US20100221747A1 - Immunochromatography detection of multidrug-resistant staphylococcus and diagnostic kit - Google Patents

Immunochromatography detection of multidrug-resistant staphylococcus and diagnostic kit Download PDF

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US20100221747A1
US20100221747A1 US12/097,460 US9746006A US2010221747A1 US 20100221747 A1 US20100221747 A1 US 20100221747A1 US 9746006 A US9746006 A US 9746006A US 2010221747 A1 US2010221747 A1 US 2010221747A1
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pbp2
cell
wall
synthesizing enzyme
bacterium
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Hiromi Ito
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Denka Seiken Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance

Definitions

  • the present invention relates to immunochromatography detection and a kit utilizing the same.
  • Staphylococci includes 40 or more bacterium types roughly classified into Staphylococcus aureus and coagulase-negative staphylococci (hereafter abbreviated to “CNS”) from the clinical point of view. Staphylococcus aureus are regarded as pathogenic organisms and CNS are regarded as nonpathogenic organisms.
  • methicillin-resistant Staphylococcus aureus are Staphylococcus aureus that show resistance to ⁇ -lactam agents, including penicillin such as methicillin. Also, many such bacteria show resistance to many drugs such as aminoglycosides and macrolides. Thus, such bacteria are clinically regarded as multidrug-resistant Staphylococcus aureus .
  • Staphylococcus aureus showing sensitivity to methicillin are referred to as methcillin-sensitive Staphylococcus aureus (hereafter referred to as “MSSA”).
  • Staphylococcus aureus produce a variety of toxins, including enterotoxin, toxic-shock syndrome toxin, hemolysin, and exfoliative toxin (Hideo IGARASHI: TSST-1, “ Shinshu to meneki (Invasion and immunity),” 3, 3-10, 1994). Infection with such toxins would cause enteritis, pneumonia, dermatitis, organic failure, or the like, and serious infection may lead to death. When Staphylococcus aureus are isolated from a patient, accordingly, whether or not the bacterium is MRSA must be inspected as quickly as possible. In the event of MRSA infections, adequate drugs, such as vancomycin or arbekacin sulfate, which are regarded as being effective against MRSA, must be selected and administered to a patient.
  • Staphylococci other than Staphylococcus aureus are indigenous bacteria, and they are nonpathogenic to healthy individuals in general.
  • an organ transplant patient takes an immunosuppressant as a measure to prevent a postoperative infection or in the case of a so-called compromised patient with a weakened immune system due to an aging-induced weakened physical strength, however, opportunistic infections may occur.
  • MRCNS methicillin-resistant coagulase-negative staphylococci
  • MRCNS multidrug-resistant coagulase-negative staphylococci
  • MRSA and MRCNS are collectively referred to as multidrug-resistant staphylococcus.
  • the drug resistance of MRSA or MRCNS is known to result from expression of a new enzyme, PBP2′, in addition to four types of penicillin-binding proteins (i.e., PBP1, PBP2, PBP3, and PBP4) that crosslink murein chains, which are constitutional elements of the cell wall of staphylococci and which synthesize the cell wall (Utsui, Y., and Yokota, T.: Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus . Antimicrobial Agents and Chemotherapy, 28, 397-403, 1985).
  • PBP1 to PBP4 proteins that staphylococci possess in common are inactivated as cell-wall-synthesizing enzymes by penicillin antibiotics, which are substrate analogues, and bacteria eventually die when synthesis of cell walls becomes unfeasible.
  • MRSA and MRCNS express a new cell-wall-synthesizing enzyme, PBP2′, which exhibits little affinity to ⁇ -lactam antibiotic substances, i.e., which would not be inactivated thereby.
  • MRSA and MRCNS are considered to proliferate by altering roles in cell wall synthesis.
  • Most MRSA and MRCNS acquire mechanisms of resistance to other antibiotics and become multidrug-resistant bacteria that are resistant to many antibiotics. Such bacteria are regarded as multidrug-resistant staphylococci instead of staphylococci having resistance merely to ⁇ -lactam antibiotics.
  • a culture solution When cultured in a liquid medium, a culture solution is sowed on an agar medium and cultured therein for colony isolation, and colonies suspected of being staphylococci are also subjected to pure culture followed by identification.
  • the bacteria that have been identified as the Staphylococcus aureus or staphylococci are subjected to a drug-sensitivity test or the like, and whether or not a bacterium of interest is MRSA, MSSA, or MRCNS is determined based on test results.
  • a drug-sensitivity test is generally carried out by culture, such as a dilution technique or a disk sensitivity test.
  • Such drug sensitivity test is known to require a culture duration of 16 to 24 hours (it would take 3 or more days from separation of clinical samples to determination if both isolation culture and pure culture are conducted) and to produce differences in test results due to, for example, the concentration of bacteria, culture temperature, medium composition, or drug to be used. Thus, a person who conducts such test is required to be highly experienced with the procedure.
  • production of PBP2′ plays a key role in the expression of multidrug resistance, and detection of PBP2′ from staphylococci can be a useful means for learning whether or not a bacterium of interest has acquired resistance.
  • PBP2′ produced specifically by bacteria of the multidrug-resistant staphylococcus is detected by an antigen-antibody-reaction-based immunological means, such as Western blotting, radioimmunoassay, or a slide latex agglutination test (JP Patent No. 3638731).
  • an antigen-antibody-reaction-based immunological means such as Western blotting, radioimmunoassay, or a slide latex agglutination test (JP Patent No. 3638731).
  • Such methods of detecting PBP2′ suffer from the following problems. Western blotting is complicated in terms of procedure, and it is difficult to rapidly process many samples.
  • Radioimmunoassay is not practical from the viewpoint of routine testing, since it involves the use of a radioisotope, it requires BF separation involving separating an antigen-antibody complex from other non-binding antigens or antibodies during assay, and it requires several hours to complete assay due to the presence of a denaturing agent used for extracting an antigen from the bacterium in the reaction system.
  • the slide latex agglutination test requires detection from axenic bacteria due to false-positive reactions caused by contaminating bacteria (e.g., false-positive reactions due to nonspecific reactions) and low sensitivity.
  • culture must be conducted at least twice, i.e., isolation culture and pure culture, which in turn requires 2 to 3 days to complete the determination, it increases the cost for mediums for pure culture, and it may cause a false-positive reaction due to agglutination after the determination time, even though such time can be as short as 3 minutes.
  • the present invention is intended to provide an immunochromatography detection device that can detect PBP2′ produced specifically by a bacterium of multidrug-resistant staphylococcus with high sensitivity in a simple and rapid manner via immunochromatography detection to determine infection with multidrug-resistant staphylococcus , a diagnostic method using such detection device, and a diagnostic kit comprising such detection device.
  • the present inventors have conducted concentrated studies regarding a method for extracting PBP2′ from a bacterium of multidrug-resistant staphylococcus and assaying the same in a simple and rapid manner without complicated procedures. They succeeded in assaying PBP2′ in a simple manner with the use of an immunochromatography detection device using a reagent that is a labeled antibody binding specifically to PBP2′ and a capture reagent that can specifically bind to and capture a complex of PBP2′ and the labeled reagent. Further, they discovered that PBP2′ could be extracted and assayed without the need for complicated centrifugation or other means by treating a sample with an alkaline solution before assay, neutralizing the same, and applying the same to the immunochromatography detection device.
  • assay can be carried out without causing a false-positive reaction by applying a surfactant such as an ampholytic surfactant to a capture reagent site of the immunochromatography detection device on which the capture reagent has been immobilized. This has led to the completion of the present invention.
  • a surfactant such as an ampholytic surfactant
  • the present invention is as follows.
  • a method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′ comprising detection of a cell-wall-synthesizing enzyme, PBP2′, via immunochromatography detection based on an antigen-antibody reaction.
  • [2] The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to [1], which involves the use of an immunochromatography detection device comprising on a sheet-like solid-phase support: a sample supply site to which a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment is supplied; a labeled reagent site that holds a reagent, which is a labeled antibody binding specifically to PBP2′, in a manner such that the reagent is able to spread across the solid-phase support; and a capture reagent site on which a capture reagent capable of specifically binding to and capturing a complex of PBP2′ and the labeled reagent has been immobilized.
  • an immunochromatography detection device comprising on a sheet-like solid-phase support: a sample supply
  • [3] The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to [1] or [2], wherein PBP2′ comes into contact with the labeled reagent at a site separated from a solid-phase support in advance, and a sample-reagent mixture comprising a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment and the labeled reagent that is a labeled antibody binding specifically to PBP2′ is supplied to the sample supply site.
  • An immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, comprising on a sheet-like solid-phase support: a sample supply site to which a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment is supplied; a labeled reagent site that holds a reagent, which is a labeled antibody binding specifically to PBP2′, in a manner such that the reagent is able to spread across the solid-phase support; and a capture reagent site on which a capture reagent capable of specifically binding to and capturing a complex of PBP2′ and the labeled reagent has been immobilized, the capture reagent site comprising an ampholytic surfactant, an anionic surfactant, and/or a nonionic surfactant.
  • the immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to [18], wherein the capture reagent site comprises a sulfobetaine-type surfactant.
  • the immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to [18] or [19], wherein the sample supply site comprises glass fibers.
  • the immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to any one of [18] to [20], wherein the bacterium producing a cell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistant staphylococcus.
  • a kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ comprising: the immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to any one of [18] to [21]; and an anion or cation solution having a high ionization tendency to be added to a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment.
  • kits for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ according to any one of [22] to [28], wherein the bacterium producing a cell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistant staphylococcus.
  • the immunochromatography detection device of the present invention enables detection of PBP2′ with high sensitivity. This then enables testing with one colony after isolation culture. Consequently, whether or not a bacterium of interest is MRSA or MRCNS can be evaluated via a single operation of culture in 1 day after separation thereof from the clinical sample. This can remarkably reduce testing duration and the cost of mediums used for pure culture. Further, blood (liquid) culture that is carried out for testing MRSA bacteremia or septicemia enables direct detection from a positive medium. This enables initiation of effective treatment at an early stage, which leads to reduction of treatment duration.
  • centrifugation becomes unnecessary by imparting filtration effects to a sample application member on a support on which an antibody has been immobilized.
  • sanitary laboratory conditions can be maintained.
  • a capture site of the immunochromatography detection device may be impregnated with a surfactant, so that binding of fractured bacteria other than antigens to the labeled reagent and/or the capture site can be prevented; i.e., false-positive reactions can be prevented, and sensitivity can be improved. This makes a previously necessary boiling procedure in the process of bacterium pretreatment unnecessary.
  • FIG. 1 shows an embodiment of the detection device of the present invention comprising a labeled reagent site.
  • FIG. 2 shows an embodiment of the detection device of the present invention comprising no labeled reagent site.
  • the present invention relates to an immunochromatography detection device that can detect PBP2′ that is present specifically in a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, such as a multidrug-resistant staphylococcus , with high sensitivity in a simple and rapid manner via immunochromatography detection to determine infection with a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, such as a multidrug-resistant staphylococcus, and a diagnostic kit.
  • PBP2′ a cell-wall-synthesizing enzyme
  • PBP2′ such as a multidrug-resistant staphylococcus
  • sample refers to a solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, and more specifically, a solution deduced to contain a multidrug-resistant staphylococcus bacterium, including a solution of bacteria suspended therein, which was prepared by smearing a growth medium, such as blood agar medium, normal agar medium, heart infusion agar medium, brain heart infusion agar medium, soybean/casein/digest agar medium, chocolate agar medium, egg-yolk-containing mannitol salt agar medium, or MRSA selection medium, with an analyte substance, such as urine, pus, spinal fluid, secreted material, or puncture fluid obtained from a suspected patient and culturing the same under aerobic conditions at 35° C.
  • a growth medium such as blood agar medium, normal agar medium, heart infusion agar medium, brain heart infusion agar medium, soybean/casein/dig
  • the term also refers to an extract deduced to contain PBP2′ released from the cell wall due to pretreatment of such a suspension of bacteria.
  • the “bacterium producing a cell-wall-synthesizing enzyme, PBP2′” includes multidrug-resistant staphylococcus, and the “multidrug-resistant staphylococcus” includes multidrug-resistant Staphylococcus aureus (MRSA) and multidrug-resistant coagulase-negative staphylococci (MRCNS).
  • a solution of an analyte substance such as a urine, pus, spinal fluid, secreted material, puncture fluid, or another sample obtained from a suspected patient suspended directly in physiological saline or phosphate buffer can also be a sample. From the viewpoint of detection sensitivity, use of a solution containing bacteria cultured in the above-described manner is preferable.
  • the immunochromatography detection device of the present invention is an immunochromatography test piece.
  • such device is composed as shown in FIG. 1 .
  • Such device comprises on sheet-like solid-phase support; a sample supply site 1 to which a sample is supplied; a labeled reagent site 2 that holds a reagent, which is a labeled antibody binding specifically to PBP2′, in a manner such that the reagent is able to spread across the solid-phase support; and a capture reagent site 3 on which a capture reagent capable of specifically binding to and capturing a complex of PBP2′ and the labeled reagent has been immobilized.
  • a sample When a sample is supplied to the sample supply site 1 , the sample passes through the a labeled reagent site 2 and the capture reagent site 3 in that order.
  • a mixture of a sample and a reagent that is a labeled antibody binding specifically to PBP2′ may be supplied to the sample supply site 1 .
  • the labeled reagent site 2 on the solid-phase support may be omitted ( FIG. 2 ).
  • PBP2′ contained in the sample comes into contact with the labeled reagent at a site separated from a solid-phase support in advance.
  • an analyte comes into contact with a labeled reagent at a site separated from a solid-phase support in advance refers to conditions such that the labeled reagent is not contained on the solid-phase support, or it is in contact with the solid-phase support, and such that a liquid is not included at a site that can communicate with the solid-phase support, such as a sample supply site, of the immunochromatography detection device.
  • an analyte comes into contact with the labeled reagent in advance, apart from the solid-phase support or a site that is in contact with the solid-phase support.
  • the immunochromatography detection device of the present invention may further comprise a control reagent and an absorption site.
  • a control reagent is not particularly limited. For example, a substance to which an antibody in a labeled reagent binds may be used.
  • a control reagent may be immobilized at a site downstream of the capture reagent site. In FIG. 1 , a control site 4 corresponds to such site.
  • An absorption site is capable of absorbing a liquid such that it absorbs a sample that has passed through the capture site to regulate the flow of the sample. Such site may be provided at the lowermost site of the detection device.
  • an absorption site 6 corresponds to such site. For example, an absorption site made of paper may be used as an absorbent pad.
  • a sample supply site may be constituted by an end of a solid-phase support as is, or it can be constituted by a member different from a solid-phase support.
  • a sample supply site is provided in contact with a solid-phase support, so that a solution can spread and migrate to the solid-phase support with the aid of capillary flow, in order that a sample supply site first absorbs a sample, or a mixture of a sample and a labeled reagent, and then supplies the absorbed sample or mixture to the solid-phase support.
  • members other than a solid-phase support include, but are not limited to, members composed of natural or synthetic polymers of nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose, or polystyrene and a mixture of such substances.
  • the “labeled reagent” of the immunochromatography detection device of the present invention is a conjugate of an antibody binding specifically to PBP2′ and an adequate label substance.
  • a label substance include metal colloids, such as gold colloid, nonmetal colloids, such as selenium colloid, and insoluble substances such as colored resin particles.
  • label substances are occasionally referred to as “insoluble carriers.”
  • insoluble carriers are negatively charged.
  • a labeled reagent is impregnated with a member different from a solid-phase support, dried, and then placed at a site continuous with the solid-phase support. Alternatively, the solid-phase support may be directly applied with the labeled reagent and then dried.
  • the labeled reagent When the sample reaches a labeled reagent site containing a labeled reagent, the labeled reagent is dissolved in the sample, and the resulting solution can then be spread across the solid-phase support. Specifically, a labeled reagent is held at a labeled reagent site in a manner extensible on the support.
  • the capture reagent of the immunochromatography detection device of the present invention is an antibody binding specifically to PBP2′, a capture reagent site can specifically bind to and capture a complex of PBP2′ and a labeled reagent, and a complex of a labeled reagent/PBP2′/a capture reagent is then formed.
  • a capture reagent is prepared via direct applying of a solid-phase support, followed by drying, although the method of preparation is not limited thereto.
  • a member other than a solid-phase support may be impregnated with a capture reagent and then dried, and the resultant may be placed on the solid-phase support.
  • a method for immobilizing a capture reagent on a solid-phase support is not limited to adsorption. Immobilization may be carried out by a conventional technique, such as chemical binding with the use of a functional group such as an amino or carboxyl group.
  • An antibody to be used as a capture reagent may be same as an antibody to be used as a labeled reagent.
  • a complex of a labeled reagent/PBP2′/a capture reagent would not be formed.
  • a capture reagent is required to bind to a site of PBP2′ that is different from a site to which a labeled reagents binds.
  • a solid-phase support may be of any substance, provided that a sample can be absorbed by such support and fluidized via capillary phenomenon.
  • a support is selected from the group consisting of a natural or synthetic polymer of nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose, or polystyrene and a mixture of such substances.
  • a solid-phase support is preferably strip-shaped.
  • the cell-wall-synthesizing enzyme, PBP2′ that is produced specifically by a bacterium of multidrug-resistant staphylococcus is present on a cell membrane located inside the cell wall. Pretreatment of breaking or melting the cell wall of multidrug-resistant staphylococcus facilitates a capture reagent and a labeled reagent to recognize PBP2′.
  • a cell-wall-digesting enzyme or a given surfactant having bactericidal action such as a given cationic surfactant or ampholytic surfactant.
  • a dilute alkaline solution is preferable as an extraction reagent for PBP2′ at the time of immunochromatography detection in the present invention.
  • a dilute alkaline solution includes an aqueous solution of 0.01N to 1.0N alkali metal hydroxide or carbonate and an aqueous solution of 0.01N to 1.0N alkaline earth metal hydroxide or carbonate.
  • the pH of such dilute alkaline solution is preferably 11 or higher.
  • a neutralization solution is not particularly limited, and examples thereof include phosphate buffer, Tris buffer, and Good's buffer, such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, or TAPS, exhibiting buffering capacity at the optimal pH of the reaction system, i.e., between 6 and 8, for immunochromatography detection.
  • pretreatment of bacteria is a process of extracting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, and more particularly, PBP2′ as an antigen by exposing a solution of bacteria deduced to contain multidrug-resistant staphylococcus to alkaline conditions.
  • Pretreatment also refers to a process of neutralizing alkaline conditions. Pretreatment may be carried out by adding an alkaline solution to a sample solution comprising bacteria for alkalinization and then adding a neutralizing solution thereto. Alternatively, bacteria collected with the use of a platinum loop or cotton swab may be suspended directly in an alkaline solution, and a neutralizing solution may then be added. The amounts of such solutions to be added are not limited, and such amounts may be determined so as to bring the pH of the neutralized sample solution to around 6 to 9. Also, a sample supply site may be impregnated with a neutralizing solution in advance and then dried.
  • the solution of bacteria may be pretreated and then neutralized, additives, such as salt, a surfactant, a protein, a polymer, an acidic compound, and a basic compound, may be added thereto, the resultant may be thoroughly mixed, and the sample may then be supplied to the sample supply site of the immunochromatography detection device.
  • additives can enhance sensitivity of an antigen-antibody reaction and can reduce false-positive reactions (e.g., false-positive reactions due to nonspecific reactions). Further, such additives may be added to the aforementioned dilute alkaline solution or neutralizing solution in advance to reduce procedural steps.
  • substances derived from non-multidrug-resistant staphylococci may occasionally bind to antibodies (i.e., as a labeled reagent and a capture reagent) and may show false-positive results.
  • a capture reagent site of the immunochromatography detection device may be impregnated with a surfactant to inhibit binding of a false-positive causative agent to a capture antibody, without inactivating the false-positive causative agent by boiling.
  • a surfactant to inhibit binding of a false-positive causative agent to a capture antibody, without inactivating the false-positive causative agent by boiling.
  • An ampholytic surfactant, an anionic surfactant, or a nonionic surfactant is preferable, and including one of them may be sufficient.
  • Inhibition of binding of a false-positive causative agent to a capture antibody with the aid of a surfactant is considered to result from masking of a site of an antibody to which a false-positive causative agent binds by a surfactant.
  • surfactants with larger molecular weights and/or greater numbers of cyclic structures are considered to be more effective.
  • any of the following nonionic surfactants are used, for example, false-positive results originating from non-multidrug-resistant Staphylococcus aureus that is not multidrug-resistant are more inhibited compared with the case where no surfactant was added: a nonionic surfactant having a molecular weight of 646 and a single 6-membered ring structure (tradename: Tx100; Nacalai Tesque); an ampholytic surfactant having a molecular weight of 364 without a 6-membered ring structure (tradename: SB3-14; Calbiochem); an ampholytic surfactant having a molecular weight of 615, three 6-membered ring structures, and a single 5-membered ring structure (tradename: CHAPS; DOJINDO LABORATORIES); or a nonionic surfactant having a molecular weight of 878, and
  • the molecular weight of a surfactant is considered to be preferably 300 or higher, and more preferably 600 or higher, and a surfactant is considered to preferably comprise a greater number of cyclic structures, such as 6-membered rings.
  • a surfactant that is charged in the same manner as with the case of an insoluble carrier is preferable.
  • an insoluble carrier is a negatively charged latex or gold colloid, it is preferable that a surfactant also be negatively charged.
  • a false-positive reaction resulting from protein A present in the cell wall of Staphylococcus aureus is of particularly serious concern.
  • Protein A strongly binds to the Fc region of immunoglobulin G.
  • Antibodies that specifically recognize PBP2′ are used as labeled reagents and capture reagents.
  • Antibodies may be immunoglobulin G or immunoglobulin M, without particular limitation. When immunoglobulin G is used, such antibody is used while eliminating the Fc region, and a capture reagent region is combined with the aforementioned surfactant. Thus, a false-positive reaction caused by protein A can be avoided.
  • the Fc region can be easily eliminated with the use of a known degrading enzyme, such as pepsin or papain.
  • Protein G contained in the cell wall of group G Streptococcus also binds specifically to the Fc region of immunoglobulin G.
  • protein G may nonspecifically react with a labeled reagent or a capture reagent, as with the case of protein A. Such false-positive reaction can also be avoided in the above-described manner.
  • an ionic surfactant is particularly affected by the concentration of anions or cations in the solution that passes through the capture reagent region. This necessitates determination of the optimal concentration of anions or cations in the solution in accordance with the type, concentration, properties, or other conditions of a surfactant to be used.
  • anions include a chloride ion, a bromide ion, and an iodide ion having a high ionization tendency.
  • cations include a potassium ion, a calcium ion, a sodium ion, and a magnesium ion having a high ionization tendency. In this case, a plurality of anions or cations may be used.
  • Detection sensitivity can be improved by adding a surfactant containing a sulfobetaine region to a capture reagent site, which is the method according to the present invention. Since PBP2′ could not be detected with high sensitivity with the use of a PBP2′ detection reagent via latex agglutination assay, the cell wall had to be broken via boiling of a solution of bacteria in the presence of an alkaline reagent in order to completely extract PBP2′. According to the present invention, detection sensitivity is improved with the addition of a surfactant containing a sulfobetaine region, which enabled detection of PBP2′ with high sensitivity even when elution of PBP2′ is incomplete in the absence of boiling. Boiling in the presence of an alkaline reagent would cause PBP2′ degradation, or would cause desensitization via denaturation of an antibody recognition site due to structural changes; however, detection sensitivity can be advantageously maintained with the absence of boiling.
  • surfactants each containing a sulfobetaine region examples include CHAPS, CHAPSO, myristylsulfobetaine (SB3-14), and dodecyl dimethyl ammonio butane sulfonate (DDABS).
  • a surfactant for inhibiting binding of the false-positive causative agent to a capture antibody, and a surfactant for improving sensitivity are added to a capture reagent in advance when a capture reagent is to be immobilized at a capture reagent site.
  • a surfactant for inhibiting binding of the false-positive causative agent to a capture antibody and a sulfobetaine-type surfactant capable of improving sensitivity may be used.
  • a surfactant for inhibiting binding of the false-positive causative agent to a capture antibody may be a sulfobetaine-type surfactant, and sensitivity can be improved in such a case.
  • the method of the present invention can produce the effect of inhibiting binding of a false-positive causative agent to a capture antibody and the effect of improving sensitivity by adding a surfactant containing a sulfobetaine region having a molecular weight of preferably 300 or higher, and more preferably 600 or higher, to a capture reagent site.
  • a surfactant containing a sulfobetaine region having a molecular weight of preferably 300 or higher, and more preferably 600 or higher a capture reagent site.
  • CHAPS and SB3-14 are preferable, with CHAPS being particularly preferable.
  • a plurality of surfactants may be added to the capture reagent site.
  • anion or cation When an anion or cation is added as described above, such anion or cation may be added to a sample solution deduced to contain multidrug-resistant staphylococcus or a sample solution deduced to contain PBP2′ released from the cell wall via sample pretreatment.
  • the method of the present invention further includes a method of preventing a false-positive causative agent from reaching the capture reagent site, thereby preventing influences of such false-positive causative agent.
  • a false-positive causative agent may be eliminated at a site upstream of the capture reagent site in the device of the present invention.
  • a sample supply site of the immunochromatography detection device preferably comprises glass fibers.
  • a bacteria-derived false-positive causative agent is adsorbed by glass fibers, which inhibits the false-positive causative agent from passing through the capture reagent. This can consequently inhibit a false-positive reaction.
  • the method of the present invention is a detection method based on an antigen-antibody reaction using a cell-wall-synthesizing enzyme, PBP2′, as an antigen and an antibody reacting therewith. If a cell-wall-synthesizing enzyme, PBP2′, is present in the sample, accordingly, PBP2′ can be detected by the method of the present invention.
  • a cell-wall-synthesizing enzyme, PBP2′ is produced specifically by a multidrug-resistant staphylococcus , and is present specifically in such bacterium.
  • multidrug-resistant staphylococcus is occasionally used as a specific example instead of the term “a bacterium producing a cell-wall-synthesizing enzyme, PBP2′.” Such use is not intended to limit the bacterium to “a multidrug-resistant staphylococcus .”
  • This term can be read as “a bacterium producing a cell-wall-synthesizing enzyme, PBP2′” within the scope that allows the method of the present invention to be implemented. That is, bacteria producing PBP2′ other than multidrug-resistant staphylococcus can be detected by the method of the present invention.
  • bacteria each containing a cell-wall-synthesizing enzyme, PBP2′ can be detected by the method of the present invention.
  • a cell-wall-synthesizing enzyme, PBP2′ is considered to be present specifically on a cell membrane inside the cell wall of a multidrug-resistant staphylococcus .
  • the method of the present invention involves the use of a PBP2′ extraction reagent used for pretreatment of breaking or melting the cell membrane. Accordingly, bacteria each comprising a cell-wall-synthesizing enzyme, PBP2′, inside the cell wall can be effectively detected by the method of the present invention.
  • An anti-PBP2′ monoclonal antibody was treated with pepsin in accordance with conventional technique to obtain F(ab′)2.
  • the resultant was subjected to sensitization of 0.4 ⁇ m of latex particles, and the resulting solution was sprayed on an unwoven polystyrene fabric.
  • the resultant was then dried under reduced pressure for 1 hour in a decompression device to prepare a dry latex antibody pad.
  • the pad was cut at intervals of 4 mm for use and used as a labeled site 2 .
  • a second anti-PBP2′ monoclonal antibody having a recognition site different from that of the anti-PBP2′ monoclonal antibody used for latex sensitization was treated with pepsin in accordance with a conventional technique to obtain F(ab′)2.
  • the resultant was diluted with a citrate buffer (pH 6) containing 0.075% CHAPS, the resultant was applied to a nitrocellulose membrane (solid-phase support 5 ), and the membrane was thoroughly dried (capture reagent site 3 ).
  • As a control reagent anti-mouse IgGs was applied to a nitrocellulose membrane in the same manner and thoroughly dried (control site 4 ).
  • a capture reagent site 3 and a solid-phase support 5 including a control site 4 were provided on a hydrophobic sheet 7 , and a labeled reagent site 2 , a glass fiber as a sample supply site 1 , and a filter paper as an absorption site 6 were provided at any positions.
  • Staphylococcus aureus 14 bacteria were cultured on blood agar medium at 35° C. overnight, and a loopful of culture was suspended directly in 0.2N NaOH. Similarly, a colony was picked up and then suspended in 100 ⁇ l of 0.2N NaOH. The resultant was then neutralized with 50 ⁇ l of 0.6M Tris-HCl buffer containing a nonionic surfactant, bovine serum albumin, and rabbit IgG.
  • the immunochromatography detection device was introduced into a 1.5-ml tube containing a solution after pretreatment of samples, color development of a capture reagent 3 was evaluated with the naked eye ten minutes later, and the capture reagent that had developed color was identified as positive. The reagent that had not developed color was identified as negative.
  • Bacteria cultured in the same manner as in (3) were suspended in a sterilized 0.9% sodium chloride solution, and the absorbance at 578 nm of the solution was adjusted to 0.3. The resulting solution was sowed on Muller-Hinton agar medium containing 6 ⁇ g/ml oxacillin and 4% sodium chloride, culture was conducted at 35° C. for 24 hours, the bacteria that had grown were determined to be MRSA, and the bacteria that had not grown were determined to be MSSA.
  • the latex particles prepared in Example 1 were used.
  • the device was prepared in the same manner as in Example 1. In this case, an device comprising a capture reagent site and 0.05% CHAPS, Tx100, SB3-14, BIGCHAP, and DTAC (dodecyl trimethyl ammonium chloride) added thereto and an device comprising no such substances were prepared and used.
  • a bacterium that had been evaluated as being drug-sensitive, i.e., MSSA, and a bacterium that had been evaluated as being drug-resistant, i.e., MRSA, via the drug sensitivity test were cultured in the same manner as in Example 1. Two and three loopfuls of MSSA and a loopful of MRSA were picked up and then pretreated.
  • Example 2 An MSSA was tested in the same manner as in Example 1 without dilution. Further, the MRSA was subjected to two-step dilution with a mixture comprising 0.2N NaOH and 0.6M Tris HCl buffer (containing a nonionic surfactant, bovine serum albumin, and rabbit IgG) at 2:1, and 150 ⁇ l thereof diluted at least 1024-fold was tested in the same manner as in Example 1.
  • Tris HCl buffer containing a nonionic surfactant, bovine serum albumin, and rabbit IgG
  • results attained with the device comprising a surfactant added to a capture reagent site and the results attained with the device comprising no surfactant were compared.
  • the results attained with the use of MSSA are shown in Table 2.
  • the negative results are represented by “ ⁇ ,” and the degree of false-positive reaction are represented by the number of “+”'s.
  • results attained with the use of MRSA are shown in Table 3.
  • a negative result is represented by “ ⁇ ,” and a positive result is represented by “+.”
  • Detection sensitivity was improved with the addition of CHAPS or SB3-14 to a capture reagent site.
  • the results attained with the addition of DTAC were considered to constitute a false-positive.
  • Example 2 demonstrate that CHAPS particularly shows satisfactory effects of inhibiting a false-positive reaction and those of improving sensitivity and that CHAPS is thus particularly useful.
  • the latex particles prepared in Example 1 were used.
  • the device was prepared in the same manner as in Example 1.
  • staphylococci that exhibit properties of staphylococci via gram staining and that had been evaluated to be positive via a catalase test and to be negative via a coagulase test
  • two bacteria that had been evaluated as being methicillin-sensitive, i.e., MSCNS, via a drug sensitivity test and a bacterium that had been evaluated as being methicillin-resistant, i.e., MRCNS, via a drug sensitivity test were cultured in the same manner as in Example 1, and a loopful thereof was picked and then subjected to pretreatment.
  • results are shown in Table 4.
  • the results of the drug sensitivity test are represented as follows: S: sensitivity; R: resistance; +: positive result; and ⁇ : negative result.
  • the two strains that had been evaluated as being sensitive, i.e., MSCNS, via the drug sensitivity test showed negative results, and a strain that had been evaluated as being resistant, i.e., MRCNS, via the drug sensitivity test showed positive results.

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US20120252004A1 (en) * 2011-03-31 2012-10-04 Fujifilm Corporation Highly sensitive immunochromatography method
US8679812B2 (en) 2009-12-28 2014-03-25 Kikkoman Corporation Method for extracting Staphylococcus aureus antigen, reagent for extracting Staphylococcus aureus antigen, and method for assessing Staphylococcus aureus
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JP2009258094A (ja) * 2008-03-18 2009-11-05 Hitachi Chem Co Ltd クロマトグラフィー分析用ストリップ及びその製造方法
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JP2019142811A (ja) * 2018-02-21 2019-08-29 コージンバイオ株式会社 イムノクロマトグラフィー用のニューデリーメタロβ−ラクタマーゼ（ＮＤＭ型ＭＢＬ）に対するモノクローナル抗体、ＮＤＭ型ＭＢＬ用のイムノクロマトグラフィー装置及びそのキット、並びにＮＤＭ型ＭＢＬの検出方法
JPWO2021193680A1 (ko) * 2020-03-25 2021-09-30
JP2022045189A (ja) * 2020-09-08 2022-03-18 デンカ株式会社 偽陽性の抑制により特異性を改善した検査キット

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US20120252004A1 (en) * 2011-03-31 2012-10-04 Fujifilm Corporation Highly sensitive immunochromatography method
CN109813898A (zh) * 2017-11-18 2019-05-28 镇江亿特生物科技发展有限公司 一种检测头孢噻呋药物残留的快速检测试纸条

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