JP7722772B2 - Immunological analysis kit for sepsis-causing bacteria - Google Patents

Description

NITE BP-03241 NITE BP-03242

The present invention relates to an analytical kit for sepsis-causing bacteria.

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

A 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). The Limulus reagent is used to diagnose or assist in the diagnosis of sepsis. However, the method using the Limulus reagent requires horseshoe crab blood, a natural resource, which raises concerns about resource depletion, and maintaining consistent quality is costly. Another drawback of this method is that it is a manual method requiring multiple steps, which can lead to variations in results.

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.

Japanese Patent Application Publication No. 04-136763

Qualitative Human Klebsiella (KBSL) ELISA Kit: Cat. No.MBS9310934

The objective 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 Limulus reagent and can detect sepsis-causing bacteria with simple operations.

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 is capable of detecting lipopolysaccharide even at even lower concentrations.
Specifically, the present invention is as follows.
<1> An immunological analysis 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 immunological analysis kit according to <1>, wherein the biological sample contains 0.023 pg/mL or more of lipopolysaccharide derived from a sepsis-causing bacterium.
<3> The immunological analysis kit according to <1> or <2>, wherein the sepsis-causing bacterium is Klebsiella pneumoniae or Escherichia coli.
<4> The immunological analysis kit according to any one of <1> to <3>, wherein the monoclonal antibody is a monoclonal antibody that specifically reacts with lipopolysaccharide derived from a sepsis-causing bacterium.
<5> The monoclonal antibody includes a first monoclonal antibody and a second monoclonal antibody,
a secondary antibody against the second monoclonal antibody, which comprises a solid phase on which the first monoclonal antibody is immobilized and a labeled substance bound to the second monoclonal antibody, or a solid phase on which the first monoclonal antibody is immobilized and a labeled substance bound to the second monoclonal antibody;
Including,
The immunological analysis kit according to any one of <1> to <4>, wherein the first monoclonal antibody and the second monoclonal antibody are monoclonal antibodies of the same type.
<6> The immunological analysis kit according to any one of <1> to <5>, wherein the biological sample is blood, plasma, or serum.
<7> The immunological analysis kit according to any one of <1> to <6>, wherein the measurement principle is ELISA.
<8> The immunological analysis kit according to any one of <1> to <7>, wherein the monoclonal antibody is an IgM antibody.
<9> The immunological analysis kit according to any one of <1> to <8>, 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.

The present invention provides an immunological analysis kit for sepsis-causing bacteria that exhibits greater sensitivity than the Limulus reagent and can detect sepsis-causing bacteria with simple operations.

1 is a schematic diagram of the structure of lipopolysaccharide in sepsis-causing bacteria. 1 is a graph showing the results of sandwich ELISA in which it was tested whether a sandwich system can be established using only the S28201R antibody. 1 is a graph showing the results of sandwich ELISA in which it was tested whether a sandwich system can be established using only the S28203R antibody. 1 is a graph showing a calibration curve for LPS of Klebsiella pneumoniae. 1 is a graph showing a calibration curve for E. coli LPS. 1 is a graph for calculating the minimum detection limit of LPS of Klebsiella pneumoniae. 1 is a graph for calculating the minimum detection limit of E. coli LPS. 1 is a graph for calculating the minimum detection limit of LPS of Klebsiella pneumoniae in a biological sample. 1 is a graph for calculating the minimum detection limit of E. coli LPS in a biological sample.

(biological samples)
The "biological sample" of the present invention mainly includes solid tissues and body fluids derived from living organisms, with body fluids being preferred. 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. Living organisms or subjects include 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.

(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 rod-shaped bacterium 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.

(lipopolysaccharide)
As used herein, "lipopolysaccharide" refers to a complex of covalently bound lipids and polysaccharides. In this specification, "lipopolysaccharide" may also be 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 O antigens, which are polysaccharide chains, extend from lipid A via oligosaccharide regions 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" in reference to a monoclonal antibody refers to a monoclonal antibody that recognizes the same epitope. The ability to form a sandwich system with a single type facilitates the construction of an experimental system. Furthermore, the ability to form a sandwich system with a single type 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 conjugated 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 conjugated to the label or indirectly conjugated to the label via a secondary antibody.

(monoclonal antibodies)
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.

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 interchangeably, 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 using antigen-immobilized ELISA, competitive ELISA, sandwich ELISA, or other methods. It can also be performed using methods utilizing the principle of surface plasmon resonance (SPR). SPR can be performed using devices, sensors, and reagents commercially available under the name Biacore®.

The phrase "does not react" with an antibody used in the present invention means that the antibody used in the present invention does not substantially react with a compound. To confirm whether or not the antibody used in the present invention "does not substantially react" with a compound, measurements can be performed using, for example, a Biacore (registered trademark) T100 or T200 based on the above-mentioned SPR method to immobilize the antibody used in the present invention. "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 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 achieved. Examples include 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.

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 are suitable. The route, dosage, and timing of administration of the adjuvant are not particularly limited, but it is desirable to select them appropriately so as to enhance the desired immune response in the animal immunized with the antigen.

The type of animal used for immunization is not particularly limited, but mammals are preferred. For example, mice, rats, cows, rabbits, goats, sheep, and alpacas can be used, and mice or rats are more preferred. Animals can be immunized according to standard techniques. For example, immunization can be performed by injecting a solution of the 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 set an immunization schedule appropriately for the animal used. It is preferable to repeat antigen administration several times after the initial immunization.

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 antigens described above (see, for example, Chapter 6 of "Antibodies, A Laboratory Manual" (Cold Spring Harbor Laboratory Press, (1988)).

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. Cells with high antibody production capacity (quality and quantity) are preferably used for cell fusion, and the myeloma-derived cell line is preferably compatible with the animal from which the antibody-producing cells are derived. Cell fusion can be performed using methods known in the art, such as the polyethylene glycol method, methods using Sendai virus, and methods utilizing electric current. The resulting hybridomas can be grown under conditions commonly used in the art. Desired hybridomas can be selected while confirming the properties of the antibodies produced. Hybridoma cloning can be performed using well-known methods, such as limiting dilution and soft agar.

Hybridoma selection can be performed efficiently at the selection stage by taking into consideration the conditions used in the actual measurement of the antibodies produced. For example, antibodies obtained by immunizing an animal are reacted with lipopolysaccharide derived from a bacterium causing sepsis 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 bacterium causing sepsis immobilized on a solid phase in the presence of components derived from a biological sample and comparing the reactivity with that in the absence of components derived from a biological sample.

After the cloning step, the binding ability of the produced antibody to lipopolysaccharide derived from the sepsis-causing bacterium can be assayed using a method such as ELISA, RIA, or fluorescent antibody technique to confirm whether the selected hybridoma produces a monoclonal antibody with the desired properties.
Monoclonal antibodies having the desired characteristics 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 abdominal 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.

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 of such fragments 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.

(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 resins, 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).

(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 a specific substrate for that enzyme (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, it is preferable to use biotin or HRP as a labeling substance, and biotin is more preferable. When biotin is used, streptavidin labeled with HRP can also be used.

As used herein, the term "insoluble carrier" refers to a substance onto which antibodies or other substances that recognize the target substance are 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." Additionally, the terms "analysis," "detection," and "measurement" include the meaning of verifying the presence and/or quantifying lipopolysaccharide derived from sepsis-causing bacteria.

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). Taking into consideration 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.

The order in which the monoclonal antibody and lipopolysaccharide are added to the analytical system does not matter, as long as the effect of the present invention is achieved.

The kit provided by the present invention preferably includes (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 includes 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 from the same hybridoma.

The solid phase onto which the first monoclonal antibody is immobilized captures lipopolysaccharide derived from the septicemia-causing bacteria 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 amount of lipopolysaccharide derived from the septicemia-causing bacteria 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, can be any method known to those skilled in the art, and can be used without limitation, in addition to the methods described herein.

The first and second monoclonal antibodies are not particularly limited as long as they are monoclonal antibodies that react with lipopolysaccharide derived from sepsis-causing bacteria.

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 support, 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.

When 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 is used, 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.

(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 nonspecific reactions, such as 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.

Electrochemiluminescence immunoassay (ECL) refers to a method for measuring the amount of a substance to be detected by passing an electric current through a labeling substance to cause it to emit light and then detecting the amount of light emitted. 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, which excites 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 contain the following (A) and (B):
(A) A labeled reagent containing a conjugate of a first monoclonal antibody (labeled antibody) that reacts with lipopolysaccharide derived from sepsis-causing bacteria and an electrochemiluminescent substance (e.g., a ruthenium complex, etc.), and (B) an insoluble carrier on 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 a solid phase, a biological sample is added to the magnetic particles on which a solid-phase antibody is immobilized, followed by reaction. 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 reacted. After washing the magnetic particles, electrical energy is applied to the particles to generate light, and the amount of light emitted by the labeled substance is measured, allowing lipopolysaccharide derived from sepsis-causing bacteria to be analyzed.

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 the captured substance is detected using an enzyme reaction.
Sandwich ELISA is preferred as the ELISA. In this specification, 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 sandwich ELISA, an insoluble carrier is used to immobilize a first monoclonal antibody (solid-phase antibody) that recognizes the substance to be detected, and a second monoclonal antibody (labeled antibody) labeled with a labeling substance. The insoluble carrier is preferably a plate (immunoplate).
Typically, an 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 the sandwich ELISA method is used, the immunological analysis kit of the present invention may comprise the following (A) and (B):
(A) A labeling reagent containing a first monoclonal antibody that reacts with lipopolysaccharide derived from a sepsis-causing bacterium, which is labeled with a labeling substance. (B) An insoluble carrier on which a solid-phase antibody is immobilized.
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. A labeling reagent is then added, followed by incubation, and 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.

A secondary antibody can also be used in the sandwich ELISA method. By using a secondary antibody, the reaction is amplified, and 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) A second monoclonal antibody as a primary antibody; (B) A solid phase on which the first monoclonal antibody is immobilized; (C) An antibody against the second monoclonal antibody, labeled with a labeling substance (e.g., HRP or ALP); and (D) A substrate for the labeling substance (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. Lipopolysaccharides derived from sepsis-causing bacteria can be analyzed by measuring the color development using a plate reader or similar device.

The present invention will be explained in more detail below using examples, but these examples are not intended to limit the scope of the present invention. Furthermore, unless otherwise specified, % indicates % by mass.

Test Example 1: Method for Producing Monoclonal Antibodies Used in the Present Invention 1. Antibody Acquisition Rats (F344/Jc1, female) were intraperitoneally immunized weekly with heat-treated Escherichia coli and Pseudomonas aeruginosa cells diluted with PBS, or heat-treated Klebsiella pneumoniae, E. coli, and Pseudomonas aeruginosa cells diluted with PBS. Blood samples were collected 10 weeks after the first immunization (after 10 immunizations) to confirm the antibody titers in the blood. The antibody titers in the blood were evaluated by antigen solid-phase ELISA using LPS derived from Pseudomonas aeruginosa, E. coli, and Klebsiella pneumoniae, the main sepsis-causing bacteria, as antigens. The specific procedure for antigen solid-phase ELISA is as follows.

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, the 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-fold 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 color developing 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 a wavelength of 492 nm was measured using a plate reader.

As a result, an increase in titer was confirmed against the immunogens, LPS derived from Escherichia coli and LPS derived from Pseudomonas aeruginosa. After confirming the increase in antibody titer, 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.

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

An 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 allowed to stand at room temperature for 1 hour.
After washing three times (400 μL/well), LPS of various concentrations that had been biotin-labeled with EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) was 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 color developing 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 a wavelength of 492 nm was measured using a plate reader.

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.

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

An antibody solution (S28201R or S28203R) diluted with PBS was dispensed into a 96-well ELISA plate (5 μg/mL, 50 μL/well) and allowed to stand at room temperature for 2 hours or at 4°C overnight. A similar 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 allowed to stand at room temperature for 1 hour.
After washing three times (400 μL/well), antibodies (S28201R or S28203R) at various concentrations that had been biotin-labeled 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 color developing 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 a wavelength of 492 nm was measured using a plate reader.

The results are shown in Tables 1 and 2, and Figures 2 and 3. The results demonstrated that it was possible to construct a sandwich system. Furthermore, 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).

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 compared with the sensitivity of the sandwich system constructed in Example 1. The experimental procedure for the Limulus reagent was carried out according to the protocol described in the accompanying 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.

The results are shown in Tables 3 and 4. With the Limulus reagent, the LPS of Klebsiella pneumoniae was 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 the LPS of Klebsiella pneumoniae.
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.

Example 2 Calculation of the Sensitivity of the Kit 2-1. Evaluation of Commercially Available LPS Solutions 2-1-1. Preparation of 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.
A quadratic approximation equation was created using the commercially available LPS concentrations based on the labeling values and the values measured using the Limulus HS-T Single Test Wako. The results are shown in Tables 5 and 6 and Figures 4 and 5.

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-Escherichia coli LPS antibody (S28203R): y=0.0001x ² +0.0126x

2-1-2 Method for calculating 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.

2-2 Evaluation of Minimum Detection Limit Measurements were performed for n=8 samples of each LPS concentration according to the procedure for evaluating sensitivity described in Example 1. The mean value and 2.6SD (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, when "sensitivity at 0 pg/mL + 2.6 SD" was less than "sensitivity of each sample - 2.6 SD," it was determined that LPS at that concentration was 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.

As a result, it was found that the anti-Klebsiella pneumoniae LPS antibody (S28201R) was detectable down to a concentration of 0.011 pg/mL, and the anti-Escherichia coli LPS antibody (S28203R) was detectable down to a concentration of 0.22 pg/mL.

[Example 3] Measurement of LPS in biological samples Whether the prepared kit can detect LPS present in biological samples and the minimum detection limit were calculated. The same method as in Example 2 was used, except that commercially available LPS was diluted with serum from a healthy person. The same samples were also measured 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.

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) was able to detect LPS in biological samples down to concentrations of 0.023 pg/mL, more than 200 times more sensitive than the Limulus reagent. The anti-Escherichia coli LPS antibody (S28203R) was able to detect LPS down to concentrations of 0.10 pg/mL, 94 times more sensitive than the Limulus reagent.

Reference Example 2: Comparison of sensitivity 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; manufactured by 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.

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.

Reference Example 3: Comparison of sensitivity with a commercially available LPS detection ELISA kit Using a commercially available LPS detection ELISA kit (Qualitative Human Klebsiella (KBSL) ELISA Kit), the OD at 450 nm of a sample of serum from a healthy person to which Klebsiella pneumoniae LPS had been added was measured according to the manual. Measurements were performed on n=3 samples for each LPS concentration. Simultaneously, the positive control and negative control provided with the kit were also measured in the same manner. The OD when measuring the positive control was 1.14, and the OD when measuring the negative control was 0.049. The mean value and 2.6 SD (standard deviation × 2.6) of the sample measurement results were calculated from the results of triplicate measurements.
The minimum detection limit was calculated using the 2.6 SD method. Specifically, when "0 pg/mL OD + 2.6 SD"<"OD of each sample - 2.6 SD," it was determined that LPS at that concentration was detectable, and the minimum LPS concentration at which all samples above a certain concentration satisfied this condition was defined as the minimum detection limit. The results are shown in Table 12.

Table 12 shows that the kit prepared in this example is capable of detecting Klebsiella pneumoniae LPS with a sensitivity more than 2,000 times higher than commercially available LPS detection ELISA kits. Furthermore, if the standard described in the kit manual, which defines a positive result as a sensitivity of 0.15 or higher than the negative control, is adopted, the samples measured this time were determined to be negative, i.e., undetectable, at all concentrations. Therefore, even when the determination method is changed, the kit prepared in this example was found to be capable of detecting Klebsiella pneumoniae LPS with a higher sensitivity.

The present invention provides an immunological analysis kit for sepsis-causing bacteria that has similar reactivity to Limulus reagent and superior sensitivity to Limulus reagent.

[Reference to deposited biological material]
(1) Hybridoma producing antibody number S28201R 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 at the depository in Loi: July 3, 2020. Accession number assigned by the depository in Hai: NITE BP-03241.
(2) Hybridoma producing antibody number S28203R 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 at the depository institution in Loi: July 3, 2020. Accession number assigned by the depository institution in Hai: NITE BP-03242.

Claims (8)

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. 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. The immunological analysis kit according to claim 1 or 2, wherein the sepsis-causing bacterium is Klebsiella pneumoniae or Escherichia coli. The immunological analysis kit according to any one of claims 1 to 3, wherein the monoclonal antibody specifically reacts with lipopolysaccharide derived from a sepsis-causing bacterium. The immunological analysis kit according to any one of claims 1 to 4 , wherein the biological sample is blood, plasma, or serum. The immunological analysis kit according to any one of claims 1 to 5 , wherein the measurement principle is ELISA. 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 claim 1 , 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.