Patent Application
20100015596
The present invention relates to a respiratory syncytial virus detecting kit using anti-respiratory syncytial virus monoclonal antibody which can detect respiratory syncytial virus with a high level of sensitivity, and an immuno-chromatographic test device. The present invention also relates to an anti-respiratory syncytial virus monoclonal antibody which can be used for manufacturing the kit or the device.
Respiratory syncytial virus (hereinafter referred to as “RSV” or “RS virus”) is a main causal virus developing respiratory infections in infants. Usually, infants infected with RSV develop cold-like slight symptoms. However, there are cases where immunocompromised infants or elderly persons when infected with RS virus develop bronchiolitis and even die of breathing difficulties. Accordingly, it has been desired to easily and rapidly detect infection with this virus.
RSV is classified into subtypes (types A and B) based mainly on serological differences. A Long strain, A2 strain or the like is known as type-A RSV. A 9320 strain, B1 wild type strain or the like is known as type-B RSV.
In an envelope of RSV, there are a glycoprotein G protein and a cell fusion-related F protein. It is known that the amino acid sequence of G protein is considerably different between the subtypes (types A and B) of RSV. On the other hand, it is known that the amino acid sequence of F protein is slightly different even between the subtypes (types A and B) of RSV.
Some anti-RSV monoclonal antibodies recognizing RSV are known. WO 98/19704 discloses modified monoclonal antibodies binding to the F protein of RSV and having a neutralizing activity. WO 03/063767 discloses human RSV antibodies. WO 96/40252 also discloses human monoclonal antibodies binding to the F protein of RSV.
Anti-RSV monoclonal antibodies binding to the F protein of RSV, which are obtained from various clones, are commercially available.
Further, RSV detection kits based on immuno-chromatographic techniques using anti-RSV monoclonal antibodies, for example BD RSV Examan (Becton Dickinson), Check RSV (Alfresa-Pharma Co. Japan), BinaxNOW (Eiken Chemical Co., Ltd.), Immunocard (TFB, Inc.), and Poctem S RSV (Sysmex Corporation), are also commercially available.
However, these conventional kits for detection of RSV, when used to detect RSV, are poor in sensitivity and sometimes judge 30% to 40% of positive samples to be negative.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
A first aspect of the present invention is a kit for detection of RSV, comprising an RS virus F protein-recognizing anti-RSV monoclonal antibody produced by hybridoma RSF2-412 deposited on Apr. 20, 2009, under Accession No. NITE BP-601, with Incorporated Administrative Agency National Institute of Technology and Evaluation Patent Microorganisms Depositary.
A second aspect of the present invention is an immuno-chromatographic test device for detection of RSV, comprising at least an RSV F protein-recognizing anti-RSV monoclonal antibody produced by hybridoma RSF2-412.
A third aspect of the present invention is an anti-RSV monoclonal antibody recognizing an RS virus F protein, which is selected from the group consisting of an antibody produced by hybridoma RSF2-412, an antibody produced by hybridoma RSF1-1565, and an antibody produced by hybridoma RSF6-255.
The preferred embodiments of the present invention are described hereinafter with reference to the drawings.
The anti-RSV monoclonal antibody used in the RSV detection kit and immuno-chromatographic test device of the present invention is produced by hybridoma RSF2-412 (referred to hereinafter as “hybridoma 412”). This antibody is referred to hereinafter as “antibody 412”.
The antibody of the present invention can be obtained from a hybridoma created by a method known per se. That is, an RSV antigen, preferably the RSV F protein, which is optionally mixed with a suitable adjuvant, is used to immunize a suitable mammal (for example, a mouse, a rat or the like). Then, antibody-producing cells such as spleen cells, lymph node cells or B lymphocytes from the animal can be fused with myeloma cells derived from a suitable mammal (for example, a mouse, a rat or the like) to yield hybridoma. Usually, the antibody-producing cells and the myeloma cells are derived from the same kind of animals.
Cell fusion can be carried out, for example, by the PEG method wherein antibody-producing cells and myeloma cells are fused with each other in the presence of polyethylene glycol (PEG) or the like in a suitable medium. After cell fusion, the resulting hybridoma is selected in a selective medium such as HAT medium and screened by a conventional method (for example, enzyme immunoassay (EIA)) for their ability to produce antibodies recognizing the RSV F protein. Then, a hybridoma producing an appropriate antibody is cloned by a conventional method (for example, limiting dilution method), thereby selecting the hybridoma producing a monoclonal antibody.
In the present invention, hybridoma 412 was obtained by fusing mouse spleen cells obtained by immunizing a mouse with the RSV F protein, with mouse myeloma cells, as shown later in the Examples. The hybridoma 412 was deposited on Jun. 26, 2008, with Incorporated Administrative Agency National Institute of Technology and Evaluation Patent Microorganisms Depositary (2-5-8 Kazusakamatari Kisarazu-shi, Chiba-ken, Japan) and converted to international deposition (Date of conversion of deposit, Apr. 20, 2009; Accession No. NITE BP-601).
In the present invention, hybridoma RSF1-1565 (hereinafter referred to as “hybridoma 1565”) and hybridoma RSF6-255 (hereinafter referred to as “hybridoma 255”) were obtained according to the same method as described above. The hybridoma 1565 was deposited on Jul. 19, 2007, with International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (AIST) (Tsukuba Central 6, 1-1, Higashi 1-chome Tsukuba-shi, Ibaraki-ken, Japan) and converted to international deposition (Date of conversion of deposit, Apr. 20, 2009; Accession No. FERM BP-11119). The hybridoma 255 was deposited on Jun. 26, 2008, with International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology (AIST) and converted to international deposition (Date of conversion of deposit, Apr. 20, 2009; Accession No. NITEBP-602). Hereinafter, the anti-RSV monoclonal antibodies produced by the hybridomas 1565 and 255 are referred to as antibodies 1565 and 255, respectively.
A specific method of obtaining the hybridomas 412, 1565 and 255 will be described later in the Examples.
The antibodies 412, 1565 and 255 produced by the respective hybridomas can bind to the F protein of RSV. The amino acid sequence of F protein of RSV Long strain is known and set forth in SEQ ID NO: 1.
As will be described later in the Examples, these 3 antibodies are considered to recognize different epitopes of the RSV F protein.
In this specification, the “antibody” includes an antibody and an antibody fragment and modified antibody having an RSV F protein-binding property equivalent to that of the antibody. The antibody fragment includes Fab fragment, F(ab′)2 fragment, Fab′ fragment and sFv fragment.
The kit for detection of RSV in the present invention contains at least antibody 412 produced by hybridoma 412.
In this specification, the “kit for detection of RSV” contains a plurality of constituent elements serving as a group wherein the plurality of constituent elements can be used in combination to detect RSV. The kit for detection of RSV in the present invention consists of a first constituent element containing the antibody 412 and other constituent elements. These constituent elements can be a solution with or without containing the antibody, a solid carrier with or without containing the antibody, or a combination thereof.
Preferably, the kit of the present invention further contains an anti-RSV antibody (hereinafter referred to as “other antibody”) recognizing an epitope of the RSV F protein which is different from the epitope recognized by the antibody 412. The other antibody may be one or more antibodies, preferably two antibodies. More preferably, the kit of the present invention contains three antibodies, that is, the antibody 412 and two other anti-RSV antibodies, thereby attaining higher sensitivity in detection of RSV than by the conventional RSV detection kits.
Whether the epitopes recognized by the other antibodies are identical with the epitope recognized by the antibody 412 can be examined by a known method. The known method includes a competitive inhibition test. The competitive inhibition test is a method wherein the ability of an objective antibody to competitively inhibit the binding of an arbitrary antibody to an antigen is examined thereby determining whether the epitopes to which the respective antibodies bind are the same or not. More specifically, the method carried out under the conditions shown later in Example 3 can be mentioned. The arbitrary antibody is not particularly limited as long as it is an antibody other than the objective antibody.
For example, when the binding, which can be inhibited by the antibody 412, of an arbitrary antibody to an RSV antigen cannot be inhibited by a certain anti-RSV antibody in the competitive inhibition test, then this anti-RSV antibody is the “other antibody” mentioned above. When the binding, which cannot be inhibited by the antibody 412, of an arbitrary antibody to an RSV antigen can be inhibited by a certain anti-RSV antibody, then this anti-RSV antibody is also the “other antibody” mentioned above.
In the competitive inhibition test, the phrase “can be inhibited” means that the binding of an arbitrary antibody to an RSV antigen is inhibited by 30% or more, preferably 50% or more, more preferably 90% or more.
In the competitive inhibition test, the phrase “cannot be inhibited” means that the binding of an arbitrary antibody to an RSV antigen is inhibited by less than 30%, preferably less than 10%, most preferably 0%.
The antibody which can be contained in the kit of the present invention as the other antibody than the antibody 412 is preferably an anti-RSV monoclonal antibody not binding to a protein having the amino acid sequence set forth in SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 is the same as the amino acid sequence (SEQ ID NO: 1) of the RSV F protein except that a lysine residue at position 421 is changed by mutation to a threonine residue. The anti-RSV monoclonal antibody not binding to a protein having the amino acid sequence of SEQ ID NO: 2 includes, for example, the antibody 1565 (see Example 5).
Other preferable antibodies include the antibody 255. Both the antibodies 1565 and 255 are contained particularly preferably as the other antibodies in the kit.
The kit of the present invention is not particularly limited as long as it contains the antibody 412 and contains constituent elements enabling an immunoassay to detect an antigen through an antigen-antibody reaction. Such constituent elements are known to those killed in the art.
The immunoassay includes immunoprecipitation and labeling immunoassay. The labeling immunoassay includes immuno-chromatography, enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA) and chemiluminescent immunoassay.
The kit of the present invention is preferably a kit intended for labeling immunoassay. In such a kit, at least one of the anti-RSV monoclonal antibodies used is labeled with a labeling substance. The antibody labeled with a labeling substance is preferably an anti-RSV monoclonal antibody not binding to a protein having the amino acid represented by SEQ ID NO: 2, particularly preferably the antibody 1565.
The labeling substance is not particularly limited as long as it is a labeling substance that can be used in usual immunoassays. Examples of the labeling substance include an enzyme, a fluorescent substance, a radioisotope, an insoluble granular substance, and the like.
The enzyme includes an alkaline phosphatase, peroxidase, glucose oxidase, tyrosinase, acid phosphatase, and the like.
The fluorescent substance includes fluorescein isothiocyanate (FITC), green fluorescence protein (GFP), luciferin, and the like.
The radioisotope includes 125I 14C, 32P, and the like.
The insoluble granular substance includes, for example, agar, agarose, crosslinked agarose, crosslinked alginic acid, crosslinked guar gum, cellulose esters such as nitrocellulose and carboxyl cellulose, gelatin, crosslinked gelatin, latex, rubber, natural or synthetic resins such as polyethylene, polypropylene, polystyrene, styrene-butadiene copolymers, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymers, polyglycidyl methacrylate and acrolein-ethyleneglycol dimethacrylate copolymers, and derivatives of these resins. Labeled particles obtained by labeling particles consisting of an inorganic material such as glass (for example activated glass), silica gel, kaolin, talc, silica-alumina, alumina or barium sulfate, with pigment molecules, fluorescent molecules, magnetic particles or the like can also be used as the insoluble granular substance. Further, metal colloid particles such as gold colloids, erythrocytes, and the like can also be used as the insoluble granular substance. The insoluble granular substance is preferably metal colloid particles or colored synthetic polymer particles prepared by labeling the synthetic polymer with pigment molecules.
The labeling immunoassay is preferably a solid phase method of using the binding of an antibody to a solid phase.
In the solid phase method, at least one of the anti-RSV monoclonal antibodies used is immobilized on a solid phase.
The antibody immobilized on a solid phase is preferably at least one selected from the antibodies 412 and 255. More preferably, these two antibodies are immobilized on a solid phase.
The solid phase used in the solid phase method includes a microtiter plate, a membrane, particles, and the like.
The material of the solid phase includes polymer materials such as latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymers, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymers, polyglycidyl methacrylate, acrolein-ethyleneglycol dimethacrylate copolymers, silicone polyvinylidene difluoride (PVDF), celluloses (nitrocellulose, cellulose acetate, and the like), nylon (for example, a modified nylon into which a carboxyl group, and an amino group which may be substituted with an alkyl group, have been introduced); agarose; gelatin; erythrocytes; and inorganic materials such as silica gel, glass, inert alumina, and magnetic substance. The solid phase can be produced from one of these materials or a combination thereof.
The sample examined with the RSV detection kit of the present invention includes biological samples collected from patients suspected of being infected with RSV as well as samples obtained by pretreatment of the biological samples. The biological samples include blood, serum, stools, urine, saliva, nasal discharge, swabs, sweat, and tears. The pretreatment involves dissolving a biological sample in a suitable pretreatment reagent and then filtering the resulting solution.
When the solid phase is a microtiter plate, the kit of the present invention contains a microtiter plate and a solution containing at least the antibody 412. Alternatively, the kit of the present invention may be a kit containing a microtiter plate on which the antibody 412 is immobilized.
When the solid phase is a membrane, the kit of the present invention contains a membrane and a solution containing at least the antibody 412. Alternatively, the kit of the present invention may be a kit containing a membrane on which the antibody 412 is carried or immobilized.
When the solid phase is particles, the kit of the present invention contains particles and a solution containing at least the antibody 412. Alternatively, the kit of the present invention may be a kit containing particles on which the antibody 412 is immobilized.
The kit of the present invention is preferably a kit intended for immuno-chromatography. That is, the kit contains an immuno-chromatographic test device on which the antibody 412 is immobilized or carried.
In this specification, the phrase “the antibody is carried” means that the antibody is hold so as to be movable.
Accordingly, the present invention also provides an immuno-chromatographic test device on which the antibody 412 is immobilized or carried.
The immuno-chromatographic test device preferably has a sample addition member to which a sample likely of having RSV is added, a label holding member carrying an anti-RSV monoclonal antibody, and a chromatographic membrane carrier (hereinafter referred to as “chromatographic carrier”) having a judgment region on which an anti-RSV monoclonal antibody different from the antibody carried on the member holding member is immobilized.
In the immuno-chromatographic test device, the sample addition member and the label holding member are preferably in contact with each other.
The sample addition member and the chromatographic carrier are preferably in contact with each other. The sample addition member is preferably in contact with the chromatographic carrier at the upstream side in the direction of sample development in the chromatographic carrier.
The label holding member is preferably disposed on the upstream side in the direction of sample development in the chromatographic carrier. The label holding member may or may not come into contact with the chromatographic carrier.
In this specification, the terms “upstream side in the direction of sample development” and “downstream side in the direction of sample development” are indicated by relative directions assuming that the side on which a sample is added is the upstream side, while the side toward which a sample is developed (flows) is the downstream side.
In the immuno-chromatographic test device described above, the antibody carried on the label holding member is preferably labeled with the labeling substance described above. The labeling substance is preferably an insoluble granular substance to enable rapid and easy observation of color change with the naked eye, and is particularly preferably colored synthetic polymer particles or metal colloid particles.
In the immuno-chromatographic test device, the antibody labeled with a labeling substance is preferably an anti-RSV monoclonal antibody not binding to a protein having the amino acid sequence of SEQ ID NO: 2, more preferably the antibody 1565.
The anti-RSV monoclonal antibody immobilized on the judgment region is preferably at least one selected from the antibodies 412 and 255, more preferably these two antibodies.
The sample addition member is made preferably of a material such as cotton, glass fibers, a porous synthetic resin such as porous polyethylene or porous polypropylene, or cellulose fibers. The sample addition member can be a product of such a material, such as woven fabric, a nonwoven fabric, a filter paper, a sheet or a film.
The label holding member is made preferably of a material such as glass fibers, cellulose fibers, or plastic (for example, polyester, polypropylene or polyethylene) fibers.
Preferably, the label holding member further holds a control labeling substance to confirm whether the sample has been suitably developed or not. The control labeling substance is preferably substantially not reactive to components in the sample. The control labeling substance can be used in combination with the following substance capable of binding to the control labeling substance. That is, the control labeling substance, and the substance capable of binding to the control labeling substance are a combination of two substances capable of binding specifically to each other. Either of the two substances may be used as the control labeling substance. The combination of two substances capable of binding specifically to each other includes a combination of a hapten and a protein capable of binding thereto, for example a combination of biotin and streptavidin or avidin and a combination of 2,4-dinitrophenol or digoxin and a protein (for example, albumin, casein, fibrinogen or the like). Such a combination of a control labeling substance and a substance capable of binding to the control labeling substance is preferably a combination of substances that are not present in the sample.
A label for the control labeling substance may be the same label as the above labeling substance. The control labeling substance may be labeled with the same labeling component as in the antibody or may be labeled with a different labeling component.
The chromatographic carrier is made preferably of a material capable of binding to a protein via physical action such as electrostatic action or hydrophobic interaction and simultaneously developing the sample by capillary phenomenon. Such material of the chromatographic carrier includes nitrocellulose, nylon (for example, a modified nylon into which a carboxyl group, and an amino group which may be substituted with an alkyl group, have been introduced), polyvinylidene difluoride (PVDF), and cellulose acetates.
The chromatographic carrier preferably has a control part on which a substance capable of binding to the control labeling substance is immobilized in order to confirm whether the sample has been suitably developed or not.
The substance capable of binding to the control labeling substance is as described above in connection with the control labeling substance.
The immuno-chromatographic test device can further include an absorption member. The absorption member may use a woven or nonwoven fabric made of a material such as cellulose or glass fibers, or porous plastics such as polyethylene or polypropylene. The absorption member is preferably disposed so as to contact with the chromatographic carrier, at a position downstream, in the direction of sample development, of the chromatographic carrier. By arranging the absorption member at such a position, the test device can increase the development speed of a sample.
The immuno-chromatographic test device preferably has a substrate so that the sample addition member, the label holding member, the chromatographic carrier, and arbitrarily an absorption member can be held together. The substrate is not particularly limited as long as it is used as a substrate for a usual immuno-chromatographic test device. For example, plastics, paper or glass may be used as the substrate. The substrate preferably has a sticky surface to dispose the respective members thereon.
The immuno-chromatographic test device of the present invention can be produced in the same manner as for known immuno-chromatographic test devices. That is, the label holding member can be prepared by dipping a member for holding a label, in a suitable buffer solution containing an antibody, preferably a labeled antibody, and then drying it. Alternatively, the label holding member can be prepared by adding the buffer solution to a member for holding a label, and then drying it. The chromatographic carrier can be prepared by allowing a suitable antibody to be held on a judgment region and drying it, thereby immobilizing the antibody on the judgment region. The label holding member, the chromatographic carrier and the sample addition member can be suitably assembled and cut to give the immuno-chromatographic test device.
The substrate 2 is used in appropriately disposing the members such as the sample addition member 3 and the label holding member 4 thereon. The substrate 2 may be a material such as paper or glass.
A sample is added to the sample addition member 3. The sample is not particularly limited as long as there is a possibility that the sample may contain RSV. The sample is particularly preferably a nasal discharge, a nasal swab or a throat swab. The sample may be added after dilution with a suitable solvent such as a buffer solution.
In this embodiment, the label holding member 4 is disposed in contact with the sample addition member 3 and carries a first antibody (antibody 1565) labeled with a labeling substance.
In this embodiment, the chromatographic carrier 5 is disposed so as not to directly contact the label holding member 4. The chromatographic carrier 5 has a judgment region 5A on which a second antibody reacting, through an antigen-antibody reaction, with a measurement object is immobilized. The second antibody is an antibody different from the first antibody. In this embodiment, both antibodies 412 and 255 are used as the second antibody. The antibody 412 when contained in either the label holding member or the judgment region can be used in combination with another anti-RSV monoclonal antibody besides the anti-RSV monoclonal antibody of the present invention.
The absorption member 6 is disposed in contact with the chromatographic carrier 5 and arranged to absorb an excess of the sample. The absorption member 6 is one capable of rapidly absorbing and maintaining a liquid. A part of the sample addition member 3 and the surface of the absorption member 6 may be covered with a transparent sheet 7 as shown in
The immuno-chromatographic test device 1 of the present invention can be prepared for example in the following manner.
The sample is mixed if necessary with a suitable solvent to form a mixed solution capable of chromatographic development. Then, the upstream (sample addition member 3) side of the immuno-chromatographic test device 1 is immersed in the mixed solution, or the mixed solution is added to the sample addition member. By so doing, the mixed solution passes through the sample addition member 3 and mixed with the labeled first antibody in the label holding member 4. If an RSV antigen is present in the mixed solution, the RSV antigen is bound, via an antigen-antibody reaction, to the first antibody to form a complex. This complex moves in the chromatographic carrier 5 and reaches the judgment region 5A. The complex reacts, via an antigen-antibody reaction, with the second antibody immobilized on the judgment region 5A, so that the complex is captured by the judgment region 5A. When colored synthetic polymer particles such as blue latex particles are used as the labeling substance, these particles accumulate thereby staining the judgment region 5A blue. The presence of RSV can thereby be immediately confirmed visually.
The chromatographic carrier 5 may be provided not only with only one judgment region but also with two or more judgment regions. The chromatographic carrier 5 may also be provided with a control portion. When the control portion is arranged, for example, a labeling substance (for example red latex particles)—labeled avidin is movably held on the label holding member 4, and biotin capable of specific binding to avidin is immobilized on the control portion in the chromatographic carrier 5.
The kit for detection of RSV and the immuno-chromatographic test device in the present invention, as compared with conventional RSV detection methods, can improve the sensitivity of RSV detection. Accordingly, the present invention enables more accurate detection of RSV to be effected rapidly and easily, thus providing an indicator for more accurate diagnosis of RSV infection.
The present invention will be described in more detail with reference to the Examples, but the present invention is not limited to these examples.
100 μL of a phosphate buffer solution (PBS) containing a commercial RSV antigen obtained from Capricon was mixed with 100 μL of Freund's complete adjuvant (FCA) and emulsified to prepare 200 μL of an FCA RSV antigen solution. Separately, 200 μL of an FIA RSV antigen solution was prepared in the same manner as described above except that Freund's incomplete adjuvant (FIA) was used in place of FCA.
A 7- to 8-week-old female Balb/c mouse was initially immunized by intraperitoneal injection of 200 μL of the FCA RSV antigen solution. After the initial immunization, booster immunization with 200 μL of the FIA RSV antigen solution was carried out every 2 to 3 weeks. Ten days and three days before removal of splenocytes, 200 μL of an RSV antigen (manufactured by Capricon) was intravenously injected. Three days after final administration, splenocytes were separated and fused with mouse myeloma cells P3X63-Ag8•653 to prepare hybridomas.
The hybridomas were suspended at a density of 2.5×106 cells/mL in HAT medium and dispensed to each well (2.5×105 cells/well) of a 96-well plate (manufactured by Corning Inc.; referred to hereinafter as culture plate). The culture plate was left in an 8% CO2 incubator at 37° C., to initiate culture of the hybridomas. When the cells were cultured for 10 or more days until hybridoma colonies appeared, a monoclonal antibody-producing hybridoma was screened.
An RSV antigen (manufactured by Capricon) was added to a concentration of 0.5 μg/mL to 0.1 M phosphate buffer solution (PBS) (pH 7.5) containing 0.1 w/v % NaN3, to prepare an RSV antigen solution for immunization. 100 μL of this RSV antigen solution for immunization was dispensed into each well of an immunomodule (manufactured by NUNC) (hereinafter, the immunomodule will be referred to as antigen-immobilized plate). The antigen-immobilized plate was left overnight at 4° C. and then washed 3 times with a PBS buffer solution containing Tween 20 at a concentration of 0.05% (hereinafter, referred to as buffer solution A). After washing, 300 μL of PBS containing BSA at a concentration of 1 w/v % (hereinafter, referred to as buffer solution B) was added to each well of the antigen-immobilized plate and left for at least 4 hours at 2° C. to 8° C. The antigen-immobilized plate was stored at 2° C. to 8° C. before use.
The buffer solution B was removed from the antigen-immobilized plate. After removal of the buffer solution, 75 μL of buffer solution B was added to each well of the antigen-immobilized plate. A culture supernatant of the hybridoma described above was removed from each well of the culture plate and then added in a volume of 25 μL to each well of the antigen-immobilized plate. After the buffer solution B and the culture supernatant were added, the plate was stirred at room temperature for 1 hour. After stirring, each well of the antigen-immobilized plate was washed 3 times with buffer solution A. After washing, 100 μL of horseradish peroxidase (POD)-labeled anti-mouse Ig polyclonal antibody (Code No. P0447, manufactured by DAKO) was added to each well of the antigen-immobilized plate and incubated at room temperature for 30 minutes. After incubation, each well of the antigen-immobilized plate was washed 3 times with buffer solution A. After washing, 100 μL of a substrate solution containing ortho-phenylene diamine (OPD) as a substrate of POD was added to each well and then left at room temperature for 10 minutes. Then, 100 μL of a reaction termination solution containing 2 N H2SO4 was added to each well of the antigen-immobilized plate. The reaction solution in each well was measured for its absorbance at 492 nm with a microplate reader (manufactured by Molecular Devices).
The resulting three hybridomas RSF1-1565, RSF2-412 and RSF6-255have been internationally deposited (Accession No. FERM BP-11119, Accession No. NITE BP-601, and Accession No. NITE BP-601).
As RSV-F antigens, Long strain (10 [6.7] TCID (50)/ml), A2 strain (10 [5.5] TCID (50)/ml), 9320 strain (10 [5.5] TCID (50)/ml) and B1 wild-type (BWV) strain (10 [4.5] TCID (50)/ml) were used. These 4 strains of RSV can be purchased from American Type Culture Collection (ATCC). Each of the RSV strains was diluted to 1/10 with a phosphate buffer solution containing bovine serum albumin (BSA) (hereinafter referred to as first buffer solution).
Each diluted antigen solution and an analyte extracting reagent (100 mM citric acid/0.4 M NaCl, 10 mM dithiothreitol, 0.1 (v/v) % polyoxyethylene(9) octylphenyl ether) were mixed with each other in equal volumes and then subjected to extraction treatment for 5 minutes to prepare each antigen extract.
A Sepharose bead solution in which Sepharose beads (manufactured by Amersham Biosciences) to which commercial anti-mouse antibody IgG had been bound were contained at a concentration of 15 v/v % was prepared.
The Sepharose bead solution, each antigen extract described above, and the first buffer solution were mixed to prepare each antigen sample. The amounts of the Sepharose bead solution and each antigen extract in each antigen sample are shown in Table 1.
The antigen sample was added in a volume of 60 μL/well to each well of a V-shaped 96-well plate (manufactured by Sanplatec Co., Ltd.). Then, the antibody 1565, antibody 412 and antibody 255 were diluted to 1 μg/ml with the first buffer solution to prepare an antibody 1565 solution, an antibody 412 solution and an antibody 255 solution, respectively. The prepared antibody solutions were added separately to wells in a volume of 30 μL/well. Thereafter, the V-shaped 96-well plate was stirred at room temperature for 60 minutes. After stirring, the V-shaped 96-well plate was left for 10 minutes.
A solution of antibody 133-1H (antibody against RSV; manufactured by CHEMICON) diluted to a concentration of 10 μg/mL with 0.1 M sodium phosphate buffer solution was dispensed in a volume of 100 μL to each well of an immunomodule (manufactured by NUNC). Each well was washed 3 times with a Tween 20-containing phosphate buffer solution (referred to hereinafter as second buffer solution). After washing, each well was blocked by dispensing 300 μL of the first buffer solution to each well (hereinafter, this immunomodule will be referred to as antibody 133-1H-sensitized plate).
The first buffer solution was removed from the antibody 133-1H-sensitized plate. After removal of the buffer solution, 50 μL of the supernatant in each well of the above V-shaped 96-well plate which had been left for 10 minutes was added to each well of the antibody 133-1H-sensitized plate. Then, the antibody 133-1H-sensitized plate was stirred at room temperature for 30 minutes, and then the antibody 133-1H-sensitized plate was washed 3 times with the second buffer solution.
100 μL of a first buffer solution containing biotin-labeled antibody B016 (final concentration of 2 μg/mL, manufactured by Serotec) and streptavidin-labeled peroxidase (POD, final concentration of 50 mU/mL) was added to each well of the antibody 133-1H-sensitized plate after washing. Then, the antibody 133-1H-sensitized plate was stirred at room temperature for 60 minutes. Thereafter, the antibody 133-1H-sensitized plate was washed 3 times with the second buffer solution.
100 μL of a substrate solution containing ortho-phenylene diamine (OPD) as a substrate of POD was added to each well of the antibody 133-1H-sensitized plate and then left at room temperature for 10 minutes. Then, 100 μL of a reaction termination solution containing 2 N H2SO4 was added to each well of the antibody 133-1H-sensitized plate, and then the reaction solution in each well was measured for its absorbance (absorbance A) at 492 nm with a microplate reader (manufactured by Molecular Devices).
In a control experiment, a control sample in which the antibody solution had been changed to the first buffer solution was prepared and examined in the same experiment as described above to determine the absorbance (absorbance B). In another control experiment, a control sample in which the antibody solution and the antigen sample had been changed to the first buffer solution was prepared and examined in the same experiment as described above to determine the absorbance (absorbance C).
Using the absorbance A to C thus obtained, the degree of absorption of each measurement sample by each hybridoma-derived anti-RSV-F-protein monoclonal antibody was determined according to the following equation (1):
Degree of absorption (%)=[(1−(A−C)/(B−C)]×100 (1)
The evaluation results of the reactivity of each anti-RSV-F-protein monoclonal antibody are shown in Table 2. In Table 2, “+++” shows an absorption of 90% or more as determined by the equation (1); “++”, 50% or more; “+”, 30% or more; and “−”, less than 30%.
As being clear from the results in Table 2, it has revealed that the antibodies 1565, 412 and 255 exhibit reactivity to any antigens of Long strain, A2 strain, 9320 strain and BWV strain.
The A2 strain (10 [5.5] TCID (50)/ml) was diluted to 1/10 with the first buffer solution. The diluted antigen solution and an analyte extracting reagent were mixed with each other in equal volumes and then subjected to extraction treatment for 5 minutes to prepare an A2 strain antigen extract. Then, the A2 strain antigen extract was diluted to 1/20 with the first buffer solution to prepare an antigen sample.
A solution of antibody 133-1H (manufactured by CHEMICON) diluted to a concentration of 10 μg/mL with 0.1 M sodium phosphate buffer solution was dispensed in a volume of 100 μL to each well of an immunomodule (manufactured by NUNC). Each well was washed 3 times with the second buffer solution. After washing, each well was blocked by dispensing300μL of the first buffer solution to each well (hereinafter, this immunomodule will be referred to as antibody 133-1H-sensitized plate).
The first buffer solution was removed from the antibody 133-1H-sensitized plate. After removal of the buffer solution, 100 μL of the antigen sample was added to each well of the antibody 133-1H-sensitized plate which was then stirred at room temperature for 30 minutes. Thereafter, each well of the antibody-sensitized plate was washed 3 times with the second buffer solution.
The antibodies 1565, 412 and 255 were diluted to 10 μg/mL with the first buffer solution respectively, to prepare an antibody 1565 solution, an antibody 412 solution, and an antibody 255 solution. The prepared antibody solutions were added separately in a volume of 50 μL/well to each well of the antibody 133-1H-sensitized plate. Then, 50 μL first buffer solution containing biotin-labeled antibody B016 (antibody against RSV; final concentration of 2 μg/ml; manufactured by UK-Serotec) and streptavidin-labeled POD (final concentration 40 mU/ml) was added to each well of the antibody 133-1H-sensitized plate, and then the antibody 133-1H-sensitized plate was stirred at room temperature for 60 minutes and then the antibody 133-1H-sensitized plate was washed 3 times with the second buffer solution. 100 μL of a substrate solution containing OPD was added to each well of the antibody 133-1H-sensitized plate and then left at room temperature for 10 minutes. Then, 100 μL of a reaction termination solution containing 2 N H2SO4 was added to each well of the antibody 133-1H-sensitized plate, and the reaction solution in each well was measured for its absorbance at 492 nm with a microplate reader (absorbance A).
In a control experiment, a control sample in which the antibody solution had been changed to the first buffer solution was prepared and examined in the same experiment as described above to determine the absorbance (absorbance B). In another control experiment, a control sample in which the antibody solution and the antigen sample had been changed to the first buffer solution was prepared and examined in the same experiment as described above to determine the absorbance (absorbance C).
Using the absorbance A to C thus obtained, the degree of inhibition of each measurement sample by each hybridoma-derived anti-RSV-F-protein monoclonal antibody was determined according to the following equation (2):
Inhibition (%)=[(1−(A−C)/(B−C)]×100 (2)
An experiment was conducted in the same manner as in the above test of inhibition on the antibody B016 except that an antibody B016-sensitized plate having an immunomodule sensitized in each well with the antibody B016 was used in place of the antibody 133-1H-sensitized plate, and a biotin-labeled antibody 133-1H was used in place of the biotin-labeled antibody B016.
The evaluation results of the inhibition of each anti-RSV-F-protein monoclonal antibody on the antibodies 133-1H and B016 are shown in Table 3. In Table 3, “+++” shows an inhibition degree of 90% or more as determined by the equation (2); “++”, 50% or more; “+”, 30% or more; and “−”, less than 30%.
From the results in Table 3, it has revealed that the antibody 412 has an antigen recognition site different from the antigen recognition sites of the antibodies 133-1H and B016.
The Long strain (10 [6.7] TCID (50)/ml) was diluted to 1/10 with the first buffer solution. The diluted antigen solution and an analyte extracting reagent were mixed with each other in equal volumes and then subjected to extraction treatment for 5 minutes to prepare a Long strain antigen extract.
The antibodies 1565, 412 and255diluted to a concentration of 5 μg/mL with 0.1 M sodium phosphate buffer solution were dispensed in a volume of 100 μL/well to each well of immunomodules, respectively. Each well was washed 3 times with the second buffer solution. After washing, each well was blocked by dispensing 300 μL of the first buffer solution to each well, to prepare an antibody 1565-sensitized plate, an antibody 412-sensitized plate and an antibody 255-sensitized plate.
The first buffer solution was removed from each of the prepared antibody-sensitized plates. After removal of the buffer solution, 50 μL of the Long strain antigen extract was added to each well of each antibody-sensitized plate. Each antibody-sensitized plate was stirred at room temperature for 30 minutes and then washed 3 times with the second buffer solution.
The antibodies 1565, 412 and 255 were labeled with biotin by a known method, thereby preparing a biotin-labeled antibody RSF1-1565, a biotin-labeled antibody RSF2-412 and a biotin-labeled antibody RSF6-255.
A first buffer solution containing each of the prepared biotin-labeled antibodies (final concentration 2 μg/ml) and streptavidin-labeled POD (final concentration 40 mU/ml) was added in a volume of 100 μL to each of the antibody-sensitized plates. Each of the antibody-sensitized plates was stirred at room temperature for 60 minutes and then washed 3 times with the second buffer solution.
100 μL of a substrate solution containing OPD was added to each well of each antibody-sensitized plate and then left at room temperature for 10 minutes. Then, 100 μL of a reaction termination solution containing 2 N H2SO4 was added to each well of the antibody-sensitized plates. The reaction solution in each well was measured for its absorbance at 492 nm with a microplate reader. The measurement results are shown in Table 4.
From the results in Table 4, it has revealed that the antibodies 1565, 412 and 255, unless the same antibody is used as antibodies on the solid phase side and on the label side, can be combined with each other to detect the RSV antigen by sandwich immunoassay.
Hep2 cells for infection with RSV were cultured on a 24-well plate (manufactured by Corning) with Eagle MEM (manufactured by SIGMA) containing 10% fetal bovine serum. When the Hep2 cells became confluent, the culture medium was exchanged with Eagle MEM containing 1% fetal bovine serum, and the cells were infected at an MOI of 0.0005 with the Long strain (10 [6.7] TCID (50)/ml). After infection, the cells were incubated for 1 hour at 37° C. in 5% CO2. After incubation for 1 hour, the antibody 1565 was added in a volume of 20 μg/well to the cells. After the antibody was added, the cells were cultured at 37° C. in 5% CO2 for 3 to 4 days.
A culture medium was removed from HeP2 cells cultured separately in Eagle MEM containing 10% fetal bovine serum, and to the HeP2 cells was added half of a culture supernatant of the above cells which had been cultured for 3 to 4 days. Further, Eagle MEM containing 1% fetal bovine serum in an amount equal to the culture supernatant of the cells was added thereto. After addition, the cells were incubated for 1 hour at 37° C. in 5% CO2. After incubation for 1 hour, the antibody 1565 was added in a volume of 20 μg/well. After the antibody was added, the cells were cultured at 37° C. in 5% CO2 for 3 to 4 days. This procedure was carried out further 5 times.
After the cells infected with RSV were subcultured 7 times in total in the presence of the antibody 1565, cytopathic effect was observed. The appearance of an antibody-1565 escape mutant could be confirmed by observing the cytopathic effect, so the whole culture of the cells was recovered and stored in a frozen state.
From a culture supernatant of the antibody-1565 escape mutant and the Long strain, cDNA was synthesized by using ReverTra Ace-α (Product Code FSK-101, manufactured by Toyobo) and a primer
Each synthesized cDNA was subjected to polymerase chain reaction (PCR) with PrimeStar (Product Code R010A, manufactured by TAKARA) and primers [sequences: ATGGATCCATGGAGTTGCCAATCCTCAAAGC (SEQ ID NO: 4) and TAGAATTCTAGTGATGGTGATGGTGATGATTTGTGGTGGATTTACCAGCATT (SEQ ID NO: 5)]. PCR involved a reaction at 95° C. for 2 minutes followed by 35 cycles each consisting of a reaction at 95° C. for 30 seconds, at 55° C. for 20 seconds and at 72° C. for 100 seconds. After the reaction was finished, the PCR product was stored in a frozen state.
Using MinElute PCR Purification Kit (QIAGEN), the primers were removed from the PCR product. The PCR product from which the primers had been removed was subjected to cycle sequencing reaction with BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied Biosystems) and primers [sequences: ATGGATCCATGGAGTTGCCAATCCTCAAAGC (SEQ ID NO: 4), TTTCTTGGTTTTTTGTTAGGTGTTGG (SEQ ID NO: 6), TTTAACATTACCAAGTGAAATAAATCT (SEQ ID NO: 7), and TCTTCTTTTCCTTTTCTTGCTTAATG (SEQ ID NO: 8)]. After the reaction, the labeled nucleotide was removed, and gene sequence analysis and predicted amino acid sequence analysis were performed with a sequencer (manufactured by Applied Biosystems) and sequence analysis software (manufactured by Hitachi Software).
The amino acid sequence of the RSV-F protein of the antibody-1565 escape mutant is set forth in SEQ ID NO: 2. The amino acid sequence of the RSV-F protein of the Long strain is set forth in SEQ ID NO: 1. As being clear from SEQ ID NOS: 1 and 2, it has revealed that in the RSV-F protein of the antibody-RSF1-1565 escape mutant, lysine (K) at position 421 had been changed to threonine (T).
(Analysis of Reactivity with the Antibody-1565 Escape Mutant)
A culture supernatant of the antibody-1565 escape mutant and the Long strain were diluted to 1/10 with the first buffer solution, respectively. Each of the diluted antigen solutions and an analyte extracting reagent were mixed with each other in equal volumes and then subjected to extraction treatment for 5 minutes to prepare each antigen extract. Then, each antigen extract was diluted to ⅕ with the first buffer solution to prepare an antigen sample.
100 μL of a solution of antibody B016 (manufactured by UK-Serotec) diluted to a concentration of 10 μg/mL with 0.1 M sodium phosphate buffer solution was dispensed to each well of an immunomodule (manufactured by NUNC). Each well was washed 3 times with a Tween 20-containing phosphate buffer solution (referred to hereinafter as second buffer solution). After washing, each well was blocked by dispensing 300 μL of the first buffer solution to each well (hereinafter, this immunomodule will be referred to as antibody B016-sensitized plate).
The first buffer solution was removed from the antibody B016-sensitized plate. After removal of the buffer solution, 100 μL of the antigen sample was added to each well of the antibody B016-sensitized plate and then stirred for 30 minutes. Thereafter, each well of the antibody B016-sensitized plate was washed 3 times with the second buffer solution.
The antibody 1565 was labeled with biotin by a known method, thereby preparing a biotin-labeled antibody RSF1-1565.
A first buffer solution containing the prepared biotin-labeled antibody 1565 (final concentration 2 μg/ml) and streptavidin-labeled POD (final concentration 40 mU/ml) was added in a volume of 100 μL to each well of the antibody B016-sensitized plate, and the antibody B016-sensitized plate was stirred at room temperature for 60 minutes. Thereafter, the antibody B016-sensitized plate was washed 3 times with the second buffer solution.
100 μL of a substrate solution containing OPD was added to each well of the antibody B016-sensitized plate and then left at room temperature for 10 minutes. Then, 100 μL of a reaction termination solution containing 2 N H2SO4 was added to each well of the antibody B016-sensitized plate, and then the reaction solution in each well was measured for its absorbance at 492 nm with a microplate reader. The measurement results of the absorbance are shown in Table 5.
From Table 5, it has revealed that the antibody 1565 does not recognize the RSV-F protein of the antibody-1565 escape mutant.
The antibody 1565 was used as the first antibody labeled with a labeling substance, and the antibodies 412 and 255 were used as the second antibody immobilized on a chromatographic carrier, and an immuno-chromatographic test device 1 (referred to hereinafter as test device) as shown in
First, the antibodies 412 and 255 were diluted to a final concentration of 1.0 mg/mL with a phosphate buffer solution, pH 7.0, to prepare a mixed solution of the antibodies 412 and 255. Then, the mixed solution of the antibodies 412 and 255 was applied in 1-mm width via an antibody applicator (BioDot Ltd.) onto a judgment part 5A of a chromatographic carrier made of a nitrocellulose membrane, and then dried at 50° C. for 30 minutes.
After drying, the chromatographic carrier 5 was blocked by dipping in a blocking solution (BSA-containing phosphate buffer solution, pH 7.0). Thereafter, the chromatographic carrier 5 was washed with a washing solution (SDS-containing phosphate buffer solution, pH 7.0) and dried at 40° C. for 120 minutes to complete the chromatographic carrier 5.
Then, blue colored polystyrene latex particles (particle size 0.3 μm) were sensitized with the antibody 1565 and then suspended in a dispersing buffer solution (phosphate buffer solution, pH 7.0, containing BSA and sucrose), to prepare antibody 1565-sensitized latex particles. In this sensitization, the antibody was used at a concentration of 200 μg IgG per mL of 1% latex particles. The antibody 1565-sensitized latex particles were added to a glass fiber pad and then dried in a vacuum drier to prepare a label holding member 4.
The chromatographic carrier 5 and the label holding member 4 were used to produce the test device of the present invention.
A control test device was prepared in the same manner as in preparation of the test device of the present invention except that the antibody B016 was used as the first antibody, the antibody 133-1H was used as the second antibody, and an antibody B016 solution diluted to a final concentration of 2.0 mg/mL was used in place of the mixed solution of the antibodies 412 and 255.
Using the test device of the present invention prepared in Example 6, its reactivity to RSV was confirmed in the following procedures.
As conventional test devices, the control test device and commercial test devices Check RSV (manufactured by Alfresa-Pharma Co. Japan) and BD RSV Examan (manufactured by Becton Dickinson) were used to confirm their reactivity to RSV.
The reactivity of the control test device to RSV was confirmed in the same manner as described above for the test device of the present invention. The reactivity of the commercial test devices to RSV was confirmed using the above viral solutions, according to the operation described in a material attached to each of the commercial test devices. The results of confirmation of the reactivity are shown in Table 6.
NT: Not tested
As being clear from Table 6, the test device of the present invention showed higher reactivity to RSV than by the conventional test devices.
| Number | Date | Country | Kind |
|---|---|---|---|
| JP 2008-187368 | Jul 2008 | JP | national |
