Immunological analysis method for bacteria causing sepsis and monoclonal antibodies used in said method

Abstract

The present invention addresses the problem of providing a method for immunologically analyzing sepsis-causing bacteria by which sepsis-causing bacteria can be identified using a simple operation.　This problem can be solved by a method for immunologically analyzing sepsis-causing bacteria in a biological specimen, the method including a step for bringing the biological specimen into contact with at least one monoclonal antibody from among a monoclonal antibody that reacts with a Klebsiella pneumoniae-derived lipopolysaccharide but does not react with Pseudomonas aeruginosa-derived lipopolysaccharide or an Escherichia coli-derived lipopolysaccharide, and a monoclonal antibody that reacts with an Escherichia coli-derived lipopolysaccharide but does not react with a Klebsiella pneumoniae-derived lipopolysaccharide or a Pseudomonas aeruginosa-derived lipopolysaccharide, wherein the sepsis-causing bacteria is either Klebsiella pneumoniae or Escherichia coli.

Description

[Technical Field]

[0001] The present invention relates to a method for immunologically analyzing sepsis-causing bacteria and a monoclonal antibody used in said method. [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 to measure LPS (lipopolysaccharides), which constitute 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. In sepsis, early identification of the causative bacteria is necessary to determine treatment strategies. However, because Limulus reagent simultaneously analyzes the LPS of many types of Gram-negative bacteria, a problem exists in that a separate protocol is required to identify the bacterial species. Cultivation methods using various media have been used to identify the causative bacteria of sepsis, but the testing process, including the culturing, requires several days. Furthermore, the method using Limulus reagent requires horseshoe crab blood, a natural resource, which raises concerns about resource depletion and is costly to maintain consistent quality. Another drawback of this method is that it is a manual method requiring multiple steps, which can lead to variability. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 04-136763 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an immunological analysis method for sepsis-causing bacteria, which can identify sepsis-causing bacteria with a simple procedure. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and have produced a monoclonal antibody that specifically recognizes lipopolysaccharide derived from Klebsiella pneumoniae and a monoclonal antibody that specifically recognizes lipopolysaccharide derived from Escherichia coli, thereby completing the present invention. Specifically, the present invention is as follows. <1> A method for immunologically analyzing sepsis-causing bacteria in a biological sample, comprising: 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; and a monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or Klebsiella pneumoniae; and contacting a biological sample with at least one monoclonal antibody selected from the group consisting of the above, wherein the sepsis-causing bacterium is Klebsiella pneumoniae or Escherichia coli. <2> the monoclonal antibody reacts with lipopolysaccharide derived from Klebsiella pneumoniae but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Escherichia coli, and the cross-reactivity with lipopolysaccharide derived from sepsis-causing bacteria other than Klebsiella pneumoniae is less than 10%; <1> The immunoassay analysis method according to claim 1. <3> the monoclonal antibody reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae, and the cross-reactivity with lipopolysaccharide derived from sepsis-causing bacteria other than Escherichia coli is less than 10%; <1> or <2> The immunoassay analysis method according to claim 1. <4> As the monoclonal antibody, a solid-phase antibody immobilized on an insoluble carrier and a labeled antibody bound to a labeling substance are used, and the solid-phase antibody and the labeled antibody are the same monoclonal antibody. <1> ~ <3> The immunological measurement method according to any one of the above. <5> the 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 is an IgM antibody; <1> ~ <4> The immunological analysis method according to any one of the above. <6> the 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 is a monoclonal antibody produced by a hybridoma with accession number NITE BP-03241; <1> ~ <5> The immunological analysis method according to any one of the above. <7> the monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae is an IgM antibody; <1> ~ <6> The immunological analysis method according to any one of the above. <8> the monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae is a monoclonal antibody produced by a hybridoma with accession number NITE BP-03242; <1> ~ <7> The immunological analysis method according to any one of the above. <9> The biological sample is blood, plasma, or serum. <1> ~ <8> The immunological analysis method according to any one of the above. <10> A monoclonal antibody that reacts with lipopolysaccharide derived from Klebsiella pneumoniae, but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or Escherichia coli. <11> The cross-reactivity to lipopolysaccharides derived from sepsis-causing bacteria other than Klebsiella pneumoniae is 10% or less. <10> A monoclonal antibody according to claim 1. <12> Produced by the hybridoma with accession number NITE BP-03241, <10> A monoclonal antibody according to claim 1. <13> A monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli, but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or Klebsiella pneumoniae. <14> The cross-reactivity to lipopolysaccharides derived from sepsis-causing bacteria other than E. coli is 10% or less. <13> A monoclonal antibody according to claim 1. <15> Produced by the hybridoma with accession number NITE BP-03242, <13> A monoclonal antibody according to claim 1. <16> measuring a signal derived from the labeling substance; comparing the measured value of the signal with a cutoff value; further comprising: <1> ~ <9> The immunological analysis method according to any one of [Effects of the Invention]

[0007] According to the present invention, it is possible to provide an immunological analysis method for sepsis-causing bacteria, which can identify sepsis-causing bacteria with a simple procedure. [Brief explanation of the drawings]

[0008] [Figure 1] 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 a test was conducted to determine whether a sandwich system can be established 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. DETAILED DESCRIPTION OF THE INVENTION

[0009] [1] Immunological analysis method for sepsis-causing bacteria in biological samples (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.

[0010] (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. The immunological analysis method of the present invention can determine not only the presence of sepsis but also that the cause is Klebsiella pneumoniae, Escherichia coli, or both. As used herein, the term "sepsis" includes both sepsis and septic shock. Sepsis refers to a condition in which severe organ damage is caused by an infection. Septic shock refers to a condition in which acute circulatory failure leads to severe cell damage and metabolic abnormalities, potentially increasing mortality. 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 advantage of being able to not only determine the presence or absence of sepsis but also identify the causative bacteria of sepsis (Klebsiella pneumoniae or Escherichia coli) with sensitivity and reactivity equivalent to or greater than that of the Limulus reagent. In other words, the present invention has the advantage of being able to replace the Limulus reagent currently used in clinical settings and to identify the causative bacteria of sepsis.

[0011] (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 present inventors performed an analytical experiment using the Limulus reagent to analyze lipopolysaccharides derived from various sepsis-causing bacteria, and found that it reacted with lipopolysaccharides derived from all of the sepsis-causing bacteria tested (Table 1). Herein, lipopolysaccharide derived from Klebsiella pneumoniae or lipopolysaccharide derived from Escherichia coli may be collectively referred to as "lipopolysaccharide derived from a specific sepsis-causing bacterium." Similarly, herein, Klebsiella pneumoniae or Escherichia coli may be collectively referred to as "specific sepsis-causing bacterium." The monoclonal antibody used in the present invention can recognize both lipopolysaccharide that has not been detached from the cell wall of a specific sepsis-causing bacterium and lipopolysaccharide that has been detached from the cell wall of a specific 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 antibody. The ability to form a sandwich system with a single antibody facilitates the construction of an experimental system. As used herein, "one type" or "the same type" in reference to a monoclonal antibody refers to monoclonal antibodies that recognize the same epitope. Furthermore, the ability to form a sandwich system with a single 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, a "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 conjugated to a label, and the analyte 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.

[0012] (monoclonal antibody) The monoclonal antibody used in the present invention is a monoclonal antibody that reacts with lipopolysaccharide derived from Klebsiella pneumoniae but does not react with either lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Escherichia coli, and / or a monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with either lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae. In this specification, a monoclonal antibody that reacts with lipopolysaccharide derived from Klebsiella pneumoniae but not with lipopolysaccharide derived from Pseudomonas aeruginosa or Escherichia coli may be referred to as a monoclonal antibody that specifically reacts with lipopolysaccharide derived from Klebsiella pneumoniae. Similarly, a monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but not with lipopolysaccharide derived from Pseudomonas aeruginosa or Klebsiella pneumoniae may be referred to as a monoclonal antibody that specifically reacts with lipopolysaccharide derived from Escherichia coli. Examples of monoclonal antibodies that specifically react with lipopolysaccharide derived from Klebsiella pneumoniae include the S28201R antibody produced by the hybridoma with accession number NITE BP-03241. Examples of monoclonal antibodies that specifically react with lipopolysaccharide derived from Escherichia coli include the S28203R antibody produced by the hybridoma with accession number NITE BP-03242.

[0013] As used herein, the terms "reacting with" lipopolysaccharide derived from a specific sepsis-causing bacterium, "recognizing" lipopolysaccharide derived from a specific sepsis-causing bacterium, and "binding to" lipopolysaccharide derived from a specific sepsis-causing bacterium are used synonymously, but are not limited to these examples and should be interpreted in the broadest sense. Whether a monoclonal 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).

[0014] The monoclonal antibody used in the present invention "does not react" with a certain compound means that the monoclonal antibody used in the present invention does not substantially react with a certain compound. To confirm whether or not a certain compound "does not substantially react," for example, the monoclonal 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. "Does not substantially react" can also be confirmed by methods or means known to those skilled in the art other than the above-mentioned SPR method. The phrase "reacts with lipopolysaccharide derived from Klebsiella pneumoniae but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Escherichia coli" preferably means that, in the coexistence of lipopolysaccharide derived from Klebsiella pneumoniae with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Escherichia coli, when the reactivity with lipopolysaccharide derived from Klebsiella pneumoniae is taken as 1, the reactivity with both lipopolysaccharide derived from Pseudomonas aeruginosa and lipopolysaccharide derived from Escherichia coli is less than 0.1. The phrase "reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae" preferably means that, in the coexistence of lipopolysaccharide derived from Escherichia coli, lipopolysaccharide derived from Pseudomonas aeruginosa, and lipopolysaccharide derived from Klebsiella pneumoniae, when the reactivity with lipopolysaccharide derived from Klebsiella pneumoniae is set to 1, the reactivity with both lipopolysaccharide derived from Pseudomonas aeruginosa and lipopolysaccharide derived from Klebsiella pneumoniae is less than 0.1. As used herein, the cross-reactivity refers to the percentage of decrease in absorbance when free "bacterial LPS with which the antibody does not specifically react" is added in competition with bacterial LPS with which the antibody specifically reacts, assuming the decrease in reactivity when free "bacterial LPS with which the antibody specifically reacts" is added as a competition of 100. In this case, the free LPS is added at a concentration at least that reduces the reactivity of the antibody with the solid-phase LPS by 75% or more when added in competition with bacterial LPS with which the antibody specifically reacts.

[0015] The monoclonal antibodies used in the immunological analysis methods 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.

[0016] The monoclonal antibodies used in the immunological analysis method of the present invention can be produced by dissolving heat-killed bacteria derived from Klebsiella pneumoniae, Pseudomonas aeruginosa, and / or Escherichia coli as antigens (immunogens) in a solvent such as phosphate-buffered saline, and administering the resulting solution to animals 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.

[0017] 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.

[0018] 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 method 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.).

[0019] 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.

[0020] Hybridoma selection can be performed efficiently at the selection stage, taking into consideration the conditions used in the actual measurement of the produced antibodies. For example, antibodies obtained by immunizing an animal are reacted with lipopolysaccharide derived from a specific 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 specific 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.

[0021] After the cloning step, the binding ability of the produced antibody to lipopolysaccharide derived from a specific sepsis-causing bacterium can be assayed using methods such as ELISA, RIA, or 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.

[0022] In addition to whole antibody molecules, antibody fragments having antigen-antibody reaction activity can also be used as antibodies in the immunological analysis method of the present invention. In addition to those obtained through the animal immunization process described above, those obtained using genetic recombination techniques or chimeric antibodies can 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.

[0023] (solid phase antibody) In the immunological analysis method of the present invention, the monoclonal antibody can be used as a solid-phase antibody immobilized on an insoluble carrier. Alternatively, in the immunological analysis method of the present invention, the monoclonal 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) the 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).

[0024] (labeled antibody) By using a label capable of binding to the monoclonal antibody used in the immunological analysis method of the present invention, the amount of antibody bound to lipopolysaccharide derived from a specific sepsis-causing bacterium can be measured. This allows detection of lipopolysaccharide derived from a specific sepsis-causing bacterium in a biological sample. Examples of labeling substances used to produce labeled antibodies include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, radioisotopes, gold colloid particles, and colored latex. Methods for binding the label to the antibody include those readily available to those skilled in the art, such as the glutaraldehyde method, maleimide method, pyridyl disulfide method, and periodic acid method. The types of solid-phase antibodies and labeled antibodies, and their production methods, are not limited to the examples of conjugation methods described above. For example, when enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) are used as labels, 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 label, avidin or enzyme-modified avidin is generally reacted. In the immunological analysis method of the present invention, biotin or HRP is preferably used as a label, and biotin is more preferably used. When biotin is used, streptavidin labeled with HRP can also be used.

[0025] As used herein, the term "insoluble carrier" refers to a substance onto which an antibody or the like that specifically recognizes the analyte 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 antibody on an insoluble carrier, or the state of being immobilized, is sometimes referred to as "fixation," "immobilization," or "solid-phased." Furthermore, the terms "analysis," "detection," and "measurement" include the meaning of verifying the presence and / or quantification of lipopolysaccharide derived from a specific sepsis-causing bacterium.

[0026] (Immunological analysis method) The immunological analysis method of the present invention includes, but is not limited to, 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 method of the present invention is preferably electrochemiluminescence immunoassay (ECL), high-performance liquid chromatography (HPLC), or enzyme-linked immunosorbent assay (ELISA), and more preferably sandwich ELISA.

[0027] In the step of contacting the monoclonal antibody with the biological sample, 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.

[0028] A monoclonal antibody that specifically reacts with lipopolysaccharide derived from Klebsiella pneumoniae and / or a monoclonal antibody that specifically reacts with lipopolysaccharide derived from Escherichia coli can be immobilized on an insoluble carrier. When both monoclonal antibodies are immobilized on an insoluble carrier, after adding a biological sample to the insoluble carrier, a monoclonal antibody that specifically reacts with Klebsiella pneumoniae-derived lipopolysaccharide and a monoclonal antibody that specifically reacts with Escherichia coli-derived lipopolysaccharide can be further added to the insoluble carrier as detection antibodies. In this case, it is preferable to use different labels for detecting Klebsiella pneumoniae-derived lipopolysaccharide and E. coli-derived lipopolysaccharide. When both monoclonal antibodies are used, it is possible to rapidly determine whether only lipopolysaccharide derived from Klebsiella pneumoniae is present, only lipopolysaccharide derived from Escherichia coli is present, or both lipopolysaccharide derived from Klebsiella pneumoniae and lipopolysaccharide derived from Escherichia coli are present.

[0029] 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 method of the present invention, both the first and second monoclonal antibodies can be monoclonal antibodies that recognize the same epitope or the same monoclonal antibodies produced by the same hybridoma. The measurement principle when magnetic particles are used as insoluble carrier particles and a ruthenium complex is used as a labeling substance is as follows: The following shows the measurement principle in one embodiment of the present invention, and does not limit the scope of the present invention in any way. 1. When a biological sample is brought into contact with magnetic particles on which solid-phase antibodies have been immobilized, lipopolysaccharides derived from specific sepsis-causing bacteria in the biological sample bind to the solid-phase antibodies. 2. After washing the magnetic particles, the labeled antibody is brought into contact with them, and the labeled antibody binds to the lipopolysaccharide derived from a specific sepsis-causing bacterium that is bound to the magnetic particles. 3. After washing the magnetic particles, when an electric current is applied, they emit light according to the amount of labeled antibody bound to the lipopolysaccharide derived from the specific sepsis-causing bacteria. By measuring the amount of this light, the amount of lipopolysaccharide derived from the specific sepsis-causing bacteria in the biological sample can be accurately measured.

[0030] Among immunological analysis methods, enzyme-linked immunosorbent assays (ELISAs) are also preferred because they allow simple and rapid measurement of targets. 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. In the case of sandwich ELISA, an insoluble carrier on 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 can be used. The insoluble carrier is preferably a plate (immunoplate). HRP or ALP can be used as the label. Typically, an enzyme-linked immunosorbent assay (ELISA) can be performed by using a first antibody as a solid-phase antibody and a second antibody that recognizes an epitope different from that of the first antibody as a labeled antibody. However, in the immunological analysis method of the present invention, antibodies that recognize the same epitope can be used as both the first antibody (solid-phase antibody) and the second antibody (labeled antibody). The measurement procedure and principle when sandwich ELISA is used as the immunological analysis method of the present invention are as follows: The following shows the measurement principle in one embodiment of the present invention, and does not limit the scope of the present invention in any way. 1. When a biological sample is added to a carrier with immobilized solid-phase antibodies and allowed to react, lipopolysaccharides derived from specific sepsis-causing bacteria in the biological sample bind to the solid-phase antibodies, forming solid-phase antibody-lipopolysaccharide complexes on the carrier. 2. When a labeled antibody that recognizes a different epitope is added to the carrier and allowed to react, the labeled antibody binds to the captured lipopolysaccharide and forms a sandwich with the solid-phase antibody-lipopolysaccharide complex. 3. After washing, the sample reacts with the enzyme substrate to develop color, and the absorbance is measured. The amount of lipopolysaccharide in the biological sample can be measured according to the amount of labeled substance measured. Specific methods for immobilizing an antibody on an insoluble carrier, binding an antibody to a labeling substance, etc., are well known to those skilled in the art and can be used without any particular limitations. It is preferable to use biotin as the labeling substance, and when biotin is used, streptavidin labeled with HRP can also be used.

[0031] A secondary antibody can also be used in sandwich ELISA. The use of a secondary antibody amplifies the reaction, increasing detection sensitivity. When using a secondary antibody, the primary antibody is a first monoclonal antibody, a solid phase immobilized with a second monoclonal antibody, an antibody against the first monoclonal antibody (secondary antibody) labeled with a label (e.g., HRP or ALP), and a substrate for the label (e.g., OPD, TMB, or p-nitrophenyl phosphate). In this analytical method, a diluted biological sample is first added to the solid phase immobilized with the second monoclonal antibody, followed by incubation, removal of the biological sample, and washing. The primary antibody is then added, incubated, and washed. An enzyme-labeled secondary antibody is then added and incubated. A substrate is then added to develop color. The resulting color can then be measured using a plate reader or other device to analyze lipopolysaccharides derived from specific sepsis-causing bacteria.

[0032] (Sepsis diagnosis, diagnostic aids, and treatment) Based on the analysis results of the immunological analysis method of the present invention, it is possible to diagnose whether a subject has sepsis caused by a specific sepsis-causing bacterium, or the results can serve as an aid in diagnosis. In analytical methods using a Limulus reagent, identification of the bacterial species required a separate protocol. However, the immunological analysis method of the present invention can determine which bacterium caused sepsis, thereby enabling early identification of the sepsis-causing bacterium. Furthermore, after carrying out the immunological analysis method of the present invention, the patient may be subjected to another analysis method for sepsis-causing bacteria and / or a therapeutic drug for sepsis may be administered to the patient. When a monoclonal antibody that reacts with lipopolysaccharide derived from Klebsiella pneumoniae but does not react with lipopolysaccharide derived from either Pseudomonas aeruginosa or Escherichia coli is used, the immunological analysis method of the present invention can comprise the following steps (A) and / or (B): (A) detecting lipopolysaccharide derived from Klebsiella pneumoniae at a concentration of 0.011 pg / mL or more, 0.011 pg / mL to 10 μg / mL, or 0.011 pg / mL to 1 μg / mL in a biological sample, preferably blood, serum, or plasma; and / or (B) A step of diagnosing, or assisting in the diagnosis of, that the subject from whom the biological sample was collected is suffering from or suspected of suffering from sepsis based on the results of the detection step. In the above step (A), the amount of lipopolysaccharide derived from Klebsiella pneumoniae in the biological sample, preferably in blood, serum, or plasma, can be 0.011 pg / mL to 10 μg / mL, 0.023 pg / mL to 10 μg / mL, 0.1 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.

[0033] When a monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from either Pseudomonas aeruginosa or Klebsiella pneumoniae is used, the immunological analysis method of the present invention can comprise the following steps (A) and / or (B): (A) detecting lipopolysaccharide derived from Escherichia coli at a concentration of 0.22 pg / mL or more, 0.22 pg / mL to 10 μg / mL, or 0.22 pg / mL to 1 μg / mL in a biological sample, preferably blood, serum, or plasma; and / or (B) The method may include a step of diagnosing, or assisting in the diagnosis of, that the subject from whom the biological sample was collected is suffering from or suspected of suffering from sepsis based on the results of the detection step. The cutoff value can be set appropriately depending on the type of biological sample or the type of immunological analysis method. The immunological analysis method of the present invention can include a step of comparing the measured value with the cutoff value. If the measured value is lower than the cutoff value, it can be determined that the subject does not suffer from sepsis caused by a specific sepsis-causing bacterium. If the measured value is higher than the cutoff value, it can be determined that the subject suffers from sepsis caused by a specific sepsis-causing bacterium. In the above step (A), the amount of lipopolysaccharide derived from Escherichia coli in the biological sample, preferably in blood, serum or plasma, can be 0.22 pg / mL to 10 μg / mL, 0.49 pg / mL to 10 μg / mL, 1 pg / mL to 10 μg / mL, or 5 pg / mL to 10 μg / mL.

[0034] 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]

[0035] [Reference Example 1] Analysis of lipopolysaccharides derived from various sepsis-causing bacteria using Limulus reagent Lipopolysaccharides derived from various sepsis-causing bacteria were analyzed using Limulus reagent (product name: Endotoxin-Single Test Wako, Fujifilm Wako Pure Chemical Industries, Ltd.). The Limulus reagent experimental procedure was performed according to the protocol described in the attached instructions. LPS derived from Klebsiella pneumoniae (Sigma-Aldrich), LPS derived from Pseudomonas aeruginosa (Fujifilm Wako Pure Chemical Industries, Ltd.), and LPS derived from Escherichia coli (Fujifilm Wako Pure Chemical Industries, Ltd.) were prepared to 1000 ng / mL and tested for detectability using the Limulus reagent.

[0036] The results are shown in Table 1. The Limulus reagent reacted with LPS derived from all bacteria, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli O111, and Escherichia coli O26. Therefore, it is difficult to identify the causative bacterial species of sepsis using the Limulus reagent. I understand. [Table 1]

[0037] [Example 1] Method for producing monoclonal antibodies used in the present invention 1. Obtaining Antibodies Rats (F344 / Jc1, female) were intraperitoneally immunized weekly 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:

[0038] 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.

[0039] 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.

[0040] 2. Screening We attempted to screen for antibodies that react with LPS from specific 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.

[0041] 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 3 times (400μL / well), EZ-Link TM Each LPS labeled with biotin using Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) was dispensed (1 μg / mL, 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.

[0042] The reactivity of the antibodies obtained by screening with various LPS was evaluated by antigen solid-phase ELISA, and the results are shown in Tables 2 and 3. 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.

[0043] [Table 2]

[0044] [Table 3]

[0045] [Example 2] Evaluation of the sensitivity of the experimental system using the monoclonal antibody obtained in 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.

[0046] A solution of S28201R or R28203R antibody 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. As a control, the same procedure was performed using a rat IgM monoclonal antibody. 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 (indicated value) was 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.

[0047] The results are shown in Tables 4 and 5 and Figures 2 and 3. The results demonstrated that a sandwich ELISA system could be constructed (Figures 2 and 3). 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).

[0048] [Table 4]

[0049] [Table 5]

[0050] [Example 3] Comparison of sensitivity with Limulus reagent The sensitivity of the Limulus reagent (product name "Endotoxin-Single 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 compared with the sensitivity of the sandwich system constructed in Example 2. The experimental procedure for the Limulus reagent was performed according to the protocol described in the attached instruction manual. 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.

[0051] The results are shown in Tables 6 and 7. With the Limulus reagent, Klebsiella pneumoniae LPS became undetectable at 125 ng / mL (labeled value). Therefore, the sandwich system constructed in Example 2 was shown to be more than 60 times more sensitive 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 2 was shown to be more than three times more sensitive than the Limulus reagent in analyzing E. coli LPS.

[0052] [Table 6]

[0053] [Table 7]

[0054] [Example 4] Calculation of the sensitivity of the analytical method of the present invention 4-1. Evaluation of commercially available LPS solutions using Limulus reagent 4-1-1 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 8 and 9 and Figures 4 and 5.

[0055] [Table 8]

[0056] [Table 9]

[0057] As a result, the approximate formula for the quadratic function was as follows: S28201R:y=6×10 -5 x 2 +0.0057x S28203R:y=0.0001x 2 +0.0126x

[0058] 4-1-2 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.

[0059] 4-2 Minimum detection limit evaluation Measurements were performed (n=8) on samples prepared by adding LPS to serum from healthy subjects at various concentrations, following the procedure for evaluating sensitivity described in Example 3. 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, LPS was determined to be detectable when "sensitivity of 0 pg / mL + 2.6 SD" was less than "sensitivity of each sample - 2.6 SD." The minimum detection limit was determined as the lowest LPS concentration that satisfied this condition. The results are shown in Tables 10 and 11 and Figures 6 and 7.

[0060] [Table 10]

[0061] [Table 11]

[0062] As a result, it was found that S28201R could be detected down to a concentration of 0.011 pg / mL, and S28203R could be detected down to a concentration of 0.22 pg / mL.

[0063] [Example 5] Measurement of crossover rate Competitive ELISA was used to confirm whether S28201R and S28203R cross-react with LPS derived from bacterial cells, with which they do not specifically react. The specific procedure for competitive ELISA is as follows.

[0064] Various LPS diluted with PBS were dispensed into a 96-well ELISA plate 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 solution diluted with diluent and various LPS 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), 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. The cross-reactivity of each antibody with bacterial LPS, which did not specifically react with each antibody, was calculated using the following method. The results of the competitive ELISA are shown in Tables 12 and 13, and the cross-reactivity calculated by the competitive ELISA is shown in Table 14. S28201R The change in absorbance in ELISA due to the addition of Pseudomonas aeruginosa LPS (absorbance at a Pseudomonas aeruginosa LPS concentration of 0 pg / mL minus the absorbance at a Pseudomonas aeruginosa LPS concentration of 1500 pg / mL) was divided by the change in absorbance in ELISA due to the addition of Klebsiella pneumoniae LPS (absorbance at a Klebsiella pneumoniae LPS concentration of 0 pg / mL minus the absorbance at a Klebsiella pneumoniae LPS concentration of 1500 pg / mL) and multiplied by 100 to obtain the cross-reactivity rate (%). However, if the calculated value was negative, the cross-reactivity rate was considered to be 0%. The cross-reactivity rate for E. coli LPS was calculated in the same way. ·S28203R The change in absorbance in ELISA due to the addition of Klebsiella pneumoniae LPS ((absorbance at a Klebsiella pneumoniae LPS concentration of 0 pg / mL) - (absorbance at a Klebsiella pneumoniae LPS concentration of 5000 pg / mL)) was divided by the change in absorbance in ELISA due to the addition of E. coli LPS ((absorbance at an E. coli LPS concentration of 0 pg / mL) - (absorbance at an E. coli LPS concentration of 5000 pg / mL)), and multiplied by 100 to obtain the cross-reactivity rate (%). However, if the calculated value was negative, the cross-reactivity rate was considered to be 0%. The cross-reactivity rate for Pseudomonas aeruginosa was calculated in the same way.

[0065] [Table 12]

[0066] [Table 13]

[0067] [Table 14]

[0068] Table 12 shows that the absorbance of S28201R reacted with Klebsiella pneumoniae LPS decreased depending on the concentration of free Klebsiella pneumoniae LPS in the reaction system. However, in the presence of Pseudomonas aeruginosa LPS or E. coli LPS, no change in absorbance was observed depending on the concentration of coexisting LPS. Table 14 also shows that the cross-reactivity of S28201R with Pseudomonas aeruginosa LPS or E. coli LPS was less than 10%. Table 13 also shows that the absorbance of S28203R reacted with E. coli LPS decreased depending on the concentration of free E. coli LPS in the reaction system. However, in the presence of Klebsiella pneumoniae LPS or Pseudomonas aeruginosa LPS, no change in absorbance was observed depending on the concentration of coexisting LPS. Table 14 also shows that the cross-reactivity of S28203R with Klebsiella pneumoniae LPS or Pseudomonas aeruginosa LPS was less than 10%.

[0069] [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 4 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 4. The results are shown in Table 15.

[0070] [Table 15] Table 15 shows that the analytical method carried out 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.

[0071] [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 was measured for healthy human serum samples spiked with Klebsiella pneumoniae LPS, 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 x 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 16.

[0072] [Table 16]

[0073] Table 16 shows that the analytical method of 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 analytical method of this example was found to be able to detect Klebsiella pneumoniae LPS with a higher sensitivity. [Industrial Applicability]

[0074] According to the present invention, it is possible to provide an immunological analysis method for sepsis-causing bacteria, which has sensitivity equal to or greater than that of a Limulus reagent and can identify sepsis-causing bacteria with a simple procedure. [Accession number]

[0075] [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. A method for immunologically analyzing sepsis-causing bacteria in a biological sample, comprising: 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; and a monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or Klebsiella pneumoniae; contacting a biological sample with at least one monoclonal antibody selected from the group consisting of the sepsis-causing bacterium is Klebsiella pneumoniae or Escherichia coli; detecting 0.023 pg / mL to 10 μg / mL of lipopolysaccharide derived from Klebsiella pneumoniae contained in the biological sample, and / or detecting 0.22 pg / mL to 10 μg / mL of lipopolysaccharide derived from Escherichia coli contained in the biological sample; Immunological analysis methods.

2. 2. The immunoassay analysis method according to claim 1, wherein the 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 has a cross-reactivity with lipopolysaccharide derived from sepsis-causing bacteria other than Klebsiella pneumoniae of less than 10%.

3. 3. The immunoassay analysis method according to claim 1, wherein the monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae has a cross-reactivity with lipopolysaccharide derived from sepsis-causing bacteria other than Escherichia coli of less than 10%.

4. The immunological measurement method according to any one of claims 1 to 3, wherein a solid-phase antibody immobilized on an insoluble carrier and a labeled antibody bound to a labeling substance are used as the monoclonal antibody, and the solid-phase antibody and the labeled antibody are the same monoclonal antibody.

5. The immunological analysis method according to any one of claims 1 to 4, wherein the 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 is an IgM antibody.

6. The immunological analysis method according to any one of claims 1 to 5, wherein the 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 is a monoclonal antibody produced by a hybridoma with accession number NITE BP-03241.

7. The immunological analysis method according to any one of claims 1 to 6, wherein the monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae is an IgM antibody.

8. The immunological analysis method according to any one of claims 1 to 7, wherein the monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or lipopolysaccharide derived from Klebsiella pneumoniae is a monoclonal antibody produced by a hybridoma with accession number NITE BP-03242.

9. The immunological analysis method according to any one of claims 1 to 8, wherein the biological sample is blood, plasma, or serum.

10. A monoclonal antibody that reacts with lipopolysaccharide derived from Klebsiella pneumoniae, but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or Escherichia coli.

11. The monoclonal antibody according to claim 10, which has a cross-reactivity to lipopolysaccharides derived from sepsis-causing bacteria other than Klebsiella pneumoniae of 10% or less.

12. The monoclonal antibody according to claim 10, which is produced by a hybridoma having accession number NITE BP-03241.

13. A monoclonal antibody that reacts with lipopolysaccharide derived from Escherichia coli, but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa or Klebsiella pneumoniae.

14. The monoclonal antibody according to claim 13, which has a cross-reactivity to lipopolysaccharides derived from sepsis-causing bacteria other than Escherichia coli of 10% or less.

15. The monoclonal antibody according to claim 13, which is produced by a hybridoma having accession number NITE BP-03242.

16. measuring a signal derived from the labeling substance; comparing the measured value of the signal with a cutoff value; The immunological analysis method according to claim 4, further comprising:

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