JP2010085126A - Determination instrument for determining neutralizing antibody titer of rabies virus, and method for measuring neutralizing antibody titer of rabies virus - Google Patents

Abstract

Provided are a determination tool for detecting neutralizing antibodies against rabies virus contained in body fluids of humans and animals, particularly serum, and a method for detecting the neutralizing antibodies.
A rabies virus neutralizing antibody determination tool comprising a liquid absorption piece made of a material capable of transferring a test sample by capillary action as the determination tool, wherein the liquid absorption piece comprises (1) a test sample. Sample collection part for absorption collection,
(2) a labeled antibody part carrying an anti-rabies virus antibody specifically reacting with the G protein of the rabies virus;
(3) A test result display section shown below, preferably a control display section, a determination section provided with an interval,
(a) an unlabeled anti-rabies virus antibody immobilized specifically reacting with the G protein of rabies virus;
(b) a labeled display—an unlabeled antibody that reacts with an anti-rabies virus antibody—an antibody (second antibody) immobilized control display;
(4) A determination tool including a liquid absorption unit that absorbs the residual liquid of the test sample that has moved through the sample collection unit, the labeled antibody unit, and the determination unit is used.
[Selection figure] None

Description

  The present invention relates to a material (determination tool and determination kit) for determining a neutralizing antibody titer against a rabies virus contained in human or animal body fluids, particularly serum, and a method for measuring and judging the neutralizing antibody titer. About.

  More specifically, the present invention provides a method for quickly and simply qualitatively detecting and determining the presence of the neutralizing antibody in human or animal serum without using an infectious virus or cell culture facility. The present invention relates to a method and a material (determination tool) suitably used in the method.

  Rabies is a neurological disease (encephalitis) caused by the rabies virus, a member of the Rhabdoviridae family and Lyssavirus, and is bitten by rabies-infected animals and wounds contaminated with saliva from rabies-infected animals. It becomes infected by being done. Once clinical symptoms appear, it has a clinical feature leading to death at a rate of almost 100%.

  For this reason, despite the widespread use of vaccines and the use of rabies immunoglobulin, approximately 55,000 people die annually worldwide. In terms of preventing rabies infection, the neutralizing antibody titer against rabies is the only standard for acquiring immunity against rabies, but it is sufficient for many vaccinated people who are said to have more than 1 billion a year. It is currently not confirmed whether or not

  The rabies virus is characteristically a bullet-shaped, enveloped particle with an average of 75 x 180 nanometers. A virion consists of a single-stranded negative-sense RNA genome and five structural proteins (nuclear protein (N) molecule, phosphoprotein (NS), polymerase (L), matrix protein (M) and viral glycoprotein (G)). Composed. Among them, glycoprotein (G) (hereinafter referred to as “G protein”) is an important binding site for antibodies that neutralize viruses (see, for example, Non-Patent Document 1).

  Infection with rabies virus has a long incubation period of about 1 to 3 months until specific clinical symptoms appear, so it is possible to inject vaccines after exposure. Once there is no cure, there is no effective treatment, so after exposure to rabies vaccination at a defined interval after being bitten by a rabies-infected animal, post-exposure onset prevention is the only effective way to avoid rabies death . In addition, vaccinations are given in advance to those who handle rabies virus, veterinarians and travelers to rabies-infested areas (pre-exposure vaccination). Such passive immunization is usually performed by preformed (pre-formed) from pooled sera of rabies immunized individuals (human rabies-immunoglobulin; HRIG) or hyperimmunized horses (equine rabies-immunoglobulin; ERIG). ) By administering a rabies virus neutralizing antibody.

  Thus, the neutralizing antibody plays an important role in preventing the onset and infection of rabies, and consequently the transmission of rabies virus. According to WHO and the Office International des Epizooties (OIE), the neutralizing antibody titer necessary to prevent infection with rabies virus is 0.5 IU / ml or more (see Non-Patent Documents 2 and 3). ).

  Rapid Fluorescent Focus Inhibition Test (RFFIT) is generally used to measure neutralizing antibodies against rabies virus in serum, such as neutralizing antibody levels after rabies vaccination in humans and animals. (Non-Patent Document 4). However, since the method uses live rabies virus and sensitive cells (such as BHK cells) in addition to fluorescent label-anti-rabies virus antibodies, cell culture equipment, fluorescence microscope equipment, biosafety cabinets, etc. Equipment for protection against infection is necessary, and a skilled laboratory technician needs to perform it in a facility equipped with such equipment. Furthermore, it takes about 2 days for the result to be obtained. In some developing countries, mouse brain inoculation tests are being conducted as an in vivo infection evaluation system. However, this method is inconvenient because it uses mice and takes 2 weeks to produce results. is there.

In this way, the conventional testing methods have limited facilities that can be implemented, and there are problems such as it takes time to obtain results, and these are evaluations of neutralizing antibody acquisition after rabies vaccination. One of the causes that makes it difficult. For this reason, the advent of a test system that can measure the presence and level of neutralizing antibodies quickly and easily is desired.
DL Lodmell, J. Virol., 61, 10: 3314-3318, 1987 World Health Organization. Expert consultation on rabies, 1st report WHO technical series 931. Geneva; 2005, 88 pp. Office International des Epizooties.OIE manual of standards of diagnostic tests and vaccines.4th ed.Paris: Office International des Epizooties: 2000, p.15-23, 276-291 Smith, et al., Bull WHO 1973; 48; 535-541

  The present invention aims to solve the above problems. That is, an object of the present invention is to provide a method for quickly and easily measuring and determining the presence or amount (neutralizing antibody titer) of neutralizing antibodies against rabies virus in human or animal body fluids, particularly serum, and It is to provide a material (determination tool) preferably used in the method.

  A further object of the present invention is to provide a monoclonal antibody that specifically recognizes the G protein of rabies virus, which is preferably used in the method and determination tool, and a material used for the preparation thereof.

  As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors previously reacted a predetermined amount of inactivated rabies virus with the body fluid of the subject, and then recognized this reaction solution for the G protein of the rabies virus. It has been found that the neutralizing antibody titer against rabies virus in the body fluid of a subject can be specifically detected and determined by subjecting it to immunochromatographic detection using a monoclonal antibody. Furthermore, according to the method, infectious virus and cell culture The present invention was completed by confirming that the amount (neutralizing antibody titer) of the neutralizing antibody against the rabies virus, which is the above problem, can be measured and judged quickly and easily without using equipment.

That is, this invention has the following aspect.
(I) Rabies virus neutralizing antibody titer determining device (I-1 ) A rabies virus neutralizing antibody titer determining device comprising a liquid-absorbing piece made of a material capable of transferring a test sample by capillary action, Liquid pieces
(1) a sample collection part for absorbing and collecting a test sample,
(2) a label that specifically reacts with the G protein of rabies virus-a labeled antibody portion carrying an anti-rabies virus antibody,
(3) A determination unit having a test result display unit as shown below,
(a) a non-labeled anti-rabies virus antibody-fixed test result display part that specifically reacts with the G protein of rabies virus,
(4) A rabies virus neutralizing antibody titer determination device comprising a liquid absorption unit that absorbs a residual liquid of a test sample that has moved through the sample collection unit, the labeled antibody unit, and the determination unit.

(I-2) The above (3) determination unit is further provided with the following control display unit (a) spaced apart from the test result display unit, as described in (I-1) Rabies virus neutralizing antibody titer:
(b) Labeled control display with immobilized unlabeled antibody that reacts with anti-rabies virus antibody.

  (I-3) An anti-rabies virus antibody used in a labeled-anti-rabies virus antibody and an unlabeled-anti-rabies virus antibody comprises an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 95% or more identity thereto A chain variable region, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having 95% or more identity thereto, and specifically binding to the G protein of rabies virus, The rabies virus neutralizing antibody titer determination device according to (I-1) or (I-2), which is a monoclonal antibody.

  (I-4) An anti-rabies virus antibody used in a labeled-anti-rabies virus antibody and an unlabeled-anti-rabies virus antibody comprises an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having 95% or more identity thereto A monoclonal antibody comprising a chain region and a light chain region comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having 95% or more identity thereto, and which specifically binds to G protein of rabies virus The rabies virus neutralizing antibody titer determination device according to (I-1) or (I-2), which is an antibody.

(II) Method for measuring neutralizing antibody titer of rabies virus (II-1) A mixture of inactivated rabies virus and subject body fluid is used as a test sample and described in any of (I-1) to (I-4) A method for measuring a rabies virus neutralizing antibody titer in a body fluid of a subject, the method comprising the steps of providing a sample collecting part of a rabies virus neutralizing antibody titer determination tool and detecting a color of a test result display section in the determination part of the determining tool .

  (II-2) The amount of inactivated rabies virus is the amount neutralized with a neutralizing antibody with a neutralizing antibody titer of 0.5 IU / ml and not neutralized with a neutralizing antibody with a neutralizing antibody titer of 1.0 IU / ml. The method described in (II-1), wherein the method is used.

  (II-3) The method according to (II-1) or (II-2), wherein the serum of an animal other than a rabies vaccinated person and a human after rabies vaccination is used as the subject serum.

(III) Monoclonal antibody recognizing G protein of rabies virus and its gene (III-1) heavy chain variable region comprising amino acid sequence described in SEQ ID NO: 1 or amino acid sequence having 95% or more identity thereto, and sequence A monoclonal antibody comprising a light chain variable region comprising the amino acid sequence shown in No. 3 or an amino acid sequence having 95% or more identity thereto, and specifically binding to a G protein of rabies virus.

  (III-2) Heavy chain region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 95% or more identity thereto, and the amino acid sequence shown in SEQ ID NO: 4 or 95% or more identity thereto The monoclonal antibody according to (III-1), which comprises a light chain region consisting of an amino acid sequence and specifically binds to a rabies virus G protein.

  (III-3) A gene encoding the monoclonal antibody described in (III-2), wherein the nucleotide sequence described in SEQ ID NO: 5 and the nucleotide sequence described in SEQ ID NO: 6 are respectively represented by the heavy chain region of the monoclonal antibody. And a nucleotide sequence encoding a light chain region.

  According to the determination tool and method of the present invention, the presence or absence and the level (neutralizing antibody titer) of a rabies virus neutralizing antibody in body fluids of humans or animals (excluding humans; the same applies hereinafter) after rabies vaccination, particularly serum. ) Can be easily measured without using infectious virus or cell culture equipment.

  In addition, according to the determination tool and method of the present invention, a result can be obtained in a short time of about 2 hours, so that it is useful as a rapid method for measuring the rabies virus neutralizing antibody titer compared to the RFFIT method which has conventionally taken 2 days. is there.

  Furthermore, when measuring the rabies virus neutralizing antibody in the serum of dogs, if the conventional RFFIT method is used, it may be toxic to cultured cells and an accurate neutralizing antibody titer may not be obtained. The method of the present invention is useful as an alternative to such RFFIT method.

  Furthermore, the monoclonal antibody provided in the present invention has the property of specifically recognizing and binding the G protein of rabies virus, and can be used effectively in the determination tool and method of the present invention.

(I) Rabies virus neutralizing antibody titer determination tool The rabies virus neutralizing antibody titer determination tool (also referred to simply as “determination tool”) targeted by the present invention is for determining the rabies virus neutralizing antibody titer of human or animal body fluids. This is a tool for carrying out the measurement, and includes a liquid-absorbing piece made of a material capable of transferring a test sample by capillary action.

The liquid absorption piece
(1) a sample collection part for absorbing and collecting a test sample,
(2) a label that specifically reacts with the G protein of rabies virus-a labeled antibody portion carrying an anti-rabies virus antibody,
(3) A determination unit having a test result display unit as shown below,
(a) a test result display section in which an unlabeled anti-rabies virus antibody that specifically reacts with the G protein of rabies virus is immobilized, and
(4) A liquid absorption unit is provided that absorbs the residual liquid of the test sample that has moved through the sample collection unit, the labeled antibody unit, and the determination unit.

In addition to the (a) test result display unit, the (3) determination unit may include a control display unit shown below at an interval from the test result display unit:
(b) Labeled control display part with immobilized non-labeled antibody that reacts with anti-rabies virus antibody.

  According to the determination tool, (a) the degree of rabies virus neutralizing antibody titer in the test sample can be determined based on the presence or absence of coloration in the test result display section. That is, according to the determination tool, (a) by using the presence or absence of color development in the test result display section as an index, whether or not the rabies virus neutralizing antibody titer of the test sample is a certain value or more, preferably WHO It can be determined whether or not it is 0.5 IU / ml or more, which OIE has determined as a sufficient neutralizing antibody titer.

  Herein, the “rabies virus neutralizing antibody” refers to an antibody that specifically binds to a glycoprotein (G protein) present on the surface of the rabies virus and eliminates the infectivity of the virus. It is known that induction of this antibody in the body after vaccination eliminates the infectivity of viruses that have invaded from the periphery and can prevent disease.

  The determination tool of the present invention includes a liquid-absorbing piece made of a material that can transfer a test sample by capillary action or chromatographic action (in the present invention, these are collectively referred to as “capillary phenomenon”). The liquid-absorbing piece may be a sheet-like strip (hereinafter referred to as “sheet-like strip”), and the sheet-like strip may be a single sheet or a plurality of sheets stacked. Or they can be formed by concatenation. Or a liquid absorption piece can be made into various forms which can be absorbed by liquid and can be transferred by capillary action, such as an elongated rod-like piece. In addition, the determination tool of the present invention may further include a support body that supports the liquid-absorbing piece.

  The material for this liquid-absorbing piece includes a solvent (water, serum, urine and other body fluids), a test component (rabies virus, rabies virus neutralizing antibody), and a complex containing the test component (eg, labeled (1) A test sample containing a test component was applied (collected, dripped, added) to a sample collection part, having a porous structure or a capillary structure that can be penetrated by a label-antirabies virus antibody-rabies virus complex) In this case, the test sample is not particularly limited as long as it is a material that can move (expand) in the porous structure or the capillary structure while containing the antibody or the complex on the way. For example, sheets such as various filter papers and chromatographic papers, various felts, cloths such as woven fabrics and nonwoven fabrics, and glass fibers can be used. Among these, an organic porous body is more preferable. Examples of organic porous materials include fiber aggregates formed of natural fibers such as cellulose, semi-synthetic fibers such as cellulose acetate, cellulose derivatives such as nitrocellulose, synthetic fibers such as polyethylene, polypropylene, nylon, and polyester. Body, porous polypropylene, porous polystyrene, porous polymethyl methacrylate, porous nylon, porous polysulfone, porous fluororesin, porous synthetic resin such as polyvinylidene fluoride introduced with hydrophilic groups, etc. Can be mentioned. Preferred are natural fibers such as cellulose, semi-synthetic fibers such as cellulose acetate, and cellulose derivatives such as nitrocellulose.

  The size of the liquid absorption piece is not particularly limited. The width (short side length) is in the range of about 2 to 20 mm, preferably about 4 to 10 mm, and the length (long side length) is in the range of 20 to 200 mm, preferably 30 to 150 mm. It is preferable that

  Hereinafter, a determination tool according to an embodiment of the present invention will be described with reference to the accompanying drawings.

  1A and 1B show one embodiment of the determination tool of the present invention. FIGS. 1A and 1B are views of the determination tool of the present invention viewed from the side. In such a determination tool, the liquid absorption piece includes a sample collection unit (1) for collecting (adding) a test sample, a labeled antibody unit (2), and a determination unit (3) including a test result display unit (a), and A liquid absorption part (4) for absorbing the residual liquid of the test sample that has moved through the sample collection part, the labeled antibody part, and the determination part is provided. FIG. 1B shows a determination tool provided with a control display section (b) in addition to the test result display section (a) in the determination section (3).

  In the example shown in FIG. 1, the liquid absorbing piece is a sheet-like strip formed by a plurality of sheet pieces, but the sheet-like strip may be a single sheet made of the same material. Alternatively, a single sheet may be formed as a whole with a plurality of sheet pieces made of the same or different materials. In addition, the whole sheet | seat may consist of a several sheet | seat, and may have a part partially formed from a several sheet | seat.

In the example shown in FIG. 1, a sheet-like strip 20 is bonded on the support 10 . The sheet-like strip 20 includes, in order from one end side to the other end side in the longitudinal direction, a sheet 21 that forms a sample collection part (1), a sheet 22 that forms a labeled antibody part (2), and a determination part (3). The sheet 23 to be formed is provided with a sheet 24 to form the liquid absorbing portion (4). Further, as shown in FIG. 2, so that the end portion of the seat 21 overlaps with the end of the sheet 22, and as the end of the seat 22 overlaps with the end portion of the sheet 23, further end of sheet 23 is a sheet It can also comprise so that it may overlap with the 24 edge part. By taking such a configuration, the liquid can be moved more smoothly.

  The sample collection unit (1) is a part (test sample supply unit) that absorbs and collects a test sample to be measured. As a test sample, the mixture of the inactivated rabies virus and the bodily fluid of the test subject which this invention measures can be mentioned. The body fluid is not particularly limited as long as it contains a neutralizing antibody against rabies virus, and examples thereof include urine, blood (serum, plasma), sweat, saliva and the like. Serum is preferred.

The inactivated rabies virus can be prepared by treating the rabies virus with formalin, betapropiolactone (BPL), ultraviolet light or the like. Among them, a method of inactivating rabies virus by formalin treatment is preferable. The formalin treatment can be carried out with reference to the description of Example 2 described below (Reference:. H Singh Laboratory technique of rabies , 1996, 4 th ed.WHO, Chapter 20, p234-242).

  The sample collection part (1) can be located at the end (starting end) of the sheet-like strip as illustrated in FIGS.

  The sample collection part (1) is preferably composed of a hydrophilic and water-absorbing material so that the test sample can be quickly absorbed among the materials employed for the sheet-like strip. Such materials include filter paper, various fibers [natural fibers such as cotton, pulp, hemp, silk, wool, and flax; regenerated fibers such as rayon, polynosic, cupra, and lyocell; semisynthetic fibers such as acetate; polyamide ( Nylon), vinylon (polyvinyl alcohol), polyester, acrylic (polyacrylonitrile, etc.), polyolefin (polyethylene, polypropylene, etc.), and synthetic fibers such as polyurethane (glass fibers), sponges, felts, non-woven fabrics, woven fabrics, papers, etc. Can be illustrated. Preferred examples include polyester, polypropylene, rayon, sponge made of glass fiber, non-woven fabric, woven fabric, and paper.

  As described above, the labeled antibody part (2) is in contact with the terminal part of the sample collecting part (1) (FIG. 1) or formed in a state where it partially overlaps the sample collecting part (1) ( Figure 2). The labeled antibody portion (2) contains an antibody that specifically reacts and binds to the rabies virus contained in the test sample in a detachable state.

Specifically, the determination tool of the present invention removes a labeled anti-rabies virus antibody that specifically binds to the G protein of the rabies virus by an antigen-antibody reaction from the sheet 22 constituting the labeled antibody portion (2). Include as possible.

  Here, the labeled anti-rabies virus antibody is preferably a labeled monoclonal antibody that specifically binds to the G protein of the rabies virus and does not cross-react with other proteins.

  Such a monoclonal antibody preferably contains a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 3, and is specific for the G protein of rabies virus And monoclonal antibodies that bind to. As long as the specific binding property to the G protein of rabies virus is not impaired, the heavy chain variable region is 85% or more, preferably 90% or more, more preferably 95% with the amino acid sequence shown in SEQ ID NO: 1. It may consist of an amino acid sequence having the above identity, and the light chain variable region is 85% or more, preferably 90% or more, more preferably 95% or more with the amino acid sequence shown in SEQ ID NO: 3. It may consist of amino acid sequences having the same identity.

  Each of these heavy chain variable region and light chain variable region has three framework regions (FWR1 to FWR3) and two complementarity determining regions (CDR1 and CDR2). Specifically, the heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 1 is FWR1 (1-30th amino acid region) and CDR1 (31-35th amino acid region) sequentially from the N-terminal side in SEQ ID NO: 1. Amino acid region), FWR2 (36th to 49th amino acid region), CDR2 (50th to 66th amino acid region), and FWR3 (67th to 98th amino acid region). The light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 is FWR1 (1-7th amino acid region), CDR1 (8-18th amino acid region) in that order from the N-terminal side in SEQ ID NO: 3. , FWR2 (19th to 33rd amino acid region), CDR2 (34th to 40th amino acid region), and FWR3 (41th to 72nd amino acid region).

  The monoclonal antibody includes a heavy chain region containing the heavy chain variable region as a heavy chain region and a light chain region containing the light chain variable region as a light chain region, and specifically binds to a rabies virus G protein. It is preferably an antibody. Here, the heavy chain region is preferably a region consisting of the amino acid sequence described in SEQ ID NO: 2, and the light chain region is preferably a region consisting of the amino acid sequence described in SEQ ID NO: 4. In addition, unless the specific binding property to the G protein of rabies virus is impaired, the heavy chain region of the monoclonal antibody is 85% or more, preferably 90% or more, more than the amino acid sequence shown in SEQ ID NO: 2. Preferably, it may consist of an amino acid sequence having 95% or more identity. The light chain region is also 85% or more, preferably 90% or more, more preferably 95% or more of the amino acid sequence shown in SEQ ID NO: 4 as long as the monoclonal antibody does not impair the specific binding property to the G protein of rabies virus. It may consist of amino acid sequences having% or more identity.

  In addition, the binding property to the G protein of rabies virus can be evaluated by eliminating the infectivity to sensitive cells by the virus bound to the monoclonal antibody. Specifically, a monoclonal antibody and a rabies virus are mixed at an appropriate ratio, and this is inoculated into susceptible cells (for example, Baby Hamster Kidney cells, neuroblastoma NA cells) that can infect and proliferate with the virus. Later, it can be judged by staining with a monoclonal antibody against anti-rabies virus N protein that the focus of infected cells is not formed.

  These monoclonal antibodies, i.e. anti-rabies virus antibodies, are used labeled with any labeling agent, i.e. as labeled-anti-rabies virus antibodies. The labeling agent used here is a metal or inorganic particle such as gold, silver, or selenium; a fluorescent labeling agent such as fluorescein or rhodamine; a dye labeling agent such as red blood cells, latex particles, colored or colored latex particles An enzyme labeling agent such as β-galactosidase, alkaline phosphatase, peroxidase; or a mixture thereof. Inorganic particles made of gold are preferable. The labeling method with a labeling agent is well known, and can be performed according to a standard method depending on the type of labeling agent to be used.

  The labeled antibody part (2) contains the label-anti-rabies virus antibody in a detachable state among the materials adopted for the sheet-like strip, and the test in the test sample moved from the sample collection part (1) The antigen-antibody complex formed by reacting with the component (rabies virus) (complex of rabies virus and label-anti-rabies virus antibody) is transferred to the determination part (3) of the test sample. 1, it is preferably composed of a hydrophilic and water-absorbing material having the property of being able to move in the direction indicated by the arrow P.

  Such materials include filter paper, various fibers [natural fibers such as cotton, pulp, hemp, silk, wool, and flax; regenerated fibers such as rayon, polynosic, cupra, and lyocell; semisynthetic fibers such as acetate; polyamide ( Nylon), vinylon (polyvinyl alcohol), polyester, acrylic (polyacrylonitrile, etc.), polyolefin (polyethylene, polypropylene, etc.), and synthetic fibers such as polyurethane (glass fibers), sponges, felts, non-woven fabrics, woven fabrics, papers, etc. Can be illustrated. Preferred examples include polyester, polypropylene, rayon, sponge made of glass fiber, non-woven fabric, woven fabric, and paper.

  The labeled antibody part (2) is not limited as a method for carrying the labeled anti-rabies virus antibody in a detachable state, but a solution containing the labeled anti-rabies virus antibody is added to the labeled antibody part (2). A method of impregnation or adhesion can be exemplified. In addition, in order to bind the test component (rabies virus) contained in the test sample to the labeled antibody part (2) without omission, the labeled antibody part (2) carries the above labeled anti-rabies virus antibody in an excessive amount. It is preferable to keep it.

  The determination unit (3) is a part that further transfers the test sample that has moved from the sample collection unit (1) through the labeled antibody unit (2) to the liquid absorption unit (4), on the transfer region. In addition, a specific component (complex of rabies virus and label-anti-rabies virus antibody) contained in the test sample to be transported is captured and the captured result is displayed [test result display section (a)]. Based on the result shown in the test result display part (a), the degree of the rabies virus neutralizing antibody titer of the test sample can be determined. Therefore, the region including the display part (a) is designated as “determination part (3 ) ".

  The determination tool of the present invention may include a control display unit (b) in addition to the test result display unit (a) in the determination unit (3). The test result display unit (a) and the control display unit (b) are sequentially arranged at intervals.

  An unlabeled antibody (unlabeled anti-rabies virus antibody) that specifically reacts with the G protein of rabies virus is fixed in an excessive amount in the test result display section (a). For this reason, the rabies virus combined with the labeled anti-rabies virus antibody by the antigen-antibody reaction in the labeled antibody portion (2) is in the state of a complex with the labeled anti-rabies virus antibody in the test result display section ( It is captured in a) and exhibits a color caused by the labeling agent with a strength according to the amount captured.

  Here, as the anti-rabies virus antibody constituting the unlabeled anti-rabies virus antibody, the aforementioned monoclonal antibody that specifically binds to the G protein of the rabies virus can be similarly used.

The amount of unlabeled anti-rabies virus antibody immobilized on the test result display section (a) is not limited. For example, in the case of a determination tool using serum as a test sample, approximately 3 × 10 ³ focus forming units (10 ^1.75 50% infectious units) / 50ml equivalent) The amount that can be combined with the above rabies virus.

  A predetermined amount of unlabeled-antibody (second antibody) that reacts with the labeled anti-rabies virus antibody contained in the labeled antibody portion (2) is fixed to the control display portion (b). The control display part (b) is spaced apart from the test result display part (a) in the direction of movement of the test sample (direction p in FIG. 1), and the test result display part (a) and the control display part ( They can be arranged in the order of b). In the control display part (b), an excessive amount of unlabeled-antibody (second antibody) that reacts with the labeled anti-rabies virus antibody contained in the test sample moving from the preceding sheet piece is fixed.

  Therefore, in the control display part (b), the labeled anti-rabies virus antibody released from the labeled antibody part (2) and released in the test sample was captured and fixed to the control display part (b). It develops color due to the labeling agent of the label-anti-rabies virus antibody with a strength according to the amount of the second antibody.

  Here, the non-labeled antibody (second antibody) may be any one that binds to the labeled anti-rabies virus antibody. Such an unlabeled-antibody (second antibody) is not limited. For example, when a mouse-derived monoclonal antibody is used as the labeled-anti-rabies virus antibody, the unlabeled anti-mouse IgG goat antibody ( For example, DAKO Inc.), or an anti-mouse IgG mouse antibody can be used.

  The determination of the rabies virus neutralizing antibody titer using the determination tool of the present invention is performed using the presence or absence of color development in the test result display section (a) in the determination section (3) as an index. At this time, if necessary, the presence / absence of coloration in the control display part (b) in the judgment part (3) can also be used for judgment. Even if no color development is observed in the test result display part (a), if color development is observed in the control display part (b), the measurement is performed normally and the measurement result is negative (rabies virus neutralizing antibody). It can be determined that the price is equal to or higher than a predetermined value. However, when color development is not recognized in the control display part (b), it indicates that the measurement is not normally performed, and it cannot be determined as negative based on the result of the test result display part (a).

  The determination unit (3) can move a test sample containing various components to the liquid absorption unit (4) by capillarity among the materials adopted for the sheet-like strip, and the test result display unit (a ) And a hydrophilic and water-absorbing material having the property of stably immobilizing the above-mentioned non-labeled anti-rabies virus antibody and the above-mentioned non-labeled antibody (second antibody) on the control display part (b) It is preferable that it is comprised.

  Such materials include filter paper, various fibers [natural fibers such as cotton, pulp, hemp, silk, wool, and flax; regenerated fibers such as rayon, polynosic, cupra, and lyocell; semisynthetic fibers such as acetate; polyamide ( Nylon), vinylon (polyvinyl alcohol), polyester, acrylic (polyacrylonitrile, etc.), polyolefin (polyethylene, polypropylene, etc.), and synthetic fibers such as polyurethane (glass fibers), sponges, felts, non-woven fabrics, woven fabrics, papers, etc. Can be illustrated. Preferred examples include sponges, nonwoven fabrics, woven fabrics, and papers made of cellulose and nitrocellulose.

  The liquid absorption part (4) includes the sample collection part (1) to the labeled antibody part (2) and the determination part (3) (test result display part (a) or test result display part (a) and control display part. (b)) is a part that absorbs the remaining liquid of the test sample that has moved in the direction of arrow P through

  The liquid absorbent part (4) is preferably excellent in hydrophilicity and absorbability among the materials adopted for the sheet-like strips, but more preferably a material having the property of not absorbing the absorbed liquid. Alternatively, it is desirable to be made of an elastic material. Specific examples of such a material include filter paper and hydrophilic fiber non-woven fabric, and a laminate of filter paper and non-woven fabric can also be used.

The determination tool of the present invention is basically composed of a sheet-like strip having the above-described configuration, but may further include a support 10 in addition to the sheet-like strip. The support 10 is not particularly limited as long as it has a material and form capable of holding the sheet-like strip. For example, it may be a sheet-like support used by being laminated on the back surface (lower layer surface) of the sheet-like strip, or may be a housing-type support that accommodates the sheet-like strip.

  As the sheet-like support, for example, various plastic sheets (plastic sheets), hard paper, metal (including alloy) sheets such as aluminum, for example, a plurality of different or homogeneous papers are laminated (bonded). ) Multi-layer paper, laminated plastic sheet and paper (laminated), laminated paper and metal sheet (laminated), laminated plastic sheet, paper and metal sheet (laminated), Examples include a paper coated with a waterproof coating. Preferably, it has a waterproof function.

  The support in the form of a housing is preferably made of a moisture-impermeable material such as polyvinyl chloride, polypropylene, polyethylene, polystyrene, acrylic acid polymer, etc., but it should be used if it is made of paper and has a water-repellent finish. Can do. The housing corresponds to at least the `` liquid collection window '' corresponding to the sample collection part (1) of the above-mentioned sheet-like piece housed inside, and to the test result display part (a) of the determination part (3). It is desirable that a “control display window” is formed corresponding to the “determination display window” and the control display section (b). The “determination display window” and the “control display window” may be formed individually, but may be formed as one window.

  In the use of the determination tool of the present invention provided with the above-described sheet-like strip, first, the sample collection part (1) is impregnated with the test sample. Then, the test sample absorbed in the sample collection part (1) penetrates the sheet-like strip by capillary action, and first, the labeled antibody part (2) that is carried so that the label-anti-rabies virus antibody can be detached. ). Here, the test component (rabies virus) in the test sample specifically binds to the label-anti-rabies virus antibody by an antigen-antibody reaction. Next, the test sample is accompanied by "complex of rabies virus and label-anti-rabies virus antibody" and "excess label-anti-rabies virus antibody that did not bind to rabies virus". The test result display part (a) is reached. In the test result display part (a), the non-labeled antibody that specifically binds to the rabies virus--the anti-rabies virus antibody is stably immobilized, so that “label labeled specifically bound to the rabies virus in the test sample” “Anti-rabies virus antibody” is captured and accumulated. As a result, the test result display unit (a) develops a color with an intensity corresponding to the amount of the rabies virus contained in the test sample based on the labeling agent of the captured label-anti-rabies virus antibody. Thereby, the presence or the quantitative ratio of the rabies virus in the test sample (more precisely, the rabies virus not neutralized with the neutralizing antibody) can be detected.

  Next, the test sample reaches the control display section (b) of the determination section (3) accompanied by “excess label-anti-rabies virus antibody not bound to rabies virus”. Since the second antibody [unlabeled-antibody] that binds to the label-anti-rabies virus antibody is stably fixed in a certain amount in the control display part (b), the above excess label in the test sample is here. -Anti-rabies virus antibodies are captured and accumulated. As a result, the control display part (b) develops color with an intensity corresponding to the amount of the second antibody immobilized on the control display part (b) (or the amount of the labeled antibody bound to the second antibody).

  In addition to these, the determination tool of the present invention can be variously modified without departing from the scope of the present invention.

  In addition to the determination tool of the present invention, specifications describing the method of using the determination tool and the determination method can be attached, and the present invention provides a determination kit in which the determination tool and the specification are a set. It is to provide.

(II) Measuring method of neutralizing antibody titer of rabies virus TECHNICAL FIELD This invention relates to the method of measuring a rabies virus neutralizing antibody titer by making a test subject's body fluid into symmetry. This method is characterized by using the aforementioned rabies virus neutralizing antibody titer determination tool.

  Specifically, in the method of the present invention, the rabies virus (inactivated rabies virus) obtained by inactivating the body fluid of the subject is first mixed, and this is used as a test sample to determine the rabies virus neutralizing antibody titer of the present invention described above. This can be carried out by performing the step provided to the sample collection unit (1) and the step of detecting the color of the test result display unit (a) in the determination unit (3) of the determination tool.

  When the sample collection part (1) is impregnated with the test sample, the test sample absorbed in the sample collection part (1) penetrates the sheet-like strip by capillary action, and the label-anti-rabies virus antibody can be detached first It reaches the labeled antibody part (2) carried as such.

  Here, when rabies virus is contained in the test sample (when inactivated rabies virus is present without being neutralized), it specifically binds to the label-anti-rabies virus antibody by antigen-antibody reaction. To do. The "complex of rabies virus and label-anti-rabies virus antibody" formed here is accompanied by "excess label-anti-rabies virus antibody that did not bind to rabies virus", and the test of the determination part (3) The result display part (a) is reached. Since the non-labeled anti-rabies virus antibody that specifically binds to the rabies virus is stably immobilized in the test result display part (a), the “complex of rabies virus and label-anti-rabies virus antibody” is here. Are captured and accumulated. As a result, the test result display unit (a) develops a color with an intensity corresponding to the amount of the rabies virus contained in the test sample based on the labeling agent of the captured label-anti-rabies virus antibody. On the other hand, when the test sample does not contain the rabies virus (when the inactivated rabies virus is completely neutralized), the label-anti-rabies virus antibody and antigen carried on the labeled antibody part (2) Since no antibody reaction occurs, the test result display part (a) does not develop color. Therefore, by the method of the present invention, it is possible to detect the presence or the quantitative ratio of the rabies virus in the test sample (more precisely, the rabies virus that has not been neutralized with the neutralizing antibody).

  As a rabies virus neutralizing antibody titer determination tool, one having only the test result display part (a) in the determination part (3) can be used, but in addition to the test result display part (a), a label-anti-antibody A determination tool provided with a control display part (b) to which a second antibody [unlabeled-antibody] that binds to a rabies virus antibody is fixed can also be used. In this case, even if the test result display part (a) does not develop color, the test sample normally flows to the liquid absorption part by the color development of the control display part (b), and the test of the present invention is normally performed. It can be determined whether it has been done.

  The body fluid of the subject used in the method of the present invention is not particularly limited as long as it contains a neutralizing antibody against rabies virus, and examples thereof include urine, blood (serum, plasma), sweat, saliva and the like. Serum is preferred.

Inactivated rabies virus can be prepared by treating rabies virus with formalin, betapropiolactone (BPL), or ultraviolet rays as described above. method of inactivating Te is preferred (literature:. H Singh Laboratory technique of rabies , 1996, 4 th ed.WHO, Chapter 20, p234-242).

  According to WHO and OIE, the neutralizing antibody titer necessary to prevent rabies virus infection is 0.5 IU / ml or more. Therefore, the amount of inactivated rabies virus mixed with the body fluid of the subject was completely neutralized with a neutralizing antibody with a neutralizing antibody titer of 0.5 IU / ml, and with a neutralizing antibody with a neutralizing antibody titer of 1.0 IU / ml. It is preferred to use an amount that is not neutralized. When a body fluid mixed with such an amount of inactivated rabies virus is used as a test sample, according to the method of the present invention, the presence or absence of color development in the test result display section (a) in the determination section (3) It can be determined whether or not there is a neutralizing antibody (neutralizing antibody titer of 0.5 IU / ml or more) necessary for preventing rabies virus infection. That is, if the test result display part (a) is colored, it may be determined that the neutralizing antibody titer is not more than 0.5 IU / ml and there is a risk of rabies virus infection (necessity of rabies vaccination) On the other hand, if the test result display part (a) does not develop color, the neutralizing antibody titer is 0.5 IU / ml or more and there is no or low risk of rabies virus infection (no need for rabies vaccination) ). Therefore, the method of the present invention is a test for confirming whether the neutralizing antibody titer has reached a predetermined value (0.5 IU / ml or more) that can prevent rabies virus infection in humans and animals vaccinated with a rabies vaccine. Can also be used effectively.

(III) Monoclonal antibody against G protein of rabies virus The monoclonal antibody of the present invention is preferably a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable consisting of the amino acid sequence shown in SEQ ID NO: 3. A monoclonal antibody comprising a region and specifically binding to the G protein of rabies virus. Further, the monoclonal antibody of the present invention has a heavy chain variable region of 85% or more, preferably 90% or more of the amino acid sequence shown in SEQ ID NO: 1 unless specific binding to the G protein of rabies virus is impaired. More preferably, it may consist of an amino acid sequence having 95% or more identity, and the light chain variable region is 85% or more, preferably 90% or more, with the amino acid sequence shown in SEQ ID NO: 3. More preferably, it may consist of an amino acid sequence having 95% or more identity.

  Each of these heavy chain variable region and light chain variable region has three framework regions (FWR1 to FWR3) and two complementarity determining regions (CDR1 and CDR2) as described above.

  The monoclonal antibody of the present invention includes a heavy chain region containing the heavy chain variable region as a heavy chain region, a light chain region containing the light chain variable region as a light chain region, and specific to the G protein of rabies virus. Preferably, the antibody binds to. Here, the heavy chain region is preferably a region consisting of the amino acid sequence described in SEQ ID NO: 2, and the light chain region is preferably a region consisting of the amino acid sequence described in SEQ ID NO: 4. As long as the specific binding property to the G protein of rabies virus is not impaired, the heavy chain region of the monoclonal antibody is not less than 85%, preferably not less than 90%, more preferably not less than the amino acid sequence shown in SEQ ID NO: 2. It may consist of an amino acid sequence having 95% or more identity. The light chain region is also 85% or more, preferably 90% or more, more preferably 95% or more of the amino acid sequence shown in SEQ ID NO: 4 as long as the monoclonal antibody does not impair the specific binding property to the G protein of rabies virus. It may consist of amino acid sequences having% or more identity.

  In addition, the binding property to the G protein of rabies virus can be evaluated by eliminating the infectivity to sensitive cells by the virus bound to the monoclonal antibody. Specifically, a monoclonal antibody and a rabies virus are mixed at an appropriate ratio, and this is inoculated into susceptible cells (for example, Baby Hamster Kidney cells, neuroblastoma NA cells) that can infect and proliferate with the virus. Later, it can be judged by staining with a monoclonal antibody against anti-rabies virus N protein that the focus of infected cells is not formed.

  The method for preparing such a monoclonal antibody can be specifically exemplified by the method described in Example 1, but in addition, a method for preparing by amino acid synthesis with reference to the amino acid sequence of the above monoclonal antibody, and a monoclonal antibody It can also be prepared by a method prepared by genetic engineering techniques with reference to the base sequence of the gene to be encoded.

  The base sequence encoding the heavy chain region (SEQ ID NO: 2) of the monoclonal antibody of the present invention is shown in SEQ ID NO: 5, and the base sequence encoding the light chain region (SEQ ID NO: 4) is shown in SEQ ID NO: 6, respectively. The base sequence encoding the heavy chain region of the monoclonal antibody of the present invention has a homology of 85% or more with the base sequence of SEQ ID NO: 5, and binds to a base sequence complementary to the base sequence under stringent conditions. It may have a base sequence. The base sequence encoding the light chain region of the monoclonal antibody of the present invention has 85% or more homology with the base sequence of SEQ ID NO: 6, and binds to the base sequence complementary to the base sequence under stringent conditions. It may have a base sequence. Here, stringent conditions include, for example, hybridization in a buffer containing 50% formamide, 5 × SSC, and 1% SDS at 42 ° C., or 5 × SSC, and 1% SDS at 65 ° C. (Both accompanied by a wash of 0.2 × SSC and 0.1% SDS at 65 ° C.).

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to such examples.

Example 1 Preparation of monoclonal antibody against G protein of rabies virus (1-1) Immunization of mouse and isolation of spleen cells Spleen cells (B cells) used for cell fusion were formalin-inactivated (described later in Example 2 of Example 2). -Refer to -2) The rabies virus CVS-11 strain was sacrificed in the abdominal cavity of Balb / c mice (6 weeks old female) every 2 weeks for a total of 3 days after administration, and prepared from the removed spleen. NS1 / 1-Ag 4.1 cells (RIKEN Cell Bank (Wako City, Saitama Prefecture)) were used as mouse myeloma cells used as cell fusion partners.

(1-2) Rabies virus In order to evaluate neutralizing ability against various rabies virus strains, antigenically different fixed (adapted) strains (Evelyn-Lokitoniki-Averces (ERA) strain, CVS-11 strain, Nishigahara strain, HEP-Flury strain) and street rabies virus (1088 field strain) were used. Each of the above-mentioned fixed strains and 1088 outdoor strains were stored at Oita University School of Medicine Microbiology.

(1-3) Cell fusion and culture method Spleen cells and myeloma cells are mixed in a 5: 1 ratio in a test tube, and the test tube is warmed in a 37 ° C water bath for 1 minute, and then warmed to 0.5 ml (37 ° C). 1 ml of 50% (wt / vol) polyethylene glycol (Sigma Chemical Co., St. Louis, MO, catalog number: P-7181), gently shaking the tube for 45 seconds Added dropwise. Thereafter, 3 ml of warm serum-free medium was slowly added over 30 seconds, and then 9 ml was added in 3 portions over 30 seconds to stop the fusion reaction. The tube was allowed to stand at room temperature for 8 minutes and then incubated for 2 minutes in a 37 ° C. water bath. The resulting fused cells are then centrifuged at 500 g for 3 minutes, and the cell pellet is gently resuspended in 30 ml of Iskov modified Dulbecco (Gibco) medium containing 10% FBS and HAT, and a 96-well flat bottom. The cells were plated in a microtiter plate at a concentration of 3.5 × 10 ⁵ cells / well and incubated in the same manner as described above. (Change the HAT medium once in the middle). Thereafter, the culture medium was replaced with HT medium, and the hybridized cells were confirmed under direct observation. The supernatant secreted from the well in which the colony was formed was transferred to the subsequent assay.

  After culturing the fused cells for about 4 weeks, the supernatant was collected and tested for the presence of rabies virus specific antibodies by ELISA. Positive wells are first transferred to 1 ml and then 2 ml cultures (48 and 24 well plates, Nunc), after which the supernatant is RFFIT (eg, see Example 3 (2-1) below). Were assayed for the presence or absence of rabies virus neutralizing antibodies. When a colony is established (cell # 4-12) that produces neutralizing antibody (cell culture for about 6 weeks), production of rabies virus-specific antibody in the supernatant by ELISA and RFFIT method It was confirmed. The antibody producing cells (cell # 4-12) were cloned at least 3 times by limiting dilution in microtiter plates. Cells were titrated in 96 well round bottom plates starting at 4 cells / well at 2-fold dilution. Cells collected from wells containing an average of 0.25 cells or less were grown and the supernatant was collected (culture supernatant sample).

(1-4) Analysis of rabies virus-specific antibody by ELISA The antibody specificity and isotype of the antibody produced from the cells (cell # 4-12) obtained above were evaluated by solid-phase ELISA. First, 5 mg / ml rabies ERA virus in which plates (PolySorb ™, Nunc) were diluted in phosphate buffered saline (PBS) overnight at room temperature in a humidified chamber Coated with glycoprotein or nucleoprotein. The plates were then blocked with 5% milk powder in PBS, washed with 0.05% Tween ₂₀ (Tween ₂₀₎ containing PBS (PBS-Tween), it was added to the culture supernatant samples of the antibody producing cells.

(1-5) Determination of antibody isotype After the supernatant prepared above was incubated at room temperature for 2 hours, this plate was washed with PBS-Tween to remove unbound antibody. An antibody specific to a chain isotype and a secondary antibody conjugated with various enzymes or a videotinylated secondary antibody was added and allowed to react at room temperature for 1 hour. After the reaction, a substrate (3,3 ′, 5,5′-tetramethylbenzidine (TMB) in phosphate-citrate buffer, or p− in 0.1 M glycine buffer, depending on the enzyme bound to the secondary antibody. Add nitrophenyl phosphate (PNPP), Sigma, or, in the case of videotinylated secondary antibody, add avidin-alkaline phosphate (30 minutes at room temperature (RT)) and PNPP substrate, secondary antibody As a result, it was found that the isotype of the monoclonal antibody produced by cell # 4-12 was IgG2.

(1-6) Purification of antibodies by affinity chromatography
Monoclonal antibodies produced by IgG2 antibody-producing cells # 4-12 were purified using a protein A column (rProtein A Sepharose ™ Fast Flow, Amersham Pharmacia Biotech). Specifically, first, the culture supernatant of each cell was purified by filtration through a 0.45 μm membrane, and the pH was adjusted to 8.0 with 1N NaOH. Next, this was passed through the column at a linear flow rate of about 100 cm / hour, washed with PBS (pH 8), and then the antibody was eluted from the column using a 0.1 M citric acid solution, and then dialyzed against PBS.

(1-7) Binding of rabies virus to G protein The monoclonal antibody obtained above is mixed with the rabies virus in an appropriate ratio, and this is inoculated into a sensitive cell (Baby Hamster Kidney cell). Using a monoclonal antibody against the rabies virus N protein, it was confirmed that the focus of infected cells was not formed. From this, it was determined that the obtained monoclonal antibody has no infectivity to sensitive cells, that is, the monoclonal antibody has binding property to G protein on the surface of rabies virus.

(1-8) Amino acid analysis of monoclonal antibody The monoclonal antibody (antibody # 4-12) produced by the cell # 4-12 thus prepared was subjected to amino acid analysis according to a conventional method.

  As a result, antibody # 4-12 was found to have a heavy chain region consisting of the 137 amino acid sequence shown in SEQ ID NO: 2 and a light chain region consisting of the 98 amino acid sequence shown in SEQ ID NO: 4. Among such heavy chain regions, the amino acid region of 11 to 108 is a heavy chain variable region. The heavy chain variable region is composed of three framework regions (FWR1 to FWR3) and two complementarity determining regions (CDR1 and CDR2). Specifically, the heavy chain variable region consists of FWR1 (11-40 amino acid region), CDR1 (31-45th amino acid region), FWR2 (46-59th amino acid region), CDR2 (60 -76th amino acid region) and FWR3 (77-108th amino acid region).

  Further, the amino acid region of 1 to 72 in the light chain region is the light chain variable region. Similar to the heavy chain variable region, the light chain variable region is composed of three framework regions (FWR1 to FWR3) and two complementarity determining regions (CDR1 and CDR2). FWR1 (1-7 amino acid region), CDR1 (8-18 amino acid region), FWR2 (19-33 amino acid region), CDR2 (34-40 amino acid region), and FWR3 (41- 72th amino acid region).

  The base sequence of the gene encoding the amino acid sequence of the heavy chain region is shown in SEQ ID NO: 5, and the base sequence of the gene encoding the amino acid sequence of the light chain region is shown in SEQ ID NO: 6, respectively.

Example 2 Preparation of determination tool (1) Preparation of colloidal gold label-anti-rabies virus antibody A colloidal gold label-anti-rabies virus antibody was prepared using the monoclonal antibody (antibody # 4-12) prepared above. Specifically, first, 25.0 mL of anti-rabies monoclonal antibody (antibody # 4-12) diluted to 0.1 mg / mL in potassium carbonate buffer was added to 500 mL of colloidal gold solution, and reacted at room temperature for 30 minutes. Next, 5 mL of a bovine serum albumin aqueous solution having a concentration of 10 g / dL was added and reacted for 20 minutes. After centrifugation, the collected sediment was made up to 100 mL with the same buffer as above, and this was used as a gold colloid-labeled anti-rabies virus antibody solution.
(2) Preparation of Rabies Virus Neutralizing Antibody Titer Determination Tool As shown in FIG. 1 (B), on the support 10 , a sample collection part (1) (sheet 21 ), a labeled antibody part (2) (sheet 22 ) ), Determination unit (3) (sheet 23 ), and determination unit (width 6 x length 69 mm) in which the liquid-absorbing pieces (4) (sheet 24 ) are successively laminated. Here, the support 10 is a YUPO sheet (manufactured by YUPO Corporation), the sample collection section (1) (sheet 21 ) and the labeled antibody section (2) (sheet 22 ) are glass fiber filter paper, and the determination section (3) (sheet 23 ). Is a nitrocellulose, and the liquid absorption part (4) (sheet 24 ) is made of glass fiber filter paper.

  Monoclonal antibody # 4-12 prepared above was immobilized at a rate of 0.75 μg / strip on the (a) test result display part (test line) of the judgment part (3) of the judgment tool. In addition, (b) on the control line, an anti-mouse IgG goat antibody (obtained from DAKO) as a non-labeled antibody (second antibody) that reacts with the labeled anti-rabies virus antibody was obtained at 0.70 μg / strip. Fixed in proportion. The antibody was fixed to each member by spraying the antibody solution at room temperature using a bio-Dot (USA) applicator and then air-drying.

The colloidal gold-labeled monoclonal antibody was adsorbed at a rate of 1 μg / strip on the (2) labeled antibody portion (sheet 22 ) of the above-described determination tool. Specifically, the antibody solution prepared above was adsorbed on a sheet 22 made of glass fiber filter paper and dried at room temperature.

Thus
(1) Sample collection part for absorbing and collecting the test sample (1),
(2) Labeled antibody part carrying anti-rabies virus antibody that specifically reacts with G protein of rabies virus (2)
(3) Judgment part (3) provided with the following test result display part (a) and control display part (b) at an interval,
(a) an unlabeled anti-rabies virus antibody immobilized specifically reacting with the G protein of rabies virus;
(b) a labeled display—an unlabeled antibody that reacts with an anti-rabies virus antibody—an antibody (second antibody) immobilized control display;
(4) Create a rabies virus neutralizing antibody determination tool equipped with a liquid absorption part that absorbs the residual liquid of the test sample that has moved through the sample collection part (1), labeled antibody part (2), and determination part (3) did.

Example 3 Measurement of Rabies Virus Neutralizing Antibody Titer All the following studies were conducted with the approval of the Oita University Medical School Ethics Committee (IRB).
(1) Preparation of sample (1-1) Preparation of test sample Human serum was used as a test sample. Specifically, serum was collected from subjects who had been vaccinated in the past (raised vaccination) (n = 27) and subjects who have never been vaccinated (vaccinated non-vaccinated) (n Collected from = 10). Most of the rabies vaccine used for the above inoculation was a tissue-cultured rabies vaccine for human tissue (PCEC) manufactured by Kakenken. In addition, 5 people who had been vaccinated were further vaccinated with rabies vaccine (PCEC), and serum was collected again from the subject. As a result, a total of 42 serum samples were used for the measurement described below.

  Serum was separated from the blood collected from each subject by centrifugation, treated at 56 ° C. for 30 minutes to inactivate (complement inactivation), and stored at −20 ° C. until use.

(1-2) Rabies virus and its inactivation The challenge virus standard (CVS-11) strain was used as a rabies virus. The CVS-11 virus was grown on Eagle's MEM medium containing 2% FBS on a baby hamster kidney epithelial cell line (BHK-21) and collected.

Inactivated CVS-11 virus (inactivated virus) was prepared as follows. BHK cells were infected with CVS-11 at moi 0.1 (0.1 infectious unit of virus per cell) and cultured in the presence of CO ₂ at 37 ° C. for 4 days. After 4 days, the culture supernatant was recovered, and then the cells were precipitated by centrifuging at 2,800 rpm for 10 minutes at 4 ° C., and the supernatant was recovered. To the resulting supernatant, 37% formaldehyde (formalin) was added to a final concentration of 0.05%, and the mixture was stirred at 37 ° C. for 48 hours. Thereafter, the formalin-treated culture supernatant was centrifuged at 16,000 g for 24 hours at high speed to remove the supernatant, and then the precipitate was suspended in 1/10 PBS of the original liquid volume. Thereafter, the mixture was dialyzed with PBS at 4 ° C. for 24 hours, and the resulting dialyzate was used as the final preparation (inactivated rabies virus).

  The protein concentration (measured by the Lowry method) of the final preparation (inactivated rabies virus) prepared in 4 batches was 1.5 to 1.8 mg / ml, which was almost constant. The final preparation (inactivated rabies virus) was added to mouse neuroblastoma NA-C1300 cells or BHK-21 cells, and 24 hours later, FITC-labeled anti-rabies virus antibody (Fujirebio Diagnostics, Inc., Malvern, PA, USA) was reacted and observed under a fluorescence microscope, and it was confirmed that no infectious ability remained, that is, inactivated.

(2) Measurement of rabies virus neutralizing antibody titer For serum collected from subjects, rabies virus neutralizing antibody titer was measured by (a) the rapid fluorescence focus suppression test method (RFFIT), which is the conventional method, and (b) this The rapid neutralizing antibody test (RAPINA test) of the present invention was performed.

(2-1) Rapid fluorescence focus suppression test (RFFIT) method
The RFFIT method was performed according to the method described in Smith et al., (Bull WHO 1973; 48; 535-541). In addition, the rabies virus neutralizing antibody titer of all the serum samples was measured in duplicate.

Specifically, each serum sample was first diluted 2-fold in a 96-well plate using Eagle's MEM medium containing 2% FBS. Serially diluted serum in each well was inoculated with 30-100 TCID ₅₀ (50% tissue culture infectious dose) of CVS-11 virus and incubated at 37 ° C. for 90 minutes in the presence of 5% CO ₂ . Thereafter, BHK-21 cells (1.0 × 10 ⁶ cells / ml) were added to each well and again incubated at 37 ° C. for 24 hours. The plates were then fixed with 90% acetone and reacted with FITC-labeled anti-rabies virus antibody (Fujirebio Diagnostics, Inc., Malvern, PA, USA) at 37 ° C. for 45 minutes.

  Next, the 50% fluorescence focus suppression concentration was calculated by the Speerman-Karber method. Neutralizing antibody titers were expressed in international units (IU / ml) by comparison with WHO standard serum.

(2-2) Rapid neutralizing antibody titer test (RAPINA test)
The RAPINA test of the present invention was performed using the rabies virus neutralizing antibody determination tool prepared in Example 1. When a test sample (sample) is dropped onto the sample collection part (1) (sheet 21) of the determination tool, the sample is placed on the liquid-absorbing piece, the labeled antibody part (2) (sheet 22), and the determination part (3) (sheet). 23), and the liquid absorption part (4) (sheet 24). At this time, if the G protein of the rabies virus is contained in the test sample, the labeled anti-antibody adsorbed to the labeled antibody part (2) It binds with the rabies virus antibody to form an antigen-antibody complex. This complex further travels on the liquid absorption piece together with the test sample, is trapped by the label-anti-rabies virus antibody fixed to the test result display section (a) of the determination section (3), and develops a red band at this site. (Nishizono A. et al. Microbiology and Immunology 2008, 52: 243-249).

  FIG. 3 shows a schematic reaction diagram when a test sample containing the G protein of rabies virus is used in the sample collection part (1) of the determination tool. On the other hand, FIG. 4 shows a reaction schematic diagram when the G protein in the test sample is neutralized with a neutralizing antibody of rabies virus.

A qualitative measurement system for rabies virus neutralizing antibody titer was constructed using this determination tool. Specifically, first of all, inactivation using the human serum of 0.5 IU / ml and 1.0 IU / ml of rabies virus neutralizing antibody titer, which is an international standard of WHO, prepared in the above (1-2) It was mixed with VS-11 virus (inactivated virus) and incubated at 37 ° C. for 90 minutes in the presence of CO ₂ .

  Subsequently, this was added to the above-described determination tool to the sample collection part (1), and after 30 minutes, the presence or absence of a colored band in the test result display part (a) of the determination part (3) was observed. As a result, a colored band was confirmed in the case of inactivated virus treated with a standard serum having a rabies virus neutralizing antibody titer of 0.5 IU / ml. This means that serum with a neutralizing antibody titer of 0.5 IU / ml cannot completely neutralize the G protein in the inactivated virus. On the other hand, in the case of the inactivated virus treated with the standard serum having a rabies virus neutralizing antibody titer of 1.0 IU / ml, a colored band could not be confirmed. This means that serum with neutralizing antibody titer 1.0 IU / ml can completely neutralize the G protein in the inactivated virus. From this, it was found that the inactivated virus (1.5 mg / ml) prepared in the above (1-2) can be neutralized with a neutralizing antibody having a neutralizing antibody titer of 0.5 to 1.0 IU / ml. That is, when the inactivated virus (1.5 mg / ml) prepared in the above (1-2) and serum are mixed and used as a test sample in the determination tool of the present invention, (a) a test result display section If a red band appears, the neutralizing antibody titer in the target serum can be determined to be 0.5 IU / ml or less. If a red band does not appear, The neutralizing antibody titer of can be determined to be 0.5 IU / ml or more.

  Based on this, an inactivated virus was prepared so as to be neutralized with a neutralizing antibody having a neutralizing antibody titer of 0.5 IU / ml. Such inactivated virus is mixed with each serum sample (n = 42), incubated at 37 ° C. for 90 minutes, and added to the sampling part (1) of the determination tool in the same manner as above, and after 30 minutes have passed. The presence or absence of a colored band in the test result display part (a) of the judgment part (3) was observed. Judgment was performed blindly by three people who did not know the results of measuring each serum sample by the RFFIT method.

  The results are shown in Table 1 and FIG.

  In FIG. 5, “+” on the horizontal axis indicates a positive reaction in RAPINA test (that is, when the serum neutralizing antibody titer is less than 0.5 IU / ml), and “−” indicates a negative reaction in RAPINA test (that is, Serum neutralizing antibody titer is 0.5 IU / ml or higher). The vertical axis means the neutralizing antibody titer (IU / ml) in serum measured by RFFIT method.

  As can be seen from these results, none of the RAPINA test confirmed a red band (positive) showed a neutralizing antibody titer of 0.5 IU / ml or more by the RFFIT method, but the RAPINA test showed a red band. Among those that were not confirmed (positive), some (1/18 cases) had a neutralizing antibody titer with RFFIT of less than 0.5 IU / ml. RFFIT neutralizing antibody titers of 1.0 IU / ml or more were all judged negative in the RAPINA test (FIG. 5).

  From this result, the detection accuracy of the neutralizing antibody titer of rabies virus by the RAPINA test of the present invention was evaluated using the neutralizing antibody titer by the RFFIT method as the Golden standard. The results are shown in Table 2.

  As shown in the table, sensitivity (probability that a serum having a neutralizing antibody titer of 0.5 IU / ml or more is judged as negative by the determination tool of the present invention) 89.5%, specificity (serum having a neutralizing antibody titer of less than 0.5 IU / ml Probability of being determined as positive by the determination tool of the present invention) 95.7%, accuracy (coincidence rate with RFFIT method) 92.9%, positive reaction rate (serum determined as positive by the determination tool of the present invention is neutralizing antibody (Probability that the titer is less than 0.5 IU / ml) and 91.7%, and negative predictive value (probability that the serum determined to be negative with the determination tool of the present invention has a neutralizing antibody titer of 0.5 IU / ml or more) is 94.4% It was. False negative was confirmed in 1 out of 18 negatives. This was a past vaccination and the neutralizing antibody titer in the RFFIT method was around 0.4 IU / ml and approximated 0.5 IU / ml. It was an example having a valence. None of the unvaccinated patients (non-vaccinated persons) were found to be false negative, and both sensitivity and specificity were 100%. Since these subjects have been vaccinated against other viruses in the past, it is considered that the method of the present invention has no cross-reactivity against viruses other than rabies virus.

  False positives were confirmed in 2 of 24 positives. For serum having a neutralizing antibody titer of 1.0 IU / ml or more, the sensitivity of the determination tool of the present invention was 100%. When rabies vaccination is performed according to a schedule, many have neutralizing antibody titers of 1.0 IU / ml or more, and thus the RAPINA test of the present invention is considered useful for rabies vaccinated persons.

(A) And (B) is a figure (figure seen from the side) which shows one aspect | mode of the rabies virus neutralization antibody titer determination tool of this invention. Fig. (A) shows a mode in which only the test result display unit (a) is provided in the determination unit (3), and Fig. (B) shows a test result display unit (a) and a control display unit (b) in the determination unit (3). It is an aspect including both. It is a figure (perspective view) which shows one aspect | mode of the rabies virus neutralization antibody titer determination tool of this invention. It is a schematic diagram which shows reaction when the test sample containing a rabies virus is used for the rabies virus neutralization antibody titer determination tool of this invention. It is a schematic diagram which shows reaction when the test sample containing the rabies virus neutralized with the neutralizing antibody is used for the rabies virus neutralizing antibody titer determination tool of the present invention. The results of measurement of the neutralizing antibody titer of rabies virus performed in Example 2 were compared between the conventional rapid fluorescence focus suppression test (RFFIT) method and the rapid neutralizing antibody titer test method (RAPINA test) of the present invention. FIG.

Explanation of symbols

10: Support 21: Sheet 22 constituting the sample collection part (1) 22: Sheet 23 constituting the labeled antibody part (2) 23: Sheet 24 constituting the determination part (3) 24: Liquid absorption part (4) Sheet
(a): Test result display area
(b): Control display area
(1): Sampling part
(2): Labeled antibody part
(3): Judgment unit
(4): Liquid absorption part P: Arrow indicating the method of flowing the test sample

Claims (10)

A rabies virus neutralizing antibody titer determination device comprising a liquid absorption piece composed of a material capable of transferring a test sample by capillary action, wherein the liquid absorption piece
(1) a sample collection part for absorbing and collecting a test sample,
(2) a label that specifically reacts with the G protein of rabies virus-a labeled antibody portion carrying an anti-rabies virus antibody,
(3) A determination unit having a test result display unit as shown below,
(a) a non-labeled anti-rabies virus antibody-fixed test result display part that specifically reacts with the G protein of rabies virus,
(4) A rabies virus neutralizing antibody titer determination device comprising a liquid absorption unit that absorbs a residual liquid of a test sample that has moved through the sample collection unit, the labeled antibody unit, and the determination unit. The rabies virus neutralizing antibody titer determination according to claim 1, wherein the (3) determination unit further comprises (a) a control display unit shown below, spaced apart from the test result display unit. Ingredients:
(b) Labeled control display part with immobilized non-labeled antibody that reacts with anti-rabies virus antibody.   An anti-rabies virus antibody used in a labeled-anti-rabies virus antibody and an unlabeled-anti-rabies virus antibody, the heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 95% or more identity thereto; and A monoclonal antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having 95% or more identity thereto, and specifically binding to a G protein of rabies virus The rabies virus neutralizing antibody titer determination device according to claim 1 or 2.   The anti-rabies virus antibody used in the labeled-anti-rabies virus antibody and the unlabeled-anti-rabies virus antibody is a heavy chain region comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having 95% or more identity thereto, and a sequence A monoclonal antibody comprising a light chain region comprising the amino acid sequence described in No. 4 or an amino acid sequence having 95% or more identity thereto, and specifically binding to a G protein of rabies virus, Item 3. The rabies virus neutralizing antibody titer according to Item 1 or 2.   The step of using the mixture of the inactivated rabies virus and the subject body fluid as a test sample for the sample collection part of the rabies virus neutralizing antibody titer determination tool according to any one of claims 1 to 4, and the determination part of the determination tool A method for measuring a neutralizing antibody titer of a rabies virus in a body fluid of a subject, the method comprising detecting a color of a test result display unit.   The amount of inactivated rabies virus is characterized by using an amount neutralized with a neutralizing antibody with a neutralizing antibody titer of 0.5 IU / ml and not neutralized with a neutralizing antibody with a neutralizing antibody titer of 1.0 IU / ml. The method according to claim 5.   The method according to claim 5 or 6, wherein the serum of an animal excluding a rabies vaccination person and a human after rabies vaccination is used as the subject serum.   It consists of the heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 95% or more identity thereto, and the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 95% identity or more. A monoclonal antibody comprising a light chain variable region and specifically binding to a G protein of rabies virus.   A heavy chain region consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 95% or more identity thereto, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 95% or more identity thereto The monoclonal antibody according to claim 8, which comprises a chain region and specifically binds to the G protein of rabies virus.   A gene encoding the monoclonal antibody according to claim 9, wherein the nucleotide sequence set forth in SEQ ID NO: 5 and the nucleotide sequence set forth in SEQ ID NO: 6 are respectively converted into a nucleotide sequence encoding a heavy chain region of the monoclonal antibody and a light sequence. The gene as described above, which comprises a base sequence encoding a chain region.

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