirus include immunochromatography using anti-adenovirus antibodies and EIA methods. However, in the field of ophthalmology, where the amount of sample that can be collected is limited, the positive rate is less than 60%, and there is a demand for more sensitive rapid diagnostic methods or anti-adenovirus monoclonal antibodies that can be used for these methods.

[0004] The National Institute of Infectious Diseases has identified information on the occurrence of patients with adenovirus-related 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, and adenoviruses types 8, 19, and 37 of species D are also responsible for outbreaks of epidemic keratoconjunctivitis in some countries, especially in East Asia and Southeast Asia. Adenoviruses types 8, 19, and 37 are well known as epidemiological causes of nosocomial infections. Recently, nosocomial infections caused by adenoviruses have become a major social problem in public health, and an economic and ethical problem in hospitals.

[0005] Currently, several monoclonal antibodies that react with adenoviruses have been produced and reported. For example, a method for detecting adenoviruses using monoclonal antibodies that react with specific subtypes of adenoviruses has been reported (Patent Documents 1 and 2). [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 2000-290298 [Patent Document 2] Patent Publication 2000-290299 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the sensitivity of the currently produced monoclonal antibodies against adenovirus is insufficient for detecting adenovirus, and a monoclonal antibody with higher sensitivity is required. Furthermore, the amino acid sequence of the epitope of the conventional anti-adenovirus monoclonal antibodies has not been specified, and therefore there is a problem of poor reproducibility. The present invention aims to provide a monoclonal antibody that can rapidly, easily, and highly sensitively detect and measure 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 intensive research into the above-mentioned problems, the inventors discovered that in order to detect adenovirus with higher sensitivity, it is effective to use a monoclonal antibody which has an epitope that is a specific amino acid sequence contained in adenovirus, and thus completed the present invention.

[0009] That is, the present invention is as follows. [1] A monoclonal antibody or an antigen-binding fragment thereof that undergoes an antigen-antibody reaction with a polypeptide having the sequence of amino acids 21 to 944 of SEQ ID NO:1. [2] The monoclonal antibody or antigen-binding fragment thereof described in [1], which undergoes an antigen-antibody reaction with a polypeptide having at least one range of sequences selected from amino acids 21 to 131, 266 to 412, and 448 to 944 of the amino acid sequence shown in SEQ ID NO:1. [3] A monoclonal antibody or an antigen-binding fragment thereof according to [1] or [2], which reacts with a polypeptide having at least one range of sequences selected from amino acids 21 to 115, 266 to 385, and 451 to 944 of the amino acid sequence of SEQ ID NO: 1. [4] The monoclonal antibody or antigen-binding fragment thereof according to [1] or [2], which reacts with a polypeptide having at least one range of sequences selected from amino acids 21 to 45, 56 to 115, 266 to 385, 451 to 485, 526 to 575, 581 to 615, 656 to 725, 766 to 795, 801 to 830, 851 to 875, and 886 to 944 in the amino acid sequence of SEQ ID NO: 1. [5] A method for immunoassaying adenovirus, comprising immunoassaying adenovirus by utilizing an antigen-antibody reaction between the monoclonal antibody or its antigen-binding fragment described in any one of [1] to [4] and adenovirus in a sample. [6] The method according to [5], wherein the immunoassay method is a sandwich method, and the monoclonal antibody or an antigen-binding fragment thereof is used as at least one of a label or a solid phase. [7] An adenovirus immunoassay device comprising the monoclonal antibody or its antigen-binding fragment described in any one of [1] to [4]. Effect of the Invention

[0010] The present invention provides a monoclonal antibody capable of quickly, easily and with high sensitivity detecting and measuring adenovirus contained in a test sample, and an immunoassay method and an immunoassay device for adenovirus using the same. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of Western blotting performed in Example 3. [Diagram 2] FIG. 1 shows the results of staining the gel with CBB after electrophoresis performed in Example 3. [Figure 3-1] FIG. 1 shows indexes 1 to 51 of a peptide library of the hexon protein of adenovirus type 3 strain GB. [Figure 3-2] FIG. 1 shows indexes 52 to 102 of a peptide library of the hexon protein of adenovirus type 3 strain GB. [Figure 3-3] FIG. 1 shows indices 103 to 152 of a peptide library of the hexon protein of adenovirus type 3 strain GB. [Diagram 3-4] FIG. 1 shows indices 153 to 187 of a peptide library of the hexon protein of adenovirus type 3 strain GB. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Hereinafter, an embodiment 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 undergoes an antigen-antibody reaction with a polypeptide having the sequence of amino acids 21 to 944 of the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the sequence of the hexon monomer protein of type 3 adenovirus GB strain (GenBank Accession No. AB330084.1) consisting of 944 amino acid residues. By using the monoclonal antibody or antigen-binding fragment thereof of the present invention for detecting adenovirus by Western blotting, a specific signal due to an antigen-antibody reaction can be detected in a band considered to be a monomer having a molecular weight of about 100 kD and a band considered to be a trimer having a molecular weight of 200 to 300 kD. However, only a very weak reaction is observed with the band considered to be a monomer having a molecular weight of about 100 kD.

[0014] In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof of the present invention undergoes an antigen-antibody reaction with a polypeptide having at least one range of a sequence selected from the amino acid sequences of positions 21 to 131, 266 to 412, and 448 to 944 of SEQ ID NO: 1. The sequences of positions 132 to 265 and 413 to 447 of the amino acid sequence of SEQ ID NO: 1 are considered to be sequences with low conservation among adenovirus subtypes. In another preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof of the present invention undergoes an antigen-antibody reaction with a polypeptide having at least one range of sequences selected from the amino acid sequences 21 to 115, 266 to 385, and 451 to 944 of SEQ ID NO:1. Furthermore, in another preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof of the present invention undergoes an antigen-antibody reaction with a polypeptide having at least one range of sequences selected from amino acids 21 to 45, 56 to 115, 266 to 385, 451 to 485, 526 to 575, 581 to 615, 656 to 725, 766 to 795, 801 to 830, 851 to 875, and 886 to 944 of the amino acid sequence of SEQ ID NO:1. By using a monoclonal antibody or an antigen-binding fragment thereof which undergoes an antigen-antibody reaction with a polypeptide having an amino acid sequence within the above range, adenovirus can be detected with high sensitivity.

[0015] 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 include the amino acid sequences of the complementarity determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region includes three CDRs together with three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4).

[0016] The monoclonal antibody of the present invention includes a four-chain antibody (e.g., two light chains and two heavy chains), a recombinant antibody, or a modified antibody (e.g., a chimeric antibody, a humanized antibody, a human antibody, a CDR-grafted antibody, a primatized antibody, a deimmunized antibody, a synhumanized antibody, a half antibody, and a bispecific antibody). In addition, the class of the monoclonal antibody is not limited to IgG, and may be IgM or IgY.

[0017] 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 of such fragments having specific antigen-binding ability include Fab, Fab', F(ab')2, and single-chain antibody (scFv), which are produced by known methods.

[0018] (Method of producing monoclonal antibodies) The monoclonal antibody of the present invention can be obtained by using a known immunological technique, immunizing an animal with a complex or extract containing an adenovirus containing the above-mentioned specific amino acid sequence with which the monoclonal antibody of the present invention reacts by antigen-antibody, or with the adenovirus or a partial peptide thereof, and producing a hybridoma using the cells of the immunized animal. The length of the peptide used for immunization is not particularly limited, but a peptide of preferably 5 amino acids or more, more preferably 10 amino acids or more can be used as an immunogen.

[0019] The immunogen can be obtained from the culture medium, or can be obtained by incorporating DNA encoding an adenovirus antigen containing the above-mentioned specific amino acid sequence with which the monoclonal antibody of the present invention reacts with an antigen-antibody into a plasmid vector, which is then introduced into a host cell and expressed. The adenovirus antigen or its partial peptide to be used as an immunogen can be expressed as a fusion protein with a protein such as those exemplified below, and used as an immunogen after purification or as is. To prepare a fusion protein, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), Nus tag, S tag, HSV tag, FRAG tag, polyhistidine tag, and the like, which are commonly used by those skilled in the art as "protein expression and purification tags," can be used. The fusion protein with these is preferably used as an immunogen after cleaving the above-mentioned adenovirus antigen or its partial peptide portion and the other tag portions using a digestive enzyme, and separating and purifying the fusion protein.

[0020] Preparation of monoclonal antibodies from immunized animals can be easily performed by the well-known method of Kohler et al. (Kohler et al., Nature, vol. 256, p. 495-497 (1975)). That is, antibody-producing cells such as spleen cells and lymphocytes are collected from the immunized animal, and these are fused with mouse myeloma cells by a conventional method to prepare hybridomas. The obtained hybridomas are cloned by limiting dilution or the like, and from the monoclonal antibodies produced by each cloned hybridoma, a monoclonal antibody that undergoes an antigen-antibody reaction with the antigen used to immunize the animal is selected.

[0021] Monoclonal antibodies can be purified from ascites or culture supernatant by known immunoglobulin purification methods, such as 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.

[0022] <Immunoassay method>

[0023] In the present invention, adenovirus can be detected with extremely high sensitivity by using the above-mentioned monoclonal antibody or an antigen-binding fragment thereof. In the following description before the examples, unless otherwise clear from the context, the term "monoclonal antibody" means "monoclonal antibody or an antigen-binding fragment thereof."

[0024] In the present invention, adenovirus is detected by immunoassay of adenovirus utilizing the antigen-antibody reaction between the monoclonal antibody and the adenovirus in a sample. The 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 where the antigen protein and the monoclonal antibody are mixed, the antibody does not react with components other than the antigen protein at a detectable level, or even if it does react with the antigen protein or the monoclonal antibody, it reacts only with a reaction that is clearly weaker than the antigen-antibody reaction between the antibody and the antigen protein.

[0025] 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.

[0026] 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. All of these sandwich methods are well known, and the method of the present invention can be carried out by the well-known sandwich method, except for using the specific monoclonal antibody described above.

[0027] In the sandwich method, two types of antibodies that recognize an antigen (an immobilized antibody immobilized on a solid phase and a labeled antibody) are used, and 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. Since the amount of the immobilized antibody immobilized on the solid phase that can be immobilized per unit area is limited, in order to better achieve the object of the present invention of improving sensitivity, it is preferable to use at least the monoclonal antibody of the present invention as the immobilized antibody. Note that when at least two antigens recognized by the monoclonal antibody are present in a single molecule or a single complex, it is also possible to carry out the sandwich method using a single type of the monoclonal antibody as the immobilized antibody and the labeled antibody.

[0028] 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, a particulate substance, a test tube, or a resin plate. In addition, substances that can be used to label an antibody include enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, and the like. Among the immunoassays using the various materials described above, a lateral flow immunoassay using a membrane is preferred, particularly from the viewpoint of the simplicity and speed of clinical testing.

[0029] In the present invention, even when adenovirus is quantified or semi-quantified using a monoclonal antibody, quantification and semi-quantification 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-quantification, and detection. EXAMPLES

[0030] 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.

[0031] Example 1: Preparation of anti-adenovirus monoclonal antibody 1. Adenovirus Antigen Preparation Adenovirus was infected into susceptible mammalian cells, which were then cultured for several days, after which the culture medium of the adenovirus-infected cells was inactivated by UV irradiation.

[0032] 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 they had been raised for a certain period of time. The spleens were then fused with mouse myeloma cells (P3×63) using the method described by Kohler et al. (Nature, vol., 256, p. 495-497 (1975)), and multiple hybridoma cell lines producing anti-adenovirus antibodies were obtained. The obtained cell line was intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites was collected about 2 weeks later. IgG was purified from the ascites by affinity chromatography using a protein A column, and multiple purified anti-adenovirus monoclonal antibodies were obtained.

[0033] In the following examples, two anti-adenovirus monoclonal antibodies, Antibody 1 and Antibody 2, were used, which were selected from the multiple anti-adenovirus monoclonal antibodies obtained, taking into consideration their reactivity and specificity.

[0034] (Example 2) Immunoassay device for measuring adenovirus 1. Immobilization of anti-adenovirus antibodies onto nitrocellulose membrane 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.

[0035] 2. Immobilization of anti-adenovirus antibodies onto colored polystyrene particles The anti-adenovirus antibody (antibody 1) prepared in Example 1 was covalently bonded to colored polystyrene particles, and the colored polystyrene particles were then suspended in a suspension. The colored polystyrene particles were then thoroughly dispersed by ultrasonic treatment to obtain anti-adenovirus antibody-bound colored polystyrene particles. In this specification, the particles obtained here are referred to as anti-adenovirus antibody-immobilized particles.

[0036] 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 called a labeled antibody pad.

[0037] 4. Preparation 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.

[0038] 5. Confirmation of reactivity of adenovirus testing 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 specimen suspension (10 mM Tris (pH 8.0), 1 w / v% polyoxyethylene octylphenyl ether, 3 w / v% arginine, 3 w / v% BSA), and 50 μL was added dropwise to the adenovirus testing device prepared in 4, and then allowed to stand for 5 minutes.

[0039] If color development can be visually confirmed at both the anti-mouse IgG antibody and anti-adenovirus antibody application positions, it 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, it is judged as - (negative). Also, if color development cannot be visually confirmed at the anti-mouse IgG antibody application position, it is judged as invalid. The minimum adenovirus concentration determined as positive was defined as the minimum detection sensitivity, and the results are shown in Table 1.

[0040] [Table 1]

[0041] 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 the subtypes 53, 54, 56, 64, 79, 81, and 85 were also confirmed.

[0042] 6. Confirmation of the specificity of the adenovirus testing device 50 μL of a specimen 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, it 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, it is judged as -. Also, if color development cannot be visually confirmed at the anti-mouse IgG antibody application position, it is judged as invalid. The results are shown in Table 2.

[0043] [Table 2]

[0044] 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 to other causative viruses of respiratory infections, confirming that it reacts specifically to adenovirus.

[0045] 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. The culture fluid of the adenovirus-infected cells was diluted with each buffer solution to prepare a 2-fold dilution series. 50 μL of the sample suspension containing the adenovirus dilution was added to this product, and the product was judged after leaving it to stand for 5 minutes. For the commercially available kits, the test was performed and judged according to the package insert of each kit using a specified amount of the adenovirus dilution as the sample. When the maximum adenovirus dilution ratio at which this product was judged to be positive was taken as "1," the maximum dilution ratio at which a commercially available adenovirus kit was judged to be positive was taken as the relative sensitivity, which is shown in Table 3.

[0046] [Table 3]

[0047] 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 also had the highest reactivity against other types of adenovirus, alongside Kit A.

[0048] (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.

[0049] 1. Preparation of Adenovirus Concentrates A549 cells were infected with adenovirus and cultured. On the seventh day of culture, the adenovirus-infected cells were harvested and disrupted by sonication. The cell lysate was centrifuged to remove cell debris, and an adenovirus concentrate was obtained.

[0050] 2. Sample preparation without reduction treatment A two-fold dilution series was prepared from the adenovirus concentrate obtained in step 1, and each reagent was added to the 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.

[0051] 3. Sample preparation with reduction treatment A two-fold dilution series was prepared from the adenovirus concentrate obtained in step 1, and each reagent was added so that the final concentrations were 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. The mixture was then heat denatured at 95°C for 1 minute, after which standard SDS-PAGE was performed.

[0052] 4. Western Blotting The electrophoretic gels obtained in 2 and 3 were transferred to a PVDF membrane. After blocking with skim milk, the membrane was thoroughly washed with PBS-Tween. Anti-adenovirus antibody adjusted to 3.8 μg / mL with PBS-Tween was reacted at room temperature for 1 hour. After thorough washing with PBS-Tween, HRP-labeled anti-mouse antibody diluted 3000-fold was reacted at room temperature for 1 hour. After thorough washing with PBS-Tween, the signal was detected using a chemiluminescence detection reagent. The results of Western blotting are shown in FIG.

[0053] 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 about 200 kD (hexon trimer) contained in the sample obtained in 2 (without reduction treatment) (left panel). It is believed that the hexon trimer was converted to a monomer by reduction treatment, and a very weak reaction was observed with a protein of about 100 kD (hexon monomer) in the sample obtained in 3 (with reduction treatment) (right panel). The results of staining the gels after electrophoresis obtained in 2 and 3 with CBB are shown in FIG.

[0054] As shown by the arrows in Figure 2, the main protein contained in the sample obtained in 2 (without reduction treatment) was around 200 kD (left panel), and in the sample obtained in 3 (with reduction treatment) it was 100-150 kD (right panel). After cutting out each stained region indicated by a square in the figure, the amino acid sequence was analyzed by LC-MS / MS after hydrolysis with trypsin. The obtained peptide fragments were analyzed using Mascot (Ver. 2.5) (Matrix Science) and Scaffold (Proteome Software), and it was revealed that the main component of both stained regions was the adenovirus hexon protein.

[0055] 1 and 2, it was found that both of the antibodies obtained in Example 1 reacted more strongly with the hexon trimer protein than with the hexon monomer, and showed a weak reaction with the monomeric hexon protein. Although the reaction between the antibody of the present invention and the hexon monomer was weak, it was demonstrated that the antibody of the present invention, which undergoes an antigen-antibody reaction with a specific sequence in the monomer, is very effective in detecting adenovirus.

[0056] Example 4: Analysis of reactivity of anti-adenovirus monoclonal antibodies to adenovirus hexon protein using peptide microarray PepStar (JPT Peptide Technologies, hereinafter referred to as "Pepstar") 1. Preparation of PepStar Based on the amino acid sequence information of the hexon protein of the adenovirus type 3 GB strain (GenBank Accession No. AB330084.1), a peptide microarray Pepstar (JPT Peptide Technologies) was purchased, on which the peptides shown in the peptide library in Table 4 were immobilized.

[0057] [Table 4-1]

[0058] [Table 4-2]

[0059] [Table 4-3]

[0060] [Table 4-4]

[0061] 2. Analysis of the reactivity of anti-adenovirus monoclonal antibodies using Pepstar The two types of anti-adenovirus antibodies prepared in Example 1 were diluted with a buffer solution, and each antibody was reacted on a Pepstar at 30°C for 1 hour. After the reaction, 1 μg / ml of a secondary fluorescently labeled anti-mouse IgG antibody was added to the corresponding well and reacted for 1 hour. After washing and drying the reaction products on the wells, the slides were scanned with a 635 nm high-resolution laser scanner to obtain a fluorescence intensity profile. The resulting images were quantified to obtain the average pixel value of each peptide. To visualize the results obtained and compare the individual binding regions, a heatmap diagram (Figure 3) was created showing the fluorescence intensity color-coded from white (no binding) to red (strong binding).

[0062] The heat map results in FIG. 3 revealed the reactive regions of the two types of anti-adenovirus monoclonal antibodies prepared in Example 1 with respect to the adenovirus hexon protein.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof which undergoes an antigen-antibody reaction with a polypeptide having the amino acid sequences 56 to 70, 316 to 330, 536 to 560, 656 to 670, 776 to 795, and 911 to 925 of the amino acid sequence of SEQ ID NO: 1, wherein the monoclonal antibody or antigen-binding fragment thereof undergoes an antigen-antibody reaction with all adenovirus types 1, 2, 3, 5, 6, 7, 8, 11, 19, 31, 37, 53, 54, 56, 64, 79, 81, and 85.

2. 10. A method for immunoassaying adenovirus, comprising immunoassaying adenovirus by utilizing an antigen-antibody reaction between the monoclonal antibody or antigen-binding fragment thereof according to claim 1 and adenovirus in a sample.

3. 3. The method according to claim 2, wherein the immunoassay method is a sandwich method, and the monoclonal antibody or an antigen-binding fragment thereof is used as at least one of a label and a solid phase.

4. An immunoassay device for adenovirus, comprising the monoclonal antibody or its antigen-binding fragment according to claim 1.

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