Immunological analysis kit for sepsis-causing bacteria

Abstract

Provided is an immunological analysis kit for septicemia-causing bacteria, wherein the immunological analysis kit exhibits the same reactivity as Limulus reagent and exhibits a better sensitivity than Limulus reagent.　The problem can be solved by an immunological analysis kit for detecting septicemia-causing bacteria in a biological sample, wherein the immunological analysis kit contains monoclonal antibody that reacts with lipopolysaccharide originating with the septicemia-causing bacteria and the monoclonal antibody detects septicemia-causing bacteria present at 0.023 pg / mL or more in a biological sample.

Description

[Technical Field]

[0001] The present invention relates to an analytical kit for sepsis-causing bacteria. [Background technology]

[0002] Sepsis is a condition in which an infection causes severe organ damage. The prognosis for sepsis varies depending on the pathogen, the background factors of the infected patient, and the quality of treatment. Although it is difficult to generalize, it is estimated that 100,000 people die from sepsis each year in Japan.

[0003] Limulus reagent, which utilizes the blood coagulation reaction of horseshoe crabs, is used as a method for measuring LPS (lipopolysaccharide), which constitutes the outer cell membrane of Gram-negative bacteria that cause sepsis (Patent Document 1). Limulus reagent is used to diagnose or assist in the diagnosis of sepsis. However, the method using Limulus reagent requires horseshoe crab blood, which is a natural resource, and there are concerns about resource depletion. In addition, maintaining a consistent quality is costly. Another drawback of this method is that it is a manual method requiring multiple steps, which is prone to variability.

[0004] In recent years, an ELISA kit (https: / / www.mybiosource.com / human-elisa-kits / klebsiella / 9310934) has been developed to measure LPS, which constitutes the outer membrane of Gram-negative bacteria (Non-Patent Document 1). However, the sensitivity of these kits is inferior to that of the Limulus reagent, making them difficult to use as a substitute for the Limulus reagent. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 04-136763 [Non-patent literature]

[0006] [Non-Patent Document 1] Qualitative Human Klebsiella (KBSL) ELISA Kit:Cat.No:MBS9310934 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide an immunological analysis kit for sepsis-causing bacteria that has sensitivity equal to or greater than that of a Limulus reagent and can detect sepsis-causing bacteria with a simple procedure. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above-mentioned problems and developed an immunological analysis kit capable of detecting lipopolysaccharide derived from sepsis-causing bacteria even when the biological sample contains very small amounts of the lipopolysaccharide. They then confirmed that this kit has superior sensitivity to the Limulus reagent, leading to the completion of the present invention. Specifically, the lower limit of measurement for the Limulus reagent is 0.35 pg / mL for lipopolysaccharide derived from Escherichia coli O111, according to the package insert. However, because the specimen is diluted 10-fold during the preparation of the measurement sample, the actual lower limit of measurement is 3.5 pg / mL. In contrast, the immunological analysis kit of the present invention can detect lipopolysaccharides present at even lower concentrations. Specifically, the present invention is as follows. <1> 1. An immunological assay kit for detecting sepsis-causing bacteria in a biological sample, comprising: It contains a monoclonal antibody that reacts with lipopolysaccharide derived from sepsis-causing bacteria, The immunological analysis kit as described above, wherein the monoclonal antibody detects sepsis-causing bacteria at 0.023 pg / mL or more in a biological sample. <2> The biological sample contains 0.023 pg / mL or more of lipopolysaccharide derived from sepsis-causing bacteria. <1> The immunological analysis kit according to claim 1. <3> The sepsis-causing bacterium is Klebsiella pneumoniae or Escherichia coli. <1> or <2> The immunological analysis kit according to claim 1. <4> The monoclonal antibody is a monoclonal antibody that specifically reacts with lipopolysaccharide derived from a sepsis-causing bacterium. <1> ~ <3> 10. The immunological analysis kit according to claim 9, wherein <5> The monoclonal antibodies include a first monoclonal antibody and a second monoclonal antibody; a solid phase on which the first monoclonal antibody is immobilized; a labeled substance bound to the second monoclonal antibody; contains, or a solid phase on which the first monoclonal antibody is immobilized; the second monoclonal antibody; a secondary antibody against the second monoclonal antibody bound to a labeling substance; Including, The first monoclonal antibody and the second monoclonal antibody are monoclonal antibodies of the same type. <1> ~ <4> 10. The immunological analysis kit according to claim 9, wherein <6> The biological sample is blood, plasma, or serum. <1> ~ <5> 10. The immunological analysis kit according to claim 9, wherein <7> ELISA method is used as the measurement principle. <1> ~ <6> 10. The immunological analysis kit according to claim 9, wherein <8> The monoclonal antibody is an IgM antibody. <1> ~ <7> 10. The immunological analysis kit according to claim 9, wherein <9> The monoclonal antibody is a monoclonal antibody produced by a hybridoma having accession number NITE BP-03241 or a monoclonal antibody produced by a hybridoma having accession number NITE BP-03242. <1> ~ <8> 10. The immunological analysis kit according to claim 9, wherein [Effects of the Invention]

[0009] According to the present invention, it is possible to provide an immunological analysis kit for sepsis-causing bacteria, which has superior sensitivity to the Limulus reagent and can detect sepsis-causing bacteria with a simple operation. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a schematic diagram of the structure of lipopolysaccharide in sepsis-causing bacteria. [Figure 2] Fig. 10 is a graph showing the results of sandwich ELISA in which it was tested whether a sandwich system can be formed using only the S28201R antibody. [Figure 3] Fig. 10 is a graph showing the results of sandwich ELISA in which it was tested whether a sandwich system can be formed using only the S28203R antibody. [Figure 4] 1 is a graph showing a calibration curve for LPS of Klebsiella pneumoniae. [Figure 5] 1 is a graph showing a calibration curve for E. coli LPS. [Figure 6] 1 is a graph for calculating the minimum detection limit of LPS of Klebsiella pneumoniae. [Figure 7] 1 is a graph for calculating the minimum detection limit of E. coli LPS. [Figure 8] 1 is a graph for calculating the minimum detection limit of LPS of Klebsiella pneumoniae in a biological sample. [Figure 9] 1 is a graph for calculating the minimum detection limit of E. coli LPS in a biological sample. DETAILED DESCRIPTION OF THE INVENTION

[0011] (biological samples) The "biological sample" of the present invention mainly includes solid tissues and body fluids derived from living organisms (organisms), and body fluids are preferably used. The biological sample of the present invention is more preferably blood, serum, plasma, urine, saliva, sputum, tears, otorrhea, or prostatic fluid, even more preferably blood, serum, or plasma, and even more preferably blood, serum, or plasma from a subject suspected of having sepsis. The living organism or subject includes humans or animals (e.g., monkeys, dogs, cats, mice, guinea pigs, rats, hamsters, etc.), preferably humans. The biological sample may be in vivo or in vitro.

[0012] (Sepsis-causing bacteria) As used herein, "sepsis-causing bacteria" refers to bacteria that cause sepsis, such as Klebsiella pneumoniae, Streptococcus, Staphylococcus, Escherichia coli, and Pseudomonas aeruginosa. As used herein, the term "sepsis" includes both sepsis and septic shock. Sepsis refers to a condition in which an infection causes severe organ damage. Septic shock refers to a condition in which acute circulatory failure leads to severe cell damage and metabolic abnormalities, potentially increasing mortality. The "sepsis-causing bacteria" is preferably Klebsiella pneumoniae or Escherichia coli. As used herein, "Klebsiella pneumoniae" refers to the gram-negative bacillus Klebsiella pneumoniae. As used herein, "E. coli" refers to Escherichia coli, a gram-negative rod-shaped bacterium. The present invention has the same reactivity as the Limulus reagent and can therefore replace the Limulus reagent currently used in clinical practice. Furthermore, the present invention has the advantage of being able to detect lipopolysaccharides derived from sepsis-causing bacteria with greater sensitivity than the Limulus reagent.

[0013] (lipopolysaccharide) As used herein, "lipopolysaccharide" refers to a complex of covalently bound lipids and polysaccharides. In this specification, "lipopolysaccharide" is sometimes simply referred to as LPS (lipopolysaccharide). Lipopolysaccharides are present in the outer membrane of Gram-negative bacteria. Structurally, lipopolysaccharides form a membrane structure in which the lipid portion, lipid A, is embedded in the outer membrane, and the O antigen, a polysaccharide chain, extends from lipid A via an oligosaccharide region called the core (outer core and inner core) (Figure 1). The antibody used in the present invention can recognize both lipopolysaccharide that has not been detached from the cell wall of a sepsis-causing bacterium and lipopolysaccharide that has been detached from the cell wall of a sepsis-causing bacterium. Sandwich analysis often uses two types of antibodies, a solid-phase antibody and a labeled antibody, that recognize different epitopes. The monoclonal antibody used in the present invention has the advantage that a sandwich system can be formed with a single type. As used herein, "one type" or "the same type" with respect to monoclonal antibodies refers to monoclonal antibodies that recognize the same epitope. The ability to form a sandwich system with a single type of antibody facilitates the construction of an experimental system. Furthermore, the ability to form a sandwich system with a single type of antibody reduces the possibility of nonspecific reactions. The monoclonal antibodies used in the present invention are preferably the same antibody, i.e., monoclonal antibodies produced by the same hybridoma. Note that, as used herein, "sandwich system" refers to an experimental system in which an antigen is sandwiched between two types of antibodies, a capture antibody (solid-phase antibody) and a detection antibody, to detect the antigen. The detection antibody is bound to a label, and the target substance can be analyzed by measuring the signal intensity derived from the label. The detection antibody may be directly bound to the label or indirectly bound to the label via a secondary antibody.

[0014] (monoclonal antibody) The monoclonal antibody used in the present invention is a monoclonal antibody that reacts with lipopolysaccharide derived from sepsis-causing bacteria, preferably a monoclonal antibody that reacts specifically with lipopolysaccharide derived from sepsis-causing bacteria, and more preferably the S28201R antibody produced by the hybridoma with accession number NITE BP-03241 or the S28203R antibody produced by the hybridoma with accession number NITE BP-03242. In this specification, an antibody that reacts with LPS derived from Klebsiella pneumoniae may be referred to as an anti-Klebsiella pneumoniae LPS antibody, and an antibody that reacts with LPS derived from Escherichia coli may be referred to as an anti-Escherichia coli LPS antibody.

[0015] As used herein, the terms "react with" lipopolysaccharide derived from sepsis-causing bacteria, "recognize" lipopolysaccharide derived from sepsis-causing bacteria, and "bind" to lipopolysaccharide derived from sepsis-causing bacteria are used synonymously, but are not limited to these examples and should be interpreted in the broadest sense. Whether an antibody "reacts" with an antigen (compound) such as lipopolysaccharide can be confirmed by antigen-immobilized ELISA, competitive ELISA, sandwich ELISA, or the like. Alternatively, the reaction can be confirmed by a method utilizing the principle of surface plasmon resonance (SPR). The SPR method can be performed using devices, sensors, and reagents commercially available under the name Biacore (registered trademark).

[0016] The expression "not reacting" between an antibody used in the present invention and a certain compound means that the antibody used in the present invention does not substantially react with a certain compound. To confirm whether or not the antibody "does not substantially react" with a certain compound, for example, the antibody used in the present invention can be immobilized and measured using Biacore (registered trademark) T100 or T200 based on the above-mentioned SPR method. "Not substantially reacting" can also be confirmed by methods or means well known to those skilled in the art other than the above-mentioned SPR method.

[0017] The monoclonal antibodies used in the immunological analysis kit of the present invention include functional fragments of the monoclonal antibodies, as long as the effects of the present invention are obtained, such as functional fragments containing the Fab portion of the monoclonal antibody obtained by enzymatic digestion of the monoclonal antibody, functional fragments containing the Fab portion of the monoclonal antibody produced by genetic recombination, and functional fragments containing scFv produced by phage display.

[0018] The antibodies used in the immunological analysis kit of the present invention can be produced by dissolving heat-killed bacteria derived from sepsis-causing bacteria, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and / or Escherichia coli, as an antigen (immunogen) in a solvent such as phosphate-buffered saline, and administering the resulting solution to an animal for immunization. If necessary, an appropriate adjuvant may be added to the solution, followed by immunization using the resulting emulsion. Commonly used adjuvants, such as water-in-oil emulsions, water-in-oil-in-water emulsions, oil-in-water emulsions, liposomes, and aluminum hydroxide gel, can be used as adjuvants. Proteins and peptides derived from biological components can also be used as adjuvants. For example, Freund's incomplete adjuvant or Freund's complete adjuvant can be suitably used. The route, dosage, and timing of administration of the adjuvant are not particularly limited, but are desirably selected appropriately to enhance the desired immune response in the animal immunized with the antigen.

[0019] The type of animal used for immunization is not particularly limited, but mammals are preferred, such as mice, rats, cows, rabbits, goats, sheep, and alpacas, and more preferably mice or rats. Animal immunization can be performed according to conventional techniques. For example, immunization can be performed by injecting a solution of an antigen, preferably a mixture with an adjuvant, into the animal subcutaneously, intradermally, intravenously, or intraperitoneally. Since immune responses generally vary depending on the type and strain of the animal being immunized, it is desirable to appropriately set the immunization schedule depending on the animal used. It is preferable to repeatedly administer the antigen several times after the initial immunization.

[0020] To obtain a monoclonal antibody, the following procedures can be subsequently carried out, but are not limited to these. Methods for producing monoclonal antibodies themselves are well known and widely used in the art, so those skilled in the art can easily produce antibodies to be used in the immunological analysis kit of the present invention by using the above-mentioned antigens (see, for example, Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988) Chapter 6, etc.).

[0021] After the final immunization, antibody-producing spleen cells or lymph node cells are extracted from the immunized animal. These cells can then be fused with a myeloma-derived cell line with high proliferation potential to produce hybridomas. For cell fusion, it is preferable to use cells with high antibody production capacity (quality and quantity), and it is also preferable that the myeloma-derived cell line is compatible with the animal from which the antibody-producing cells to be fused are derived. Cell fusion can be performed according to methods known in the art, such as the polyethylene glycol method, a method using Sendai virus, or a method using electric current. The resulting hybridomas can be grown under conditions commonly used in the art. Desired hybridomas can be selected by confirming the properties of the produced antibodies. Hybridoma cloning can be performed by well-known methods, such as limiting dilution and soft agar.

[0022] Hybridoma selection can be performed efficiently at the selection stage, taking into consideration the conditions to be used in the actual measurement of the produced antibodies. For example, antibodies obtained by immunizing an animal are reacted with lipopolysaccharide derived from a septicemic bacterium immobilized on a solid phase in the presence of a compound whose cross-reactivity is to be confirmed. Hybridomas producing the desired antibodies can then be selected more efficiently by comparing the reactivity with that in the absence of the compound whose cross-reactivity is to be confirmed. Furthermore, hybridomas producing the desired antibodies can also be selected more efficiently by reacting antibodies obtained by immunizing an animal with lipopolysaccharide derived from a septicemic bacterium immobilized on a solid phase in the presence of a component derived from a biological sample and comparing the reactivity with that in the absence of the component derived from a biological sample.

[0023] After the cloning step, the binding ability of the produced antibody to lipopolysaccharide derived from the sepsis-causing bacterium can be assayed using methods such as ELISA, RIA, and fluorescent antibody assay to confirm whether the selected hybridoma produces a monoclonal antibody with the desired properties. Monoclonal antibodies with desired properties can be produced by mass-culturing the hybridomas selected as described above. The mass-culturing method is not particularly limited, but examples include culturing the hybridomas in an appropriate medium to produce the monoclonal antibody in the medium, or injecting the hybridomas into the peritoneal cavity of a mammal to allow them to grow and produce the antibody in the ascites. Purification of the monoclonal antibody can be carried out by appropriately combining the above-mentioned methods for purifying antibodies from antisera, such as DEAE anion exchange chromatography, affinity chromatography, ammonium sulfate fractionation, PEG fractionation, and ethanol fractionation.

[0024] The antibodies used in the immunological analysis kit of the present invention may be whole antibody molecules or antibody fragments having antigen-antibody reaction activity. In addition to those obtained through the animal immunization process described above, those obtained using genetic recombination techniques or chimeric antibodies may also be used. Functional antibody fragments are preferred, and examples include F(ab')2, Fab', and scFv. These fragments can be produced by treating the antibodies obtained as described above with protease (e.g., pepsin or papain), or by cloning the antibody DNA and expressing it in a culture system using Escherichia coli or yeast.

[0025] (solid phase antibody) In the immunological analysis kit of the present invention, the antibody can be used as a solid-phase antibody immobilized on an insoluble carrier. Alternatively, in the immunological analysis kit of the present invention, the antibody can be used as a labeled antibody labeled with a labeling substance commonly known to those skilled in the art, as described below. For example, a solid-phase antibody can be produced by physically adsorbing or chemically binding (possibly via an appropriate spacer) a monoclonal antibody to an insoluble carrier, or by binding via an antibody bound to the insoluble carrier. Examples of insoluble carriers that can be used include insoluble carriers made of polymeric substrates such as polystyrene resin, inorganic substrates such as glass, and polysaccharide substrates such as cellulose and agarose. The shape of the insoluble carrier is not particularly limited, and any shape can be selected, including plates (e.g., microplates and membranes), beads, particles (e.g., latex particles, magnetic particles), and cylinders (e.g., test tubes).

[0026] (labeled antibody) By using a label capable of binding to the antibody used in the immunological analysis kit of the present invention, the amount of antibody bound to lipopolysaccharide derived from sepsis-causing bacteria can be measured. This allows detection of lipopolysaccharide derived from sepsis-causing bacteria in a biological sample. Examples of labeling substances for producing labeled antibodies include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, radioisotopes, gold colloid particles, and colored latex. Methods for binding the labeling substance to the antibody that are readily available to those skilled in the art, such as the glutaraldehyde method, maleimide method, pyridyl disulfide method, or periodic acid method, can be used. The types of solid-phase antibodies and labeled antibodies, and their production methods, are not limited to the examples of the above-mentioned binding methods. For example, when an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) is used as a labeling substance, the enzyme activity can be measured using the enzyme's specific substrate (e.g., O-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB) for HRP, or p-nitrophenyl phosphate for ALP). When biotin is used as a labeling substance, avidin or enzyme-modified avidin can be reacted. In the immunological analysis kit of the present invention, biotin or HRP is preferably used as a labeling substance, and biotin is more preferred. When biotin is used, streptavidin labeled with HRP can also be used.

[0027] As used herein, the term "insoluble carrier" refers to a substance onto which an antibody or the like that recognizes a target substance is immobilized. Examples include, but are not limited to, immunoplates, membranes, latex particles, and magnetic particles. As used herein, "insoluble carrier" is sometimes referred to as "solid phase," and the physical or chemical immobilization of an antigen or antibody on an insoluble carrier, or the state of being immobilized, is sometimes referred to as "immobilization." Furthermore, the terms "analysis," "detection," and "measurement" include the meaning of verifying the presence and / or quantification of lipopolysaccharide derived from sepsis-causing bacteria.

[0028] The immunological analysis kit of the present invention includes, but is not limited to, analysis kits using electrochemiluminescence immunoassay (ECL), enzyme-linked immunosorbent assay (ELISA), latex agglutination immunoassay (LTIA), chemiluminescence immunoassay, fluorescent antibody assay, and high-performance liquid chromatography (HPLC). In consideration of measurement sensitivity and ease of operation, the immunological analysis kit of the present invention is preferably an analysis kit using electrochemiluminescence immunoassay (ECL), high-performance liquid chromatography (HPLC), or enzyme-linked immunosorbent assay (ELISA), and more preferably an analysis kit for sandwich ELISA.

[0029] The order in which the monoclonal antibody and lipopolysaccharide are added to the analysis system may be any, as long as the effects of the present invention can be obtained.

[0030] The kit provided by the present invention preferably comprises (a) a solid phase, such as a plate, on which a first monoclonal antibody is immobilized, and (b) a second monoclonal antibody labeled with a labeling substance. Alternatively, the kit provided by the present invention preferably comprises a solid phase on which a first monoclonal antibody is immobilized, a second monoclonal antibody, and a secondary antibody against the second monoclonal antibody, which is bound to a labeling substance. In this case, the first monoclonal antibody and the second monoclonal antibody can be monoclonal antibodies of the same type. Monoclonal antibodies of the same type refer to monoclonal antibodies that recognize the same epitope. The monoclonal antibodies used in the kit of the present invention are preferably the same antibody, i.e., monoclonal antibodies produced by the same hybridoma.

[0031] The solid phase on which the first monoclonal antibody is immobilized captures lipopolysaccharide derived from the septicemia-causing bacterium in the biological sample, forming a lipopolysaccharide-antibody complex. The second monoclonal antibody labeled with a labeling substance reacts with this lipopolysaccharide-antibody complex to form a sandwich. The lipopolysaccharide derived from the septicemia-causing bacterium in the sample can be measured by measuring the amount of the labeling substance using a method appropriate for the labeling substance. Specific methods for constructing the kit, such as the method for immobilizing the first monoclonal antibody on the solid phase and the method for labeling the second monoclonal antibody with a labeling substance, may be any method known to those skilled in the art, as well as those described herein.

[0032] The first and second monoclonal antibodies are not particularly limited as long as they are monoclonal antibodies that react with lipopolysaccharide derived from a bacterium that causes sepsis.

[0033] The labeling substance may be, for example, a fluorescent substance, a chemiluminescent substance, biotin, avidin, or any other labeling substance known to those skilled in the art. The method for binding the labeling substance to the antibody may be appropriately selected from known binding methods depending on the labeling substance and antibody used, and may include, for example, the glutaraldehyde method, the maleimide method, the pyridyl disulfide method, or the periodic acid method. Biotin or HRP is preferably used as the labeling substance, and biotin is more preferably used. (Sepsis diagnosis, diagnostic aids, and treatment) Based on the analysis results of the immunological analysis kit of the present invention, it can be diagnosed whether or not a subject is suffering from sepsis caused by a sepsis-causing bacterium, or the results can serve as an aid in diagnosis.

[0034] When using a monoclonal antibody that reacts with lipopolysaccharide derived from Klebsiella pneumoniae but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Escherichia coli, the immunological analysis kit of the present invention can detect lipopolysaccharide derived from sepsis-causing bacteria at 0.023 pg / mL to 10 μg / mL, 0.1 pg / mL to 10 μg / mL, 0.3 pg / mL to 10 μg / mL, 0.5 pg / mL to 10 μg / mL, 1 pg / mL to 10 μg / mL, or 5 pg / mL to 10 μg / mL in a biological sample, preferably blood, serum, or plasma. The cutoff value can be set appropriately depending on the type of biological sample or the type of analysis kit. If the measured value is lower than the cutoff value, it can be determined that the subject does not suffer from sepsis caused by sepsis-causing bacteria. If the measured value is higher than the cutoff value, it can be determined that the subject suffers from sepsis caused by sepsis-causing bacteria.

[0035] (others) The immunological analysis kit of the present invention may contain, in addition to the above components, a buffer component (buffer solution). Buffer solutions that can be used in the present invention may be any commonly used solution, such as Tris-HCl, boric acid, phosphoric acid, acetic acid, citric acid, succinic acid, phthalic acid, glutaric acid, maleic acid, glycine, and salts thereof, as well as Good's buffers such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, and HEPES. Furthermore, the immunological analysis kit of the present invention may contain sugars, proteins, etc. as needed to improve measurement sensitivity and suppress non-specific reactions. Examples of such ingredients include components that promote antigen-antibody reactions (polymers such as polyethylene glycol, polyvinylpyrrolidone, and phospholipid polymers), proteins and peptides (albumin, casein, etc.), amino acids, sugars (sucrose, cyclodextrin, etc.), and preservatives (sodium azide, ProClin 300, etc.). The reagents used in the immunological analysis kit of the present invention can be adjusted to appropriate concentrations by those skilled in the art. The immunological analysis kit of the present invention may also contain instructions for use, a stabilizer, a reaction vessel, a pretreatment solution, a sample extract, and the like.

[0036] Electrochemiluminescence immunoassay (ECL) is a method for measuring the amount of a substance to be detected by detecting the amount of light emitted by a labeling substance when an electric current is applied. In ECL, a ruthenium complex can be used as the labeling substance. An electrode is placed on a solid phase (such as a microplate) and radicals are generated on the electrode, exciting the ruthenium complex to emit light. The amount of light emitted by the ruthenium complex can then be detected. Typically, electrochemiluminescence immunoassay (ECL) can be performed by using a first monoclonal antibody as a solid-phase antibody and a second monoclonal antibody that recognizes an epitope different from that of the first monoclonal antibody as a labeled antibody. On the other hand, in the immunological analysis kit of the present invention, antibodies that recognize the same epitope can be used as both the first and second monoclonal antibodies. When the ECL method is used, the immunological analysis kit of the present invention may comprise the following (A) and (B): (A) a labeling reagent containing a conjugate of a first monoclonal antibody (labeled antibody) that reacts with lipopolysaccharide derived from a sepsis-causing bacterium and an electrochemiluminescent substance (e.g., a ruthenium complex, etc.); and (B) An insoluble carrier onto which a second monoclonal antibody (solid-phase antibody) that reacts with lipopolysaccharide derived from sepsis-causing bacteria is immobilized. The first and second monoclonal antibodies may each be monoclonal antibodies that recognize the same epitope. For example, in a kit using magnetic particles as the solid phase, a biological sample is added to the magnetic particles on which a solid-phase antibody is immobilized, and the reaction is allowed to proceed. The biological sample is then removed and washed. A conjugate of a labeled antibody and an electrochemiluminescent substance (e.g., a ruthenium complex) is then added and allowed to react. After washing the magnetic particles, electrical energy is applied to cause light emission, and the amount of light emitted by the labeled substance is measured, allowing lipopolysaccharide derived from sepsis-causing bacteria to be analyzed.

[0037] Enzyme-linked immunosorbent assays (ELISAs) using enzyme labels are also preferred because they allow for simple and rapid target measurement. As used herein, ELISA refers to a method in which an antigen or antibody, which is a substance to be detected contained in a sample, is captured using an antibody or antigen against the substance to be detected, and then detected using an enzyme reaction. Sandwich ELISA is preferred as the ELISA. As used herein, sandwich ELISA refers to an ELISA in which an antigen is sandwiched between two types of antibodies, a capture antibody (solid-phase antibody) and a detection antibody, to detect and quantify the antigen. Sandwich ELISA uses an insoluble carrier onto which a first monoclonal antibody (solid-phase antibody) that recognizes the substance to be detected is immobilized, and a second monoclonal antibody (labeled antibody) labeled with a labeling substance. The insoluble carrier is preferably a plate (immunoplate). Typically, enzyme-linked immunosorbent assay (ELISA) can be performed using a first monoclonal antibody as a solid-phase antibody and a second monoclonal antibody that recognizes an epitope different from that of the first monoclonal antibody as a labeled antibody. However, in the immunological analysis kit of the present invention, antibodies that recognize the same epitope can be used as both the first and second monoclonal antibodies. When a sandwich ELISA method is used, the immunological analysis kit of the present invention can contain the following (A) and (B): (A) A labeling reagent containing a first monoclonal antibody that reacts with lipopolysaccharide derived from a sepsis-causing bacterium, labeled with a labeling substance. (B) Insoluble carrier with immobilized solid-phase antibody. In such a kit, a biological sample is first added to an insoluble carrier on which a solid-phase antibody has been immobilized, followed by incubation, followed by removal of the sample and washing. Next, a labeling reagent is added, followed by incubation, and then a substrate is added to develop color. The substance to be detected can be analyzed by measuring the color development using a plate reader or the like. Biotin is preferably used as the labeling substance, and when biotin is used, streptavidin labeled with HRP can also be used.

[0038] In the sandwich ELISA method, a secondary antibody can also be used. By using a secondary antibody, the reaction is amplified, and the detection sensitivity can be further increased. When a secondary antibody is used, the analysis kit of the present invention can include the following (A) to (D). (A) Secondary monoclonal antibody as primary antibody (B) Solid phase on which the first monoclonal antibody is immobilized (C) An antibody against the second monoclonal antibody, labeled with a labeling substance (HRP, ALP, etc.) (D) Substrate for labeling (e.g., OPD, TMB, or p-nitrophenyl phosphate) In such a kit, a suitably treated and diluted biological sample is first added to a solid phase on which a first monoclonal antibody has been immobilized, followed by incubation, followed by removal of the biological sample and washing. Next, a primary antibody (second monoclonal antibody) is added, followed by incubation and washing, and then an enzyme-labeled secondary antibody is added and incubated. A substrate is then added to develop color. Lipopolysaccharide derived from sepsis-causing bacteria can be analyzed by measuring the color development using a plate reader or similar device.

[0039] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention. Unless otherwise specified, % indicates % by mass. [Example]

[0040] [Test Example 1] Method for producing monoclonal antibodies used in the present invention 1. Obtaining Antibodies Rats (F344 / Jc1, female) were immunized weekly intraperitoneally with heat-treated Escherichia coli and Pseudomonas aeruginosa cells diluted in PBS, or heat-treated Klebsiella pneumoniae, E. coli, and Pseudomonas aeruginosa cells diluted in PBS. Blood samples were taken 10 weeks after the first immunization (after 10 immunizations) to confirm the antibody titers. The antibody titers were evaluated by antigen solid-phase ELISA using LPS derived from Pseudomonas aeruginosa, E. coli, and Klebsiella pneumoniae, the main sepsis-causing bacteria. The specific procedure for antigen solid-phase ELISA is as follows:

[0041] Various LPS diluted with PBS were dispensed into a 96-well ELISA plate (1 μg / mL, 50 μL / well) and left to stand at room temperature for 2 hours or at 4°C overnight. After washing three times (400 μL / well), blocking solution was dispensed (100 μL / well) and the plate was left to stand at room temperature for 1 hour or at 4°C overnight. After removing the blocking solution, culture supernatant or serum was dispensed (50 μL / well) and allowed to stand at room temperature for 1 hour. After washing three times (400 μL / well), HRP-labeled goat anti-rat IgG (H+L) antibody diluted 17,000 times with antibody diluent was dispensed (50 μL / well) and allowed to stand at room temperature for 1 hour. After washing three times (400 μL / well), OPD coloring solution was dispensed (50 μL / well) and allowed to react at room temperature for 10 minutes. The reaction was stopped by dispensing stop solution (50 μL / well). The absorbance at 492 nm was measured using a plate reader.

[0042] As a result, an increase in antibody titers against the immunogens E. coli-derived LPS and Pseudomonas aeruginosa-derived LPS was confirmed. After confirming the increase in antibody titers, spleen cells and iliac lymph node cells were fused with myeloma cells SP2 / O by electrofusion. Cells that were not subjected to fusion were frozen and stored. The fused cells were cultured in 96-well plates, and the culture supernatant was collected 7 days after fusion and screened. The medium was changed the day before screening.

[0043] 2. Screening We attempted to screen for antibodies that react with LPS from sepsis-causing bacteria by liquid-phase ELISA using LPS from Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa, which cause sepsis. The specific procedure for liquid-phase ELISA is as follows.

[0044] Anti-rat IgG or anti-rat IgM antibody solution diluted with PBS was dispensed into a 96-well ELISA plate (5 μg / mL, 50 μL / well) and left to stand at room temperature for 2 hours or at 4°C overnight. After washing three times (400 μL / well), blocking solution was dispensed (100 μL / well) and the plate was left to stand at room temperature for 1 hour or at 4°C overnight. After removing the blocking solution, antibody solutions diluted to various concentrations were dispensed (50 μL / well) and left to stand at room temperature for 1 hour. After washing three times (400 μL / well), LPS of various concentrations biotin-labeled with EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) was dispensed (50 μL / well) and left to stand at room temperature for 1 hour. After washing three times (400 μL / well), HRP-labeled streptavidin was dispensed (0.2 μg / mL, 50 μL / well) and allowed to stand at room temperature for 30 minutes. After washing three times (400 μL / well), OPD coloring solution was dispensed (50 μL / well) and allowed to react at room temperature for 10 minutes. The reaction was stopped by dispensing stop solution (50 μL / well). The absorbance at 492 nm was measured using a plate reader.

[0045] As a result of liquid-phase ELISA, we successfully established one antibody strain (IgM type: S28201R) that reacts with LPS derived from Klebsiella pneumoniae, which was not included in the immunogen and did not show an insufficient increase in titer, and one antibody strain (IgM type: S28203R) that reacts with LPS derived from Escherichia coli.

[0046] [Example 1] Evaluation of the sensitivity of the experimental system using the monoclonal antibody obtained in Test Example 1 We evaluated whether a sandwich system could be constructed using the same antibody for S28201R and S28203R by sandwich ELISA. The specific procedure for sandwich ELISA is as follows.

[0047] Antibody solution (S28201R or S28203R) diluted with PBS was dispensed into a 96-well ELISA plate (5 μg / mL, 50 μL / well) and left to stand at room temperature for 2 hours or at 4°C overnight. The same procedure was also performed using a rat IgM monoclonal antibody as a control. After washing three times (400 μL / well), blocking solution was dispensed (100 μL / well) and the plate was left to stand at room temperature for 1 hour or at 4°C overnight. After removing the blocking solution, LPS diluted to each concentration was dispensed (50 μL / well) and left to stand at room temperature for 1 hour. After washing three times (400 μL / well), various concentrations of antibody (S28201R or S28203R) biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) were dispensed (50 μL / well) and allowed to stand at room temperature for 1 hour. After washing three times (400 μL / well), HRP-labeled streptavidin was dispensed (0.2 μg / mL, 50 μL / well) and allowed to stand at room temperature for 30 minutes. After washing three times (400 μL / well), OPD coloring solution was dispensed (50 μL / well) and allowed to react at room temperature for 10 minutes. The reaction was stopped by dispensing stop solution (50 μL / well). The absorbance at 492 nm was measured using a plate reader.

[0048] The results are shown in Tables 1 and 2 and Figures 2 and 3. The results demonstrated that sandwich ELISA systems could be constructed. The S28201R sandwich ELISA system was capable of detecting antigens at concentrations up to approximately 2 ng / mL (labeled value). The S28203R sandwich ELISA system was capable of detecting antigens at concentrations up to approximately 30 ng / mL (labeled value).

[0049] [Table 1]

[0050] [Table 2]

[0051] [Reference Example 1] Comparison of sensitivity with Limulus reagent The sensitivity of the Limulus reagent (product name: Limulus ES-J Test Wako, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was measured using LPS derived from Klebsiella pneumoniae and LPS derived from Escherichia coli, and the sensitivity was compared with that of the sandwich system constructed in Example 1. The experimental procedure for the Limulus reagent was performed according to the protocol described in the attached instructions. LPS derived from Klebsiella pneumoniae (Sigma-Aldrich) and LPS derived from Escherichia coli (Fujifilm Wako Pure Chemical Industries, Ltd.) were serially diluted, and each concentration was tested for detectability using the Limulus reagent.

[0052] The results are shown in Tables 3 and 4. With the Limulus reagent, Klebsiella pneumoniae LPS became undetectable at 125 ng / mL (labeled value). Therefore, the sandwich system constructed in Example 1 was shown to have higher sensitivity than the Limulus reagent in analyzing Klebsiella pneumoniae LPS. Furthermore, the Limulus reagent was unable to detect E. coli LPS at 100 ng / mL (labeled value). Therefore, the sandwich system constructed in Example 1 was shown to have higher sensitivity than the Limulus reagent in analyzing E. coli LPS.

[0053] [Table 3]

[0054] [Table 4]

[0055] [Example 2] Calculation of kit sensitivity 2-1 Pricing of commercially available LPS solutions 2-1-1 Creating a calibration curve Commercially available LPS (5 mg / mL (labeled value)) was diluted with PBS to various concentrations (400, 300, 200 ng / mL). The diluted LPS was measured using a Limulus HS-T Single Test Wako according to the manufacturer's instructions. Using the commercially available LPS concentrations based on labeling values and the values measured using the Limulus HS-T Single Test Wako, we created an approximate quadratic function. The results are shown in Tables 5 and 6 and Figures 4 and 5.

[0056] [Table 5]

[0057] [Table 6]

[0058] As a result, the approximate formula for the quadratic function was as follows: ·Anti-Klebsiella pneumoniae LPS antibody (S28201R): y=6×10 -5 x 2 +0.0057x ·Anti-E. coli LPS antibody (S28203R):y=0.0001x 2 +0.0126x

[0059] 2-1-2 Calculation method for the concentration of commercially available LPS solutions The indicated concentration of commercially available LPS was substituted for x in the above-mentioned calibration curve formula to calculate the endotoxin concentration. Example: When converting 100 ng / mL of Klebsiella pneumoniae LPS into a concentration, applying x = 100 results in y = 1.17. Therefore, the amount of endotoxin in 100 ng / mL of Klebsiella pneumoniae LPS is 1.17 pg / mL.

[0060] 2-2 Minimum detection limit evaluation Measurements were performed for samples of each LPS concentration (n=8) according to the procedure for evaluating sensitivity described in Example 1. The mean value and 2.6 SD (standard deviation × 2.6) were calculated from the results of the eight measurements. The minimum detection limit was calculated using the 2.6 SD method. Specifically, if the sensitivity of 0 pg / mL + 2.6 SD was less than the sensitivity of each sample - 2.6 SD, the LPS concentration was determined to be detectable, and the lowest LPS concentration that satisfied this condition was defined as the minimum detection limit. The results are shown in Tables 7 and 8 and Figures 6 and 7.

[0061] [Table 7]

[0062] [Table 8] As a result, it was found that anti-Klebsiella pneumoniae LPS antibody (S28201R) could be detected down to a concentration of 0.011 pg / mL, and anti-Escherichia coli LPS antibody (S28203R) could be detected down to a concentration of 0.22 pg / mL.

[0063] [Example 3] Measurement of LPS in biological samples The ability of the prepared kit to detect LPS present in biological samples was examined, and the minimum detection limit was calculated. The same procedure as in Example 2 was followed, except that commercially available LPS was diluted with serum from healthy individuals. The same samples were also assayed using the Limulus HS-T Single Test Wako to confirm whether LPS could be detected. The results are shown in Tables 9 and 10 and Figures 8 and 9.

[0064] [Table 9]

[0065] [Table 10]

[0066] Tables 9 and 10 and Figures 8 and 9 show that the developed kit can detect LPS in biological samples with the same sensitivity as LPS in PBS. Furthermore, the anti-Klebsiella pneumoniae LPS antibody (S28201R) could detect LPS in biological samples down to a concentration of 0.023 pg / mL, more than 200 times more sensitive than the Limulus reagent. The anti-Escherichia coli LPS antibody (S28203R) could detect LPS down to a concentration of 0.10 pg / mL, 94 times more sensitive than the Limulus reagent.

[0067] [Reference Example 2] Sensitivity comparison with commercially available anti-LPS antibodies The antibody solution and biotin-labeled antibody used in the sandwich ELISA in Example 1 were replaced with a commercially available anti-LPS antibody (anti-lipopolysaccharide, mouse IgG2a, recombinant monoclonal antibody, clone WN1 222-5; Absolute Antibody), and the minimum detection limit for detecting E. coli LPS in the serum of healthy subjects was calculated in the same manner as in Example 3. The results are shown in Table 11.

[0068] [Table 11] Table 11 shows that the kit prepared in this example is capable of detecting E. coli LPS with a sensitivity at least five times higher than that of ELISA using a commercially available anti-LPS antibody.

[0069] [Reference Example 3] Sensitivity comparison with commercially available LPS detection ELISA kits Using a commercially available LPS detection ELISA kit (Qualitative Human Klebsiella (KBSL) ELISA Kit), the OD at 450 nm of healthy human serum samples spiked with Klebsiella pneumoniae LPS was measured according to the manual. Measurements were performed in triplicate for each LPS concentration. The positive and negative controls included with the kit were also measured in the same manner. The OD for the positive control was 1.14, and the OD for the negative control was 0.049. The mean and 2.6 SD (standard deviation × 2.6) were calculated from the results of triplicate measurements of the samples. The minimum detection limit was calculated using the 2.6SD method. Specifically, if "OD of 0 pg / mL + 2.6SD" was less than "OD of each sample - 2.6SD," that concentration of LPS was deemed detectable. The minimum detection limit was determined as the lowest LPS concentration at which all samples above a certain concentration satisfied this condition. The results are shown in Table 12.

[0070] [Table 12]

[0071] Table 12 shows that the kit prepared in this example can detect Klebsiella pneumoniae LPS with a sensitivity more than 2000 times higher than that of commercially available LPS detection ELISA kits. Furthermore, when adopting the standard described in the kit manual, in which a positive result is determined to be a sensitivity of 0.15 or higher than the OD of the negative control, the samples measured in this example were determined to be negative, i.e., undetectable, at all concentrations. Therefore, even when the determination method was changed, the kit prepared in this example was found to be able to detect Klebsiella pneumoniae LPS with a higher sensitivity. [Industrial Applicability]

[0072] According to the present invention, it is possible to provide an immunological analysis kit for sepsis-causing bacteria that has the same reactivity as a Limulus reagent and exhibits superior sensitivity to a Limulus reagent. [Accession number]

[0073] [Reference to deposited biological material] (1) Hybridoma producing antibody number S28201R a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material in a depository institution in the Republic of Ireland July 3, 2020 The accession number assigned to the deposit by the depository institution NITE BP-03241 (2) Hybridoma producing antibody number S28203R a) The name and address of the depository institution that deposited the biological material National Institute of Technology and Evaluation 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material in a depository institution in the Republic of Ireland July 3, 2020 The accession number assigned to the deposit by the depository institution NITE BP-03242

Claims

1. 1. An immunological assay kit for detecting sepsis-causing bacteria in a biological sample, comprising: It contains a monoclonal antibody that reacts with lipopolysaccharide derived from sepsis-causing bacteria, The monoclonal antibodies include a first monoclonal antibody and a second monoclonal antibody; a solid phase to which the first monoclonal antibody is immobilized and a labeling substance to which the second monoclonal antibody is bound, or a solid phase on which the first monoclonal antibody is immobilized, the second monoclonal antibody, and a secondary antibody against the second monoclonal antibody bound to a labeling substance; the first monoclonal antibody and the second monoclonal antibody are monoclonal antibodies of the same type; The immunological analysis kit as described above, wherein the monoclonal antibody detects sepsis-causing bacteria at 0.023 pg / mL or more in a biological sample.

2. 2. The immunological analysis kit according to claim 1, wherein the biological sample contains 0.023 pg / mL or more of lipopolysaccharide derived from a sepsis-causing bacterium.

3. 3. The immunological analysis kit according to claim 1, wherein the sepsis-causing bacterium is Klebsiella pneumoniae or Escherichia coli.

4. 4. The immunological analysis kit according to claim 1, wherein the monoclonal antibody specifically reacts with lipopolysaccharide derived from a sepsis-causing bacterium.

5. The immunological analysis kit according to any one of claims 1 to 4, wherein the biological sample is blood, plasma, or serum.

6. The immunological analysis kit according to any one of claims 1 to 5, wherein the measurement principle is ELISA.

7. The immunological analysis kit according to any one of claims 1 to 6, wherein the monoclonal antibody is an IgM antibody.

8. The immunological analysis kit according to any one of claims 1 to 7, wherein the monoclonal antibody is a monoclonal antibody produced by a hybridoma having accession number NITE BP-03241 or a monoclonal antibody produced by a hybridoma having accession number NITE BP-03242.

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