WO2022054753A1

WO2022054753A1 - Immunological analysis kit for septicemia-causing bacteria

Info

Publication number: WO2022054753A1
Application number: PCT/JP2021/032629
Authority: WO
Inventors: 勇輝 ▲高▼良; 知啓三浦; 修宮崎
Original assignee: 積水メディカル株式会社
Priority date: 2020-09-08
Filing date: 2021-09-06
Publication date: 2022-03-17
Also published as: JPWO2022054753A1

Abstract

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

Description

Immunological analysis kit for sepsis-causing bacteria

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

Sepsis means a condition in which an infectious disease causes serious organ damage. The prognosis of sepsis varies depending on the pathogenic microorganism, background factors of the infected patient, and the quality of intervention. Although it cannot be said unconditionally, it is estimated that 100,000 people die in one year in Japan.

As a method for measuring LPS (lipopolysaccharide) constituting the outer cell membrane of Gram-negative bacteria that cause sepsis, a Limulus reagent using the blood coagulation reaction of horseshoe crab is used (Patent Document 1). Limulus reagents have been used to diagnose or assist in the diagnosis of sepsis. However, the method using the Limulus reagent requires blood of horseshoe crab, which is a natural resource, and in addition to the concern of resource depletion, it is costly to maintain a certain quality. Another drawback of this method is that it tends to vary because it is a method that requires a plurality of steps.

In recent years, an ELISA kit (https://www.mybiosource.com/human-elisa-kits/klebsiella/9310934) for measuring LPS constituting the outer cell membrane of Gram-negative bacteria has been developed (Non-Patent Document 1). .. However, the sensitivity of these kits was inferior to that of the Limulus reagent, making it difficult to use as a substitute for the Limulus reagent.

Japanese Unexamined Patent Publication No. 04-137663

An object of the present invention is to provide an immunological analysis kit for sepsis-causing bacteria, which has a sensitivity equal to or higher than that of a Limulus reagent and can detect sepsis-causing bacteria with a simple operation.

The present inventors have diligently studied to solve the above-mentioned problems, and have created an immunological analysis kit capable of detecting the lipopolysaccharide even if the biological sample contains a very small amount of lipopolysaccharide derived from the causative bacterium of sepsis. .. Then, it was confirmed that this kit was superior in sensitivity to the Limulus reagent, and the present invention was completed. Specifically, the lower limit of measurement of the Limulus reagent is 0.35 pg / mL for lipopolysaccharide derived from Escherichia coli O111, according to the package insert. However, since the sample is diluted 10-fold at the stage of preparing the measurement sample, the actual lower limit of measurement is 3.5 pg / mL. On the other hand, the immunological analysis kit of the present invention can detect even lower concentrations of lipopolysaccharide.
Specifically, the present invention is as follows.
<1> An immunological analysis kit for detecting sepsis-causing bacteria in a biological sample.
Contains monoclonal antibodies that react with lipopolysaccharides derived from sepsis-causing bacteria
The immunological analysis kit in which the monoclonal antibody detects sepsis-causing bacteria of 0.023 pg / mL or more in a biological sample.
<2> The immunological analysis kit according to <1>, wherein the biological sample is a biological sample containing lipopolysaccharide derived from a sepsis-causing bacterium of 0.023 pg / mL or more.
<3> The immunological analysis kit according to <1> or <2>, wherein the causative bacterium for sepsis 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.
It contains a solid phase on which the first monoclonal antibody is immobilized and a labeling substance to which the second monoclonal antibody is bound, or is labeled with the solid phase on which the first monoclonal antibody is immobilized and the second monoclonal antibody. A secondary antibody against the second monoclonal antibody to which the substance was bound, and
Including
The immunological analysis kit according to any one of <1> to <4>, wherein the first monoclonal antibody and the second monoclonal antibody are the same type of monoclonal antibody.
<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>, which uses an ELISA method as a measurement principle.
<8> The immunological analysis kit according to any one of <1> to <7>, wherein the monoclonal antibody is an IgM antibody.
<9> Either <1> to <8>, wherein the monoclonal antibody is a monoclonal antibody produced by a hybridoma of accession number NITE BP-03241 or a monoclonal antibody produced by a hybridoma of accession number NITE BP-03242. The immunological analysis kit described in.

According to the present invention, it is possible to provide an immunological analysis kit for sepsis-causing bacteria, which exhibits superior sensitivity to Limulus reagent and can detect sepsis-causing bacteria with a simple operation.

It is a schematic diagram of the structure of lipopolysaccharide in the causative bacterium of sepsis. It is a graph which shows the result of the sandwich ELISA which tested whether or not the sandwich system can be formed only by S28201R antibody. It is a graph which shows the result of the sandwich ELISA which tested whether or not the sandwich system can be formed only by S28203R antibody. It is a graph which shows the calibration curve of LPS of Klebsiella pneumoniae. It is a graph which shows the calibration curve of LPS of Escherichia coli. It is a graph for calculating the minimum detection limit of LPS of Klebsiella pneumoniae. It is a graph for calculating the minimum detection limit of LPS of Escherichia coli. It is a graph for calculating the minimum detection limit in a biological sample of LPS of Klebsiella pneumoniae. It is a graph for calculating the minimum detection limit in a biological sample of LPS of Escherichia coli.

(Biological sample)
Examples of the "biological sample" in the present invention include solid tissues and body fluids derived from living organisms (organisms), and it is preferable to use body fluids. The biological sample in the present invention is more preferably blood, serum, plasma, urine, saliva, sputum, tears, ear leak, or prostatic fluid, more preferably blood, serum or plasma, and even more preferably septicemia. Blood, serum or plasma of the subject suspected of having. Living organisms or subjects include humans or animals (eg, monkeys, dogs, cats, mice, guinea pigs, rats, hamsters, etc.) and are preferably humans. The biological sample may be in vivo or in vitro.

(Bacteria causing sepsis)
As used herein, the term "sepsis-causing bacterium" means a bacterium that causes sepsis, such as pneumoniae, streptococcus, staphylococcus, Escherichia coli, and Pseudomonas aeruginosa. The term "sepsis" herein includes both sepsis and septic shock. Sepsis refers to a condition in which an infection causes serious organ damage. Septic shock refers to a condition in which acute circulatory insufficiency causes severe cell damage and metabolic disorders that can increase mortality. The "sepsis-causing bacterium" is preferably Klebsiella pneumoniae or Escherichia coli.
As used herein, "pneumonia pneumoniae" means Klebsiella pneumoniae, which is a gram-negative bacillus.
As used herein, "Escherichia coli" means Escherichia coli, which is a gram-negative bacillus.
In the present invention, the reactivity is similar to that of the Limulus reagent, and therefore, it can be replaced with the Limulus reagent actually used in the medical field at present. Further, the present invention has an advantage that lipopolysaccharide derived from a sepsis-causing bacterium can be detected with higher sensitivity than the Limulus reagent.

(Lipopolysaccharide)
As used herein, the term "lipopolysaccharide" means a complex of a lipid and a polysaccharide linked by a covalent bond. In the present specification, "lipopolysaccharide" may be simply referred to as LPS (lipopolysaccharide). Lipopolysaccharide is present on the outer membrane of Gram-negative bacteria. In lipopolysaccharide, a membrane structure is structurally formed so that lipid A, which is a lipid portion, is buried in the outer membrane, and the lipid A is mediated by oligosaccharide regions called cores (Outer Core and Inner Core). Therefore, the O antigen, which is a polysaccharide chain, is elongated (Fig. 1).
The antibody used in the present invention can recognize both lipopolysaccharide not detached from the cell wall of the sepsis-causing bacterium and lipopolysaccharide detached from the cell wall of the sepsis-causing bacterium.
Sandwich analysis often uses two types of antibodies that recognize different epitopes in the solid phase antibody and the labeled antibody. The monoclonal antibody used in the present invention has the advantage that one type can form a sandwich system. As used herein, the term "one type" or "same type" with respect to a monoclonal antibody means a monoclonal antibody that recognizes the same epitope. Being able to form a sandwich system with one type facilitates the construction of an experimental system. In addition, the ability to form a sandwich system with one type reduces the possibility of non-specific reactions occurring. The monoclonal antibody used in the present invention is preferably the same antibody, i.e., a monoclonal antibody produced from the same hybridoma. In the present specification, the “sandwich system” means an experimental system in which an antigen is sandwiched between two types of antibodies, an antibody for capture (solid phase antibody) and an antibody for detection, to detect the antigen. A labeling substance is bound to the antibody for detection, and the detection target can be analyzed by measuring the intensity of the signal derived from the labeling substance. The detection antibody may bind directly to the labeling substance or indirectly to the labeling substance via the secondary antibody.

(Monclonal antibody)
The monoclonal antibody used in the present invention is a monoclonal antibody that reacts with lipopolysaccharide derived from septicemia-causing bacteria, preferably a monoclonal antibody that specifically reacts with lipopolysaccharide derived from septicemia-causing bacteria, and is more preferably contracted. The S28201R antibody produced by the hybridoma of No. NITE BP-03241 or the S28203R antibody produced by the hybridoma of accession No. NITE BP-03242.
In the present specification, an antibody that reacts with LPS derived from pneumonia rod may be referred to as an anti-Pneumococcal LPS antibody, and an antibody that reacts with LPS derived from Escherichia coli may be referred to as an anti-E. coli LPS antibody.

In the present specification, "reacting" with lipopolysaccharide derived from sepsis-causing bacteria, "recognizing" lipopolysaccharide derived from sepsis-causing bacteria, and "binding" with lipopolysaccharide derived from sepsis-causing bacteria are used interchangeably. , Not limited to these examples, but need to be interpreted in the broadest sense. Whether or not the antibody "reacts" with an antigen (compound) such as lipopolysaccharide can be confirmed by an antigen-immobilized ELISA method, a competitive ELISA method, a sandwich ELISA method, or the like. Further, it can also be performed by a method (SPR method) using the principle of surface plasmon resonance. The SPR method can be carried out using devices, sensors and reagents commercially available under the name of Biacore®.

"No reaction" between the antibody used in the present invention and a certain compound means that the antibody used in the present invention does not substantially react with a certain compound. In order to confirm whether or not it "substantially does not react" with a certain compound, for example, Biacore® T100 or T200 is used based on the above SPR method, and the antibody used in the present invention is immobilized. Measurements can be made. It can be confirmed that "substantially no reaction" can be confirmed by a method or means well known to those skilled in the art other than the above SPR method.

The monoclonal antibody used in the immunological analysis kit of the present invention contains a fragment having the function of the monoclonal antibody as long as the effect of the present invention can be obtained. For example, a functional fragment containing the Fab portion of the monoclonal antibody obtained by enzymatic digestion of the monoclonal antibody, a functional fragment containing the Fab portion of the monoclonal antibody produced by gene recombination, and a phage display method. Included are functional fragments containing scFv.

The antibody used in the immunological analysis kit of the present invention is a phosphate buffered saline containing heat-killed cells derived from sepsis-causing bacteria such as pneumonia rod, pyogenic, and / or Escherichia coli as an antigen (immunogen). It can be produced by dissolving it in a solvent such as water and administering this solution to an animal to immunize it. If necessary, an appropriate adjuvant may be added to the solution, and then the emulsion may be used for immunization. As the adjuvant, a general-purpose adjuvant such as a water-in-oil emulsion, a water-in-oil-in-water emulsion, an oil-in-water emulsion, liposomes, and aluminum hydroxide gel can be used. As an adjuvant, a protein derived from a biological component, a peptide substance, or the like may be used. For example, Freund's incomplete adjuvant or Freund's complete adjuvant can be preferably used. The route of administration, dose, and timing of administration of the adjuvant are not particularly limited, but it is desirable to appropriately select the adjuvant 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 preferable, and for example, mice, rats, cows, rabbits, goats, sheep, alpaca and the like can be used, and more preferably mice or rats can be used. Animal immunization may be performed according to a general method. Immunization can be performed, for example, by injecting a solution of the antigen, preferably a mixture with an adjuvant, subcutaneously, intradermally, intravenously or intraperitoneally in the animal. Since the immune response generally depends on the type and strain of the animal to be immunized, it is desirable to set the immune schedule appropriately according to the animal used. Antigen administration is preferably repeated several times after the first immunization.

In order to obtain a monoclonal antibody, the following operations can be continuously performed, but the procedure is not limited to this. Since the method for producing the monoclonal antibody itself is well known and widely used in the art, those skilled in the art can easily produce the antibody used in the immunological analysis kit of the present invention by using the above-mentioned antigen. (See, for example, Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988), Chapter 6 and the like).

After the final immunization, spleen cells or lymph node cells, which are antibody-producing cells, are removed from the immunized animal. Then, a hybridoma can be produced by fusing these cells with a cell line derived from myeloma having high proliferative ability. It is preferable to use cells having a high antibody-producing ability (quality / quantity) for cell fusion, and it is preferable that the cell line derived from myeloma is compatible with the animal from which the antibody-producing cells to be fused are derived. Cell fusion can be performed according to a method known in the art, and for example, a polyethylene glycol method, a method using Sendai virus, a method using an electric current, or the like can be adopted. The resulting hybridoma can be propagated according to conditions general in the art. A desired hybridoma can be selected while confirming the properties of the antibody produced. Cloning of hybridomas can be performed by well-known methods such as the limiting dilution method and the soft agar method.

Hybridoma selection can also be done efficiently at the selection stage, taking into account the conditions used for actual measurement of the antibody produced. For example, an antibody obtained by immunizing an animal is reacted with a lipopolysaccharide derived from a septic disease-causing bacterium immobilized on a solid phase in the presence of a compound whose cross-reactivity is desired to be confirmed. Then, by comparing with the reactivity in the absence of the compound for which cross-reactivity is desired to be confirmed, a hybridoma producing a desired antibody can be selected more efficiently. In addition, the antibody obtained by immunizing an animal is reacted with lipopolysaccharide derived from a septic disease-causing bacterium immobilized on a solid phase in the presence of a component derived from a biological sample, and in the absence of the component derived from the biological sample. Hybridomas that produce the desired antibody can also be selected more efficiently by comparison with reactivity.

After the cloning step, the hybridoma selected is desired by assaying the binding ability of the produced antibody to the lipopolysaccharide derived from the bacterial cause of blood loss by using a method such as ELISA method, RIA method, or fluorescent antibody method. It can be confirmed whether or not a monoclonal antibody having properties is produced.
By mass-culturing the hybridomas selected as described above, a monoclonal antibody having desired characteristics can be produced. The method of mass culturing is not particularly limited, but for example, a method of appropriately culturing a hybridoma in a medium to produce a monoclonal antibody in the medium, or a method of injecting a hybridoma into the abdominal cavity of a mammal to proliferate the antibody in ascites. Can be mentioned as a method for producing. Purification of the monoclonal antibody shall be carried out by appropriately combining the above-mentioned methods for purifying the antibody from the antiserum, for example, DEAE anion exchange chromatography, affinity chromatography, sulfur fractionation method, PEG fractionation method, ethanol fractionation method and the like. Can be done.

As the antibody used in the immunological analysis kit of the present invention, it is also possible to use a fragment of an antibody having an antigen-antibody reaction activity in addition to the entire antibody molecule. In addition to those obtained through the immunization step to animals as described above, those obtained by using genetic recombination technology or chimeric antibodies can also be used. The antibody fragment is preferably a functional fragment, and examples thereof include F (ab') ₂ , Fab', and scFv. These fragments can be obtained by treating the antibody obtained as described above with a proteolytic enzyme (for example, pepsin or papain), or by cloning the DNA of the antibody and expressing it in a culture system using Escherichia coli or yeast. Can be manufactured by

(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. Further, in the immunological analysis kit of the present invention, the antibody can be used as a labeled antibody labeled with a labeling substance well known to those skilled in the art described later. For example, a solid phase antibody by physically adsorbing a monoclonal antibody to an insoluble carrier, chemically binding (may be via an appropriate spacer), or binding via an antibody bound to an insoluble carrier. Can be manufactured. As the insoluble carrier, an insoluble carrier made of a polymer base material such as polystyrene resin, an inorganic base material such as glass, or a polysaccharide base material such as cellulose or agarose can be used. The shape of the insoluble carrier is not particularly limited, and any shape such as a plate (for example, a microplate or a membrane), beads or particles (for example, latex particles or magnetic particles), or a tubular shape (for example, a test tube) can be selected. ..

(Labeled antibody)
By using a label that can bind to the antibody used in the immunological analysis kit of the present invention, the amount of the antibody bound to the lipopolysaccharide derived from the sepsis-causing bacterium can be measured. Thereby, lipopolysaccharide derived from the causative bacterium of sepsis disease can be detected in the biological sample. Examples of the labeling substance for producing a labeled antibody include enzymes, fluorescent substances, chemical luminescent substances, biotin, avidin, radioisotopes, colloidal gold particles, colored latex and the like. As a method for binding the labeling substance to the antibody, a method such as a glutaraldehyde method, a maleimide method, a pyridyl disulfide method, or a periodic acid method, which can be used by those skilled in the art, can be used. The types of the solid phase antibody and the labeled antibody, and the method for producing them are not limited to the above-mentioned examples of the binding method. For example, when an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) is used as a labeling substance, the specific substrate of the enzyme (when the enzyme is HRP, for example, O-phenylenediamine (OPD)). Alternatively, enzyme activity can be measured using 3,3', 5,5'-tetramethylbenzidine (TMB), p-nitrophenyl phosphate in the case of ALP, etc.). 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 it is more preferable to use biotin. When biotin is used, HRP-labeled streptavidin can be further used.

In the present specification, the "insoluble carrier" means a substance in which an antibody or the like that recognizes the detected substance is immobilized on the detected substance. Examples thereof include, but are not limited to, immunoplates, membranes, latex particles, magnetic particles and the like. In the present specification, the term "insoluble carrier" is referred to as "solid phase", and the state in which an antigen or antibody is physically or chemically supported on an insoluble carrier, or the state in which the carrier is supported is referred to as "fixation" or "immobilization". There is. In addition, the terms "analysis", "detection", or "measurement" include meanings such as proof and / or quantification of the presence of lipopolysaccharide derived from a septic disease-causing bacterium.

The immunological analysis kit of the present invention includes electrochemiluminescence immunoassay (ECL method), enzyme immunoassay (ELISA method), latex aggregation immunoassay (LTIA method), chemiluminescence immunoassay, and immunofluorescence method. , And analysis kits using high performance liquid chromatography (HPLC), but are not limited to these. The immunological analysis kit of the present invention is an electrochemical luminescence immunoassay (ECL method), a high performance liquid chromatograph method (HPLC), or an enzyme-linked immunosorbent assay (ELISA method) in consideration of measurement sensitivity and ease of operation. ) Is preferable, and it is more preferable that the analysis kit is for sandwich ELISA.

The order of adding the monoclonal antibody and the lipopolysaccharide to the analysis system may be any first as long as the effect of the present invention can be obtained.

The kit provided by the present invention preferably comprises (a) a solid phase such as a plate on which the first monoclonal antibody is immobilized, and (b) a second monoclonal antibody labeled with a labeling substance, or the present invention. The kit provided by is preferably comprising a solid phase on which the first monoclonal antibody is immobilized, a second monoclonal antibody, and a secondary antibody against the second monoclonal antibody to which a labeling substance is bound. In this case, the first monoclonal antibody and the second monoclonal antibody can be the same type of monoclonal antibody. The same type of monoclonal antibody means a monoclonal antibody that recognizes the same epitope. The monoclonal antibody used in the kit of the present invention is preferably the same antibody, i.e., a monoclonal antibody produced from the same hybridoma.

The solid phase on which the first monoclonal antibody is immobilized captures the lipopolysaccharide derived from the septic disease-causing bacterium in the biological sample to form the lipopolysaccharide-antibody complex. The second monoclonal antibody labeled with the labeling substance reacts with this lipopolysaccharide-antibody complex to form a sandwich. By measuring the amount of the labeling substance using a method according to the labeling substance, lipopolysaccharide derived from the septic disease-causing bacterium in the sample can be measured. Specific methods for constructing the kit, such as a method for immobilizing the first monoclonal antibody on the solid phase and a method for labeling the second monoclonal antibody with a labeling substance, are described in the methods described in the present specification. In addition, methods well known to those skilled in the art can be used without limitation.

The first monoclonal antibody and the second monoclonal antibody are not particularly limited as long as they are monoclonal antibodies that react with lipopolysaccharide derived from the causative bacterium of sepsis.

As the labeling substance, for example, a labeling substance known to those skilled in the art such as a fluorescent substance, a chemical luminescent substance, biotin, and avidin can be used. The binding method between the labeling substance and the antibody can be appropriately selected from known binding methods depending on the labeling substance and the antibody to be used, for example, glutaraldehyde method, maleimide method, pyridyl disulfide method, or periodic acid. A method such as an acid method can be used. It is preferable to use biotin or HRP as the labeling substance, and it is more preferable to use biotin.
(Diagnosis, diagnostic assistance, and treatment of sepsis)
Based on the analysis results of the immunological analysis kit of the present invention, it is possible to diagnose whether or not the subject has sepsis caused by a sepsis-causing bacterium, or it can be used as an aid to the diagnosis.

When the immunological analysis kit of the present invention uses a monoclonal antibody that reacts with lipopolysaccharide derived from Pseudomonas aeruginosa but does not react with lipopolysaccharide derived from Pseudomonas aeruginosa and lipopolysaccharide derived from Escherichia coli, a biological sample is used. Medium, preferably 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 contained in blood, serum or plasma. It is possible to detect lipopolysaccharide derived from sepsis-causing bacteria at / mL, 1 pg / mL to 10 μg / mL, or 5 pg / mL to 10 μg / mL.
The cutoff value can be appropriately set according to 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 have sepsis due to the causative agent of sepsis. If the measured value is higher than the cutoff value, it can be determined that the subject has sepsis derived from the sepsis-causing bacterium.

(others)
The immunological analysis kit of the present invention may contain a buffer component (buffer solution) in addition to the above. The buffer solution that can be used in the present invention may be any commonly used buffer, such as tris-hydrogen, boric acid, phosphoric acid, acetic acid, citric acid, succinic acid, phthalic acid, glutaric acid, and maleic acid. , Glycin and salts thereof, and Gut buffers such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES and the like.
In addition, the immunological analysis kit of the present invention contains saccharides, proteins and the like, if necessary, for the purpose of improving measurement sensitivity and suppressing non-specific reactions. For example, 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.), preservatives. (Sodium azide, ProClin300, etc.) can be mentioned.
The reagent used in the immunological analysis kit of the present invention can be adjusted to an appropriate concentration and used by those skilled in the art.
The immunological analysis kit of the present invention may also include instructions for use, stabilizers, reaction vessels, pretreatment liquids, sample extracts and the like.

The electrochemiluminescence immunoassay (ECL method) means a method of measuring the amount of a substance to be detected by causing a labeled substance to emit light by energization and detecting the amount of the light emitted. In the electrochemical luminescence immunoassay (ECL method), a ruthenium complex can be used as a labeling substance. An electrode is placed on a solid phase (microplate, etc.) and radicals are generated on the electrode to make the ruthenium complex excited and emit light. Then, the amount of light emitted from this ruthenium complex can be detected.
Usually, an electrochemical luminescence immunoassay (ECL method) is performed using a first monoclonal antibody as a solid phase antibody and a second monoclonal antibody that recognizes an epitope different from the first monoclonal antibody as a labeled antibody. Can be done. On the other hand, in the immunological analysis kit of the present invention, an antibody that recognizes the same epitope can be used for both the first monoclonal antibody and the second monoclonal antibody.
When using the ECL method, the immunological analysis kit of the present invention can include (A) and (B) below.
(A) Labeling reagent containing a conjugate of a first monoclonal antibody (labeled antibody) that reacts with lipopolysaccharide derived from septicemia-causing bacteria and an electrochemical luminescent substance (for example, ruthenium complex, etc.), and (B) derived from septicemia-causing bacteria. An insoluble carrier on which a second monoclonal antibody (solid phase antibody) that reacts with lipopolysaccharide is immobilized.
Monoclonal antibodies that recognize the same epitope can be used for both the first monoclonal antibody and the second monoclonal antibody.
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 and reacted, and then the biological sample is removed and washed. Subsequently, a conjugate of the labeled antibody and an electrochemically chemiluminescent substance (for example, a ruthenium complex or the like) is added and reacted. After washing the magnetic particles, electric energy is applied to emit light, and the amount of light emitted from the labeling substance is measured, whereby lipopolysaccharide derived from the causative bacterium of sepsis can be analyzed.

An enzyme-linked immunosorbent assay (ELISA method) using an enzyme label is also preferable because the target can be measured easily and quickly. As used herein, the term ELISA means a method of capturing an antigen or an antibody, which is a substance to be detected, contained in a sample by using an antibody or an antigen against the substance to be detected, and then detecting the substance by using an enzymatic reaction. do.
As the ELISA, sandwich ELISA is preferable. As used herein, the sandwich ELISA means an ELISA in which an antigen is sandwiched between two types of antibodies, an antibody for capture (solid phase antibody) and an antibody for detection, 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 a substance to be detected is immobilized and a second monoclonal antibody (labeled antibody) labeled with a labeling substance are used. The insoluble carrier is preferably a plate (immunoplate).
Usually, an enzyme immunoassay (ELISA method) can be performed using a first monoclonal antibody as a solid phase antibody and a second monoclonal antibody that recognizes an epitope different from the first monoclonal antibody as a labeled antibody. However, in the immunological analysis kit of the present invention, an antibody that recognizes the same epitope can be used for both the first monoclonal antibody and the second monoclonal antibody.
When using the sandwich ELISA method, the immunological analysis kit of the present invention can include the following (A) and (B).
(A) Labeling reagent containing a first monoclonal antibody that reacts with lipopolysaccharide derived from sepsis-causing bacteria labeled with a labeling substance (B) An insoluble carrier on which a solid-phase antibody is immobilized.
In such a kit, first, a biological sample is added to an insoluble carrier on which a solid phase antibody is immobilized, and then the sample is incubated, and the sample is removed and washed. Next, the labeling reagent is added and then incubated, and the 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. It is preferable to use biotin as the labeling substance, and when biotin is used, HRP-labeled streptavidin can be further used.

Secondary antibodies can also be used in the sandwich ELISA method. By using the secondary antibody, the reaction is amplified and the detection sensitivity can be further increased. When a secondary antibody is used, the analysis kit of the present invention can include the following (A) to (D).
(A) Second monoclonal antibody as the primary antibody (B) Solid phase (C) labeled substance (HRP, ALP, etc.) on which the first monoclonal antibody is immobilized, antibody to the second monoclonal antibody (D) Labeled substance Substrate (OPD, TMB, or p-nitrophenyl phosphate, etc.)
In such a kit, first, the first monoclonal antibody is appropriately treated on the immobilized solid phase, a diluted biological sample is added, and then the incubation is performed, and the biological sample is removed and washed. Subsequently, a primary antibody (secondary monoclonal antibody) is added for incubation and washing, and an enzyme-labeled secondary antibody is further added for incubation. Then, the substrate is added to develop the color. By measuring the color development using a plate reader or the like, lipopolysaccharide derived from the causative bacterium of sepsis can be analyzed.

Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention. Further, unless otherwise specified,% indicates mass%.

[Test Example 1] Method for producing a monoclonal antibody used in the present invention 1. Acquisition of antibody Heat-treated cells of Escherichia coli and Pseudomonas aeruginosa diluted with PBS or heat-treated cells of Pneumonia bacillus, Escherichia coli, and Pseudomonas aeruginosa diluted with PBS were placed in the abdominal cavity of a rat (F344 / Jc1, ♀). I was immunized every week. Test blood was collected 10 weeks after the initial immunization (after 10 immunizations), and the antibody titer in the blood was confirmed for each. The antibody titer in blood was evaluated by an antigen solid-phase ELISA using LPS derived from each of the main causative bacteria of sepsis, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. The specific procedure of the antigen solid phase ELISA is as follows.

Various LPS diluted with PBS were dispensed into a 96-well plate for ELISA (1 μg / mL, 50 μL / well) and allowed to stand at room temperature for 2 hours or at 4 ° C. overnight.
-After washing 3 times (400 μL / well), the blocking solution was dispensed (100 μL / well) and allowed 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 3 times (400 μL / well), HRP-labeled goat anti-rat IgG (H + L) antibody diluted 17,000 times with an antibody diluted solution was dispensed (50 μL / well) and allowed to stand at room temperature for 1 hour.
-After washing 3 times (400 μL / well), the OPD color-developing solution was dispensed (50 μL / well) and reacted at room temperature for 10 minutes.
-The stop solution was dispensed (50 μL / well) and the reaction was stopped. The absorbance at a wavelength of 492 nm was measured with a plate reader.

As a result, it was confirmed that the titers of LPS derived from Escherichia coli, which is an immunogen, and LPS derived from Pseudomonas aeruginosa increased. After confirming the increase in antibody titer, spleen cells and iliac lymph node cells were fused with myeloma cells SP2 / O by an electric fusion method. Cells that were not subjected to fusion were cryopreserved. The fused cells were cultured on 96-well plates, and the culture supernatant was collected and screened 7 days after the fusion. The medium was exchanged the day before the screening.

2. 2. Screening We attempted to screen for antibodies that react with LPS derived from the bacteria that cause sepsis by liquid phase ELISA using LPS derived from pneumonia rod, Escherichia coli, and Pseudomonas aeruginosa that cause sepsis as antigens. The specific procedure of the 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 plate for ELISA (5 μg / mL, 50 μL / well) and allowed to stand at room temperature for 2 hours or 4 ° C overnight.
-After washing 3 times (400 μL / well), the blocking solution was dispensed (100 μL / well) and allowed to stand at room temperature for 1 hour or at 4 ° C. overnight.
-After removing the blocking solution, the antibody solution diluted to each concentration was dispensed (50 μL / well) and allowed to stand at room temperature for 1 hour.
-After 3 washes (400 μL / well), biotin-labeled LPS at each concentration was dispensed with EZ-LinkTM Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) (50 μL / well) and allowed to stand at room temperature for 1 hour. Placed.
-After washing 3 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 3 times (400 μL / well), the OPD color-developing solution was dispensed (50 μL / well) and reacted at room temperature for 10 minutes.
-The stop solution was dispensed (50 μL / well) and the reaction was stopped. The absorbance at a wavelength of 492 nm was measured with a plate reader.

As a result of the liquid-phase ELISA, one antibody (IgM type: S28201R) that reacts with LPS derived from pneumonia rod and LPS derived from Escherichia coli, which was not included in the immunogen and had an insufficient increase in titer. We succeeded in establishing one antibody (IgM type: S28203R) that reacts.

[Example 1] Evaluation of sensitivity of an experimental system using the monoclonal antibody obtained in Test Example 1 Whether a sandwich system can be formed with the same antibody in each of S28201R and S2803R was evaluated by sandwich ELISA. The specific procedure of the sandwich ELISA is as follows.

An antibody solution (S28201R or S28203R) diluted with PBS was dispensed (5 μg / mL, 50 μL / well) into a 96-well plate for ELISA and allowed to stand at room temperature for 2 hours or 4 ° C. overnight. In addition, the same operation was performed using a rat IgM monoclonal antibody under control.
-After washing 3 times (400 μL / well), the blocking solution was dispensed (100 μL / well) and allowed 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), biotin-labeled antibody (S28201R or S28023R) at each concentration with EZ-LinkTM Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) was dispensed (50 μL / well). It was allowed to stand at room temperature for 1 hour.
-After washing 3 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 3 times (400 μL / well), the OPD color-developing solution was dispensed (50 μL / well) and reacted at room temperature for 10 minutes.
-The stop solution was dispensed (50 μL / well) and the reaction was stopped. The absorbance at a wavelength of 492 nm was measured with a plate reader.

The results are shown in Tables 1 and 2, and FIGS. 2 and 3. As a result, it was shown that it is possible to form a sandwich system. In addition, the S28201R sandwich ELISA system was able to detect the antigen up to a concentration of about 2 ng / mL (display value). The S28203R sandwich ELISA system was able to detect the antigen up to a concentration of about 30 ng / mL (displayed value).

[Reference Example 1] Comparison of Sensitivity with Limulus Reagent The sensitivity of Limulus Reagent (product name: Limulus ES-J Testwaco Fujifilm Wako Pure Chemical Industries, Ltd.) was measured using LPS derived from Klebsiella pneumoniae and LPS derived from Escherichia coli. , The sensitivity of the sandwich system constructed in Example 1 was compared. The experimental procedure for the Limulus reagent was performed according to the protocol described in the attached manual. LPS derived from pneumonia rod (Sigma-Aldrich) and LPS derived from Escherichia coli (Fuji Film Wako Pure Chemical Industries, Ltd.) were serially diluted, and each concentration was tested for detection using Limulus reagent.

The results are shown in Tables 3 and 4. With the Limulus reagent, LPS of Klebsiella pneumoniae became undetectable at 125 ng / mL (displayed value). Therefore, it was shown that the sandwich system constructed in Example 1 was more sensitive than the Limulus reagent in the analysis of LPS of Klebsiella pneumoniae.
In addition, with the Limulus reagent, LPS of Escherichia coli became undetectable at 100 ng / mL (displayed value). Therefore, it was shown that the sandwich system constructed in Example 1 was more sensitive than the Limulus reagent in the analysis of LPS of Escherichia coli.

[Example 2] Calculation of kit sensitivity 2-1 Pricing of commercially available LPS solution 2-1-1 Preparation of calibration curve ・ Commercially available LPS (5 mg / mL (displayed value)) at each concentration (400, 300) in PBS , 200 ng / mL).
The diluted LPS was measured using Limulus HS-T Single Test Wako according to the instruction manual.
An approximate expression of the quadratic function was created using the commercially available LPS concentration based on the displayed value and the measured value by Limulus HS-T Single Test Wako. The results are shown in Tables 5 and 6 and FIGS. 4 and 5.

As a result, the approximate expression of the quadratic function is as follows.
-Anti-pneumonia Klebsiella LPS antibody (S28201R): y = 6 x ^10-5 x ² + 0.0057 x
-Anti-E. coli LPS antibody (S28203R): y = 0.0001x ² + 0.0126x

2-1-2 Concentration calculation method of commercially available LPS solution The endotoxin concentration was calculated by substituting the display value concentration of the commercially available LPS into x of the above-mentioned calibration curve formula.
Example) When converting the concentration of 100 ng / mL Klebsiella pneumoniae LPS, when x = 100 is applied, y = 1.17. Therefore, the amount of endotoxin in 100 ng / mL Klebsiella pneumoniae LPS is 1.17 pg / mL.

2-2 Minimum detection limit evaluation According to the procedure for evaluating the sensitivity described in Example 1, measurement was performed at n = 8 for each LPS concentration sample. From the results of eight measurements, the mean value and 2.6 SD (standard deviation x 2.6) were calculated.
The minimum detection limit was calculated by the 2.6SD method. Specifically, when "sensitivity of 0 pg / mL + 2.6 SD"<"sensitivity of each sample-2.6 SD", it is judged that LPS at that concentration can be detected, and the minimum condition satisfying the above conditions is satisfied. The LPS concentration was set as the minimum detection limit. The results are shown in Tables 7 and 8 and FIGS. 6 and 7.

As a result, it was found that the anti-Klebsiella pneumoniae LPS antibody (S28201R) can be detected up to a concentration of 0.011 pg / mL, and the anti-E. coli LPS antibody (S28203R) can be detected up to a concentration of 0.22 pg / mL. rice field.

[Example 3] Measurement of LPS in a biological sample Whether or not the produced kit can detect LPS present in a biological sample, and the minimum detection limit at that time were calculated. The procedure was the same as in Example 2 except that the commercially available LPS was diluted with the serum of a healthy person. The same sample was also measured with Limulus HS-T Single Test Wako to confirm whether LPS could be detected. The results are shown in Tables 9 and 10 and FIGS. 8 and 9.

From Tables 9 and 10 and FIGS. 8 and 9, it was found that the prepared kit can detect LPS in a biological sample with the same sensitivity as LPS in PBS. Furthermore, the anti-Klebsiella pneumoniae LPS antibody (S28201R) was able to detect LPS in a biological sample up to a concentration of 0.023 pg / mL, which was more than 200 times more sensitive than the Limulus reagent. In addition, the anti-E. coli LPS antibody (S28203R) was able to detect LPS up to a concentration of 0.10 pg / mL, 94 times more sensitive than the Limulus reagent.

[Reference Example 2] Sensitivity comparison with commercially available anti-LPS antibody The antibody solution of sandwich ELISA and the biotin-labeled antibody in Example 1 were used as a commercially available anti-LPS antibody (Anti-Lipoporysaccharide, Mouse IgG2a, Recombinant Monoclonal Antibody, clone22N1). The minimum detection limit for detecting Escherichia coli LPS in the serum of healthy subjects was calculated in the same manner as in Example 3 by changing to Absolute antibody (manufactured by Absolute antibody). The results are shown in Table 11.

From Table 11, it was found that the kit prepared in this example can detect Escherichia coli LPS with a sensitivity 5 times or more higher than that of ELISA using a commercially available anti-LPS antibody.

[Reference Example 3] Sensitivity comparison with commercially available LPS detection ELISA kit When using a commercially available LPS detection ELISA kit (Qualitative Human Klebsiella (KBSL) ELISA Kit) and using a healthy human serum supplemented with pneumonia rod LPS as a sample according to the manual. The OD at 450 nm was measured at n = 3 for each LPS concentration sample. At the same time, the Positive Control and Negative Control included in the kit were measured in the same manner. The OD at the time of the Positive Control measurement was measured. It was 1.14, and the O.D. at the time of Negative Control measurement was 0.049. Regarding the measurement results of the sample, from the results of three measurements, the average value and 2.6 SD (standard deviation × 2.6) ) Was calculated.
The minimum detection limit was calculated by the 2.6SD method. Specifically, when "0 pg / mL O.D. + 2.6 SD"<"O.D.-2.6 SD of each sample", it is judged that LPS at that concentration can be detected. The minimum detection limit was set to the minimum LPS concentration in which all the samples having a certain concentration or higher satisfy the above conditions. The results are shown in Table 12.

From Table 12, it was found that the kit prepared in this example can detect Klebsiella pneumoniae LPS with 2000 times or more higher sensitivity than the commercially available LPS detection ELISA kit. If the criteria described in the kit manual for positive judgment with a sensitivity of OD + 0.15 or higher of the Negative Control are adopted, the sample measured this time is negative judgment, that is, undetectable at any concentration. Is. Therefore, it was found that the kit prepared in this example can detect Klebsiella pneumoniae LPS with higher sensitivity even when the determination method is changed.

According to the present invention, it is possible to provide an immunological analysis kit for sepsis-causing bacteria, which has the same reactivity as the Limulus reagent and shows superior sensitivity to the Limulus reagent.

[Reference to deposited biomaterials]
(1) Hybridoma that produces antibody number S28201R Name and address of the depositary institution that deposited the biological material Independent Administrative Institution Product Evaluation Technology Infrastructure Organization
2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan (Postal code 292-0818)
Date of deposit of biomaterials to Loi's depositary agency July 3, 2nd year The deposit number NITE BP-03241 given by Hai's depositary agency for deposits.
(2) Hybridoma that produces antibody number S2803R Name and address of the depositary institution that deposited the biological material Independent Administrative Institution Product Evaluation Technology Infrastructure Organization
2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan (Postal code 292-0818)
Date of deposit of biomaterials to Loi's depositary agency July 3, 2nd year Deposit number NITE BP-03242 assigned by Hai's depositary agency for deposits

Claims

An immunological analysis kit for detecting sepsis-causing bacteria in biological samples.
Contains monoclonal antibodies that react with lipopolysaccharides derived from sepsis-causing bacteria
The immunological analysis kit in which the monoclonal antibody detects sepsis-causing bacteria of 0.023 pg / mL or more in a biological sample.
The immunological analysis kit according to claim 1, wherein the biological sample is a biological sample containing lipopolysaccharide derived from a sepsis-causing bacterium of 0.023 pg / mL or more.
The immunological analysis kit according to claim 1 or 2, wherein the causative bacterium for sepsis is Klebsiella pneumoniae or Escherichia coli.
The immunological analysis kit according to any one of claims 1 to 3, wherein the monoclonal antibody is a monoclonal antibody that specifically reacts with lipopolysaccharide derived from a sepsis-causing bacterium.
The monoclonal antibody includes a first monoclonal antibody and a second monoclonal antibody.
It contains a solid phase on which the first monoclonal antibody is immobilized and a labeling substance to which the second monoclonal antibody is bound, or is labeled with the solid phase on which the first monoclonal antibody is immobilized and the second monoclonal antibody. A secondary antibody against the second monoclonal antibody to which the substance was bound, and
Including
The immunological analysis kit according to any one of claims 1 to 4, wherein the first monoclonal antibody and the second monoclonal antibody are the same type of monoclonal antibody.
The immunological analysis kit according to any one of claims 1 to 5, wherein the biological sample is blood, plasma, or serum.
The immunological analysis kit according to any one of claims 1 to 6, wherein the ELISA method is used as a measurement principle.
The immunological analysis kit according to any one of claims 1 to 7, wherein the monoclonal antibody is an IgM antibody.
The immunology according to any one of claims 1 to 8, wherein the monoclonal antibody is a monoclonal antibody produced by a hybridoma of Accession No. NITE BP-03241 or a monoclonal antibody produced by a hybridoma of Accession No. NITE BP-03242. Analysis kit.