Immunoassay method for β-D-glucan in biological samples, and β-D-glucan assay kit

Abstract

The problem to be solved is to perform an immunoassay for BG in a biological sample, which method has the same level of sensitivity and reactivity as a horseshoe crab amebocyte lysate reagent. The problem can be solved by an immunoassay for β-D-glucan in a biological sample, the method comprising measuring β-D-glucan in a biological sample using a monoclonal antibody that binds to (1→3)-β-D-glucan with a degree of polymerization of 4.

Description

Technical Field

[0001] The present invention relates to an immunoassay method for β-D-glucan (hereinafter sometimes referred to as BG) in a biological sample. The present invention also relates to an analysis kit for β-D-glucan for measuring BG in a biological sample. The present invention also relates to a monoclonal antibody that binds to (1→3)-β-D-glucan having a degree of polymerization of 4. Background Art

[0002] β-D-glucan is a glucose polymer in which multiple glucose molecules are bonded by β-bonds. The carbon atom at position 1 of glucose can bond to each of the five carbons of other glucose, i.e., carbons at positions 1, 2, 3, 4, and 6. It is reported that bonding combinations of positions 1 and 3 (1→3), positions 1 and 4 (1→4), or positions 1 and 6 (1→6) often occur in natural β-D-glucan.

[0003] β-D-glucan is a component of the cell wall of fungi and is a characteristic substance not found in other microorganisms such as bacteria. Due to this characteristic, β-D-glucan analysis has been used for the examination of deep mycosis. Deep mycosis is an opportunistic infection in which patients develop immune dysfunction due to weakened resistance and become seriously ill. Typical examples of pathogenic fungi of deep mycosis include Candida and Aspergillus. Since the presence of BG is common in the cell walls of these pathogenic fungi, the measurement of BG in body fluids has been used as an auxiliary diagnosis for deep mycosis infection. The structure of β-glucan in clinical samples is believed to differ depending on the pathogenic fungi of deep mycosis, and particularly large amounts of these pathogenic fungi are believed to have cell walls composed of (1→3)-β-D-glucan (hereinafter sometimes abbreviated as (1→3)BG) and (1→3)(1→6)-β-D-glucan (hereinafter sometimes abbreviated as (1→3)(1→6)BG).

[0004] At present, a horseshoe crab amebocyte lysate reagent that utilizes the protective reaction of horseshoe crab to BG is used for the inspection of deep fungal diseases (Patent Documents 1 and 2). This horseshoe crab amebocyte lysate reagent is approved for in vitro diagnosis. However, the method using this horseshoe crab amebocyte lysate reagent requires the blood of horseshoe crab, a natural resource, so in addition to the worry of causing resource depletion, the cost of maintaining a certain quality is also high. Another drawback of this method is that the measured value is easily discrete because this technology requires multiple manual operation steps.

[0005] In recent years, in order to eliminate the defects of the horseshoe crab amebocyte lysate reagent and measure BG more quickly and easily, attempts have been made to measure BG using antibodies that recognize BG (Patent Document 3, Non-Patent Documents 1, 2). However, the antibodies used in these methods have poor measurement sensitivity or are difficult to use in so-called sandwich assays, in which the antigen to be measured is sandwiched between two antibodies (immobilized antibody and labeled antibody). In addition, there has been no research on a measurement system using antibodies that recognize BG, which has a sensitivity comparable to that of the horseshoe crab amebocyte lysate reagent and a similar reactivity, and there is currently no immunoassay method for measuring BG that can replace the horseshoe crab amebocyte lysate reagent.

[0006] Reference List

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No.: 5089375

[0009] Patent Document 2: Japanese Patent No.: 3553656

[0010] Patent Document 3: Japanese Patent Publication No.: 4-346791

[0011] Non-patent literature

[0012] Non-patent literature 1: Use of beta-1,3-glucan-specific antibody to study the cyst wall of Pneumocystis carinii and effects of pneumocandin B0 analog L-733,560. Antimicrobial Agents and Chemotherapy 1994Oct; 38(10):2258-2265

[0013] Non-patent document 2: Development of a two-site enzyme immunoassay based on monoclonal antibodies to measure airborne exposure to (1-3)-beta-D-glucan. Journal of Immunological Methods 2008Aug 20;337(1):55-62 Summary of the invention

[0014] Technical issues

[0015] As described above, conventional techniques for measuring BG using antibodies that recognize BG have many problems. Therefore, there is a need for a BG immunoassay method using an antibody that has the same detection sensitivity as a horseshoe crab amebocyte lysate reagent, exhibits the same reactivity as a horseshoe crab amebocyte lysate reagent, and can be used to perform a high-sensitivity sandwich assay.

[0016] Solution to the problem

[0017] The present inventors have analyzed the reactivity of the horseshoe crab amebocyte lysate reagent to various BGs in detail. As a result, it was confirmed that the horseshoe crab amebocyte lysate reagent reacts with (1→3)BG and (1→3)(1→6)BG among BGs.

[0018] As described above, the structure of BG in a biological sample is considered to be different depending on the pathogenic species of deep mycosis, and in particular, (1→3)BG and (1→3)(1→6)BG exist in large quantities. In the structure of (1→3)(1→6)BG, it is considered that a certain number of β(1→6) bonds exist in the β(1→3) bonds. The present inventors have conducted intensive studies to solve this problem and have found the possibility that an antibody that recognizes a long β(1→3) polymer as an epitope may not be able to recognize (1→3)(1→6)BG because the β(1→6) bond becomes an obstacle. The present inventors believe that in order to have the same reactivity as a horseshoe crab amebocyte lysate reagent and detect pathogenic organisms of deep mycosis with high sensitivity, it is effective to produce an antibody that recognizes a short β(1→3) polymer as an epitope that has the potential to recognize both (1→3)BG and (1→3)(1→6)BG. The present inventors also believe that a highly sensitive sandwich assay can be constructed by recognizing a short β(1→3) polymer as an epitope.

[0019] Therefore, various antibodies recognizing short β(1→3) aggregates as epitopes were generated. By constructing a sandwich assay using these antibodies, the present inventors succeeded in constructing a BG measurement system having comparable sensitivity and similar reactivity to that of the horseshoe crab amebocyte lysate reagent, thereby completing the present invention.

[0020] Specifically, the present invention is as follows.

[0021] <1> The invention discloses an immunoassay method for β-D-glucan in a biological sample, comprising: using a monoclonal antibody that binds to (1→3)-β-D-glucan to measure the β-D-glucan in the biological sample, wherein the (1→3)-β-D-glucan has a polymerization degree of 4.

[0022] <2> according to <1> The immunoassay method, wherein the biological sample is blood, plasma or serum.

[0023] <3> according to <1> or <2> The immunoassay method, wherein the β-D-glucan is (1→3)-β-D-glucan and (1→3)(1→6)-β-D-glucan.

[0024] <4> according to <1> to <3> The immunoassay method according to any one of the above aspects, wherein the concentration of the β-D-glucan is 1 μg / mL or less.

[0025] <5> according to <1> to <4> The immunoassay method described in any one of the preceding claims, wherein ELISA is used.

[0026] <6> according to <1> to <5> The immunoassay method described in any one of the preceding claims, wherein two monoclonal antibodies that bind to (1→3)-β-D-glucan having a degree of polymerization of 4 are used.

[0027] <7> according to <1> to <6> The immunoassay method of any one of the above further comprises determining whether the subject suffers from deep fungal disease, and wherein the determined cutoff value is 20 pg / mL.

[0028] <8> according to <1> to <7> The immunoassay method described in any one of the preceding claims, wherein the monoclonal antibody has a competitive inhibition effect in a horseshoe crab amebocyte lysate reagent in an assay of β-D-glucan.

[0029] <9> A kit for determining β-D-glucan in biological samples, comprising:

[0030] (a) A solid phase having immobilized thereon a first monoclonal antibody that binds to (1→3)-β-D-glucan having a degree of polymerization of 4.

[0031] <10> according to <9> The assay kit further comprises:

[0032] (b) A second monoclonal antibody that binds to (1→3)-β-D-glucan having a degree of polymerization of 4 and is labeled with a labeling substance.

[0033] <11> according to <9> or <10> The assay kit, wherein the biological sample is blood, plasma or serum.

[0034] <12> according to <9> to <11> The assay kit described in any one of the preceding claims, wherein the β-D-glucan is (1→3)-β-D-glucan and (1→3)(1→6)-β-D-glucan.

[0035] <13> according to <9> to <12> The assay kit described above is characterized in that the concentration of the β-D-glucan is 1 μg / mL or less.

[0036] <14> according to <9> to <13> The assay kit described in any one of the preceding claims, wherein the kit is used to determine whether a subject suffers from deep fungal disease, and wherein the cutoff value for the determination is 20 pg / mL.

[0037] <15> A monoclonal antibody that binds to (1→3)-β-D-glucan, wherein the (1→3)-β-D-glucan has a degree of polymerization of 4.

[0038] Advantageous Effects of the Invention

[0039] According to the present invention, an immunoassay method for BG in a biological sample having the same sensitivity and similar reactivity as a horseshoe crab amebocyte lysate reagent can be performed. According to the present invention, an immunoassay kit for BG in a biological sample having the same sensitivity and the same reactivity as a horseshoe crab amebocyte lysate reagent can be provided.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] [ Figure 1 ] Figure 1 Schematic diagram showing the advantages of antibodies recognizing short β(1→3) aggregates as epitopes in testing clinical samples.

[0042] [ Figure 2 ] Figure 2 The figure shows the results of competitive ELISA for screening antibodies that recognize short β(1→3) aggregates as epitopes.

[0043] [ Figure 3 ] Figure 3 It is a figure which shows the result of epitope analysis of 86202R antibody.

[0044] [ Figure 4 ] Figure 4 This is a diagram showing the results of epitope analysis of the 86207 antibody.

[0045] [ Figure 5 ] Figure 5 The graph shows the results of sandwich ELISA using the 86202R antibody and the 86207 antibody and various BGs.

[0046] [ Figure 6 ] Figure 6 is a graph showing the results of analyzing various BGs using commercially available reagent 1.

[0047] [ Figure 7 ] Figure 7 is a graph showing the results of analyzing various BGs using commercially available reagent 2.

[0048] [ Figure 8 ] Figure 8 This is a graph showing the results of studying the lower limit of detection of the immunoassay method for BG of the present invention.

[0049] [ Fig. 9 ] Fig. 9 It is a graph showing the correlation between the immunoassay method of BG of the present invention and commercially available reagent 1.

[0050] [ Fig.10 ] Fig.10 It is a graph showing the correlation between the immunoassay method of BG of the present invention and commercially available reagent 2.

[0051] [ Fig.11 ] Fig.11 This is a graph showing the results of a competitive assay experiment performed using the antibody used in the immunoassay method for BG of the present invention and commercially available reagent 2.

[0052] [ Fig.12 ] Fig.12 is a graph showing the results of determining the presence or absence of fungal infection in healthy human plasma samples using the cutoff value (20 pg / mL) of the Limulus amebocyte lysate reagent. DETAILED DESCRIPTION

[0053] [1] Immunoassay method for β-D-glucan in biological samples

[0054] (Biological Samples)

[0055] Examples of "biological samples" in the present invention mainly include solid tissues and body fluids from living bodies (organisms), and body fluids are preferably used. The biological samples of the present invention are more preferably blood, serum, plasma, urine, saliva, sputum, tears, ear discharge or prostatic fluid, further preferably blood, serum or plasma, further preferably blood, serum or plasma of a subject suspected of having deep fungal disease, and most preferably blood, serum or plasma of a subject suspected of having deep fungal disease caused by pathogenic fungi of the genus Candida and / or Aspergillus. Examples of living bodies or subjects include humans or animals (e.g., monkeys, dogs, cats, mice, guinea pigs, rats, hamsters, horses, cattle, pigs, birds and fish), and are preferably humans.

[0056] In the immunoassay method of the present invention, the biological sample is pretreated as needed. Examples of pretreatment include alkali treatment and heat treatment, or these treatments can be combined. The treatment time, pH value of the alkaline solution, type of alkaline reagent, treatment temperature, etc. can be appropriately set according to the type of biological sample, etc.

[0057] (β-D-Glucan)

[0058] In the present specification, "β-D-glucan" and "BG" refer to a glucose polymer in which multiple glucose molecules are bound by β-bonds. BG is a component of the fungal cell wall, and the determination of BG in body fluids has been used for auxiliary diagnosis of deep fungal infections. Examples of BG may include (1→3)-β-D-glucan, (1→3)(1→6)-β-D-glucan, (1→3)(1→4)-β-D-glucan (β-glucan from barley, lichen polysaccharide, etc.). In the immunoassay method of the present invention, the β-D-glucan in the biological sample to be tested is preferably (1→3)-β-D-glucan and / or (1→3)(1→6)-β-D-glucan.

[0059] ((1→3)-β-D-glucan)

[0060] In the present specification, "(1→3)-β-D-glucan" and "BG" refer to glucans in which the 1-carbon of glucose and the 3-carbon of other glucose are bound in a β-type bond. BG has a unique structure called a triple helix structure.

[0061] Examples of (1→3)BG include pachyman, curdlan, laminaritotetraose, paramylon, carboxymethyl pachyman, carboxymethyl curdlan, (1→3)BG present in the cell wall of Aspergillus fungi, and (1→3)BG present in the cell wall of Candida fungi.

[0062] In the present specification, (1→3)BG having glucose bound to the outer chain of the triple helix structure in the form of (1→6) in the structure of (1→3)BG is referred to as (1→3)(1→6)-β-D-glucan or (1→3)(1→6)BG. Examples of (1→3)(1→6)-β-D-glucan include laminarin, sizoran, (1→3)(1→6)BG present in the cell wall of Aspergillus fungi, and (1→3)(1→6)BG present in the cell wall of Candida fungi. (1→3)(1→6)BG has a structure in which glucose is bound to the outer chain of the triple helix structure in the form of (1→6), and an antibody that recognizes a long β(1→3) polymer may not recognize (1→3)(1→6)BG because the binding of the antibody to glucose bound to the outer chain of the triple helix structure in the form of (1→6) is inhibited. Figure 1 ).

[0063] The monoclonal antibody used in the present invention preferably does not react with any of the structures of (1→4)-β-D-glucan, (1→6)-β-D-glucan, (1→6)-α-D-glucan, (1→4)(1→6)-α-D-glucan, (1→6)(1→6)-α-D-glucan and (1→4)-α-D-glucan.

[0064] Specifically, the monoclonal antibody used in the present invention preferably does not react with any of the following: mannan and carboxymethylcellulose, which are (1→4)-β-D-glucans; palstan, which is (1→6)-β-D-glucans; glucan, which is (1→6)-α-D-glucans; glycogen and amylopectin, which are (1→4)(1→6)-α-D-glucans; pullulan, which is (1→6)(1→6)-α-D-glucans; and amylose, which is (1→4)-α-D-glucans.

[0065] As used herein, "deep mycosis" refers to a condition in which fungi enter deep parts of the body, such as the lungs, liver, kidneys, or brain, causing infection. Deep mycosis mainly occurs in patients who have received organ transplants and are receiving immunosuppressive drug therapy. Examples of pathogenic fungi of deep mycosis include Aspergillus and Candida, and the immunoassay method of the present invention can effectively analyze BG present in the cell walls of these fungi.

[0066] (Monoclonal antibody that binds to (1→3)-β-D-glucan with a degree of polymerization of 4)

[0067] The monoclonal antibody used in the present invention is a monoclonal antibody that binds to (1→3)-β-D-glucan having a degree of polymerization of 4. “Binding to (1→3)-β-D-glucan having a degree of polymerization of 4” means recognizing a β(1→3) bond having a degree of polymerization of 4 as an epitope. In the present specification, “determining β-D-glucan in a biological sample by using a monoclonal antibody that binds to (1→3)-β-D-glucan having a degree of polymerization of 4” means contacting a biological sample with a monoclonal antibody and comparing the obtained measured value with a cutoff value.

[0068] A degree of polymerization of "4" means that four glucose molecules are polymerized.

[0069] The anti-(1→3)BG antibody used in the present invention preferably reacts with BG, preferably (1→3)BG and / or (1→3)(1→6)BG, preferably at a free antigen concentration of 1 μg / mL or less, more preferably at a free antigen concentration of 100 ng / mL or less.

[0070] Specific examples of the monoclonal antibody used in the present invention include 86202R antibody, which is a monoclonal antibody produced by hybridoma NITE BP-02723, and 86207 antibody, which is a monoclonal antibody produced by hybridoma NITE BP-02724.

[0071] Although "reacting" with (1→3)-β-D-glucan having a degree of polymerization of 4, "recognizing" (1→3)-β-D-glucan having a degree of polymerization of 4, and "binding" to (1→3)-β-D-glucan having a degree of polymerization of 4 are used synonymously in this specification, they must be interpreted in the broadest sense and are not limited to these examples. Whether an antibody "reacts" with an antigen (compound) can be confirmed by antigen solid phase ELISA method, competitive ELISA method, sandwich ELISA method, etc., and a method using the surface plasmon resonance principle (SPR method), etc. The SPR method can be performed using an apparatus, sensor, and reagent commercially available under the name of Biacore (registered trademark).

[0072] Claiming that the antibody used in the present invention "does not react with a certain compound" means that the antibody used in the present invention does not substantially react with a certain compound, and claiming "substantially non-reactive" means that, for example, when Biacore (registered trademark) T100 or T200 is used to fix and measure the antibody of the present invention based on the SPR method, the enhanced reactivity of the antibody used in the present invention is not recognized. Specifically, this means that the reactivity between the antibody and the compound is not significantly different from the reactivity of the control (no compound added). Obviously, in addition to the SPR method, it is also possible to confirm that the antibody and the compound are "substantially non-reactive" by methods or means well known to those skilled in the art.

[0073] The monoclonal antibody used in the immunoassay method of the present invention includes a fragment having the function of the monoclonal antibody as long as the effect of the present invention can be obtained. Examples of the fragment include a functional fragment containing the Fab portion of a monoclonal antibody obtained by enzymatic digestion of the monoclonal antibody, a functional fragment containing the Fab portion of a monoclonal antibody prepared by gene recombination, and a functional fragment containing scFv prepared by phage display method, etc.

[0074] The antibody used in the immunoassay method of the present invention can be produced by dissolving (1→3)BG with a degree of polymerization of 4, such as laminarin, as an antigen (immunogen) in a solvent such as phosphate buffered saline, and administering the solution to immunize an animal. Hybridomas that produce antibodies that bind to (1→3)BG with a degree of polymerization of 4 can be screened by using (1→3)BG with a degree of polymerization of 5 or more, such as laminarin, as an antigen (immunogen). After adding an appropriate adjuvant to the solution as needed, an emulsion can be used for immunization. The adjuvant can be a widely used adjuvant, such as an oil-in-water emulsion, a water-in-oil-in-water emulsion, an oil-in-water emulsion, a liposome or an aluminum hydroxide gel, and a protein or peptide substance derived from a biological component. For example, Freund's incomplete or complete adjuvant can be preferably used. Although not particularly limited, it is desirable to appropriately select the route of administration, the dosage and the time of administration of the adjuvant so that the desired immune response can be enhanced in an animal immunized with the antigen.

[0075] Although not particularly limited, the type of animal used for immunization is preferably a mammal, which may be a mouse, rat, cattle, rabbit, goat, sheep and alpaca, more preferably a mouse or rat. Animals can be immunized according to common techniques, for example, immunization can be achieved by injecting an antigen solution subcutaneously, intradermally, intravenously or intraperitoneally to the animal, preferably with a mixture of an adjuvant. Since the immune response is usually different depending on the type and strain of the animal to be immunized, it is desirable to appropriately set the immunization schedule according to the animal to be used. It is preferred to repeat the administration of the antigen several times after the initial immunization.

[0076] Although the following operations are performed continuously to obtain monoclonal antibodies, the operations are not limited thereto, and the method of producing monoclonal antibodies itself is well known in the art and widely used, so the antibodies used in the immunoassay method of the present invention can be easily produced by using the above-mentioned antigen (see, for example, Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988), Chapter 6)).

[0077] After the final immunization, by extracting antibody-producing cells--spleen or lymph node cells from immune animals, and by merging these cells with a cell line derived from a myeloma with high proliferation capacity, hybridomas are produced. Cells with high antibody production capacity (quantitative and qualitative) are preferably used for cell fusion, and the cell line derived from a myeloma is preferably compatible with the source animal of the antibody-producing cells to be fused. Cell fusion can be carried out according to methods well known in the art, and a polyethylene glycol method, a method using Sendai virus, or a method utilizing electric current can be used. The hybridoma obtained can be obtained according to the widely used conditional propagation in this area, and the desired hybridoma can be selected while confirming the characteristics of the antibody produced. The cloning of hybridomas can be carried out by known methods such as limiting dilution method, soft agar method.

[0078] By taking into account the conditions under which the produced antibodies are actually used for measurement, hybridomas can be efficiently selected at the selection stage. For example, an antibody obtained by immunizing an animal is reacted with (1→3)BG having a degree of polymerization of 4 fixed on a solid phase in the presence of a compound whose cross-reactivity needs to be confirmed, and the reactivity can be compared with the reactivity in the absence of the compound whose cross-reactivity needs to be confirmed, so as to more efficiently select a hybridoma producing the desired antibody. Alternatively, an antibody obtained by immunizing an animal is reacted with (1→3)BG having a degree of polymerization of 4 fixed on a solid phase in the presence of a biological sample-derived component, and the reactivity can be compared with the case in which the biological sample-derived component is not present, so as to more efficiently select a hybridoma producing the desired antibody.

[0079] After the cloning step, the ability of the produced antibody to bind to (1→3)BG can be detected by, for example, ELISA, RIA, or fluorescent antibody method to confirm whether the selected hybridoma produces a monoclonal antibody with desired properties.

[0080] By large-scale cultivation of hybridomas selected as described above, monoclonal antibodies with desired characteristics can be produced. Although the method of mass cultivation is not particularly limited, examples thereof may include methods for producing monoclonal antibodies in culture medium by culturing hybridomas in appropriate culture medium and methods for producing antibodies in ascites by injecting hybridomas into the peritoneal cavity of animals for proliferation. Monoclonal antibodies can be purified by appropriately combining the above-mentioned methods for purifying antibodies from antiserum, such as DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, and ethanol fractionation.

[0081] The monoclonal antibody used in the immunoassay method of the present invention may be a complete antibody molecule or an antibody fragment having antigen-antibody reactivity. The antibody may be obtained by the above-mentioned animal immunization step or by genetic recombination technology, or may be a chimeric antibody. Antibody fragments are preferably functional fragments; examples thereof include F(ab') 2 , Fab', scFv, etc.; and these fragments can be obtained by treating the antibody obtained above with a proteolytic enzyme (eg, pepsin or papain), or by cloning the DNA of the antibody and expressing it in a culture system using Escherichia coli or yeast.

[0082] The antibody used in the immunoassay method of the present invention can be used as an immobilized (solid phase) antibody fixed on an insoluble carrier, or can be used as a labeled antibody labeled with a labeling substance known to those skilled in the art as described later. For example, the fixed antibody can be produced by physically adsorbing or chemically binding an insoluble carrier to a monoclonal antibody (a suitable spacer may be present in between). The insoluble carrier can be made of a polymer substrate such as a polystyrene resin, an inorganic substrate such as glass, a polysaccharide substrate such as cellulose and agarose, and its shape is not particularly limited and can be selected from any shape, such as a plate (such as a microplate and a membrane), a bead or granular (such as a latex particle) and a tubular (such as a test tube).

[0083] By using a labeled antibody (secondary antibody) that can bind to the antibody used in the immunoassay method of the present invention, the amount of antibody bound to BG can be measured, thereby detecting BG in a biological sample. BG in a biological sample is preferably (1→3)BG and / or (1→3)(1→6)BG. Examples of labeling substances used to produce labeled antibodies include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, radioactive isotopes, colloidal gold particles or colored latex. The method for binding the labeling substance to the antibody can be, for example, the glutaraldehyde method, maleimide method, pyridyl disulfide method, or periodic acid method that can be used by those skilled in the art, and the fixed and labeled antibodies and the method for producing the antibodies are not limited to these examples. For example, when an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) is used as a labeling substance, it can be reacted by using a specific substrate of the enzyme (e.g., when the enzyme is HRP, o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB), in the case of ALP, p-nitrophenyl phosphate), and when biotin is used as a labeling substance, at least avidin or enzyme-modified avidin is generally reacted. In the immunoassay method of the present invention, it is preferred to use biotin or HRP as a labeling substance.

[0084] In this specification, "insoluble carrier" may be expressed as "solid phase", an antigen or antibody may be physically or chemically supported by an insoluble carrier, or the support state may be expressed as "immobilized", "fixed" or "solid phase". The terms "determining", "detecting" or "measuring" must be interpreted in the broadest sense, including the demonstration and / or quantification of the presence of BG, and must not be interpreted in any restrictive manner.

[0085] (Immunoassay method)

[0086] Examples of the immunoassay method of the present invention include, but are not limited to, ELISA, enzyme immunoassay, immunohistochemical staining, surface plasmon resonance, latex agglutination immunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation method, immunoblotting method, immunochromatography, high performance liquid chromatography (HPLC), etc. ELISA is preferably used as the immunoassay method of the present invention. In ELISA, a sandwich ELISA using a first monoclonal antibody immobilized on a solid phase and a labeled second monoclonal antibody is preferred.

[0087] In this case, the 86207 antibody can be used as the first monoclonal antibody and the 86202R antibody can be used as the second monoclonal antibody. The same antibody can be used as the first monoclonal antibody and the second monoclonal antibody. The antibody fixed on the solid phase or the labeled antibody can be a mixture of multiple antibodies. Biotin or HRP is preferably used as a labeling substance for labeling antibodies.

[0088] (Diagnosis, diagnostic aids and treatment of deep fungal diseases)

[0089] Based on the analysis results of the immunoassay method of the present invention, it is possible to diagnose whether the subject suffers from deep mycosis, or to assist in the diagnosis. Conventional immunoassay methods using antibodies are not sensitive enough, and patients with deep mycosis in the early stages of the disease may have negative results. Early detection and early treatment are important for deep mycosis. Using the highly sensitive immunoassay method of the present invention, early detection and early treatment of deep mycosis can be achieved.

[0090] After performing the immunoassay method of the present invention, if necessary, based on the results of the step of analyzing BG in the biological sample, another analysis method for deep mycosis may be performed on the patient, and / or a drug for treating deep mycosis may be administered to the patient.

[0091] The immunoassay method of the present invention may include a step of detecting 4 pg / mL or more BG contained in a biological sample (e.g., blood, serum or plasma, preferably plasma) and / or a step of diagnosing a subject corresponding to the collected biological sample as having or suspected of having deep fungal disease or a step of assisting diagnosis based on the result of the detection step. The cutoff value may be appropriately set according to the type of biological sample or the type of immunoassay method. For example, the immunoassay method of the present invention may include a step of detecting 6 pg / mL or more, 7 pg / mL or more, 8 pg / mL or more, 9 pg / mL or more, 10 pg / mL or more, 11 pg / mL or more, 20 pg / mL or more, 50 pg / mL or more, 80 pg / mL or more, 100 pg / mL or more, 150 pg / mL or more, 200 pg / mL or more, 250 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 1 ng / mL or more, 5 ng / mL or more, or 10 ng / mL or more BG in a biological sample (e.g., blood, serum or plasma, preferably plasma), and / or a step of diagnosing a subject corresponding to the collected biological sample as having or suspected of having deep fungal disease or a step of assisting diagnosis based on the result of the detecting step.

[0092] In the commercially available horseshoe crab amebocyte lysate reagent, (1→3)BG and / or (1→3)(1→6)BG in the sample binds to factor G. This binding initiates a cascade to analyze (1→3)BG and / or (1→3)(1→6)BG. The monoclonal antibody used in the immunoassay method of the present invention preferably recognizes the factor G binding site in (1→3)BG and / or (1→3)(1→6)BG.

[0093] The monoclonal antibody used in the immunoassay method of the present invention preferably has a competitive inhibition effect with the horseshoe crab amebocyte lysate reagent in the analysis of β-D-glucan, preferably (1→3)BG and / or (1→3)(1→6)BG. In this case, the competitive inhibition effect specifically means that the binding of the monoclonal antibody to β-D-glucan inhibits the binding of β-D-glucan to G-factor, thereby inhibiting the progress of the horseshoe crab amebocyte lysate reagent reaction cascade.

[0094] When the monoclonal antibody used in the immunoassay method of the present invention has a competitive inhibition effect with a horseshoe crab amebocyte lysate reagent in the analysis of β-D-glucan, the concentration of the monoclonal antibody in the reaction system is 0.1 μg / mL to 1000 μg / mL, preferably 1 μg / mL to 100 μg / mL.

[0095] [2] β-D-Glucan Analysis Kit in Biological Samples

[0096] The kit provided by the present invention includes (a) a solid phase on which a first monoclonal antibody that binds to (1→3)-β-D-glucan with a degree of polymerization of 4 is immobilized, and preferably includes (b) a second monoclonal antibody that binds to (1→3)-β-D-glucan with a degree of polymerization of 4 and is labeled with a labeling substance. The solid phase on which the first monoclonal antibody is immobilized captures BG in a biological sample and forms a BG-antibody complex. The second anti-BG monoclonal antibody labeled with a labeling substance reacts with the BG-antibody complex to form a sandwich. BG in the sample can be measured by measuring the amount of the labeling substance by a method corresponding to the labeling substance. For the specific construction method of the kit, such as a method for immobilizing the first monoclonal antibody on a solid phase and a method for labeling the second monoclonal antibody with a labeling substance, the methods described in this specification and methods well known to those skilled in the art can be used without restriction.

[0097] The first monoclonal antibody and the second monoclonal antibody are not particularly limited as long as the antibodies are monoclonal antibodies that bind to (1→3)-β-D-glucan having a degree of polymerization of 4. For example, 86207 antibody can be used as the first monoclonal antibody, and 86202R antibody can be used as the second monoclonal antibody.

[0098] For the labeling substance, for example, labeling substances well known to those skilled in the art such as fluorescent substances, chemiluminescent substances, biotin and avidin can be used. According to the labeling substance and antibody to be used, the method for combining the labeling substance and the antibody can be appropriately selected from known binding methods, and can be, for example, glutaraldehyde method, maleimide method, pyridyl disulfide method or periodic acid method. Preferably, biotin or HRP is used as the labeling substance.

[0099] The kit of the present invention may further include instructions for use, etc. The kit may include any constituent elements, such as a buffer, a stabilizer, and a reaction container.

[0100] The kit of the present invention can detect 4 pg / mL or more of BG contained in a biological sample (e.g., blood, serum or plasma, preferably plasma), and based on the result, the subject corresponding to the collected biological sample can be diagnosed as having or suspected of having deep fungal disease. The cutoff value can be appropriately set according to the type of biological sample, etc. For example, the kit of the present invention can detect 6 pg / mL or more, 7 pg / mL or more, 8 pg / mL or more, 9 pg / mL or more, 10 pg / mL or more, 11 pg / mL or more, 15 pg / mL or more, 20 pg / mL or more, 24.9 pg / mL or more, 25 pg / mL or more, 30 pg / mL or more, 50 pg / mL or more, 80 pg / mL or more, 100 pg / mL or more, 150 pg / mL or more, 200 pg / mL or more, 250 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 1 ng / mL or more, 5 ng / mL or more, or 10 ng / mL or more of BG contained in a biological sample (e.g., blood, serum or plasma, preferably plasma), and based on the result, the subject corresponding to the collected biological sample can be diagnosed as having or suspected of having deep fungal disease.

[0101] The present invention is specifically described below by way of examples, but these examples do not limit the scope of the present invention.

[0102] Example

[0103] [Test Example 1] Method for preparing the monoclonal antibody used in the present invention.

[0104] 1. Preparation of Immunizing Antigens

[0105] Laminaria heptaose (manufactured by Seikagaku Biobusiness Corporation) is a (1→3) BG having a structure of 7 glucose molecules polymerized into a line, and is used as an immunizing antigen. Laminaria heptaose-transferrin conjugate was prepared by the same preparation method as described in Non-Patent Document 1 and used as an immunizing antigen.

[0106] 2. Hybridoma Generation and Antibody Collection

[0107] The total amount of suspension consisting of 100 μL of the prepared laminarin seven sugar-transferase conjugate solution (1 mg / mL), 0.25 mL 0.1 M PBP (pH 7.2) and 0.25 mL Freund's adjuvant (complete adjuvant or incomplete adjuvant) was administered subcutaneously or intraperitoneally to the back of BALB / c mice (female) and F344 / Jc1 rats (female) for a total of 3 to 6 times. The administration interval was 2 weeks, and the first administration was with Freund's complete adjuvant, and the last four administrations were with Freund's incomplete adjuvant. One week after the fifth administration, the abdominal cavity of mice and rats was opened, their spleens were removed, and single cells were obtained by pipetting.

[0108] These spleen cells were washed twice with serum-free PRMI1640 medium and incubated with mouse myeloma cells (X-63-Ag8-6.5.3) cultured and washed separately at 1×10 7 Myeloma cells vs 5×10 7 The mixture was centrifuged to remove the supernatant. After the precipitate was fully dissolved, polyethylene glycol 1540 (1 mL) was slowly added as a fusion promoter at 37°C for 1 minute, and the mixture was stirred for 1 minute to fuse. These fused cells (hybridomas) were suspended in 10 mL of RPMI1640 medium supplemented with fetal bovine serum, centrifuged, and the residue was inoculated on a single 96-well culture plate at 37°C and 5% CO. 2 After culturing in HAT medium at 37°C for one week, only hybridomas were selectively collected.

[0109] 3. Antigen solid phase ELISA (preliminary screening)

[0110] The preliminary screening of hybridomas was performed by the following method. Laminarin was used as an antigen. Laminarin is a (1→3)(1→6)BG, having a structure in which glucose molecules are bound to the outer chains of the triple helix structure in the form of (1→6). It is believed that antibodies that recognize laminarin can recognize short β(1→3) bonds between glucose molecules bound in the form of (1→6).

[0111] 1) Dispense the laminarin solution into the plate (50 μL / well) and let it stand at room temperature for 2 hours or at 4°C overnight.

[0112] 2) After washing three times (400 μL / well), blocking solution (100 μL / well) was dispensed and allowed to stand at room temperature for 1 hour.

[0113] 3) After washing three times (400 μL / well), serial dilutions of the culture supernatant were dispensed (50 μL / well) and allowed to stand at room temperature for 1 hour.

[0114] 4) After washing three times (400 μL / well), HRP-labeled goat anti-mouse IgG polyclonal antibody (50 μL / well) was dispensed and allowed to stand at room temperature for 1 hour.

[0115] 5) After washing three times (400 μL / well), OPD color development solution (50 μL / well) was dispensed and reacted at room temperature for 10 minutes.

[0116] 6) Dispense stop solution (50 μL / well) to stop the reaction and measure using a plate reader.

[0117] 7) 50 hybridomas producing antibodies highly reactive with laminarin were selected and used in the following competitive ELISA.

[0118] 4. Competitive ELISA (secondary screening)

[0119] Secondary screening of hybridomas (confirmation of the reactivity of established candidate strains) was performed by the following method.

[0120] 1) Dispense the laminarin solution into an ELISA plate (50 μL / well) and let stand at room temperature for 2 hours or at 4°C overnight.

[0121] 2) After washing three times (400 μL / well), blocking solution (100 μL / well) was dispensed and allowed to stand at room temperature for 1 hour.

[0122] 3) After washing three times (400 μL / well), sugar chains (glucan, laminarin or laminaria heptaose) (25 μL / well) were dispensed. Subsequently, the culture supernatant was dispensed (25 μL / well) and allowed to stand at room temperature for 1 hour.

[0123] 4) After washing three times (400 μL / well), HRP-labeled goat anti-mouse IgG polyclonal antibody (50 μl / well) was dispensed and allowed to stand at room temperature for 1 hour.

[0124] 5) After washing three times (400 μL / well), OPD color development solution (50 μL / well) was dispensed and reacted at room temperature for 10 minutes.

[0125] 6) Dispense stop solution (50 μL / well) to stop the reaction and measure using a plate reader.

[0126] 7) The results are as follows Figure 2 86207 (from mouse) and 86202R (from rat) were chosen because both would cause competition when laminarin and laminaria heptaose were used as free antigens.

[0127] 5. Antibody Purification Procedure

[0128] The monoclonal antibodies were purified using the two hybridomas obtained. Ascites was extracted from mice injected with hybridoma cells intraperitoneally and stored frozen at -80°C. Ascites solution was added to the ascites, thawed in a water bath, and the supernatant was collected after centrifugation.

[0129] 50% ammonium sulfate was added to the recovered supernatant. Centrifuge again and add a redissolving solution to the precipitate to dissolve the precipitate. Centrifuge the dissolved solution and collect the supernatant. The supernatant was dialyzed overnight at 4°C using 100 times the amount of supernatant (20mM NaPi pH7.5, 100mM NaCl, 1mM EDTA 2Na). For dialysis, a Slide-A-Lyzer Diarysis box 10k (manufactured by Thermo Fisher Scientific) was used.

[0130] The next day, protein G column Hi Trap protein G HP 5mL (GE Healthcare production) was connected to a low pressure chromatography system AKTA Prime plus (GE Healthcare production), and the sample solution after dialysis was separated and purified. The protein G column was preliminarily balanced with a column equilibration solution. After the sample solution was added to the column, the antibody bound to the column was eluted with an eluent of pH 4. After pH 4 elution, further elution was performed with pH 3 eluents. The sample solution after elution with pH 4 eluent and pH 3 eluent was dialyzed by antibody preservation solution (PBS) respectively. Dialysis was performed twice with 100 times the sample solution at 4 ° C. After dialysis, sodium azide with a final concentration of 0.09% was added, and the mixture was divided into 400 μL aliquots and stored at -80 ° C. Two antibodies (86202R antibody and 86207 antibody) were obtained by the above procedure. The antibody sample eluted at pH 4 was used for the following measurement.

[0131] [Example 2] Verification of antibody reactivity to poly (1→3) BG

[0132] Competitive ELISA was performed using (1→3)BG with a degree of polymerization of 1 to 7 to verify the reactivity of 86202R antibody and 86207 antibody to (1→3)BG with each degree of polymerization. For (1→3)BG with a degree of polymerization of 1 to 7, glucose (degree of polymerization of 1), laminaribiose (degree of polymerization of 2), laminaritriose (degree of polymerization of 3), laminariotetraose (degree of polymerization of 4), laminaripentaose (degree of polymerization of 5), and laminariheptaose (degree of polymerization of 7) were used. Laminarihexaose (degree of polymerization of 6) was not used for verification because the raw material was difficult to obtain. Glucose was purchased from FUJIFILM Wako Pure Chemical Corporation, and other (1→3)BGs were purchased from Santa Cruz Biotechnology.

[0133] 1) Laminarin was dispensed into an ELISA plate (1 μg / ml in PBS, 50 μL / well) and allowed to stand overnight at 4°C.

[0134] 2) After washing three times (400 μL / well), blocking solution (100 μL / well) was dispensed and allowed to stand at room temperature for 1 hour.

[0135] 3) After washing 3 times (400 μL / well), each (1→3) BG (25 μL / well, each (1→3) BG concentration is 100 μg / ml) was dispensed. Subsequently, 86202R antibody solution or 86207 antibody solution (25 μL / well) was dispensed and left to stand at room temperature for 1 hour.

[0136] 4) After washing three times (400 μL / well), HRP-labeled goat anti-rat IgG polyclonal antibody or HRP-labeled goat anti-mouse IgG polyclonal antibody (50 μL / well) was dispensed and allowed to stand at room temperature for 1 hour.

[0137] 5) After washing three times (400 μL / well), OPD color development solution (50 μL / well) was dispensed and reacted at room temperature for 10 minutes.

[0138] 6) Dispense stop solution (50 μL / well) to stop the reaction and measure using a plate reader.

[0139] 7) The results of 86202R antibody are shown in Figure 3 , the results of 86207 antibody are shown in Figure 4 .

[0140] The 86202R antibody recognizes (1→3)BG with a degree of polymerization of 4 to 7. Therefore, this antibody is considered to be an antibody that recognizes β(1→3) bonds with a degree of polymerization of 4.

[0141] The 86207 antibody recognized (1→3)BG with a degree of polymerization of 4 to 7. Therefore, this antibody was considered to be an antibody that recognizes β(1→3) bonds with a degree of polymerization of 4. Considering the difference in reactivity between the two antibodies, the recognition sites of the two antibodies may be slightly different.

[0142] [Example 3] Confirmation of reactivity to BG with different binding forms

[0143] By using 86207 antibody as a solid phase antibody and 86202R antibody as an HRP-labeled antibody (liquid phase antibody) for sandwich ELISA, the reactivity of various BGs was compared with the horseshoe crab amebocyte lysate reagent. For the horseshoe crab amebocyte lysate reagent, β-glucan Test Wako (FUJIFILM Wako Pure Chemical Corporation) (hereinafter referred to as "commercially available reagent 1") and Fungitec (registered trademark) G test ES "Nissui" (Nissui Pharmaceutical Co., Ltd.) (hereinafter referred to as "commercially available reagent 2") were used, and the experiment was performed according to the attached protocol. The antigens used are as follows.

[0144] [Table 1]

[0145] antigen Combination form Carboxymethylated curdlan β(1-3) Carboxymethylated Pachymannan β(1-3) Laminaria polysaccharide (1-3)β(1-6) Lichen polysaccharide β(1-3)β(1-4) Carboxymethylated cellulose β(1-4) Glycogen α(1-4)α(1-6) Pullulan α(1-6)α(1-6) Mannan β(1-4) Galactomannan Mβ(1-4)Mβ(1-4)Gβ(1-4)

[0146] The procedure of sandwich ELISA will be described below.

[0147] 1) Solid phase antibody (5 μg / mL, 100 μL, diluted with PBS) was dispensed into each well of a 96-well plate (nunc immunoplate, part number 442404, Thermo Fisher Scientific) and allowed to stand at room temperature for 2 hours or at 4°C overnight.

[0148] 2) After removing the liquid from each well, 200 μL PBST was dispensed into each plate well twice by using an 8-channel pipette (400 μL in total). The added PBST was removed, and these operations were defined as one washing operation. This operation was performed 3 times (= 3 washes with 400 μL PBST; the same washing procedure was performed with the following operations).

[0149] 3) After washing, dispense 200 μL of blocking solution into each well and let stand at room temperature for 1 hour or more.

[0150] 4) After discarding the blocking solution, add each serial dilution of BG and react for 60 minutes.

[0151] 5) After the reaction, the liquid in each well was removed and then washed three times with 400 μL PBST.

[0152] 6) HRP-labeled antibody (0.5 μg / mL, 100 μL, diluted with blocking solution) was dispensed and reacted for 60 minutes.

[0153] 7) After the reaction, the liquid in each well was removed and then washed three times with 400 μL PBST.

[0154] 8) Dispense 100 μL of color development solution into each well and react at room temperature for 10 minutes.

[0155] 9) Add 100 μL of reaction stop solution to each well.

[0156] 10) Measure the absorbance at 492 nm using a plate reader (Multican FC, thermo science).

[0157] The results are as follows Figures 5 to 7 As shown. In the sandwich ELISA using an antibody that recognizes BG, when (1→3)BG was used as an antigen, a reaction was observed from an antigen concentration of about 0.1 ng / mL. When (1→3)(1→6)BG was used as an antigen, a reaction was observed from an antigen concentration of about 10 ng / mL ( Figure 5). 5.) No reaction occurred with other BG.

[0158] In the case of commercially available reagent 1, when (1→3)BG was used as an antigen, a reaction was observed from an antigen concentration of about 0.1 ng / mL. When (1→3)(1→6)BG was used as an antigen, a reaction was observed from an antigen concentration of about 10 ng / mL. Figure 6 ). No reaction occurred with other BG.

[0159] In the case of commercially available reagent 2, when (1→3)BG was used as an antigen, a reaction was observed from an antigen concentration of about 0.01 ng / mL. When (1→3)(1→6)BG was used as an antigen, a reaction was observed from an antigen concentration of about 1 ng / mL. Figure 7 ). No reaction occurred with other BG.

[0160] Thus, it was demonstrated that sandwich ELISA using an antibody recognizing BG had the same reactivity to (1→3)BG and (1→3)(1→6)BG as that of the horseshoe crab amebocyte lysate reagent manufactured by Wako Pure Chemical Industries, Ltd (commercially available reagent 1) and the horseshoe crab amebocyte lysate reagent manufactured by Nissui Pharmaceutical Co., Ltd (commercially available reagent 2).

[0161] [Example 4] Examination of the lower limit of BG detection in biological samples

[0162] In Example 4, the lower limit of detection of sandwich ELISA was examined using biological samples. 86207 antibody was used as a solid phase antibody, and HRP-labeled 86202R antibody was used as a liquid phase antibody.

[0163] 4-1. Alkali pretreatment

[0164] The biological sample (human plasma containing 24 pg / mL of BG from fungi, measured by commercially available reagent 2) was diluted in stages with PBST to prepare a dilution series. 309.6 μL of alkaline treatment solution (150 mM KOH: potassium hydroxide) was added to 176 μL of the sample and incubated at 37°C for 15 minutes. Subsequently, 394.4 μL of 1M Tris / HCl (pH 7.5) was added to neutralize the alkaline treatment solution (a total of 880 μL, 5-fold dilution of the sample, for 8 measurements). These samples were used as alkali pre-treated samples.

[0165] 4-2. HRP labeling of antibodies

[0166] HRP labeling of 86202R antibody was performed by using an HRP labeling kit (Peroxidase Labeling Kit-SH, LK09, manufactured by Dojindo Laboratories). Protein quantification after labeling was performed by the BCA method (Kit name: Micro BCA Protein Assay Kit, manufactured by Thermo Scientific, Part No.: 23235).

[0167] 4-3. Sandwich ELISA

[0168] 86207 antibody for solid phase (5 μg / mL, 100 μL, diluted with PBS) was dispensed into each well of a 96-well plate (nunc immunoplate, part number 442404, manufactured by Thermo Fisher Scientific) and left to stand at 4°C overnight.

[0169] After removing the liquid from each well, 200 μL PBST was dispensed into each plate well twice using an 8-channel pipette (400 μL in total). The added PBST was removed, and these operations were defined as one washing operation. This operation was performed 3 times (washed 3 times with 400 μL PBST; the same washing procedure was performed with the following operations).

[0170] After washing, 200 μL of blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour or more.

[0171] After discarding the blocking solution, 100 μL of each pre-treated sample was added to each well and reacted for 90 minutes.

[0172] After the reaction, the liquid in each well was removed and then washed three times with 400 μL of PBST.

[0173] The HRP-labeled 86202R antibody prepared in 3-2 (0.5 μg / mL, 100 μL, diluted with blocking solution) was dispensed into each well and reacted for 30 minutes.

[0174] After the reaction, the liquid in each well was removed and then washed three times with 400 μL of PBST.

[0175] Dispense 100 μL of color development solution into each well and react at room temperature for 10 minutes.

[0176] Add 100 μL of reaction stop solution to each well.

[0177] The absorbance at 492 nm was measured using a plate reader (Multiskan FC, manufactured by Thermo Fisher Scientific). n=8 were measured for each sample.

[0178] 4-4. Results

[0179] The results are shown in Table 2 and Figure 8 The absorbance in Table 2 represents the mean value of n=8 measurements, and SD represents the standard deviation.

[0180] [Table 2]

[0181]

[0182] The value (0.1142) obtained by adding 2.6SD of the sample to the mean absorbance of the sample with an actual concentration of 0.0 pg / mL was smaller than the value (0.1217) obtained by subtracting 2.6SD of the sample from the mean absorbance of the sample with an actual concentration of 4.0 pg / mL, and thus it was found that BG can be detected in ELISA as an embodiment of the immunoassay method of the present invention even if the BG derived from the fungus contained in the actual sample is 4.0 pg / mL (measured value of commercially available reagent 2). The cutoff value of commercially available reagent 2 is 20 pg / mL, and this result indicates that ELISA as an embodiment of the immunoassay method of the present invention has a sensitivity comparable to that of the horseshoe crab amebocyte lysate reagent.

[0183] [Example 5] Examination of correlation with horseshoe crab amebocyte lysate reagent

[0184] In Example 5, the correlation between the measured values ​​of sandwich ELISA and the measured values ​​of commercially available reagents 1 and 2 was examined by using human plasma samples (n=39). 86207 antibody was used as a solid phase antibody, and HRP-labeled 86202R antibody was used as a liquid phase antibody.

[0185] 5-1. Alkali pretreatment

[0186] In the correlation comparison with the commercially available reagent 1, the sample was subjected to the following pretreatment. After adding 14.7 μL of human plasma sample and 29.3 μL of PBST, 77.4 μL of alkaline pretreatment solution (150 mM KOH: potassium hydroxide) was added and incubated at 37°C for 15 minutes. Subsequently, 98.6 μL of 1M Tris / HCl (pH 7.5) was added to the alkaline pretreatment solution to neutralize the alkaline pretreatment solution (a total of 220 μL; 15-fold dilution of the sample; pH after neutralization was less than 7.9).

[0187] In the correlation comparison with the commercially available reagent 2, the sample was subjected to the following pretreatment. 77.4 μL of an alkali pretreatment solution (150 mM KOH: potassium hydroxide) was added to 44 μL of a human plasma sample and incubated at 37°C for 15 minutes. Subsequently, 98.6 μL of 1 M Tris / HCl (pH 7.5) was added to the alkali pretreatment solution to neutralize the alkali pretreatment solution (a total of 220 μL; 5-fold dilution of the sample; pH after neutralization was less than 7.9). This was used as an alkali pretreated sample.

[0188] 5-2. HRP labeling of antibodies

[0189] The same procedure as in Example 3 was followed to carry out HRP labeling of the antibody.

[0190] 5-3. Sandwich ELISA

[0191] Sandwich ELISA was performed with the same procedure as in Example 3. n=2 were measured for each sample, and the average value was used as the measured value.

[0192] 5-4. Limulus amebocyte lysate reagent

[0193] BG was measured according to the protocol described in each package insert of commercially available reagents 1 and 2.

[0194] 5-5. Results

[0195] Fig. 9 The correlation between the measured value of the commercially available reagent 1 and the absorbance according to the ELISA method is shown. Fig.10 The correlation between the measured value of the commercially available reagent 2 and the absorbance according to the ELISA method is shown.

[0196] The absorbance measurement value of the sandwich ELISA according to the embodiment of the immunoassay method of the present invention showed correlation with both measurement values ​​of the horseshoe crab amebocyte lysate reagent according to commercially available reagents 1 and 2.

[0197] [Example 6] Competitive test with horseshoe crab amebocyte lysate reagent

[0198] In order to examine whether the 86207 antibody and the 86202R antibody used in Example 5 inhibit the cascade reaction of the horseshoe crab amebocyte lysate reagent of the commercially available reagent 2, a competitive assay was performed between these two monoclonal antibodies and the commercially available reagent 2.

[0199] When the experiment was carried out, the protocol of the accompanying commercially available reagent 2 was slightly modified. The measurement sample was PBS-Tween containing 1% BSA, in which CM-Pachyman was dissolved. The measurement sample was incubated at 25°C for 60 minutes in the presence or absence of 10μg / mL antibody. Subsequently, 200μL of distilled water was added to 50μL of the measurement sample (pretreatment solution of commercially available reagent 2 was not used to prevent antibody denaturation). After incubation at 37°C for 10 minutes, 50μL of the measurement sample was added to 300μL of commercially available reagent 2. The cascade reaction of commercially available reagent 2 was measured at 37°C for 30 minutes using an ES analyzer (manufactured by Nissui Pharmaceutical Co., Ltd.).

[0200] For the control experiment of mouse IgG antibody, anti-surfactant protein D antibody (manufactured by Abcam) was used. Each experiment was performed 3 times, and the average value was calculated.

[0201] The results are as follows Fig.11 As shown. The 86207 antibody and the 86202R antibody inhibited the activity of the commercial reagent 2 by 80.0% and 95.5%, respectively. Compared with the absence of the antibody, the control IgG antibody did not inhibit the activity of the commercial reagent 2. The results show that both the 86207 antibody and the 86202R antibody recognize the structure of the G factor in the reaction cascade that activates the commercial reagent 2 in BG.

[0202] [Example 7] Measurement of healthy human plasma samples using the cutoff value (20 pg / mL) of the horseshoe crab amebocyte lysate reagent

[0203] An experiment was conducted using 32 healthy human plasma samples to confirm whether fungal infection can be diagnosed by ELISA using 86207 antibody and 86202R antibody in the same manner as the horseshoe crab amebocyte lysate reagent. The experimental protocol was the same as in Example 5 (correlation comparison with commercially available reagent 2). The results are shown in Fig.12 shown.

[0204] The measured values ​​of the samples were all less than the cutoff value of 20 pg / mL of the horseshoe crab amebocyte lysate reagent, and the median was 3.6 pg / mL. Therefore, it was demonstrated that fungal infection can be diagnosed by ELISA using the 86207 antibody and the 86202R antibody in the same manner as the horseshoe crab amebocyte lysate reagent.

[0205] Industrial Applicability

[0206] The present invention enables an immunoassay method for BG in a biological sample to have the same sensitivity and similar reactivity as a horseshoe crab amebocyte lysate reagent. The present invention can provide an immunoassay kit for BG in a biological sample, which has comparable sensitivity and similar reactivity as a horseshoe crab amebocyte lysate reagent.

[0207] Accession number

[0208] [Citation of Deposited Biological Material]

[0209] (1) Hybridoma 86202R producing antibody No. 86202R

[0210] i) the name and address of the depositary institution where the biological material has been deposited.

[0211] National Institute of Technology and Evaluation (NITE) Patented Microorganisms Collection Center

[0212] Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan 2920818

[0213] ii) the date of deposit of the biological material with the depository referred to in i).

[0214] May 17, 2018

[0215] iii) the accession number assigned by the depository in i).

[0216] NITE BP-02723

[0217] (2) Hybridoma 86207 producing antibody No. 86207

[0218] i) the name and address of the depositary institution where the biological material has been deposited.

[0219] National Institute of Technology and Evaluation (NITE) Patented Microorganisms Collection Center

[0220] Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan 2920818

[0221] ii) the date of deposit of the biological material with the depository referred to in i).

[0222] May 17, 2018

[0223] iii) the accession number assigned by the depository in i).

[0224] NITE BP-02724.

Claims

1. Use of monoclonal antibodies in the preparation of immunoassays for β-D-glucan in biological samples, in, The monoclonal antibody is at least one selected from the group consisting of 86202R antibody produced by hybridoma NITE BP-02723 and 86207 antibody produced by hybridoma NITE BP-02724, and the β-D-glucan is (1→3)-β-D-glucan and (1→3)(1→6)-β-D-glucan.

2. The use according to claim 1, wherein the biological sample is blood, plasma or serum.

3. The use according to claim 1 or 2, in, The concentration of the β-D-glucan is 1 μg / mL or less.

4. The use according to claim 1 or 2, wherein ELISA is used.

5. The use according to claim 1 or 2, wherein the monoclonal antibody has a competitive inhibition effect in a Limulus amebocyte lysate reagent in a β-D-glucan assay.

6. A kit for determining β-D-glucan in a biological sample, comprising: (a) a solid phase on which the 86202R antibody produced by the hybridoma NITE BP-02723 is immobilized, and (b) 86207 antibody produced by hybridoma NITE BP-02724, which is labeled with a labeling substance.

7. The assay kit according to claim 6, wherein the biological sample is blood, plasma or serum. 8 . The assay kit according to claim 6 , wherein the β-D-glucan is (1→3)-β-D-glucan and (1→3)(1→6)-β-D-glucan. 9 . The assay kit according to claim 6 or 7 , wherein the concentration of the β-D-glucan is 1 μg / mL or less.

10. The assay kit according to claim 6 or 7, wherein the kit is used to determine whether a subject suffers from deep fungal disease, and wherein the determined cutoff value is 20 pg / mL.

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