TWI606237B - Methods and identification kits for the identification of sensitized drugs for drug allergy - Google Patents

Description

Method and identification kit for identifying sensitizing drugs for drug allergic reactions

The invention relates to a method and a kit for identifying a sensitizing drug, in particular to a method and an identification kit for identifying a sensitizing drug for a drug allergic reaction, and co-cultivating a lymphocyte with a suspect drug or a metabolite thereof to form a reactant. By quantifying the content of granulysin protein, polypeptide or mRNA in the reactants and comparing with the control value, the sensitizing drug which triggers the allergic reaction of the drug can be identified, so that the invention has a single easy-to-execute technology, fast and economical. Excellent features such as high sensitivity and high specificity.

Drug allergies are a class of potentially fatal immune diseases caused by drugs, including minor skin rash (MPE), erythema multiforme majus (EMM), and fixed drug eruptions ( Fixed drug eruption (FDE), to severe cutaneous adverse reactions (SCAR), including drug rash with eosinophilia and systemic symptoms (DRESS) , Stevens-Johnson syndrome (SJS), and Toxic epidermal necrolysis (TEN).

SCAR is thought to be associated with drug-specific T-cells. The traditional in vitro assay method lymphocyte transformation test (LTT) is currently used in the detection of T cell-mediated drug allergic reactions. This method is a cell culture method for the activation and proliferation of T lymphocytes in vitro. vitro cell culture from patient blood of the separated lymphocytes to stimulation of suspicion sensitizing drugs, radiation measuring elements heavy hydrogen ⁽³ H) thymidine Down flag of the amount of DNA after one week, to react T lymphocytes The case of globular cell proliferation. However, the sensitivity of this method for SCAR is very low, and LTT is often negative for drug testing in SJS/TEN patients. In addition, radiological analysis experiments require experienced technicians and expensive instruments, potential health threats from radiation, and radiation-operated personnel and restricted radiation allow the operating environment to limit this analysis. Implementation.

Another method for diagnosing drug allergic reactions is to measure the amount of synthesis and secretion of interferon gamma (IFN- γ ), interleukin (IL)-2, 5, 13 and other cytokines. This method is to determine the cytokine content in the cell culture supernatant when T lymphocyte activation, or to determine the intracellular cytokine content by flow cytometry, or by polymerase chain reaction (Polymerase Chain Reaction, The amount of intracellular cytokine gene expression was determined by PCR or reverse transcription-PCR (RT-PCR). Although these cytokines can be used to identify sensitizing drugs for drug-allergic patients, they have no specificity of drug allergy and low sensitivity, and are not suitable for clinical use. Moreover, the method for detecting immune molecules on the surface of the blood cell requires more blood samples and the use of more expensive flow cytometers, low sensitivity, increased expenditure on purchasing reagents, and increased time and labor consumption.

The inventors have for the first time found that granulysin (GNLY) plays an important regulatory role in epidermal cell death in St. Johnson's Johnson & Johnson syndrome and toxic epidermal lysis. This finding can be applied to the diagnosis and treatment of Stevenson's Johnson & Johnson syndrome and toxic epidermal lysis (Patent No. TW I333978). However, this patent does not disclose how to use particulate lysin to identify sensitizing drugs that cause severe skin drug allergy disorders.

In summary, the traditional LTT method is a method that only has limited sensitivity and is costly. And the implementation of traditional LTT also depends on experienced technicians and holds licenses for radioactive substances and environmental restrictions. In addition, due to the low sensitivity of intracellular granulysin measured by flow cytometry, other methods are needed to identify sensitizing drugs that trigger SCAR. At present, there is no method with high sensitivity, high reliability, and low cost to identify sensitizing drugs that cause drug allergic reactions. Therefore, there is still room for improvement in the prior art.

The invention relates to a method for identifying a sensitizing drug for a drug allergic reaction, comprising the following steps: Step 1: co-culturing a bio-sample lymphocyte with a suspect drug or a metabolite thereof to form a reactant; Step 2: detecting the reactant The amount of granulysin protein, polypeptide or mRNA is compared with the control value. If the expression level of granulysin protein, polypeptide or mRNA is more than 1.2 times higher than the control value, the suspect drug or its Metabolites activate lymphocytes to a greater extent and are sensitizing drugs and may trigger drug allergic reactions.

The above-mentioned control value refers to the value of the granulysin expression amount of the biological sample lymphocyte cultured in the culture solution or the culture solution containing the solvent in which the drug is dissolved.

The second step is to react the granulysin protein or the multi-peptide of the reaction with a specific capture antibody and a detection antibody to detect the granulysin expression.

Wherein, the second step is to react the granulysin mRNA in the reaction with a specific oligonucleotide primer or probe to detect the amount of granulysin expression.

Among them, the method for capturing antibody and detecting antibody to identify the amount of granulysin in the reaction is enzyme-binding immunosorbent assay or enzyme-binding immunospot assay.

Wherein, the lymphocyte cells include body fluids isolated from peripheral blood cells or from biological individuals, preferably from peripheral blood.

Among them, the drug allergic reaction includes Stevenson Johnson & Johnson syndrome, toxic epidermal lysis, drug rash with eosinophilia and systemic symptoms, skin rash, severe erythema multiforme, and fixed drug eruption.

Among them, sensitizing drugs include western medicines, traditional Chinese medicines, vaccines, and antigen molecules that can cause T cell activation.

An identification kit for identifying a sensitizing drug that triggers an allergic reaction to a drug, comprising the following reagents: a test kit for separating a reagent from a biopsy lymphocyte and a suspect drug or a metabolite thereof Co-cultivating to form a reactant; a probe set, reacting the probe set with the reactant formed by the test set, and detecting the amount of granulysin protein, polypeptide or mRNA in the reaction, if the granule lysin The expression level of the protein, the polypeptide or the mRNA is more than 1.2 times higher than the control value, and the suspicious drug or its metabolite can be judged to be a sensitizing drug.

Wherein, the detection kit comprises a capture antibody specific for granulysin and a detection antibody to detect the expression amount of the granulysin protein or the multi-peptide in the reaction.

Wherein, the detection kit comprises a pair of oligonucleotide primers or probes specific to granulysin mRNA or genomic DNA to detect the amount of granulysin mRNA in the reaction.

Among them, the method for identifying the amount of granulysin in the reaction by using the capture antibody and the detection antibody is an enzyme-binding immunosorbent assay or an enzyme-binding immunospot assay.

Among them, drug allergic reactions include Stevenson Johnson & Johnson syndrome, toxic epidermal lysis, drug rash with eosinophilia and systemic symptoms, skin rash, severe erythema multiforme and Fixed drug rash.

Among them, sensitizing drugs include western medicines, traditional Chinese medicines, vaccines, and antigen molecules that can cause T cell activation.

The present invention combines a lymphocyte with a suspect drug or a metabolite thereof to form a reactant in vitro, and utilizes an oligonucleotide primer, a probe, an anti-granulysin protein or a multi-peptide capture antibody and a detection antibody. The step of binding the granulysin expressed by the activated lymphocytes comprises the concentration of the suspected sensitizing drug or its metabolite, the composition of the cell culture solution, and the time of cell culture. The invention has the advantages of single easy-to-execute technology, fast, economical, high sensitivity and high specificity.

The above described objects and features of the present invention will be apparent from the following description of the preferred embodiments of the invention.

Figure 1 is a block diagram of the detection process of the present invention.

Figure 2 is a fragmentation of lymphocytes and sensitizing drugs/metabolites or tolerant drugs of severe dermatological allergic diseases, tolerant patients and healthy subjects of the present invention after one to two weeks of in vitro culture. The amount of granule lysin measured by ELISA; the fold change of granule lysin is calculated by dividing the obtained data by the solvent control data.

Figure 3 is a dermal cell and a sensitizing drug/metabolite or tolerant drug of a severe dermatological allergic disease, a tolerant patient and a healthy subject of the present invention, cultured in vitro for one week to two weeks after IFN- The amount of expression of γ, wherein the amount of IFN-γ is measured by ELISA; the ratio of IFN-γ The number change calculation is obtained by dividing the obtained data by the solvent control data.

Figure 4 is a lysin mRNA of a lymphoblast cell and a 1 or 10 times sensitizing drug/metabolite or tolerant drug in a severe dermatological allergy disease caused by allopurinol in the present invention. The amount of expression, wherein the amount of granulysin mRNA is measured by RT-PCR; the fold change of granulysin is calculated by dividing the obtained data by the solvent control data.

Referring to Figures 1 to 4, the present invention is a method for identifying a sensitizing drug for a drug allergic reaction, comprising the following steps: Step 1: Biopsies lymphocytes and a suspect drug or a metabolite thereof are co-cultured in vitro to form a reactant. Step 2: Detect the amount of granulysin protein, polypeptide or mRNA in the reaction and compare it with the control value. If the expression of granulysin protein, polypeptide or mRNA is more than 1.2 times higher than the control value, It can be determined that this suspicious drug or its metabolite activates lymphocytes to a greater extent, is a sensitizing drug and may cause a drug allergic reaction.

The above-mentioned control value refers to the amount of granulysin expression amount in which the biopsy lymphocytes are cultured in the culture solution or the culture solution containing the solvent in which the drug is dissolved.

The second step is to react the granulysin protein or the multi-peptide of the reaction with a specific capture antibody and a detection antibody to detect the granulysin expression.

Wherein, the second step is to react the granulysin mRNA in the reaction with a specific oligonucleotide primer or probe to detect the amount of granulysin expression.

Among them, sensitizing drugs include western medicines, traditional Chinese medicines, vaccines, and antigen molecules that can cause T cell activation.

The above identification method is performed by measuring the granulysin expressed in the reactants. Identification of sensitizing drugs that trigger drug allergic reactions. Drug allergic reactions include: Stevens-Johnson syndrome (SJS), Toxic epidermal necrolysis (TEN), drug rash with eosinophilia Drug reaction with eosinophilia and systemic symptoms (DRESS), skin rash (maculopapular eruptions (MPE), erythema multiforme majus (EMM) and fixed drug eruption (FDE).

The performance and content of the granulysin protein or nucleic acid in the lymphocyte culture solution can be evaluated from the results of co-culture of the lymphocyte sample of the test subject with the suspected sensitizing drug/metabolite or tolerant drug. Lymphocytes include body fluids derived from cells in the peripheral blood or from biological individuals, preferably from peripheral blood. The amount of granulysin expression can be determined in several ways, including but not limited to the method of determining the mRNA transcribed from the granulysin gene, the amount of protein transduced by the gene, or the activity of the gene to translate the protein.

The mRNA isolated from the cells in the reaction can be detected by hybridization or amplification assays, including but not limited to the following methods: Northern blot analyses, polymerase chain reaction , and probe arrays. A preferred method for determining the mRNA content is to contact the isolated mRNA with a nucleic acid molecule (probe) that hybridizes to the mRNA of the gene to be tested. In the present invention, the nucleic acid molecule probe may be an intact granulysin nucleic acid having a full gene sequence length, or an oligonucleotide containing at least 7, 15, 30, 50, 100, or 250 nucleotides in length (oligonucleotide). ) and can fully hybridize to granulysin mRNA or genomic DNA under stringent conditions.

The granulysin mRNA content in the reaction can also be determined by nucleotide amplification techniques, such as reverse transcription polymerase chain reaction (RT-PCR), ligase chain reaction, autonomous sequence replication system, transcriptional amplification system, Q-Beta After amplification of the nucleotides by Replicase, rolling circle replication or any other method, the amplified molecules are determined using known techniques. The amplification primer used in the present invention is a pair of nucleic acid molecules which can be bonded to the 5' or 3' end of the gene region (plus plus minus strands, or vice-versa, respectively). In general, the amplification primer is composed of 10 to 30 nucleotide molecules and can be laterally extended to a region of 50 to 200 nucleotides in length. When appropriate conditions and reagents are available, the primer can amplify the nucleic acid molecule according to its own nucleotide sequence.

In the embodiment of the present invention, the method comprises the following steps: hybridizing a control sample with an amplification primer for detecting granulysin mRNA or genomic DNA, and comparing the performance of granulysin mRNA or genomic DNA in a control group and a test sample. .

The amount of granulysin protein in the reactants (supernatants) can be assayed by a number of methods, including the use of an agent that selectively binds to the granulysin protein or its antigen or its immunological fragment ( For example, the antibody is combined with the sample to evaluate the granulysin protein content of the sample. The technology includes enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT) assays, immunoprecipitations, immunofluorescence, and enzymes. Immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.

In the embodiment of the present invention, further comprising contacting the control sample with an antibody detecting granulysin, and comparing the granulysin protein expression in the control group and the test group. The method further comprises comparing the experimental values to a reference value (eg, the culture of the sample in culture medium/solvent).

The invention also includes an identification kit for identifying a sensitizing drug that triggers an allergic reaction to a drug, comprising the following reagents: a test kit, the test kit is a reagent and a biopsy lymph The globular cells are co-cultured with the suspect drug or its metabolite in vitro to form a reactant; a probe set is used to react the probe set with the reaction formed by the test set to detect the granulysin protein in the reaction, The multi-peptide or mRNA expression amount, if the expression amount of the granulysin protein, the multi-peptide or the mRNA is more than 1.2 times higher than the control value, it can be determined that the suspect drug or its metabolite is a sensitizing drug.

Wherein, the detection kit comprises a pair of oligonucleotide primers or probes specific to granulysin mRNA or genomic DNA to detect the amount of granulysin mRNA in the reaction.

Wherein, the detection kit comprises a capture antibody specific for granulysin and a detection antibody to detect the expression amount of the granulysin protein or the multi-peptide in the reaction.

Among them, the method for identifying the amount of granulysin in the reaction by using the capture antibody and the detection antibody is an enzyme-binding immunosorbent assay or an enzyme-binding immunospot assay.

Among them, sensitizing drugs include western medicines, traditional Chinese medicines, vaccines, and antigen molecules that can cause T cell activation.

The identification method described in the present invention can identify a sensitizing drug which can be triggered in an individual or has a risk of causing a drug allergic reaction, and the above-mentioned drug allergic reaction includes maculopapular eruptions (MPE) and fixed drug eruptions (fixed). Drug eruption, FDE), erythema multiforme majus (EMM), severe cutaneous adverse reactions (SCAR), drug rash with eosinophilia and systemic symptoms (Drug reaction with eosinophilia and Systemic symptoms (DRESS), Stevens-Johnson syndrome (SJS), and Toxic epidermal necrolysis (TEN).

This method may further comprise comparing the experimental values to the control values (eg, the culture of the sample in the culture medium/solvent). The results of granulysin expression can be obtained from any of the methods described herein. For example, the nucleic acid obtained in the reaction is contacted with an oligonucleotide primer or a probe, or the reactant is contacted with an anti-granulolytic antibody.

The present application comprises a method of co-culturing lymphocytes in vitro with a suspected drug or a metabolite thereof, measuring its granulysin expression by ELISA or ELISPOT, or measuring its lysin mRNA by real-time quantitative PCR. The method steps are as follows: lymphocytes are isolated from the whole blood of the patient, and the lymphocytes are cultured in 96-well microplates containing RPMI-1640 medium at 37 ° C and 5% CO ₂ . In addition, the co-cultured drugs or drug metabolites, and tolerant drugs are diluted to a physiologically therapeutic level: 1 fold (1-fold, 1X), 0.1 fold (0.1-fold, 0.1X) ), and 10 times (10-fold, 10X), co-culture for one to two weeks.

Lymphocytes and suspected drugs/metabolites were co-cultured: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood samples by Fischer-Paque (Pharmacia Fine Chemicals, USA) by density gradient centrifugation. PBMC (1.0x10 ⁶ /well) was cultured in a 96-well culture dish containing 10% autologous serum and IL7 (1 ng/ml) in RPMI-1640 medium (GIBCO Invitrogen, Life Technologies, Carlsbad, CA) at 37 ° C and 5 The conditions of % CO ₂ are cultured for one to two weeks. Suspicious sensitizing drugs, drug metabolites, and tolerant drugs co-cultured are diluted to one, one tenth, or ten times the physiologically therapeutic level. For example, the cells are cultured in a tolerant drug culture medium containing oxypurinol (10 μg/mL, 100 μg/mL) or containing the patient for more than three months without causing a drug allergic reaction. In addition, the negative control is added to the culture solution using a solvent for dissolving the drug, and the positive control is a culture solution to which a concentration of 10 μg/mL of phytohemagglutinin (PHA) is added.

ELISA quantification of granulysin performance: After culture, the supernatant was collected on the seventh and fourteenth days, and the granulysin was measured by ELISA. Briefly, the culture plate (Nunc, Roskilde, Denmark) was first coated with 50 μg/ml anti-granulysin monoclonal antibody G011, and then washed with 10% FBS washing buffer (PBS containing 0.1% Tween-20). Next, the following series of reactions were carried out at room temperature: the reaction to be detected was added for two hours; 1 μg/ml of biotin-labeled anti-granulin monoclonal antibody G052 was added to the blocking buffer for one hour; 2 μg/ml of horseradish peroxidase- The conjugated streptavidin rinses the plate with a washing buffer in the washing buffer. Finally, the culture plate is added to the substrate solution containing H ₂ O ₂ reaction, and the tetramethylbenzinine reaction is added after the plate is rinsed. The analytical sensitivity of granulysin was 1.56 ng/ml. In addition, the analytical sensitivity of IFN- γ in the sample to IFN- γ ELISA kits (Invitrogen, Carlsbad, CA) was 1.56 pg/ml.

Quantitative Real-Time RT-PCR: RNA (Total RNA) isolated from cultured lymphocytes. The granulysin mRNA content was measured using a Light Cycler (Roche molecular Biochemicals)-based master SYBR Green 1 kit. The absolute number of sets will be confirmed by the method described by Matsushita et al. (Matsushita et al. Br J Haematol. 2001 March; 112(4): 916-26). The following are the oligonucleotides used in the method: granulysin: 5'-TCTCTCGTCTGAGCCC-3', 5'-GCAGCATTGGAAACACT-3'; β-actin: 5'-ACATCCGCAAAGACCT-3', 5' -AGGG TGTAACGCAACTA-3'.

Statistical analysis: Lymphocytes are stabbed by suspicious drugs/metabolites or tolerant drugs After the agitation, the fold change of the granulysin in the culture supernatant was calculated by dividing the obtained data by the solvent control data. Significant differences between groups were analyzed by one sample t test or student's t test. The calculation of sensitivity and specificity is based on its standard definition. All P values were two-tailed, with a P value <0.05 indicating a statistically significant difference in this analysis. Statistical analysis was performed with SPSS software Version 18.0 (SPSS Inc, Chicago, IL).

In order to detect whether granulysin can be used to identify sensitizing drugs that trigger drug allergic reactions, this example collected 22 patients with severe cutaneous adverse drug reactions, including: SJS, TEN, DRESS, FDE and EMM and so on. After stimulating the lymphocytes for one week or two weeks with sensitizing drugs or metabolites, the granulysin and IFN- γ content in the samples were measured by ELISA, and the sensitivity was compared. The results of the data show that the sensitivity of granulysin can reach 77.3-81.8%; while the sensitivity of IFN- γ is only less than 20% (Table 1). Therefore, this experiment demonstrates that the use of ELISA to detect granulysin is highly sensitive in the application of the granulysin-based drug lymphocyte stimulation test, and can be used to identify sensitizing drugs that trigger drug allergic reactions.

After one to two weeks of stimulation, the sensitivity comparison results of the released granulysin and IFN- γ were calculated by ELISA.

The present invention further examines the specificity of granulysins for identifying sensitizing drugs that trigger a drug allergic reaction. In order to confirm that in this evaluation method, only the lymphocytes and sensitizing drugs/metabolites of drug-sensitive patients are cultured in vitro, the amount of granulysin is increased, and drug-tolerant patients and healthy subjects The lymphocytes do not have this phenomenon. In this example, 11 drug-tolerant patients and 10 healthy subjects were collected, and their lymphocytes were stimulated with their tolerant drugs or metabolites for one week or two. After week, the content of granule lysin and IFN- γ in the biopsies was measured by ELISA, and the specificity was compared. The data showed that in the tolerant patient test, the specificity of granulysin was 95.7% after one week of culture, while the specificity of IFN- γ was only 76.2%; the specificity of granulysin after two weeks of culture was 92.9%, the specificity of IFN- γ was 77.8% (Table 2). As for the healthy subjects, the specificity of granulysin was 86.7% after two weeks of culture; the specificity of IFN- γ was 75.6% (Table 2). Therefore, this experiment demonstrates that the use of ELISA to detect granulysin is highly specific in the application of the granulysin-based drug lymphocyte stimulation test, and can be used to identify sensitizing drugs that trigger drug allergic reactions.

Specificity comparison results.

Lymphocytes and sensitizing drugs/metabolites or tolerant drugs in severe dermatological allergic patients, tolerant patients and healthy subjects are stimulated in vitro for one to two weeks, and their granules are measured by ELISA. The amount of IFN- γ expression is shown in Figures 2 and 3. The results showed that the lymphocyte cells of severe dermatological allergic patients showed a significant increase in granulysin after stimulation with sensitizing drugs/metabolites, while there was no significant difference between tolerant patients and healthy subjects (Fig. 2A) And Figure 2B). Although the expression of IFN- γ is also increased after stimulation of lymphocytes in some patients with severe skin drug allergies (Fig. 3A and Fig. 3B), it is only limited in a few cases and is not sufficient as specificity. Biomarker. Therefore, this result again points out that the use of ELISA to detect granulysin is the best method for identifying sensitizing drugs that trigger drug allergic reactions in a granulysin-based drug lymphocyte stimulation test.

Referring to Figure 4, at the mRNA level, this example collected 3 patients with severe dermatological drug allergy caused by allopurinol, and their lymphocytes were sensitized with 1 or 10 times sensitizing drugs/metabolites or other tolerances. One week after the drug was cultured in vitro, the amount of granulysin mRNA was measured by RT-PCR. The results also showed that the granulysin had better sensitivity (66.7%) and better specificity (100%), whether it was 1 or 10 times sensitizing drug/metabolite stimulation.

All of the above examples show that granulysin (detected by RT-PCR or ELISA) is highly sensitive and specific for use in the in vitro detection of the granulysin-based drug lymphocyte stimulation test. To identify sensitizing drugs that trigger a drug allergic reaction. The invention provides a method for measuring the granulysin mRNA and protein expression in a drug lymphocyte activation test in vitro; and also provides a granulysin-based drug lymphocyte stimulation test in vitro. Test) A kit or kit for measuring the expression of granulysin mRNA and protein, including primers and probes that can be used to detect granulysin mRNA, and antibodies that recognize lysin.

The present invention has been described with reference to the preferred embodiments of the present invention. However, those skilled in the art can change and modify the present invention without departing from the spirit and scope of the invention. Such changes and modifications shall be covered in the scope defined by the following patent application.

<110> Zhong Wenhong

<120> Methods and identification kits for identifying allergic drugs for drug allergic reactions

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Claims (11)

A method for identifying a sensitizing drug that may cause an allergic reaction to a severe skin drug, comprising the following steps: Step 1: Biopsies lymphocytes and a suspected drug or a metabolite thereof diluted from one tenth to ten times the physiological concentration Forming a supernatant in a co-culture in vitro for one week to two weeks; Step 2: detecting the amount of granulysin protein or multi-peptide in the supernatant and comparing it with a control value if the supernatant granulysin protein Or the performance of the multi-peptide is higher than the control value, and it can be determined that the suspect drug or its metabolite is a sensitizing drug which may cause an allergic reaction to a severe skin drug. A method for identifying a sensitizing drug which may cause an allergic reaction to a severe skin drug, as described in claim 1, wherein the second step is to specifically capture the granulysin protein or the peptide in the supernatant. The antibody and the detection antibody react to detect the amount of granulysin expression. A method for identifying a sensitizing drug which may cause an allergic reaction to a severe skin drug, as described in claim 1, wherein the lymphocyte comprises a body fluid isolated from a peripheral blood cell or from a biological individual. A method for identifying a sensitizing drug which may cause an allergic reaction to a severe skin drug, as described in claim 2, wherein the method for capturing antibody and detecting the amount of granulysin in the antibody is an enzyme-binding immunosorbent assay. Method or enzyme combined with immunospot assay. A method for identifying a sensitizing drug that may cause an allergic reaction to a severe skin drug, as described in claim 1, wherein a severe skin drug allergic reaction includes a history Empowered Johnson & Johnson syndrome, toxic epidermal lysis, drug rash with eosinophilia and systemic symptoms, skin rash, severe erythema multiforme, or fixed drug eruption. A method for identifying a sensitizing drug which may cause an allergic reaction to a severe skin drug, as described in claim 1, wherein the sensitizing drug comprises a western medicine, a Chinese medicine, a vaccine, and an antigen molecule capable of causing T cell activation. An identification kit for identifying sensitizing drugs that may cause severe skin drug allergic reactions, including the following reagents: a test kit for one-fold to ten-fold dilution of a reagent, biopsy lymphocyte The physiotherapeutic concentration of the suspicious drug or its metabolite is co-cultured in vitro to form a supernatant in a week to two weeks; a detection kit is used to react the probe set with the supernatant formed by the test kit And detecting the expression amount of the granulysin protein or the multi-peptide in the supernatant, and if the expression amount of the granulysin protein or the multi-peptide of the supernatant is higher than the control value, the suspect drug or its metabolite may be determined as Sensitizing drugs that may cause severe skin drug allergic reactions. The identification kit of claim 7, wherein the detection kit comprises a capture antibody specific for granulysin and a detection antibody to detect the amount of granulysin protein expression. The identification kit of claim 8, wherein the method for identifying the amount of granulysin in the reaction by using the capture antibody and the detection antibody is an enzyme-binding immunosorbent assay or an enzyme-binding immunospot assay. For example, the identification kit described in claim 7 of the patent scope, wherein the severe skin drug allergic reaction includes Stevenson Johnson & Johnson syndrome, toxic epidermal lysis, drug rash Combined with eosinophilia and systemic symptoms, skin rash, severe erythema multiforme, or fixed drug eruption. The identification kit of claim 7, wherein the sensitizing drug comprises a western medicine, a traditional Chinese medicine, a vaccine, and an antigen molecule capable of causing T cell activation.