JP2010286375A - Marker, method and kit for diagnosing sensibility to severe drug eruption - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a marker, a method and a kit for diagnosing sensibility to severe drug eruption, for quickly and easily detecting the sensibility to the severe drug eruption for a subject having no drug eruption or a subject having a touch of drug eruption before developing the severe drug eruption. <P>SOLUTION: The marker is an index of the severe drug eruption for the subject having no drug eruption or the subject having the touch of drug eruption, and granulysin contained in serum extracted from the subject is used as the marker. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention is a marker for diagnosing severe drug rash susceptibility for diagnosing severe drug rash susceptibility before onset of severe drug eruption in a subject who has not developed drug eruption or a subject who has developed mild drug eruption, The present invention relates to a method for diagnosis of susceptibility to severe drug eruption and a kit for diagnosis of susceptibility to severe drug eruption.

  Drug eruption refers to skin eruption that occurs in response to a drug, and all drugs can cause drug eruption. Severe drug eruptions that are life-threatening due to drug eruptions are called severe drug eruptions, and the most severe forms of Stevens-Johnson syndrome (SJS) and toxic epidermal necrosis (TEN) are high fever and extensive epidermis It is a very poor prognosis characterized by cell necrosis. In SJS, severe mucosal eruptions and erythema of the skin are seen in the transmucosal area of the skin such as the lips, ocular mucosa, and vulva, and erosion, blistering, and peeling of the body surface are 10% or less of the body surface area. SJS often moves to TEN. TEN also includes subtypes that have migrated from SJS, the symptoms of which are similar to SJS, but are more severe than SJS as a whole, and erosion, blistering, and epidermal detachment are seen in more than 10% of the body surface area. The lethality of TEN is 20-25%.

  The initial symptoms of these severe drug eruptions are very similar to the symptoms of mild drug eruptions that show polymorphic exudative erythema on the epidermis, making it difficult to diagnose early. It contributes to the fatality rate. Therefore, there is an urgent need to establish a diagnostic method capable of diagnosing whether or not it is likely to suffer from severe drug eruption before the onset of severe drug eruption, and research and development have been made.

  For example, the soluble Fas ligand concentration in the serum of a subject developing SJS / TEN is compared to the soluble Fas ligand concentration in the serum of a subject developing mild drug eruption or a subject not developing drug eruption. It was found by the present inventors that the level was significantly increased, and a drug having a high incidence of severe drug eruption was added to the lymphocytes of patients scheduled to be administered in advance and cultured, and the soluble Fas ligand in the culture medium was measured. A method for predicting the onset of severe drug eruption has been proposed (Non-patent Document 1). Moreover, the soluble Fas ligand concentration in the serum before the onset of the subject who develops SJS or TEN is the concentration of the soluble Fas ligand in the serum of the subject who has developed mild drug eruption or the subject who has not developed drug eruption. It is significantly increased compared to J. A method for early diagnosis of the onset of severe drug eruption, which is found by Murata et al. And measures soluble Fas ligand in serum, has been proposed (Non-patent Document 2).

  On the other hand, granulysin is a cell killing activity that natural killer cells (NK cells) and cytotoxic T lymphocytes (CTLs) release to target cells such as cancer cells, transplanted cells, or microorganisms to cause apoptosis. It is a molecule and is known to play an important role in biological defense.

  Several methods have been proposed for detecting granulysin and diagnosing a subject's immune status. For example, Japanese Patent Laid-Open No. 2001-249126 discloses a method for diagnosing the immune status of a specimen provider by detecting granulysin in NK cells and CTLs (Patent Document 1), and WO 2003/052417. Discloses a method for diagnosing an immune state of a specimen donor by detecting granulysin in a body fluid (Patent Document 2).

  In addition, it is described that granulysin is highly expressed in the blisters of the affected area of subjects who develop SJS or TEN, and that granulysin is a causative substance that causes necrosis of epidermal cells. H. Chung et al. (Non-patent Document 3).

JP 2001-249126 A International Publication WO2003 / 052417 Pamphlet

Ribeichiro Abe, Hashi Allergy, Vol. 13, pp. 53-57, 2005 J. et al. Murata et al., J ALLERGY CLIN IMMUNOL, Vol. 122, pp. 992-1000, 2008 W. H. Chung et al., NATURE MEDICINE ADVANCE ONLINE PUBLICATION, published online 23 November 2008

  As described above, there is a demand for a reliable method for diagnosing whether or not a drug eruption is likely to occur before the onset of the drug eruption. Since it takes time and labor for the cell culture of the prospective person, it is difficult to diagnose the susceptibility to severe drug eruption in a short time. In the diagnostic method proposed in Non-Patent Document 2, Detection is difficult because the absolute amount of soluble Fas ligand present is small. In addition, the diagnostic methods disclosed in Patent Document 1 and Patent Document 2 are not used in a method for diagnosing whether or not a serious drug eruption is likely to occur before the onset of severe drug eruption. There is no description that the concentration of granulysin in the serum of a subject before the onset of severe drug eruption is higher than that of a subject who does not develop severe drug eruption in the future.

  The present invention has been made in order to solve such a problem, and the susceptibility to severe drug eruption in a subject who has not developed drug eruption or a subject who has developed mild drug eruption, It is an object of the present invention to provide a marker for diagnosing severe drug rash susceptibility, a diagnostic method for susceptibility to severe drug eruption, and a kit for diagnosing severe drug rash susceptibility for rapid and easy detection before the onset of rash.

  The present inventors have found that the concentration of granulysin in the serum of a subject before the onset of severe drug eruption is significantly higher in the future compared to subjects who do not develop a severe drug eruption, and completed.

(1) A marker serving as an index of susceptibility to severe drug eruption in a subject who has not developed a drug eruption or a subject who has developed a mild drug eruption, wherein the marker is contained in serum collected from the subject A marker for diagnosis of susceptibility to severe drug eruption.

(2) Severe drug erection susceptibility using the amount of granulysin in serum collected from subjects who have not developed drug eruption or those who have developed mild drug eruption as an index of susceptibility to severe drug eruption Diagnosis method.

(3) a step of diluting serum collected from a subject who has not developed drug eruption or a subject who has developed mild drug eruption, and the diluted serum in the labeled antibody which is an anti-granulinic antibody labeled with a labeling substance A step of forming a labeled antibody-granulinicin complex by binding granulysin contained therein, and a step of binding the labeled antibody-granulinicin complex to a capture antibody that is an anti-granulinicin antibody immobilized on a carrier. The method for diagnosing susceptibility to severe drug eruption according to (2), comprising using immunochromatography comprising a step of detecting a labeled antibody-granulysin-capture antibody complex.

(4) The method for diagnosing susceptibility to severe drug eruption according to (3), wherein the dilution ratio of serum is 5 times or more.

(5) From (2) to (4), the amount of granulysin contained in the serum collected from a subject who has not developed drug eruption or a subject who has developed mild drug eruption is not less than 10 ng / mL. ) The method for diagnosing susceptibility to severe drug eruptions according to any one of the above.

(6) A kit for diagnosis of susceptibility to severe drug rash comprising a carrier, a labeled antibody that is an anti-granulinicin antibody labeled with a labeling substance, and a capture antibody that is an anti-granulinicin antibody immobilized on the carrier. .

(7) The labeled antibody is an anti-granulinic antibody produced by a mouse-derived antibody-producing cell clone RB1, and the capture antibody is an anti-granulinic antibody produced by a mouse-derived antibody-producing cell clone RC8. A kit for diagnosis of susceptibility to severe drug eruption.

  According to the marker for diagnosing severe drug rash susceptibility, the method for diagnosing severe drug rash susceptibility and the kit for diagnosing severe drug rash susceptibility according to the present invention, a subject who has not developed drug eruption or mild drug eruption It is possible to quickly and easily detect the susceptibility to a severe drug eruption in a test subject before the onset of the severe drug eruption.

Subjects who have developed Stevens-Johnson syndrome (SJS) or toxic epidermal necrosis (TEN) (hereinafter sometimes referred to as “severe drug eruption”), subjects who have not developed drug eruption, and mild drugs It is a figure which shows the density | concentration of granulysin contained in the serum extract | collected from the test subject who stayed on the onset of a rash. In the figure, “day-4 to −2” is the subject from the 4th day before the onset of severe drug eruption to the 2nd day before the onset, and “day-1 to 2” is the first day from the day before the onset of severe drug eruption to the 2nd day of onset. "Days 3 to 5" were collected from subjects on the 3rd to 5th days of onset of severe drug eruption, and "days 6 to 10" were collected from subjects on 6th to 10th day of onset of severe drug eruption. Serum (specimen) is shown. It is the top view and schematic diagram which show the structure of the granulysin detection kit in a present Example. It is the top view and side view showing the structure of the granulysin detection kit in a present Example. It is a state figure at the time of showing a positive reaction and a negative reaction in the granulysin detection kit in a present Example.

  Hereinafter, the marker for diagnosing severe drug rash susceptibility, the diagnostic method for susceptibility to severe drug eruption and the kit for diagnosing severe drug rash susceptibility according to the present invention will be described in detail. The marker for diagnosis of susceptibility to severe drug eruption according to the present invention is a marker serving as an index of susceptibility to severe drug eruption in a subject who has not developed drug eruption or a subject who has developed mild drug eruption, The marker is granulysin contained in serum collected from a subject.

  That is, the marker for diagnosis of susceptibility to severe drug eruption according to the present invention is granulysin contained in serum collected from a subject who does not develop drug eruption or a subject who develops mild drug eruption, `` Becoming an index of susceptibility to rash '' means, for example, measuring or detecting the concentration or amount of granulysin in the collected serum, and subjects with high granulysin concentration or subjects with a high amount of granulysin suffer from severe drug eruption. It means that it can be determined that it is easy. When comparing the concentration and amount of granulysin, any statistical test method can be used, such as chi-square test, F test, Z test, T test, Bayes test, Turkey Kramer method, etc. Multiple comparison tests and the like can be used.

  Here, “severe drug rash susceptibility” in the present invention means that it is easy to suffer from severe drug eruption. However, there is a case where a state in which severe drug eruption can be affected is maintained. Further, “mild drug eruption” generally refers to a mild drug eruption that does not pose a risk to life due to drug eruption and cures in a short period of time. No drug eruption.

  In addition, granulysin in vivo is known as a precursor having a molecular weight of 15 kDa and an active substance having a molecular weight of 9 kDa produced by processing, but the granulysin in the present invention is not limited to any of these. .

  Next, the method for diagnosing the susceptibility to severe drug eruption according to the present invention comprises determining the amount of granulysin contained in serum collected from a subject who has not developed drug eruption or a subject who has developed mild drug eruption. It is characterized by being an index of susceptibility to rash.

  In the present invention, the time when serum should be collected from the subject is not particularly limited as long as it does not develop severe drug eruption, but when it is collected from day 4 before onset to day 2 before onset of severe drug eruption Since the inventors have obtained remarkable results, it is preferable to continuously collect serum.

  In the present invention, the concentration of granulysin contained in the serum of a subject diagnosed as likely to suffer from severe drug eruption is preferably 10 ng / mL or more. In addition, when diluting serum collected from a subject, the dilution factor is preferably 3 times or more, and in order to reduce the possibility of false positives and ensure detection sensitivity, it is more preferably 5 times or more. preferable. The diluent to be used is not particularly limited as long as the diagnostic method for susceptibility to severe drug eruption is not impaired, but a blocking buffer prepared according to a conventional method is preferred.

  In the present invention, a method for detecting the amount of granulysin contained in serum can be appropriately selected by those skilled in the art, and is not particularly limited. For example, a reagent is added to serum to cause a chemical reaction, and color change is analyzed. The method of measuring the concentration of granulysin, the method of measuring the concentration of granulysin by electrophoresis, fractionating the serum protein, the anti-granulinis antibody specifically binding to granulysin and the granulysin contained in the serum And a method using an antigen-antibody reaction. Examples of the method utilizing an antigen-antibody reaction include an immunochromatography method, an ELISA method, a radioimmunoassay method, an immunoprecipitation method, and an analysis method utilizing a Western blot method, and the immunochromatography method is preferred.

  When using an immunochromatography method, for example, a capture antibody is fixed in advance to the center of the membrane carrier to form a test line, and a conjugate antibody is impregnated with a labeled antibody labeled with a dye particle to end one end of the membrane carrier. (Regardless of the front and back), drop the liquid sample (serum) into the conjugate pad and infiltrate the conjugate pad, and mix the sample and labeled antibody in the membrane carrier to the test line. The method of developing for it can be mentioned. When the sample contains an antigen, in the conjugate pad, the antigen and the labeled antibody form a complex by the antigen-antibody reaction, and then, in the test line, the complex is captured by the capture antibody by the antigen-antibody reaction, Accumulate and develop color. Therefore, the degree of coloration on the test line can be visually observed, and the presence or absence of the antigen in the sample can be determined.

  In addition, the developing solvent used when using the immunochromatography method can be prepared by suspending, emulsifying, or dissolving various additives as necessary.

  In the present invention, when using a method utilizing an antigen-antibody reaction such as an immunochromatography method, an anti-granulinic antibody that specifically binds to granulysin is used, but the anti-granulinic antibody can be produced according to a conventional method, The type is not particularly limited. Examples of such anti-granulinic antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, fragments produced by Fab expression libraries, and mimetics thereof.

  The anti-granulinic antibody in the present invention can be labeled with a labeling substance and used as a labeled antibody according to a conventional method, if necessary. Examples of the labeling substance include colored particles, enzymes, fluorescent substances, and isotopes. Moreover, the anti-granulinic antibody in the present invention can be used in a state of being immobilized on a carrier. Examples of the carrier include microplates, beads, agarose, cellulose membrane and the like, and the fixing method can be performed according to a standard method depending on the type of the carrier. For example, when a microplate is used, an antibody can be immobilized on this using a physical non-specific adsorption method according to a conventional method. In addition, when using beads or agarose, a method of immobilizing an antibody with a chemical cross-linking agent or a protein having affinity for the antibody such as an immobilized anti-immunoglobulin antibody or protein A is used. The method of fixing can be mentioned.

  The immunochromatography method in the present invention includes a step of diluting serum collected from a subject who has not developed drug eruption or a subject having mild drug eruption, and a labeled antibody which is an anti-granulinic antibody labeled with a labeling substance. A step of binding granulysin contained in the diluted serum to form a labeled antibody-granulinicin complex, and binding of the labeled antibody-granulinicin complex to a capture antibody that is an anti-granulinicin antibody immobilized on a carrier. And a step of detecting the labeled antibody-granulinicin-capture antibody complex formed.

  “Labeled antibody-granulinicin complex” means that the labeled antibody paratope and the epitope of granulysin are non-covalently bound by hydrogen bond, ionic bond, hydrophobic bond, or van der Waals bond, And granulysin are combined to form a single labeled molecule.

  In addition, “labeled antibody-granulinicin-capture antibody complex” means that a paratope of a supplementary antibody and an epitope of granulysin in a labeled antibody-granulinicin complex are hydrogen bonds, ionic bonds, hydrophobic bonds, or van der By binding non-covalently by the Waals bond, the labeled antibody-granulinicin complex and the supplementary antibody are combined to form a single molecule immobilized on a carrier.

  The method for diagnosing severe drug rash susceptibility according to the present invention may have other steps as long as the characteristics of the method for diagnosing severe drug rash susceptibility according to the present invention are not impaired. You may have an incubation process, a washing process, etc.

  Next, the severe drug erection susceptibility diagnostic kit according to the present invention includes a carrier, a labeled antibody that is an anti-granulinicin antibody labeled with a labeling substance, and a capture antibody that is an anti-granulinicin antibody immobilized on the carrier. And comprising. The kit for diagnosis of susceptibility to severe drug eruption according to the present invention detects a drug eruption or a mild drug eruption by detecting granulysin contained in the serum of the subject using an immunochromatography method. It is used to detect the susceptibility of a severe drug eruption before the onset of severe drug eruption.

  The kit for diagnosis of susceptibility to severe drug eruption according to the present invention comprises a carrier, a labeled antibody that is an anti-granulinicin antibody labeled with a labeling substance, and a capture antibody that is an anti-granulinicin antibody immobilized on the carrier. The structure is not particularly limited, but, for example, a membrane overlaps on a mount, and a sample pad, a conjugate pad, and a water supply paper are arranged in that order on the conjugate pad, and the conjugate pad is labeled with anti-granulinicin labeled with red particles. An antibody (labeled antibody) is included, and an anti-granulinic antibody (capture antibody) is immobilized on the test line formed on the membrane, and an anti-mouse antibody is immobilized on the control line formed on the membrane. be able to.

  Examples of the sample pad used in such a configuration include a sheet or film of porous synthetic resin such as porous polyethylene and porous polypropylene, paper or woven fabric such as filter paper and cotton cloth, and non-woven fabric.

  The water absorbent paper may be any material that can absorb and retain liquid, and examples thereof include filter paper, cotton cloth, porous plastic nonwoven fabric made of polyethylene polypropylene, and the like.

  The conjugate pad only needs to be impregnated with a labeled antibody and infiltrated with serum, and examples thereof include cellulose cloth, glass fiber nonwoven fabric, porous plastic cloth such as polyethylene and polypropylene, and the like. Can do.

  Any membrane can be used as long as it can fix the capture antibody and can develop the sample (serum). For example, a nitrocellulose membrane, a cellulose membrane, a nylon membrane, a glass fiber membrane, or the like can be used.

  The kit for diagnosis of susceptibility to severe drug eruptions according to the present invention may include all components as long as the characteristics thereof are not impaired. For example, a substance or buffer useful for carrying out immunological detection means for granulysin. A liquid etc. may be included.

  Hereinafter, the marker for diagnosing severe drug rash susceptibility, the diagnostic method for susceptibility to severe drug eruption and the kit for diagnosing severe drug rash susceptibility according to the present invention will be described based on Examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1> Measurement of serum granulysin concentration collected from a subject who developed SJS or TEN No drug eruption or mild drug eruption but later Stevens-Johnson syndrome (SJS) or moderate Measurement of serum granulysin concentration from subjects who developed toxic epidermal necrosis (TEN), subjects who did not develop drug eruption, and subjects who had only mild drug eruption. Entrusted to.

  The number of days from the day when the subject developed SJS or TEN was calculated as the first day of onset when the eye symptoms such as skin and mucosal ulcers or redness of the eyes were first confirmed. For subjects who did not develop drug eruption or developed mild drug eruption but subsequently developed SJS or TEN, SJS or TEN of 12 subjects who developed SJS and 6 subjects who developed TEN From the 4th day before the onset to the 2nd day before the onset (from the 4th day to the -2nd day), from the 1st day before the onset to the 2nd day (on the 1st day to the 2nd day), from the 3rd day onset Serum collected at one or more times selected from the onset day 5 (day 3 to day 5) and the onset day 6 to the onset day 10 (day 6 to day 10) The granulysin concentration was measured. The number of specimens was 3 for the 4th day to the -2nd day, 12 for the 1st day to the 2nd day, 10 for the 3rd day to the 5th day, and 11 for the 6th day to the 10th day. It was. Further, granulysin concentration was measured using sera collected from 32 subjects who did not develop drug eruption and 25 subjects who only developed mild drug eruption. The result is shown in FIG. In addition, the multiple comparison between each group was performed by the Turkey Kramer method. In FIG. 1, * indicates P <0.05.

  As a result, 3 samples from -4 days to -2 days, 12 samples from -1 days to 2 days, 10 samples from 3 days to 5 days, 11 samples from 6 days to 10 days, The average value of granulysin concentration in 32 samples of subjects who did not develop drug eruption and 25 samples of subjects who developed mild drug eruption was 23.1 ng / mL, 12.7 ng / mL, and 4.2 ng, respectively. / ML, 4.5 ng / mL, 1.6 ng / mL and 3.5 ng / mL, with standard deviations of ± 16.6 ng / mL, ± 16.4 ng / mL, ± 3.0 ng / mL and ± 4. 5 ng / mL, ± 0.6 ng / mL and ± 3.4 ng / mL.

  In addition, as shown in FIG. 1, the granulysin concentration of the sample in a subject who did not develop drug eruption or developed mild drug eruption but subsequently developed severe drug eruption, It was confirmed that a high value of 10 ng / mL or more was exhibited on the fourth day to the second day (P value <0.01). Moreover, in the -1st day to the 2nd day, although a sample showing a high value is confirmed as compared with the -4th day to the -2nd day, there are many samples showing a low value of less than 10 ng / mL. In the 3rd to 5th days and the 6th to 10th days, almost all specimens show a lower value of less than 10 ng / mL compared with the -4th to -2 days. It was confirmed. In addition, the granulysin concentrations of the samples of subjects who did not develop drug eruption and subjects who only developed mild drug eruption were also 10 ng / mL for all samples compared to -4 days to -2 days. It was confirmed to show a low value of less than.

  From the above results, among the subjects who did not develop drug eruption or developed mild drug eruption but subsequently developed severe drug eruption, before the onset of severe drug eruption, the fourth day to the second day The serum granulysin concentration collected from the eye subjects was limited to subjects on the first day before onset of severe drug eruption, subjects after onset of severe drug eruption, subjects who did not develop drug eruption, and mild drug eruption. Since it shows a high value of 10 ng / mL or more compared to the test subject, it is possible to diagnose whether or not the patient is likely to suffer from severe drug eruption by using the amount of granulysin contained in the collected serum as an index. Indicated.

<Example 2> Preparation and examination of granulysin detection kit A kit for detecting granulysin contained in serum collected from a subject using immunochromatography was prepared, and the dilution rate of serum when using this kit and The minimum detectable concentration of granulysin was examined.

(1) Preparation of granulysin detection kit A kit for detecting granulysin contained in serum collected from a subject was prepared at ImmunoDiagnostic Laboratory. The structure is shown in FIGS.

  As shown in FIG. 2, the immunochromatography method is used for the kit, and as shown in FIG. 3, the membrane is placed on the mount so as to be overlapped. In front of the membrane, a sample pad and a conjugate pad are stacked in order from the top, and further, water supply paper is stacked behind the membrane. The upper surface of the kit is covered with a transparent film called a cover seal (not shown). As shown in FIGS. 2 and 3, the conjugate pad is impregnated with an anti-granulinic antibody (labeled antibody) labeled with red particles, and the test line formed on the membrane has an anti-granulysin antibody. A granulysin antibody (capture antibody) and an anti-mouse antibody are immobilized on the control line. The antibodies used in the kit in this example are shown in Table 1.

  The serum dripped onto the sample pad infiltrates and develops in the order of the sample pad, conjugate pad, membrane, and water supply paper. When serum contains granulysin, the label first labeled with red particles on the conjugate pad A labeled antibody-granulaicin complex is formed by the antibody and granulysin. Next, the formed labeled antibody-granulinicin complex is infiltrated from the conjugate pad into the membrane and spreads, and in the test line formed on the membrane, it is labeled with the capture antibody fixed to the test line and the labeled antibody-granulinicin. An antibody-granulysin-capture antibody complex is formed. Since the labeled antibody is labeled with red particles, when a labeled antibody-granulinicin-capture antibody complex is formed, a red line appears on the test line, so it is possible to visually confirm granulysin positive. . Furthermore, the excess labeled antibody or labeled antibody-granulinicin complex is developed on a control line formed on the membrane. Since the anti-mouse antibody is immobilized on the control line, a labeled antibody-anti-mouse antibody complex is formed by the extra labeled antibody or the labeled antibody-granulinicin complex and the anti-mouse antibody. A red line appears in the control line. The state of granulysin positive or negative and coloration in the control line is shown in FIG.

(2) Examination of dilution rate of serum when using granulysin detection kit The dilution rate of serum for suppressing nonspecific reaction in the granulysin detection kit prepared in this Example (1) was examined.

  To the serum collected from subjects who did not develop drug eruption, granulysin (Recombinant Human Granulinsin MAb; code 3138-GN / CF; lot OHB0508041; R & D Systems) was added to a concentration of 250 ng / mL. This was diluted with 3-fold dilution (granulinisin) using blocking buffer prepared with 0.1 M Tris buffer (pH 8.5), 0.1 v / v% Tween 20 and 0.2 w / v% skim milk, respectively. Serum at a concentration of about 83 ng / mL), a 10-fold dilution (granulinisin concentration of 25 ng / mL), and a 30-fold dilution (granulinisin concentration of about 8.3 ng / mL) were prepared. In addition, without adding granulysin to serum collected from subjects who did not develop drug eruption, similarly prepared using blocking buffer without dilution, 3-fold dilution, 10-fold dilution, and 30-fold dilution, respectively. And used as a control. 70 μL of each was dropped onto the sample pad of the granulysin detection kit prepared in Example (1), and after 15 minutes, the presence or absence of a red line on the test line, ie, granulysin positive or negative was confirmed. The results are shown in Table 2.

  As shown in Table 2, in the case of no dilution and 3-fold dilution, a red line was detected in the test line regardless of whether serum was added with granulysin, and it was confirmed to be positive for granulysin. At the 10-fold dilution and the 30-fold dilution, the red line was detected in the test line for the serum added with granulysin and confirmed to be positive for granulysin. However, for the serum added with granulysin, the red line was detected on the test line. Not detected and confirmed to be granulysin negative.

  From the above results, in the granulysin detection kit prepared in this Example (1), non-diluted serum and 3-fold diluted serum are detected non-specifically as granulysin positive, but diluted 10-fold and 30-fold. In the diluted serum, false positives were suppressed, and it was confirmed that positive was detected specifically for granulysin.

(3) Examination of dilution rate of serum when using granulysin detection kit and minimum detection concentration of granulysin contained in serum In order to suppress nonspecific reaction in granulysin detection kit prepared in this Example (1) The serum dilution ratio was examined in more detail, and the minimum detectable concentration was examined by changing the serum granulysin concentration.

  Sera collected from subjects who did not develop drug eruption were treated with granulysin (Recombinant Human Granulinsin MAb; code 3138-GN / CF; lot) to be 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively. OHB0508041; R & D Systems). This was diluted 3 times, 5 times, 10 times with blocking buffer prepared with 0.1 M Tris buffer (pH 8.5), 0.1 v / v% Tween 20 and 0.2 w / v% skim milk, respectively. Double dilution serum was prepared. In addition, without adding granulysin to serum collected from subjects who did not develop drug eruption, similarly, using a blocking buffer, prepare serum diluted 3 times, 5 times and 10 times, respectively, and control. Used as. 70 μL of each was dropped onto the sample pad of the granulysin detection kit prepared in Example (1), and after 15 minutes, the presence or absence of a red line on the test line, ie, granulysin positive or negative was confirmed. The results are shown in Table 3.

  As shown in Table 3, in the serum diluted 3-fold, when the serum granulysin concentration before dilution was 0 ng / mL and 5 ng / mL, a slight amount of granulysin was detected, and the same concentration was 10 ng / mL or more. In both cases, positive for granulysin was detected. In serum diluted 5-fold, granulysin positive was not detected when the serum granulysin concentration before dilution was 0 ng / mL and 5 ng / mL, and both were positive for granulysin when the concentration was 10 ng / mL or more. was detected. In serum diluted 10-fold, no positive for granulysin was detected when the serum granulysin concentration before dilution was 0 ng / mL, 5 ng / mL and 10 ng / mL, and when the same concentration was 20 ng / mL or more, Granulysin positive was detected.

   From the above results, in the granulysin detection kit prepared in this Example (1), the serum diluted 3 times may be detected as false positives. In order to suppress this false positive reaction, the serum diluted 5 times is used. It was confirmed that it was preferable to use it. In addition, in serum diluted 5 times, when the serum granulysin concentration before dilution is 5 ng / mL, it is not detected as positive, and when the concentration is 10 ng / mL or more, it is detected as positive, so it is not diluted. It was confirmed that the concentration of granulysin contained in the serum of the subject was preferably 10 ng / mL or more.

Claims (7)

  A marker serving as an index of susceptibility to severe drug eruption in a subject who does not develop drug eruption or a subject who develops mild drug eruption, wherein the marker is granulysin contained in serum collected from the subject, Marker for diagnosis of susceptibility to severe drug eruption.   A method for diagnosing severe drug rash susceptibility, using the amount of granulysin contained in serum collected from subjects who have not developed drug eruption or subjects who have developed mild drug eruption as an indicator of susceptibility to severe drug eruption . Diluting serum collected from a subject who has not developed a drug eruption or a subject who has developed a mild drug eruption; and
A step of forming a labeled antibody-granulaicin complex by binding granulysin contained in the diluted serum to a labeled antibody that is an anti-granulinic antibody labeled with a labeling substance;
And detecting a labeled antibody-granulinicin-capture antibody complex formed by binding the labeled antibody-granulinicin complex to a capture antibody that is an anti-granulinic antibody immobilized on a carrier. The method for diagnosing susceptibility to severe drug eruption according to claim 2.   The method for diagnosing susceptibility to severe drug eruptions according to claim 3, wherein the dilution ratio of serum is 5 times or more.   Any one of claims 2 to 4, wherein the amount of granulysin contained in serum collected from a subject who has not developed drug eruption or a subject who has developed mild drug eruption is susceptible when the amount of granulysin is 10 ng / mL or more. A method for diagnosing susceptibility to severe drug eruption according to claim 1.   A kit for diagnosis of susceptibility to severe drug eruption, comprising a carrier, a labeled antibody that is an anti-granulinicin antibody labeled with a labeling substance, and a capture antibody that is an anti-granulinicin antibody immobilized on the carrier.   The severe drug eruption according to claim 6, wherein the labeled antibody is an anti-granulinic antibody produced by a mouse-derived antibody-producing cell clone RB1, and the capture antibody is an anti-granulinic antibody produced by a mouse-derived antibody-producing cell clone RC8. Susceptibility diagnostic kit.

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