JP2017129549A - Antibody xiii/13 factor b subunit antibody detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting antibody against XIII/13 factor B subunit (FXIII/13-B) in blood, and a kit used for the detection.SOLUTION: This method for detecting an isolated antibody against a XIII/13 factor B subunit (FXIII/13-B) in a blood sample includes: bringing a sample and a marked antihuman IgG antibody into contact with a support medium in which the XIII/13 factor B subunit (FXIII/13-B) has been solid-phased; forming a composite of the XIII/13 factor B subunit (FXIII/13-B)-the antibody against the XIII/13 factor B subunit (FXIII/13-B)-the marked antihuman IgG antibody on the support medium; and detecting a signal from the marked antihuman IgG antibody. A kit used for this method is also provided.SELECTED DRAWING: None

Description

  The present invention relates to a method for detecting an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody.

  Autoimmune hemorrhagic disease XIII / 13 (AH13; Ministry of Health, Labor and Welfare designated intractable disease 288) caused by a marked decrease in coagulation factor XIII / 13 (FXIII / 13) is a serious condition that requires early diagnosis and early treatment It is a hemorrhagic disease and is known to develop due to various causes (see Non-Patent Documents 1 to 3). Among them, autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) based on the appearance of antibodies against its own factor XIII / 13 B subunit (FXIII / 13-B) requires immunosuppressive therapy Therefore, rapid antibody detection is essential. Until now, since there was no reliable method for detecting anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibodies, the ratio of this disease among all autoimmune hemorrhagic diseases XIII / 13 (AH13) Since it was unknown, establishment of a highly reproducible detection method was desired.

Ichinose A, et al .; Haemophilia. 2015 Sep; 21 (5): 653-8. Souri M, et al .; J Thromb Haemost. 2015 May; 13 (5): 802-14. Wada H, et al .; Thromb Haemost. 2013 Apr; 109 (4): 661-8.

  An object of the present invention is to provide a method for detecting an antibody against factor XIII / 13 B subunit (FXIII / 13-B) in blood and a kit used for the detection.

  In order to accurately detect autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) based on the appearance of antibodies against self factor XIII / 13 B subunit (FXIII / 13-B) The present inventors conducted extensive studies on a method for detecting autoantibodies against factor XIII / 13 B subunit (FXIII / 13-B).

  The present inventors are in a state where the autoantibodies against factor XIII / 13 B subunit (FXIII / 13-B) are free antibodies, or complex with factor XIII / 13 B subunit (FXIII / 13-B). In view of the fact that it is present in the blood in the state of forming a factor XIII / 13 factor B subunit (FXIII / 13-B) is immobilized on a support and the factor XIII / 13 factor B subunit in blood (FXIII / 13-B) Immunoassay based on the principle of sandwich method to capture and measure free autoantibodies, and anti-Factor XIII / 13 B subunit antibody immobilized on a support Based on the principle of sandwich method to capture autoantibodies against factor XIII / 13 B subunit (FXIII / 13-B) present in blood in a complex with factor B subunit (FXIII / 13-B) Based immunoassay based on sensitive and specific autoantibodies against factor XIII / 13 B subunit (FXIII / 13-B) in blood It found that it is possible to detect, and completed the present invention.

That is, the present invention is as follows.
[1] A method for detecting a free antibody against a factor XIII / 13 factor B subunit (FXIII / 13-B) in a blood sample, wherein the factor XIII / 13 factor B subunit (FXIII / 13-B) is detected A sample and a labeled anti-human IgG antibody are brought into contact with the solid-phased support, and the factor XIII / 13 factor B subunit (FXIII / 13-B) -factor XIII / 13 factor B subunit (FXIII / 13. A method comprising forming an antibody-labeled anti-human IgG antibody complex against 13-B) and detecting a signal from the labeled anti-human IgG antibody.
[2] The method according to [1], wherein the free antibody against the factor XIII / 13 B subunit (FXIII / 13-B) is an autoantibody.
[3] A method for detecting an antibody against a factor XIII / 13 factor B subunit (FXIII / 13-B) complexed with a factor XIII / 13 factor B subunit (FXIII / 13-B) in a blood sample A sample and a labeled anti-human IgG antibody are brought into contact with a support on which an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody is immobilized, and the anti-factor XIII / 13 on the support. B subunit (FXIII / 13-B) antibody-antibody complex-labeling of factor XIII / 13 factor B subunit (FXIII / 13-B) and factor XIII / 13 factor B subunit (FXIII / 13-B) Forming a complex of the anti-human IgG antibody, and detecting a signal from the labeled anti-human IgG antibody.
[4] The antibody against factor XIII / 13 B subunit (FXIII / 13-B) complexed with factor XIII / 13 B subunit (FXIII / 13-B) is an autoantibody, [3] the method of.
[5] The method according to any one of [1] to [4], which is selected from the group consisting of Western blotting, ELISA, and immunochromatography.
[6] The method according to any one of [1] to [5], which is a method for obtaining auxiliary data for detection of autoimmune bleeding disease XIII / 13 (AH13).
[7] The method of [6], wherein the autoimmune bleeding disease XIII / 13 (AH13) is autoimmune bleeding disease XIII / 13 type B (AH13-B).
[8] A support comprising a solid-phased factor XIII / 13 B subunit (FXIII / 13-B) and a labeled anti-human IgG antibody, wherein the factor XIII / 13 B subunit (FXIII / 13-B)-by forming an antibody-labeled anti-human IgG antibody complex against factor XIII / 13 B subunit (FXIII / 13-B) and detecting the signal from the labeled anti-human IgG antibody A kit for detecting free antibody against factor XIII / 13 B subunit (FXIII / 13-B) in a blood sample.
[9] A support on which an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody is immobilized and a labeled anti-human IgG antibody, the anti-factor XIII / 13 B subunit on the support (FXIII / 13-B) antibody-antibody complex against factor XIII / 13 B subunit (FXIII / 13-B) and factor XIII / 13 B subunit (FXIII / 13-B) -labeled anti-human Form XIII complexed with factor XIII / 13 factor B subunit (FXIII / 13-B) in blood sample by forming IgG antibody complex and detecting signal from labeled anti-human IgG antibody Kit for detecting an antibody against the / 13 factor B subunit (FXIII / 13-B).
[10] The kit according to [8] or [9], which is selected from the group consisting of a kit for Western blotting, a kit for ELISA, and a kit for immunochromatography.
[11] The kit according to any one of [8] to [10], which is a kit for obtaining auxiliary data for detection of autoimmune bleeding disease XIII / 13 (AH13).
[12] The kit according to [11], wherein the autoimmune bleeding disease XIII / 13 (AH13) is autoimmune bleeding disease XIII / 13 type B (AH13-B).

  A support on which the factor XIII / 13 B subunit (FXIII / 13-B) of the present invention is immobilized or a support on which an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody is immobilized Using an immunoassay based on the principle of the sandwich method, the antibody against factor XIII / 13 B subunit (FXIII / 13-B) in blood can be detected with high sensitivity and specificity. . Includes autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) by detecting autoantibodies or alloantibodies against factor XIII / 13 B subunit (FXIII / 13-B) in the subject's blood Detection of autoimmune bleeding disease XIII / 13 (AH13), “Anti-factor XIII / 13 factor B subunit (FXIII / 13-B) in hereditary factor XIII / 13 B subunit (FXIII / 13-B) deficiency cases ) Homologous antibody "can be detected.

It is a figure which shows the result of the western blotting method for selection of an antibody with the high sensitivity and specificity with respect to a factor XIII / 13 B subunit (FXIII / 13-B) detection. It is a figure which shows the result of having examined the reactivity of the anti factor XIII / 13 B subunit (FXIII / 13-B) antibody by ELISA method. It is a figure which shows the result of having examined the detection sensitivity of the anti factor XIII / 13 B subunit (FXIII / 13-B) antibody by ELISA method. It is a figure which shows the result of the diagnosis of the autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) by the immunochromatography method using six types of monoclonal antibodies. It is a figure which shows the result of the diagnosis of the autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) by the immunochromatography using the monoclonal antibody 1-3C diluted in steps. It is a figure which shows the result of the diagnosis of the autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) by the immunochromatography method using the monoclonal antibody 1-3C which carried out the enzyme process. It is a figure which shows the result of the anti- factor XIII / 13 B subunit (FXIII / 13-B) antibody detection immunochromatography method (direct method) of an autoantibody positive case, an alloantibody positive example, and an autoantibody negative case. It is a figure which shows the result of the anti- factor XIII / 13 B subunit (FXIII / 13-B) antibody detection immunochromatography method (mixing method and dilution mixing method). It is a figure which shows the result of the anti- factor XIII / 13 B subunit (FXIII / 13-B) antibody detection immunochromatography method (mixing method).

Hereinafter, the present invention will be described in detail.
The present invention is a method for measuring an antibody against factor XIII / 13 B subunit (FXIII / 13-B protein) in blood by an immunoassay based on the sandwich method. Here, the immunoassay based on the sandwich method of the present invention is to measure a target substance by capturing the target substance as an antibody so as to be sandwiched by using an antigen or an antibody that binds to the target substance. This method is called simply the sandwich method. In an immunoassay based on the sandwich method, a solid phase bound to an antibody or antigen capable of binding to a target substance and capturing the target substance is used. Examples of the immunoassay based on the sandwich method include immunochromatography, enzyme immunoassay adsorption (ELISA), and Western blot.

In the present invention, the term “measurement” includes any of quantitative, semi-quantitative, and detection.
As an antibody against the factor XIII / 13 B subunit (FXIII / 13-B protein) to be measured in the present invention, an autoantibody, that is, its own factor XIII / 13 B subunit (FXIII / 13-B protein) ) As well as alloantibodies, ie non-self factor XIII / 13 B subunit (FXIII / 13-B protein), eg factor XIII / 13 factor B subunit in plasma-derived FXIII / 13 concentrate Antibodies against (FXIII / 13-B protein) are also encompassed.

  Antibody to factor XIII / 13 B subunit in blood (FXIII / 13-B) exists as free antibody or forms a complex with factor XIII / 13 B subunit (FXIII / 13-B) Exists in the state. Complex of factor XIII / 13 B subunit (FXIII / 13-B) and autoantibody (or alloantibody) when the amount of factor XIII / 13 B subunit (FXIII / 13-B) in the blood is high (Factor XIII / 13 B subunit (FXIII / 13-B) -autoantibody (or alloantibody) complex) is likely to be formed. Therefore, in the method of the present invention, an antibody against free factor XIII / 13 B subunit (FXIII / 13-B) or a complex of factor XIII / 13 B subunit (FXIII / 13-B) and an antibody is used. To detect.

  When the free antibody against factor XIII / 13 B subunit (FXIII / 13-B) is measured by the sandwich method, it is supported on the support on which the factor XIII / 13 B subunit (FXIII / 13-B) is immobilized. A sample and a labeled anti-human IgG antibody are contacted. The contact method consists of an antibody against a factor XIII / 13 factor B subunit (FXIII / 13-B) -factor XIII / 13 factor B subunit (FXIII / 13-B) -conjugated anti-human IgG antibody on a support. Although there is no restriction | limiting in particular if it is a method by which a body can be formed, Preferably it processes as follows.

  That is, the factor XIII / 13 factor B subunit (FXIII / 13-B) is bound to the solid phase, and a sample containing an antibody against the factor XIII / 13 factor B subunit (FXIII / 13-B) is added to the solid phase. To do. Antibodies against the factor XIII / 13 B subunit (FXIII / 13-B) bind to and are captured by the factor XIII / 13 B subunit (FXIII / 13-B) bound to the solid phase. Next, a labeled antibody that is an antibody against the autoantibody (or alloantibody) is added and allowed to bind to the autoantibody (or alloantibody). As a result, on the solid phase, an antibody against factor XIII / 13 factor B subunit (FXIII / 13-B) -factor XIII / 13 factor B subunit (FXIII / 13-B) -an antibody against the antibody labeled The antibody against the factor XIII / 13 factor B subunit (FXIII / 13-B) in the sample can be measured by measuring the signal emitted from the labeled antibody after the antibody complex is formed. This method is called “antigen coating method”.

  When measuring autoantibodies (or alloantibodies) present in a complex with factor XIII / 13 B subunit (FXIII / 13-B) by the sandwich method, anti-factor XIII / 13 B subunit ( FXIII / 13-B) A sample and a labeled anti-human IgG antibody are brought into contact with a support on which an antibody is immobilized. The contact method consists of anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody-factor XIII / 13 B subunit (FXIII / 13-B) and factor XIII / 13 B subunit on the support. Although there is no particular limitation as long as it can form a complex of an antibody against (FXIII / 13-B) -labeled anti-human IgG antibody, it is preferably treated as follows.

  That is, an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody is bound to a solid phase, and a factor XIII / 13 B subunit (FXIII / 13-B) and an autoantibody (or A sample containing the complex of the same type antibody is added. The complex of factor XIII / 13 B subunit (FXIII / 13-B) and autoantibody (or alloantibody) becomes anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody bound to the solid phase. The factor XIII / 13 B subunit (FXIII / 13-B) moiety binds and is captured. Next, a labeled antibody that is an antibody against the autoantibody (or alloantibody) is added and allowed to bind to the autoantibody (or alloantibody). As a result, anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody-factor XIII / 13 B subunit (FXIII / 13-B) and autoantibody (or alloantibody) complex on the solid phase A complex of labeled antibody, which is an antibody against the antibody, is formed, and by measuring the signal emitted from the labeled antibody, the factor XIII / 13 factor B subunit (FXIII / 13-B) in the sample and Antibodies against the complexed factor XIII / 13 B subunit (FXIII / 13-B) can be measured. This method is called “antibody coating method”.

  In immunoassay based on the sandwich method, factor XIII / 13 B subunit (FXIII / 13-B) or anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody is used as a solid phase. Any solid phase that can immobilize antibodies and antigens by known techniques can be used as the solid phase, for example, a porous thin film (membrane) having a capillary action, a microtiter plate, particulate matter, a test tube, a resin. A known material such as a flat plate can be arbitrarily selected. A membrane may be used for immunochromatography or Western blotting, and a microtiter plate may be used for ELISA.

  Binding of the XIII / 13 factor B subunit (FXIII / 13-B) or anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody to the solid phase may utilize adsorption, You may couple | bond by a covalent bond using functional groups, such as an amino group and a carboxyl group.

  The anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody to be immobilized may be a polyclonal antibody or a monoclonal antibody, but preferably a monoclonal antibody is used. The antibody can be produced by a known method. Commercially available antibodies may also be used.

  An anti-human IgG antibody may be used as an antibody against the antibody against the factor XIII / 13 B subunit (FXIII / 13-B) used after labeling. The antibody against the anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody and the antibody against the factor XIII / 13 B subunit (FXIII / 13-B) may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. Use antibodies. The antibody can be produced by a known method. Commercially available antibodies may also be used.

The antibody used after immobilization or labeling is an immunoglobulin fragment such as Fab or F (ab ′) ₂ , or a recombinant antibody such as scFv, dsFv, diabody, minibody expressed as a recombinant. Also good. In the present invention, the term “antibody” also includes these fragments. These fragments can be prepared by a known method.

  Substances that label labeled antibodies include enzymes such as alkaline phosphatase (ALP) and horseradish peroxidase (HRP), radioactive isotopes, fluorescent substances, luminescent substances, colored particles such as colored polystyrene particles, and colloidal particles such as gold colloids. Can be used. Labeling of the antibody can be performed by a known method.

  Samples to be used include whole blood, serum, plasma and the like of a subject. In the present invention, the detection of an antibody against factor XIII / 13 B subunit (FXIII / 13-B) in blood includes methods using whole blood, serum, or plasma as a sample.

For example, the ELISA method is performed in the following steps.
A sample is added to a microtiter plate made of polystyrene or the like on which factor XIII / 13 B subunit (FXIII / 13-B) is solid-phased, followed by an antigen-antibody reaction, and then an enzyme-labeled anti-human IgG antibody. Add, allow antigen-antibody reaction, wash, react with enzyme substrate, develop color, measure absorbance to detect free antibody against factor XIII / 13 factor B subunit (FXIII / 13-B) in sample can do. Alternatively, a sample is added to a microtiter plate made of polystyrene or the like on which an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody is solid-phased, allowed to undergo antigen-antibody reaction, and further enzyme-labeled anti-antibody Add human IgG antibody, react with antigen / antibody, wash, react with enzyme substrate, develop color, measure absorbance and complex with factor XIII / 13 factor B subunit (FXIII / 13-B) in sample Autoantibodies (or alloantibodies) that have formed bodies can be detected. Alternatively, fluorescence may be measured after an antigen-antibody reaction using a fluorescently labeled anti-human IgG antibody instead of the enzyme-labeled anti-human IgG antibody.

  In the ELISA method, the amount of sample to be added is several tens of μL to several hundreds of μL. The sample may be used after being diluted several to tens of times.

  The antigen-antibody reaction can be carried out at 4 ° C. to 45 ° C., preferably 20 ° C. to 40 ° C., more preferably 25 ° C. to 38 ° C., and the reaction time is 10 minutes to 18 hours, more preferably 10 minutes to It is about 1 hour, more preferably about 30 minutes to 1 hour.

The immunochromatography method is performed in the following steps.
When measuring free autoantibodies (or alloantibodies) against factor XIII / 13 B subunit (FXIII / 13-B), factor XIII / 13 B subunits that capture the autoantibodies (or alloantibodies) ( FXIII / 13-B) is a solid phase support (membrane) having a detection region, and a labeling substance having a labeled anti-human IgG antibody that can be deployed and labeled with a labeling substance such as colored polystyrene particles or gold colloid. An immunochromatographic immunoassay device comprising an area, a sample pad to which a sample is added, an absorption band for absorbing the developed sample liquid, and a backing sheet for bonding these members together may be used. In this method, an appropriate labeling substance such as colored polystyrene particles or colloidal gold is used by using capillary action on a solid support in which factor XIII / 13 B subunit (FXIII / 13-B) is immobilized. A complex of a labeled anti-human IgG antibody and a free autoantibody (or alloantibody) against factor XIII / 13 factor B subunit (FXIII / 13-B) is developed and transferred onto the membrane. As a result, the immobilized autoantibody (or alloantibody) -labeled against the immobilized factor XIII / 13 B subunit (FXIII / 13-B) -factor XIII / 13 B subunit (FXIII / 13-B) The anti-human IgG antibody complex is formed on the solid support, and the signal of the labeling substance emitted from the complex (in the case of gold colloid, the solid support on which the substance capable of binding to the target substance is immobilized) By detecting that the part turns red, free autoantibodies (or alloantibodies) can be detected. The immunoassay can be performed at 5 to 35 ° C., preferably at room temperature, and can be determined within a few minutes after the sample is added.

  When detecting autoantibodies (or alloantibodies) complexed with factor XIII / 13 B subunit (FXIII / 13-B), anti-factor XIII / 13 factor B subunit (FXIII) is supported on the solid support. / 13-B) The antibody may be immobilized. The antibody to be immobilized may be one type, but preferably a plurality of types are used. On the solid support, an immobilized anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody-factor XIII / 13 B subunit (FXIII / 13-B) and an autoantibody (or Homogeneous antibody) -labeled anti-human IgG antibody complex is formed.

  In the immunochromatography method, the amount of the sample to be added is several tens to several hundreds μL. The sample may be used after being diluted several to tens of times.

  The immunochromatography method can be performed at 4 ° C. to 45 ° C., preferably 20 ° C. to 40 ° C., more preferably 25 ° C. to 38 ° C., and the time from sample addition to determination is about several minutes to several tens of minutes.

  The material of the solid support used in the immunochromatography method is not limited as long as the sample can be absorbed and developed and flowed by capillary action. For example, natural, synthetic polymers such as nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose, polystyrene, or a mixture thereof can be used. The solid support preferably has the shape of a strip of strips.

  Immobilization when measuring free autoantibodies (or alloantibodies) against factor XIII / 13 B subunit (FXIII / 13-B) by immunoassay utilizing the measurement principle of the sandwich method of the present invention The factor XIII / 13 B subunit (FXIII / 13-B) used in the invention is preferably a highly purified product that does not cause cross-reaction, and in particular, the recombinant factor XIII / 13 B subunit (FXIII / It is preferable to use 13-B). Alternatively, a fragment protein of factor XIII / 13 B subunit (FXIII / 13-B) may be used.

  Further, a sensitizer may be added to the measurement system at the time of measurement. Examples of the sensitizer include polyethylene glycol, polyvinyl pyrrolidone, carboxymethyl cellulose, polyamino acid, aminomethanesulfonic acid derivative and the like.

  The method of the present invention reliably detects "anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibodies" in the blood of suspected cases of autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) can do. The present invention is useful for point-of-care testing of autoimmune hemorrhagic disease XIII / 13 (AH13), including autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) It is. In addition, since the measurement principle is the same, “anti-factor XIII / 13 factor B subunit (FXIII / 13-B) alloantibodies in hereditary factor XIII / 13 B subunit (FXIII / 13-B) deficiency cases Is also useful for the detection of [FIGS. 4-7].

  That is, the present invention relates to a method for detecting autoimmune bleeding disease XIII / 13 (AH13) including autoimmune bleeding disease XIII / 13 type B (AH13-B), or autoimmune bleeding disease XIII / 13 type B ( A method for acquiring auxiliary data for diagnosing autoimmune hemorrhagic disease XIII / 13 (AH13) including AH13-B) is included. Furthermore, the present invention provides a method for detecting an “anti-factor XIII / 13 B subunit (FXIII / 13-B) alloantibody” in a hereditary factor XIII / 13 B subunit (FXIII / 13-B) deficiency case including.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

  Autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) is a relatively rare hemorrhagic acquired coagulation disorder that presents a condition similar to factor XIII / 13 deficiency. Among them, autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) caused by anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibodies is However, although 5 cases have been confirmed, there is no measurement method for detecting anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibodies, so it is estimated that more cases are missed. The Therefore, (1) antibodies and antigens for detecting anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibodies are selected by Western blotting, and (2) anti-factor XIII / 13 is detected by ELISA. A factor B subunit (FXIII / 13-B) autoantibody measurement system was constructed, and (3) an anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibody detection immunochromatography method was developed.

1. Selection of antibodies with high sensitivity and specificity for detection of factor XIII / 13 B subunit (FXIII / 13-B) (1) Western blotting [method]
Using various lengths of recombinant factor XIII / 13 B subunit (FXIII / 13-B) as factor XIII / 13 B subunit (FXIII / 13-B) antigen, anti-factor XIII / 13 as an antibody For the seven types of factor B subunit (FXIII / 13-B) monoclonal antibodies (Hoast: mouse, Species reactivity: human), we examined the strength of reactivity to the antigen and determined the best antibody by Western blotting. did.

FIG. 1 shows the results of Western blotting.
1-3C recognizes all lengths of factor XIII / 13 B subunit (FXIII / 13-B), so it is optimal in both sensitivity and specificity, and 1-3B and 5-6C are suboptimal. It turned out to be.

(2) ELISA method (a) ELISA method (antigen amount fixed)
〔Method〕
Using full-length recombinant factor XIII / 13 B subunit (FXIII / 13-B) as factor XIII / 13 B subunit (FXIII / 13-B) antigen and anti-factor XIII / 13 B subunit as antibody (FXIII / 13-B) Monoclonal antibody (Hoast: mouse, Species reactivity: human) The optimal antibody is determined by ELISA method by gradually changing the amount of the culture solution containing the antibody. did.

Fig. 2-1 shows the results of the ELISA method.
1-3C, 1-3B, 6-5F is directly proportional to the amount added, and the reactivity to full length factor XIII / 13 B subunit (FXIII / 13-B) is enhanced, so it is optimal in terms of sensitivity. , 5-6C was found to be the second best.

(B) ELISA method (antibody amount fixed)
〔Method〕
Using a full-length recombinant factor XIII / 13 B subunit (FXIII / 13-B) as a factor XIII / 13 B subunit (FXIII / 13-B) antigen, a fixed amount of anti-factor XIII / 13 as an antibody For B subunit (FXIII / 13-B) monoclonal antibody (Host: mouse, Species reactivity: human), the antigen concentration was diluted stepwise and the optimal antibody was determined by ELISA.

Fig. 2-2 shows the result of ELISA method.
6-5F, 1-3B, and 1-3C are optimal in terms of sensitivity because their reactivity has increased in direct proportion to the added amount of full length factor XIII / 13 B subunit (FXIII / 13-B), 5-6C was found to be the second best.

2. Anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibody detection immunochromatography (1) Untreated antibody direct coating method Untreated anti-factor XIII / 13 B subunit (FXIII / 13-B) monoclonal An immunochromatography method was prototyped by applying antibodies (6 types) to the membrane side. Evaluation is based on rapid immunochromatographic test for detection of anti-factor XIII A subunit antibodies can diagnose 90% of cases with autoimmune haemorrhaphilia XIII / 13 .; Osaki T, Sugiyama D, Magari Y, Souri M, Ichinose A .; Thromb Haemost. 2015 Jun; 113 (6): 1347-56. Doi: 10.1160 / TH14-09-0745. Epub 2015 Mar 5. PMID: 25740658; Then, the 10-fold diluted specimen was run, the washing solution was run, and finally the gold colloid sensitized with anti-human IgG antibody was run.
Separately, the specimen was diluted stepwise by immunochromatography with a monoclonal antibody (1-3C), and the sensitivity and specificity were examined.

The results are shown in FIGS. 3-1 and 3-2.
In the measurement system in which the case specimen and the healthy control plasma were run as they were on the immunochromatographic strip, a difference was visually observed between the positive control and the negative control, but a non-specific reaction was observed (specimen untreated direct method; Fig. 3- 1). In order to investigate the sensitivity to positive samples and attenuate the non-specific reaction of negative samples to improve the signal / noise ratio, the sample was diluted in stages, and the difference between visual positive and negative samples was It was almost unchanged (Figure 3-2).

(2) Enzyme-treated antibody direct coating method
An immunochromatography method was prototyped by applying an anti-factor XIII / 13 B subunit (FXIII / 13-B) monoclonal antibody (1-3C) treated with an enzyme (Nacalai Tesque Pepsin) to the membrane side. The evaluation was performed in the same manner as the anti-FXIII / 13-A subunit autoantibody detection immunochromatography method, a 10-fold diluted sample was run, the washing solution was run, and finally the colloidal gold sensitized with anti-human IgG antibody Was run. The performance of two membranes used for immunochromatographic strips was compared.

The results are shown in FIG.
Treatment of the antibody with the enzyme significantly improved the nonspecific reaction and increased specificity (FIG. 4). In all cases, a clear difference was obtained between the positive control and the negative control. There was no difference depending on the membrane used.

(3) Enzyme-treated antibody coating direct / mixing / dilution mixing method [method]
Using immunochromatographic strips coated with anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody (1-3C) on the membrane side, autoantibody positive cases, negative cases, alloantibody positive cases, healthy controls Case samples that do not have factor XIII / 13 B subunit (FXIII / 13-B) antigen, such as hereditary factor XIII / 13 B subunit (FXIII / 13-B) deficiency The mixture was preliminarily mixed with healthy control plasma at a ratio of 1: 1 (mixing method) or 1:10, 1: 100 (dilution mixing method). The evaluation was performed in the same manner as the anti-FXIII / 13-A subunit autoantibody detection immunochromatography method, a 10-fold diluted sample was run, the washing solution was run, and finally the colloidal gold sensitized with anti-human IgG antibody Was run.

The results are shown in FIGS.
Autoantibody positive cases (H prefecture F hospital, T city ST hospital, K prefecture T university) are also positive by immunochromatography, and autoantibody negative cases (G prefecture G university, T city N university, T prefecture S hospital) are immunochromatography. But it was negative (Figure 5). However, in the case of positive alloantibodies (K University, O Prefecture), all three samples with different stages were negative and were not detected.
The initial specimen (Fig. 6, 110111) of alloantibody positive cases in which factor XIII / 13 B subunit (FXIII / 13-B) is congenitally deficient is mixed with healthy plasma in a 1: 1 ratio. Replenishment with XIII / 13 factor B subunit (FXIII / 13-B) resulted in antibody positivity (Fig. 6, left), but specimens at the stage where free antibody increased significantly (Fig. 7, 120327, 120820) Then, it remained negative (FIG. 7).
When the specimens of the stage in which the free antibodies were markedly increased in the alloantibody positive cases were diluted and mixed with healthy plasma at a ratio of 1:10, 1: 100, the antibodies became positive (right in FIG. 6).

  By the method of the present invention, “anti-factor XIII / 13 B subunit (FXIII / 13-B) autoantibody” is reliably detected in the blood of suspected autoimmune hemorrhagic disease XIII / 13 type B (AH13-B) can do. It is also possible to detect "anti-factor XIII / 13 B subunit (FXIII / 13-B) alloantibodies" in hereditary factor XIII / 13 B subunit (FXIII / 13-B) deficiency cases.

Claims (12)

  A method for detecting a free antibody against a factor XIII / 13 factor B subunit (FXIII / 13-B) in a blood sample, wherein the factor XIII / 13 factor B subunit (FXIII / 13-B) is immobilized. The sample and labeled anti-human IgG antibody are brought into contact with the supported support, and the factor XIII / 13 factor B subunit (FXIII / 13-B) -factor XIII / 13 factor B subunit (FXIII / 13-B) is supported on the support. ) -Labeled anti-human IgG antibody complex and detecting the signal from the labeled anti-human IgG antibody.   The method according to claim 1, wherein the free antibody against the factor XIII / 13 B subunit (FXIII / 13-B) is an autoantibody.   A method for detecting an antibody against a factor XIII / 13 factor B subunit (FXIII / 13-B) complexed with a factor XIII / 13 factor B subunit (FXIII / 13-B) in a blood sample, The sample and labeled anti-human IgG antibody are brought into contact with a support on which an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody is immobilized, and the anti-factor XIII / 13 factor B subunit is supported on the support. (FXIII / 13-B) antibody-antibody complex against factor XIII / 13 B subunit (FXIII / 13-B) and factor XIII / 13 B subunit (FXIII / 13-B) -labeled anti-human Forming a IgG antibody complex and detecting a signal from the labeled anti-human IgG antibody.   The method according to claim 3, wherein the antibody against the factor XIII / 13 B subunit (FXIII / 13-B) complexed with the factor XIII / 13 B subunit (FXIII / 13-B) is an autoantibody. .   The method according to any one of claims 1 to 4, wherein the method is selected from the group consisting of Western blotting, ELISA, and immunochromatography.   The method according to any one of claims 1 to 5, which is a method for obtaining auxiliary data for detection of autoimmune bleeding disease XIII / 13 (AH13).   7. The method of claim 6, wherein the autoimmune bleeding disease XIII / 13 (AH13) is autoimmune bleeding disease XIII / 13 type B (AH13-B).   A support comprising a solid-phased factor XIII / 13 B subunit (FXIII / 13-B) and a labeled anti-human IgG antibody, wherein the factor XIII / 13 factor B subunit (FXIII / 13-B) is supported on the support. ) -Antibodies against factor XIII / 13 B subunit (FXIII / 13-B)-In a blood sample by forming a complex of labeled anti-human IgG antibody and detecting the signal from the labeled anti-human IgG antibody A kit for detecting a free antibody against factor XIII / 13 B subunit (FXIII / 13-B).   A support comprising an anti-factor XIII / 13 B subunit (FXIII / 13-B) antibody immobilized thereon and a labeled anti-human IgG antibody, the anti-factor XIII / 13 B subunit (FXIII / 13-B) Antibody-antibody complex against factor XIII / 13 B subunit (FXIII / 13-B) and factor XIII / 13 B subunit (FXIII / 13-B)-labeled anti-human IgG antibody Factor XIII / 13 complexed with factor XIII / 13 B subunit (FXIII / 13-B) in the blood sample by forming a complex and detecting the signal from the labeled anti-human IgG antibody Kit for detecting an antibody against B subunit (FXIII / 13-B).   The kit according to claim 8 or 9, wherein the kit is selected from the group consisting of a kit for Western blotting, a kit for ELISA, and a kit for immunochromatography.   The kit according to any one of claims 8 to 10, which is a kit for obtaining auxiliary data for detection of autoimmune bleeding disease XIII / 13 (AH13).   The kit according to claim 11, wherein the autoimmune bleeding disease XIII / 13 (AH13) is autoimmune bleeding disease XIII / 13 type B (AH13-B).

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